Academic Appointments

Honors & Awards

  • Heroic Achievement in Information Technology, The ComputerWorld Smithsonian Award (June 4, 1996)
  • Symposium Chair & Speaker, International Congress of Immunology (1986)
  • Member, Genetics Society of America (0)
  • Member, American Association of Immunologists (0)
  • Eleanor Roosevelt Cancer Fellowship,, American Cancer Society (1986)

Professional Education

  • D.Sc-equiv., Universioty Paris V Sorbonne, Immunolgy (1981)

Research & Scholarship

Current Research and Scholarly Interests

We maintain jointly functioning laboratory groups. Our focus is on gene regulation in the immune system, development and function of B cell subpopulations, and applications of Fluorescence-Activated Cell Sorting(FACS).

We have been interested for many years in the genetics and development of the immune system in mice. A major tool we have developed to aid us in this work is the Fluorescence-Activated Cell Sorter(FACS), which permits analysis and sorting of viable cells very rapidly on the basis of fluorescence marking. Recently we have devised a method to study gene regulation at the individual cell level using the FACS and a fluorogenic substrate for E. coli lac Z-encoded -galactosidase (-gal).

Current Studies include:

1) A new population of B cells, Ly-1B, which follow different rules of differentiation than conventional B-cells, have different V gene repertoires and provide a model for autoimmune diseases and chronic lymphocytic leukemia (CLL).

2) Regulation of lymphocyte development, especially B and T cell development.

3) Cis and trans-acting elements regulating the variation of -gal expresstion in populations of lymphoid cells infected with lacZ recombinant retroviruses in which various viral and cellular promoters "drive" lacZ.

4) Cell lineage studies using introduced lacZ rDNA retroviruses as cellular markers with the FACS / b-gal assay.

5) The cell biology of AIDS in model systems including activation of b-gal expression in cells carrying the lacZ gene under control of HIV promoters and regulatory elements.

6) What transgenic mice can and can't tell us about cellular and molecular development of the immune system.

In addition to improving FACS operations with a dedicated group of engineers, computer scientists, programmers, and a physicist, opportunities always exist for technologically oriented students interested in molecular biology and immunology.


2014-15 Courses

Graduate and Fellowship Programs


All Publications

  • AutoGate: automating analysis of flow cytometry data IMMUNOLOGIC RESEARCH Meehan, S., Walther, G., Moore, W., Orlova, D., Meehan, C., Parks, D., Ghosn, E., Philips, M., Mitsunaga, E., Waters, J., Kantor, A., Okamura, R., Owumi, S., Yang, Y., Herzenberg, L. A., Herzenberg, L. A. 2014; 58 (2-3): 218-223


    Nowadays, one can hardly imagine biology and medicine without flow cytometry to measure CD4 T cell counts in HIV, follow bone marrow transplant patients, characterize leukemias, etc. Similarly, without flow cytometry, there would be a bleak future for stem cell deployment, HIV drug development and full characterization of the cells and cell interactions in the immune system. But while flow instruments have improved markedly, the development of automated tools for processing and analyzing flow data has lagged sorely behind. To address this deficit, we have developed automated flow analysis software technology, provisionally named AutoComp and AutoGate. AutoComp acquires sample and reagent labels from users or flow data files, and uses this information to complete the flow data compensation task. AutoGate replaces the manual subsetting capabilities provided by current analysis packages with newly defined statistical algorithms that automatically and accurately detect, display and delineate subsets in well-labeled and well-recognized formats (histograms, contour and dot plots). Users guide analyses by successively specifying axes (flow parameters) for data subset displays and selecting statistically defined subsets to be used for the next analysis round. Ultimately, this process generates analysis "trees" that can be applied to automatically guide analyses for similar samples. The first AutoComp/AutoGate version is currently in the hands of a small group of users at Stanford, Emory and NIH. When this "early adopter" phase is complete, the authors expect to distribute the software free of charge to .edu, .org and .gov users.

    View details for DOI 10.1007/s12026-014-8519-y

    View details for Web of Science ID 000336333700007

  • The early history of Stanford Immunology IMMUNOLOGIC RESEARCH Jones, P. P., Herzenberg, L. A. 2014; 58 (2-3): 164-178


    From its 1960 beginnings in a pair of windowless Genetics Department laboratories under the Stanford Medical School Dean's Office to its current broad-based program, which joins faculty members from departments across the Medical School, the Stanford Immunology Program has played a central role in shaping both basic and clinical immunology thinking. In this article, we tell the story of the beginnings of this odyssey in a reminiscence-based format that brings the flavor of the time in the words of people who lived and built the history.

    View details for DOI 10.1007/s12026-014-8518-z

    View details for Web of Science ID 000336333700002

    View details for PubMedID 24804901

  • A Conversation with Leonard and Leonore Herzenberg ANNUAL REVIEW OF PHYSIOLOGY, VOL 76 Herzenberg, L. A., Herzenberg, L. A., Roederer, M. 2014; 76: 1-20


    Leonard and Leonore Herzenberg have left an indelible mark on the fields of immunology and cell biology, both in research and clinical aspects. They are perhaps best known for developing the technologies of fluorescence flow cytometry and hybridomas. Over six decades, they made a number of important and fundamental discoveries in lymphocyte biology by applying these technologies. During this era, they immersed themselves in the sociopolitical environment, interjecting scientific rationale into public discourse about McCarthyism, nuclear fallout, war, genetics, and other politically charged topics. Their unique philosophy has shaped their lives, their science, and ultimately the scientific community. In this Conversation, we explore some of these driving forces and the impact on the laboratory.

    View details for DOI 10.1146/annurev-physiol-021113-170355

    View details for Web of Science ID 000336055000002

    View details for PubMedID 23957332

  • Our NIH Years: A Confluence of Beginnings JOURNAL OF BIOLOGICAL CHEMISTRY Herzenberg, L. A., Herzenberg, L. A. 2013; 288 (1): 687-702

    View details for DOI 10.1074/jbc.X112.426742

    View details for Web of Science ID 000313197200069

    View details for PubMedID 23066019

  • Epitope-specific regulation: the elephant in the bathtub NATURE IMMUNOLOGY Herzenberg, L. A. 2007; 8 (8): 783-786

    View details for DOI 10.1038/ni0807-783

    View details for Web of Science ID 000248169400002

    View details for PubMedID 17641654

  • Interpreting flow cytometry data: a guide for the perplexed NATURE IMMUNOLOGY Herzenberg, L. A., Tung, J., Moore, W. A., Herzenberg, L. A., Parks, D. R. 2006; 7 (7): 681-685

    View details for Web of Science ID 000238377700002

    View details for PubMedID 16785881

  • Genetics, facs, immunology, and redox: A tale of two lives intertwined ANNUAL REVIEW OF IMMUNOLOGY Herzenberg, L. A., Herzenberg, L. A. 2004; 22: 1-31
  • Long-term treatment with oral N-acetylcysteine: Affects lung. function but not sputum inflammation in cystic fibrosis subjects. A phase II randomized placebo-controlled trial JOURNAL OF CYSTIC FIBROSIS Conrad, C., Lymp, J., Thompson, V., Dunn, C., DAVIES, Z., CHATFIELD, B., Nichols, D., Clancy, J., Vender, R., Egan, M. E., Quittell, L., Michelson, P., Antony, V., Spahr, J., Rubenstein, R. C., Moss, R. B., Herzenberg, L. A., Goss, C. H., Tirouvanziam, R. 2015; 14 (2): 219-227


    To evaluate the effects of oral N-acetylcysteine (NAC), which replenishes systemic glutathione, on decreasing inflammation and improving lung function in CF airways.A multicenter, randomized, double-blind proof of concept study in which 70 CF subjects received NAC or placebo orally thrice daily for 24weeks.primary, change in sputum human neutrophil elastase (HNE) activity; secondary, FEV1 and other clinical lung function measures; and safety, the safety and tolerability of NAC and the potential of NAC to promote pulmonary hypertension in subjects with CF.Lung function (FEV1 and FEF25-75%) remained stable or increased slightly in the NAC group but decreased in the placebo group (p=0.02 and 0.02). Log10 HNE activity remained equal between cohorts (difference 0.21, 95% CI -0.07 to 0.48, p=0.14).NAC recipients maintained their lung function while placebo recipients declined (24week FEV1 treatment effect=150mL, p<0.02). However no effect on HNE activity and other selected biomarkers of neutrophilic inflammation were detected. Further studies on mechanism and clinical outcomes are warranted.

    View details for DOI 10.1016/j.jcf.2014.08.008

    View details for Web of Science ID 000352662600008

    View details for PubMedID 25228446

  • Cysteine Oxidation Targets Peroxiredoxins 1 and 2 for Exosomal Release through a Novel Mechanism of Redox-Dependent Secretion MOLECULAR MEDICINE Mullen, L., Hanschmann, E., Lillig, C. H., Herzenberg, L. A., Ghezzi, P. 2015; 21


    Nonclassical protein secretion is of major importance as a number of cytokines and inflammatory mediators are secreted via this route. Current evidence indicates that there are several mechanistically distinct methods of nonclassical secretion. We have shown recently that peroxiredoxin (Prdx) 1 and Prdx2 are released by various cells upon exposure to inflammatory stimuli such as lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-?). The released Prdx then acts to induce production of inflammatory cytokines. However, Prdx1 and 2 do not have signal peptides and therefore must be secreted by alternative mechanisms, as has been postulated for the inflammatory mediators interleukin-1? (IL-1?) and high mobility group box-1 (HMGB1). We show here that circulating Prdx1 and 2 are present exclusively as disulfide-linked homodimers. Inflammatory stimuli also induce in vitro release of Prdx1 and 2 as disulfide-linked homodimers. Mutation of cysteines Cys51 or Cys172 (but not Cys70) in Prdx2, and Cys52 or Cys173 (but not Cys71 or Cys83) in Prdx1 prevented dimer formation and this was associated with inhibition of their TNF-?-induced release. Thus, the presence and oxidation of key cysteine residues in these proteins are a prerequisite for their secretion in response to TNF-?, and this release can be induced with an oxidant. By contrast, the secretion of the nuclear-associated danger signal HMGB1 is independent of cysteine oxidation, as shown by experiments with a cysteine-free HMGB1 mutant. Release of Prdx1 and 2 is not prevented by inhibitors of the classical secretory pathway, instead, both Prdx1 and 2 are released in exosomes from both human embryonic kidney (HEK) cells and monocytic cells. Serum Prdx1 and 2 also are associated with the exosomes. These results describe a novel pathway of protein secretion mediated by cysteine oxidation that underlines the importance of redox-dependent signaling mechanisms in inflammation.

    View details for DOI 10.2119/molmed.2015.00033

    View details for Web of Science ID 000355726900010

    View details for PubMedID 25715249

  • Linkage of inflammation and oxidative stress via release of glutathionylated peroxiredoxin-2, which acts as a danger signal PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Salzano, S., Checconi, P., Hanschmann, E., Lillig, C. H., Bowler, L. D., Chan, P., Vaudry, D., Mengozzi, M., Coppo, L., Sacre, S., Atkuri, K. R., Sahaf, B., Herzenberg, L. A., Herzenberg, L. A., Mullen, L., Ghezzi, P. 2014; 111 (33): 12157-12162


    The mechanism by which oxidative stress induces inflammation and vice versa is unclear but is of great importance, being apparently linked to many chronic inflammatory diseases. We show here that inflammatory stimuli induce release of oxidized peroxiredoxin-2 (PRDX2), a ubiquitous redox-active intracellular enzyme. Once released, the extracellular PRDX2 acts as a redox-dependent inflammatory mediator, triggering macrophages to produce and release TNF-?. The oxidative coupling of glutathione (GSH) to PRDX2 cysteine residues (i.e., protein glutathionylation) occurs before or during PRDX2 release, a process central to the regulation of immunity. We identified PRDX2 among the glutathionylated proteins released in vitro by LPS-stimulated macrophages using mass spectrometry proteomic methods. Consistent with being part of an inflammatory cascade, we find that PRDX2 then induces TNF-? release. Unlike classical inflammatory cytokines, PRDX2 release does not reflect LPS-mediated induction of mRNA or protein synthesis; instead, PRDX2 is constitutively present in macrophages, mainly in the reduced form, and is released in the oxidized form on LPS stimulation. Release of PRDX2 is also observed in human embryonic kidney cells treated with TNF-?. Importantly, the PRDX2 substrate thioredoxin (TRX) is also released along with PRDX2, enabling an oxidative cascade that can alter the -SH status of surface proteins and thereby facilitate activation via cytokine and Toll-like receptors. Thus, our findings suggest a model in which the release of PRDX2 and TRX from macrophages can modify the redox status of cell surface receptors and enable induction of inflammatory responses. This pathway warrants further exploration as a potential novel therapeutic target for chronic inflammatory diseases.

    View details for DOI 10.1073/pnas.1401712111

    View details for Web of Science ID 000340438800063

    View details for PubMedID 25097261

  • Selective uptake of single-walled carbon nanotubes by circulating monocytes for enhanced tumour delivery NATURE NANOTECHNOLOGY Smith, B. R., Ghosn, E. E., Rallapalli, H., Prescher, J. A., Larson, T., Herzenberg, L. A., Gambhir, S. S. 2014; 9 (6): 481-487


    In cancer imaging, nanoparticle biodistribution is typically visualized in living subjects using 'bulk' imaging modalities such as magnetic resonance imaging, computerized tomography and whole-body fluorescence. Accordingly, nanoparticle influx is observed only macroscopically, and the mechanisms by which they target cancer remain elusive. Nanoparticles are assumed to accumulate via several targeting mechanisms, particularly extravasation (leakage into tumour). Here, we show that, in addition to conventional nanoparticle-uptake mechanisms, single-walled carbon nanotubes are almost exclusively taken up by a single immune cell subset, Ly-6C(hi) monocytes (almost 100% uptake in Ly-6C(hi) monocytes, below 3% in all other circulating cells), and delivered to the tumour in mice. We also demonstrate that a targeting ligand (RGD) conjugated to nanotubes significantly enhances the number of single-walled carbon nanotube-loaded monocytes reaching the tumour (P < 0.001, day 7 post-injection). The remarkable selectivity of this tumour-targeting mechanism demonstrates an advanced immune-based delivery strategy for enhancing specific tumour delivery with substantial penetration.

    View details for DOI 10.1038/NNANO.2014.62

    View details for Web of Science ID 000336971600021

    View details for PubMedID 24727688

  • Metabolic Adaptation of Neutrophils in Cystic Fibrosis Airways Involves Distinct Shifts in Nutrient Transporter Expression JOURNAL OF IMMUNOLOGY Laval, J., Touhami, J., Herzenberg, L. A., Conrad, C., Taylor, N., Battini, J., Sitbon, M., Tirouvanziam, R. 2013; 190 (12): 6043-6050


    Inflammatory conditions can profoundly alter human neutrophils, a leukocyte subset generally viewed as terminally differentiated and catabolic. In cystic fibrosis (CF) patients, neutrophils recruited to CF airways show active exocytosis and sustained phosphorylation of prosurvival, metabolic pathways. Because the CF airway lumen is also characterized by high levels of free glucose and amino acids, we compared surface expression of Glut1 (glucose) and ASCT2 (neutral amino acids) transporters, as well as that of PiT1 and PiT2 (inorganic phosphate transporters), in blood and airway neutrophils, using specific retroviral envelope-derived ligands. Neither nutrient transporter expression nor glucose uptake was altered on blood neutrophils from CF patients compared with healthy controls. Notably, however, airway neutrophils of CF patients had higher levels of PiT1 and Glut1 and increased glucose uptake compared with their blood counterparts. Based on primary granule exocytosis and scatter profiles, CF airway neutrophils could be divided into two subsets, with one of the subsets characterized by more salient increases in Glut1, ASCT2, PiT1, and PiT2 expression. Moreover, in vitro exocytosis assays of blood neutrophils suggest that surface nutrient transporter expression is not directly associated with primary (or secondary) granule exocytosis. Although expression of nutrient transporters on CF blood or airway neutrophils was not altered by genotype, age, gender, or Pseudomonas aeruginosa infection, oral steroid treatment decreased Glut1 and PiT2 levels in blood neutrophils. Thus, neutrophils recruited from blood into the CF airway lumen display augmented cell surface nutrient transporter expression and glucose uptake, consistent with metabolic adaptation.

    View details for DOI 10.4049/jimmunol.1201755

    View details for Web of Science ID 000320373700017

    View details for PubMedID 23690474

  • Blastocyst complementation generates exogenic pancreas in vivo in apancreatic cloned pigs PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Matsunari, H., Nagashima, H., Watanabe, M., Umeyama, K., Nakano, K., Nagaya, M., Kobayashi, T., Yamaguchi, T., Sumazaki, R., Herzenberg, L. A., Nakauchi, H. 2013; 110 (12): 4557-4562


    In the field of regenerative medicine, one of the ultimate goals is to generate functioning organs from pluripotent cells, such as ES cells or induced pluripotent stem cells (PSCs). We have recently generated functional pancreas and kidney from PSCs in pancreatogenesis- or nephrogenesis-disabled mice, providing proof of principle for organogenesis from PSCs in an embryo unable to form a specific organ. Key when applying the principles of in vivo generation to human organs is compensation for an empty developmental niche in large nonrodent mammals. Here, we show that the blastocyst complementation system can be applied in the pig using somatic cell cloning technology. Transgenic approaches permitted generation of porcine somatic cell cloned embryos with an apancreatic phenotype. Complementation of these embryos with allogenic blastomeres then created functioning pancreata in the vacant niches. These results clearly indicate that a missing organ can be generated from exogenous cells when functionally normal pluripotent cells chimerize a cloned dysorganogenetic embryo. The feasibility of blastocyst complementation using cloned porcine embryos allows experimentation toward the in vivo generation of functional organs from xenogenic PSCs in large animals.

    View details for DOI 10.1073/pnas.1222902110

    View details for Web of Science ID 000317521600039

    View details for PubMedID 23431169

  • H-Y antigen-binding B cells develop in male recipients of female hematopoietic cells and associate with chronic graft vs. host disease PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sahaf, B., Yang, Y., Arai, S., Herzenberg, L. A., Herzenberg, L. A., Miklos, D. B. 2013; 110 (8): 3005-3010


    B cells are known to play an important role in pathogenesis of human chronic graft vs. host disease (cGVHD). Our group has previously shown that IgG allo-antibodies recognize Y chromosome-encoded proteins (H-Y) and a dominant H-Y epitope, DEAD box protein (DBY-2) detectable 6-12 mo after transplant in male patients who receive grafts from female donors (F?M) hematopoietic cell transplantation (HCT). Here we present FACS studies of peripheral blood mononuclear cells collected 6 mo after transplant showing that 16 of 28 (57%) F?M HCT patients have circulating donor B cells that express B-cell receptor (mainly IgM and Ig?) specific for DBY-2. The detection of these DBY-2 B cells 6 mo after HCT are associated with cGVHD development (P = 0.004). Specifically, 15 of 16 F?M with DBY-2 B cells developed cGVHD. In contrast, cGVHD developed in only 5 of the 12 who did not have DBY-2 B cells detected. This demonstrates circulating human B cells binding an alloantigen (DBY-2) and that these DBY-2-specific B cells appear before development of cGVHD in roughly half of the F?M patients. Our study suggests that detection of anti-DBY-2 B cells may predict cGVHD and that this prediction may have clinical utility. Validation of this hypothesis will require larger prospective studies.

    View details for DOI 10.1073/pnas.1222900110

    View details for Web of Science ID 000315954400081

    View details for PubMedID 23382226

  • Blood basophils from cystic fibrosis patients with allergic bronchopulmonary aspergillosis are primed and hyper-responsive to stimulation by aspergillus allergens JOURNAL OF CYSTIC FIBROSIS Gernez, Y., Dunn, C. E., Everson, C., Mitsunaga, E., Gudiputi, L., Krasinska, K., Davies, Z. A., Herzenberg, L. A., Tirouvanziam, R., Moss, R. B. 2012; 11 (6): 502-510


    Fifteen to sixty percent of cystic fibrosis patients harbor Aspergillus fumigatus (Af) in their airways (CF-AC) and some will develop allergic bronchopulmonary aspergillosis (CF-ABPA). Since basophils play a key role in allergy, we hypothesized that they would display alterations in CF-ABPA patients compared to CF-AC or patients without Af colonization (CF).Using flow cytometry, we measured CD203c, CD63 and CD123 levels on basophils from CF-ABPA (N=11), CF-AC (N=14), and CF (N=12) patients before and after ex vivo stimulation with Af allergens.Baseline CD203c was increased in basophils from CF-ABPA compared to CF-AC and CF patients. Af extract and recombinant Aspf1 stimulated basophils from CF-ABPA patients to markedly upregulate CD203c, along with modest upregulation of CD63 and a CD123 downward trend. Plasma TARC/CCL17 at baseline and post-stimulation cell supernatant histamine levels were similar in the three groups.In CF-ABPA, blood basophils are primed and hyperresponsive to Af allergen stimulation.

    View details for DOI 10.1016/j.jcf.2012.04.008

    View details for Web of Science ID 000312115900004

    View details for PubMedID 22608296

  • Reversal of Paralysis and Reduced Inflammation from Peripheral Administration of beta-Amyloid in T(H)1 and T(H)17 Versions of Experimental Autoimmune Encephalomyelitis SCIENCE TRANSLATIONAL MEDICINE Grant, J. L., Ghosn, E. E., Axtell, R. C., Herges, K., Kuipers, H. F., Woodling, N. S., Andreasson, K., Herzenberg, L. A., Herzenberg, L. A., Steinman, L. 2012; 4 (145)


    ?-Amyloid 42 (A?42) and ?-amyloid 40 (A?40), major components of senile plaque deposits in Alzheimer's disease, are considered neurotoxic and proinflammatory. In multiple sclerosis, A?42 is up-regulated in brain lesions and damaged axons. We found, unexpectedly, that treatment with either A?42 or A?40 peptides reduced motor paralysis and brain inflammation in four different models of experimental autoimmune encephalomyelitis (EAE) with attenuation of motor paralysis, reduction of inflammatory lesions in the central nervous system (CNS), and suppression of lymphocyte activation. A?42 and A?40 treatments were effective in reducing ongoing paralysis induced with adoptive transfer of either autoreactive T helper 1 (T(H)1) or T(H)17 cells. High-dimensional 14-parameter flow cytometry of peripheral immune cell populations after in vivo A?42 and A?40 treatment revealed substantial modulations in the percentage of lymphoid and myeloid subsets during EAE. Major proinflammatory cytokines and chemokines were reduced in the blood after A? peptide treatment. Protection conferred by A? treatment did not require its delivery to the brain: Adoptive transfer with lymphocytes from donors treated with A?42 attenuated EAE in wild-type recipient mice, and A? deposition in the brain was not detected in treated EAE mice by immunohistochemical analysis. In contrast to the improvement in EAE with A? treatment, EAE was worse in mice with genetic deletion of the amyloid precursor protein. Therefore, in the absence of A?, there is exacerbated clinical EAE disease progression. Because A?42 and A?40 ameliorate experimental autoimmune inflammation targeting the CNS, we might now consider its potential anti-inflammatory role in other neuropathological conditions.

    View details for DOI 10.1126/scitranslmed.3004145

    View details for Web of Science ID 000307159500004

    View details for PubMedID 22855462

  • A Randomized Controlled Pilot Trial of Oral N-Acetylcysteine in Children with Autism BIOLOGICAL PSYCHIATRY Hardan, A. Y., Fung, L. K., Libove, R. A., Obukhanych, T. V., Nair, S., Herzenberg, L. A., Frazier, T. W., Tirouvanziam, R. 2012; 71 (11): 956-961


    An imbalance in the excitatory/inhibitory systems with abnormalities in the glutamatergic pathways has been implicated in the pathophysiology of autism. Furthermore, chronic redox imbalance was also recently linked to this disorder. The goal of this pilot study was to assess the feasibility of using oral N-acetylcysteine (NAC), a glutamatergic modulator and an antioxidant, in the treatment of behavioral disturbance in children with autism.This was a 12-week, double-blind, randomized, placebo-controlled study of NAC in children with autistic disorder. Subjects randomized to NAC were initiated at 900 mg daily for 4 weeks, then 900 mg twice daily for 4 weeks and 900 mg three times daily for 4 weeks. The primary behavioral measure (Aberrant Behavior Checklist [ABC] irritability subscale) and safety measures were performed at baseline and 4, 8, and 12 weeks. Secondary measures included the ABC stereotypy subscale, Repetitive Behavior Scale-Revised, and Social Responsiveness Scale.Thirty-three subjects (31 male subjects, 2 female subjects; aged 3.2-10.7 years) were randomized in the study. Follow-up data was available on 14 subjects in the NAC group and 15 in the placebo group. Oral NAC was well tolerated with limited side effects. Compared with placebo, NAC resulted in significant improvements on ABC irritability subscale (F = 6.80; p < .001; d = .96).Data from this pilot investigation support the potential usefulness of NAC for treating irritability in children with autistic disorder. Large randomized controlled investigations are warranted.

    View details for DOI 10.1016/j.biopsych.2012.01.014

    View details for Web of Science ID 000303814800007

    View details for PubMedID 22342106

  • Modulation of mTOR Effector Phosphoproteins in Blood Basophils from Allergic Patients JOURNAL OF CLINICAL IMMUNOLOGY Gernez, Y., Tirouvanziam, R., Reshamwala, N., Yu, G., Weldon, B. C., Galli, S. J., Herzenberg, L. A., Nadeau, K. C. 2012; 32 (3): 565-573


    The mammalian target of rapamycin (mTOR) pathway contributes to various immunoinflammatory processes. Yet, its potential involvement in basophil responses in allergy remains unclear. In this pilot study, we quantified two key mTOR effector phosphoproteins, the eukaryotic initiation factor 4E (peIF4E) and S6 ribosomal protein (pS6rp), in blood basophils from nut allergy patients (NA, N?=?16) and healthy controls (HC, N?=?13). Without stimulation in vitro, basophil peIF4E levels were higher in NA than HC subjects (P?=?0.014). Stimulation with nut (offending) but not chicken / rice (non-offending) extract increased basophil peIF4E and pS6rp levels (+32%, P?=?0.018, and +98%, P?=?0.0026, respectively) in NA but not HC subjects, concomitant with increased surface levels of CD203c and CD63, both known to reflect basophil activation. Pre-treatment with the mTOR inhibitor rapamycin decreased pS6rp and CD203c responses in nut extract-stimulated basophils in NA subjects. Thus, basophil responses to offending allergens are associated with modulation of mTOR effector phosphoproteins.

    View details for DOI 10.1007/s10875-012-9651-x

    View details for Web of Science ID 000305982100019

    View details for PubMedID 22350221

  • Distinct Plasma Profile of Polar Neutral Amino Acids, Leucine, and Glutamate in Children with Autism Spectrum Disorders JOURNAL OF AUTISM AND DEVELOPMENTAL DISORDERS Tirouvanziam, R., Obukhanych, T. V., Laval, J., Aronov, P. A., Libove, R., Banerjee, A. G., Parker, K. J., O'Hara, R., Herzenberg, L. A., Herzenberg, L. A., Hardan, A. Y. 2012; 42 (5): 827-836


    The goal of this investigation was to examine plasma amino acid (AA) levels in children with Autism Spectrum Disorders (ASD, N=27) and neuro-typically developing controls (N=20). We observed reduced plasma levels of most polar neutral AA and leucine in children with ASD. This AA profile conferred significant post hoc power for discriminating children with ASD from healthy children. Furthermore, statistical correlations suggested the lack of a typical decrease of glutamate and aspartate with age, and a non-typical increase of isoleucine and lysine with age in the ASD group. Findings from this limited prospective study warrant further examination of plasma AA levels in larger cross-sectional and longitudinal cohorts to adequately assess for relationships with developmental and clinical features of ASD.

    View details for DOI 10.1007/s10803-011-1314-x

    View details for Web of Science ID 000302771500017

    View details for PubMedID 21713591

  • Development of B Cells and Erythrocytes Is Specifically Impaired by the Drug Celastrol in Mice PLOS ONE Kusy, S., Ghosn, E. E., Herzenberg, L. A., Contag, C. H. 2012; 7 (4)


    Celastrol, an active compound extracted from the root of the Chinese medicine "Thunder of God Vine" (Tripterygium wilfordii), exhibits anticancer, antioxidant and anti-inflammatory activities, and interest in the therapeutic potential of celastrol is increasing. However, described side effects following treatment are significant and require investigation prior to initiating clinical trials. Here, we investigated the effects of celastrol on the adult murine hematopoietic system.Animals were treated daily with celastrol over a four-day period and peripheral blood, bone marrow, spleen, and peritoneal cavity were harvested for cell phenotyping. Treated mice showed specific impairment of the development of B cells and erythrocytes in all tested organs. In bone marrow, these alterations were accompanied by decreases in populations of common lymphoid progenitors (CLP), common myeloid progenitors (CMP) and megakaryocyte-erythrocyte progenitors (MEP).These results indicate that celastrol acts through regulators of adult hematopoiesis and could be used as a modulator of the hematopoietic system. These observations provide valuable information for further assessment prior to clinical trials.

    View details for DOI 10.1371/journal.pone.0035733

    View details for Web of Science ID 000305343200053

    View details for PubMedID 22545133

  • Distinct B-cell lineage commitment distinguishes adult bone marrow hematopoietic stem cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ghosn, E. E., Yamamoto, R., Hamanaka, S., Yang, Y., Herzenberg, L. A., Nakauchi, H., Herzenberg, L. A. 2012; 109 (14): 5394-5398


    The question of whether a single hematopoietic stem cell (HSC) gives rise to all of the B-cell subsets [B-1a, B-1b, B-2, and marginal zone (MZ) B cells] in the mouse has been discussed for many years without resolution. Studies here finally demonstrate that individual HSCs sorted from adult bone marrow and transferred to lethally irradiated recipients clearly give rise to B-2, MZ B, and B-1b, but does not detectably reconstitute B-1a cells. These findings place B-2, MZ, and B-1b in a single adult developmental lineage and place B-1a in a separate lineage derived from HSCs that are rare or missing in adults. We discuss these findings with respect to known developmental heterogeneity in other HSC-derived lymphoid, myeloid, and erythroid lineages, and how HSC developmental heterogeneity conforms to the layered model of the evolution of the immune system that we proposed some years ago. In addition, of importance to contemporary medicine, we consider the implications that HSC developmental heterogeneity may have for selecting HSC sources for human transplantation.

    View details for DOI 10.1073/pnas.1121632109

    View details for Web of Science ID 000302294700059

    View details for PubMedID 22431624

  • Antigen-specific memory in B-1a and its relationship to natural immunity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Yang, Y., Ghosn, E. E., Cole, L. E., Obukhanych, T. V., Sadate-Ngatchou, P., Vogel, S. N., Herzenberg, L. A., Herzenberg, L. A. 2012; 109 (14): 5388-5393


    In the companion article by Yang and colleagues [Yang Y, et al. (2012) Proc Natl Acad Sci USA, 109, 10.1073/pnas.1121631109], we have shown that priming with glycolipid (FtL) from Francisella tularensis live-vaccine strain (i) induces FtL-specific B-1a to produce robust primary responses (IgM >IgG); (ii) establishes persistent long-term production of serum IgM and IgG anti-FtL at natural antibody levels; and (iii) elicits FtL-specific B-1a memory cells that arise in spleen but rapidly migrate to the peritoneal cavity, where they persist indefinitely but divide only rarely. Here, we show that FtL rechallenge alone induces these PerC B-1a memory cells to divide extensively and to express a unique activation signature. However, FtL rechallenge in the context of a Toll-like receptor 4 agonist-stimulated inflammatory response readily induces these memory cells to migrate to spleen and initiate production of dominant IgM anti-FtL secondary responses. Thus, studies here reveal unique mechanisms that govern B-1a memory development and expression, and introduce B-1a memory as an active participant in immune defenses. In addition, at a practical level, these studies suggest previously unexplored vaccination strategies for pathogen-associated antigens that target the B-1a repertoire.

    View details for DOI 10.1073/pnas.1121627109

    View details for Web of Science ID 000302294700058

    View details for PubMedID 22421135

  • Antigen-specific antibody responses in B-1a and their relationship to natural immunity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Yang, Y., Ghosn, E. E., Cole, L. E., Obukhanych, T. V., Sadate-Ngatchou, P., Vogel, S. N., Herzenberg, L. A., Herzenberg, L. A. 2012; 109 (14): 5382-5387


    B-1a cells are primarily thought of as natural antibody-producing cells. However, we now show that appropriate antigenic stimulation induces IgM and IgG B-1a antibody responses and long-lived T-independent antigen-specific B-1a memory that differs markedly from canonical B-2 humoral immunity. Thus, we show here that in the absence of inflammation, priming with glycolipid (FtL) from Francisella tularensis live vaccine strain induces splenic FtL-specific B-1a to mount dominant IgM and activation-induced cytidine deaminase-dependent IgG anti-FtL responses that occur within 3-5 d of FtL priming and fade within 1 wk to natural antibody levels that persist indefinitely in the absence of secondary FtL immunization. Equally surprising, FtL priming elicits long-term FtL-specific B-1a memory cells (IgM>IgG) that migrate rapidly to the peritoneal cavity and persist there indefinitely, ready to respond to appropriately administrated secondary antigenic stimulation. Unlike B-2 responses, primary FtL-specific B-1a responses and establishment of persistent FtL-specific B-1a memory occur readily in the absence of adjuvants, IL-7, T cells, or germinal center support. However, in another marked departure from the mechanisms controlling B-2 memory responses, rechallenge with FtL in an inflammatory context is required to induce B-1a secondary antibody responses. These findings introduce previously unexplored vaccination strategies for pathogens that target the B-1a repertoire.

    View details for DOI 10.1073/pnas.1121631109

    View details for Web of Science ID 000302294700057

    View details for PubMedID 22421134

  • Glut1-mediated glucose transport regulates HIV infection PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Loisel-Meyer, S., Swainson, L., Craveiro, M., Oburoglu, L., Mongellaz, C., Costa, C., Martinez, M., Cosset, F., Battini, J., Herzenberg, L. A., Herzenberg, L. A., Atkuri, K. R., Sitbon, M., Kinet, S., Verhoeyen, E., Taylor, N. 2012; 109 (7): 2549-2554


    Cell cycle entry is commonly considered to positively regulate HIV-1 infection of CD4 T cells, raising the question as to how quiescent lymphocytes, representing a large portion of the viral reservoir, are infected in vivo. Factors such as the homeostatic cytokine IL-7 have been shown to render quiescent T cells permissive to HIV-1 infection, presumably by transiently stimulating their entry into the cell cycle. However, we show here that at physiological oxygen (O(2)) levels (2-5% O(2) tension in lymphoid organs), IL-7 stimulation generates an environment permissive to HIV-1 infection, despite a significantly attenuated level of cell cycle entry. We identify the IL-7-induced increase in Glut1 expression, resulting in augmented glucose uptake, as a key factor in rendering these T lymphocytes susceptible to HIV-1 infection. HIV-1 infection of human T cells is abrogated either by impairment of Glut1 signal transduction or by siRNA-mediated Glut1 down-regulation. Consistent with this, we show that the susceptibility of human thymocyte subsets to HIV-1 infection correlates with Glut1 expression; single-round infection is markedly higher in the Glut1-expressing double-positive thymocyte population than in any of the Glut1-negative subsets. Thus, our studies reveal the Glut1-mediated metabolic pathway as a critical regulator of HIV-1 infection in human CD4 T cells and thymocytes.

    View details for DOI 10.1073/pnas.1121427109

    View details for Web of Science ID 000300489200077

    View details for PubMedID 22308487

  • Nerve Growth Factor A Key Local Regulator in the Pathogenesis of Inflammatory Arthritis ARTHRITIS AND RHEUMATISM Raychaudhuri, S. P., Raychaudhuri, S. K., Atkuri, K. R., Herzenberg, L. A., Herzenberg, L. A. 2011; 63 (11): 3243-3252


    The effect of nerve growth factor (NGF) and its receptor (NGFR) in inflammatory diseases is a novel research field. The purpose of this study was to investigate the role of NGF/NGFR in human T cell subpopulations and fibroblast-like synovial cells (FLS) and examine its pathophysiologic significance in psoriatic arthritis (PsA) and rheumatoid arthritis (RA).Expression of NGF/NGFR was examined in synovial fluid (SF), FLS, peripheral blood (PB)-derived T cells, and SF-derived T cells from patients with PsA, RA, and osteoarthritis (OA). NGF levels were determined by enzyme-linked immunosorbent assay. NGF-induced T cell/FLS proliferation was examined by MTT assay. Low-affinity (p75)/high-affinity (TrkA) NGFR expression was determined by high-dimensional fluorescence-activated cell sorting. A monochlorobimane assay was used to determine the effect of NGF on T cell survival.Levels of NGF were higher in SF samples from PsA and RA patients as compared to SF samples from OA patients. NGF-induced FLS proliferation was more marked in PsA and RA patients. TrkA was up-regulated on activated SF T cells from PsA (mean SD 22 6.2%) and RA (8 1.3%) patients, whereas in SF samples from OA patients, TrkA+CD3+ T cells were not detectable. NGF induced the proliferation of PB T cells, induced the phosphorylation of Akt in activated T cells, and consistent with known pAkt activity, inhibited tumor necrosis factor ?-induced cell death in these T cells.Based on our findings, we propose a model in which NGF secreted by FLS into PsA and RA synovium promotes the survival of activated autoreactive T cells as well as FLS proliferation. Thus, NGF has the potential to sustain the chronic inflammatory cascades of arthritis of autoimmune origin.

    View details for DOI 10.1002/art.30564

    View details for Web of Science ID 000297221100008

    View details for PubMedID 21792838

  • Distinct progenitors for B-1 and B-2 cells are present in adult mouse spleen PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ghosn, E. E., Sadate-Ngatchou, P., Yang, Y., Herzenberg, L. A., Herzenberg, L. A. 2011; 108 (7): 2879-2884


    Recent studies by Dorshkind, Yoder, and colleagues show that embryonic (E9) B-cell progenitors located in the yolk sac and intraembryonic hemogenic endothelium before the initiation of circulation give rise to B-1 and marginal zone B cells but do not give rise to B-2 cells. In studies here, we confirm and extend these findings by showing that distinct progenitors for B-1 and B-2 cells are present in the adult spleen. Furthermore, we show that the splenic B-cell progenitor population (lin(-)CD19(+)/B220(lo/-)/CD43(-)) that gives rise to B-1 cells is likely to be heterogeneous because, in some recipients, it also gives rise to B cells expressing the marginal zone phenotype (B220(hi)IgM(hi)IgD(lo)CD21(hi)) and to some (CD19(-)CD5(hi)) T cells. In addition to the well-known function differences between B-1 and B-2, our studies demonstrate that substantial developmental differences separate these B-cell lineages. Thus, consistent with the known dependence of B-2 development on IL-7, all B-2 progenitors express IL-7R. However, >30% of the B-1 progenitors do not express this marker, enabling the known IL-7 independent development of B-1 cells in IL-7(-/-) mice. In addition, marker expression on cells in the early stages of the B-2 development pathway (CD19(-)/c-Kit(lo/-)/Sca-1(lo/-)) in adult bone marrow distinguish it from the early stages of B-1 development (CD19(hi)/c-Kit(+)/Sca-1(+)), which occur constitutively in neonates. In adults, in vivo inflammatory stimulation (LPS) triggers B-1 progenitors in spleen to expand and initiate development along this B-1 developmental pathway.

    View details for DOI 10.1073/pnas.1019764108

    View details for Web of Science ID 000287377000049

    View details for PubMedID 21282663

  • Basophil CD203c Levels Are Increased at Baseline and Can Be Used to Monitor Omalizumab Treatment in Subjects with Nut Allergy INTERNATIONAL ARCHIVES OF ALLERGY AND IMMUNOLOGY Gernez, Y., Tirouvanziam, R., Yu, G., Ghosn, E. E., Reshamwala, N., Tammie Nguyen, T., Tsai, M., Galli, S. J., Herzenberg, L. A., Herzenberg, L. A., Nadeau, K. C. 2011; 154 (4): 318-327


    Basophils contribute to anaphylaxis and allergies. We examined the utility of assessing basophil-associated surface antigens (CD11b/CD63/CD123/CD203c/CD294) in characterizing and monitoring subjects with nut allergy.We used flow cytometry to analyze basophils at baseline (without any activation) and after ex vivo stimulation of whole blood by addition of nut or other allergens for 2, 10, and 30 min. We also evaluated whether basophil expression of CD11b/CD63/CD123/CD203c/CD294 was altered in subjects treated with anti-IgE monoclonal antibody (omalizumab) to reduce plasma levels of IgE.We demonstrate that basophil CD203c levels are increased at baseline in subjects with nut allergy compared to healthy controls (13 subjects in each group, p < 0.0001). Furthermore, we confirm that significantly increased expression of CD203c occurs on subject basophils when stimulated with the allergen to which the subject is sensitive and can be detected rapidly (10 min of stimulation, n = 11, p < 0.0008). In 5 subjects with severe peanut allergy, basophil CD203c expression following stimulation with peanut allergen was significantly decreased (p < 0.05) after 4 and 8 weeks of omalizumab treatment but returned toward pretreatment levels after treatment cessation.Subjects with nut allergy show an increase of basophil CD203c levels at baseline and following rapid ex vivo stimulation with nut allergen. Both can be reduced by omalizumab therapy. These results highlight the potential of using basophil CD203c levels for baseline diagnosis and therapeutic monitoring in subjects with nut allergy.

    View details for DOI 10.1159/000321824

    View details for Web of Science ID 000288529200007

    View details for PubMedID 20975283

  • METABOLITE PROFILING OF CF AIRWAY FLUID SUGGESTS A ROLE FOR CATECHOLAMINES IN EARLY AND CHRONIC DISEASE Gudiputi, L., Aronov, P., Makam, M., Zirbes, J., Conrad, C., Milla, C., Herzenberg, L., Moss, R., Tirouvanziam, R. WILEY-BLACKWELL. 2011: 240-240
  • Genetic immunization in the lung induces potent local and systemic immune responses PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Song, K., Bolton, D. L., Wilson, R. L., Camp, J. V., Bao, S., Mattapallil, J. J., Herzenberg, L. A., Herzenberg, L. A., Andrews, C. A., Sadoff, J. C., Goudsmit, J., Pau, M. G., Seder, R. A., Kozlowski, P. A., Nabel, G. J., Roederer, M., Rao, S. S. 2010; 107 (51): 22213-22218


    Successful vaccination against respiratory infections requires elicitation of high levels of potent and durable humoral and cellular responses in the lower airways. To accomplish this goal, we used a fine aerosol that targets the entire lung surface through normal respiration to deliver replication-incompetent recombinant adenoviral vectors expressing gene products from several infectious pathogens. We show that this regimen induced remarkably high and stable lung T-cell responses in nonhuman primates and that it also generated systemic and respiratory tract humoral responses of both IgA and IgG isotypes. Moreover, strong immunogenicity was achieved even in animals with preexisting antiadenoviral immunity, overcoming a critical hurdle to the use of these vectors in humans, who commonly are immune to adenoviruses. The immunogenicity profile elicited with this regimen, which is distinct from either intramuscular or intranasal delivery, has highly desirable properties for protection against respiratory pathogens. We show that it can be used repeatedly to generate mucosal humoral, CD4, and CD8 T-cell responses and as such may be applicable to other mucosally transmitted pathogens such as HIV. Indeed, in a lethal challenge model, we show that aerosolized recombinant adenoviral immunization completely protects ferrets against H5N1 highly pathogenic avian influenza virus. Thus, genetic immunization in the lung offers a powerful platform approach to generating protective immune responses against respiratory pathogens.

    View details for DOI 10.1073/pnas.1015536108

    View details for Web of Science ID 000285521800052

    View details for PubMedID 21135247

  • Two physically, functionally, and developmentally distinct peritoneal macrophage subsets PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bou Ghosn, E. E., Cassado, A. A., Govoni, G. R., Fukuhara, T., Yang, Y., Monack, D. M., Bortoluci, K. R., Almeida, S. R., Herzenberg, L. A., Herzenberg, L. A. 2010; 107 (6): 2568-2573


    The peritoneal cavity (PerC) is a unique compartment within which a variety of immune cells reside, and from which macrophages (M) are commonly drawn for functional studies. Here we define two M subsets that coexist in PerC in adult mice. One, provisionally called the large peritoneal M (LPM), contains approximately 90% of the PerC M in unstimulated animals but disappears rapidly from PerC following lipopolysaccharide (LPS) or thioglycolate stimulation. These cells express high levels of the canonical M surface markers, CD11b and F4/80. The second subset, referred to as small peritoneal M (SPM), expresses substantially lower levels of CD11b and F4/80 but expresses high levels of MHC-II, which is not expressed on LPM. SPM, which predominates in PerC after LPS or thioglycolate stimulation, does not derive from LPM. Instead, it derives from blood monocytes that rapidly enter the PerC after stimulation and differentiate to mature SPM within 2 to 4 d. Both subsets show clear phagocytic activity and both produce nitric oxide (NO) in response to LPS stimulation in vivo. However, their responses to LPS show key differences: in vitro, LPS stimulates LPM, but not SPM, to produce NO; in vivo, LPS stimulates both subsets to produce NO, albeit with different response patterns. These findings extend current models of M heterogeneity and shed new light on PerC M diversity, development, and function. Thus, they introduce a new context for interpreting (and reinterpreting) data from ex vivo studies with PerC M.

    View details for DOI 10.1073/pnas.0915000107

    View details for Web of Science ID 000274408100039

    View details for PubMedID 20133793

  • Human Basophils: New Mechanisms Of Action And Role In Food Allergy Gernez, Y., Tirouvanziam, R., Yu, G., Reshamwala, N., Tsai, M., GALLI, S. J., Herzenberg, L. A., Nadeau, K. C. MOSBY-ELSEVIER. 2010: AB86-AB86
  • SMALL METABOLITE PROFILING OF AIRWAY FLUID UNCOVERS SPECIFIC ALTERATIONS IN CF Tirouvanziam, R., Aronov, P., Laval, J., Makam, M., Liu, C., Chien, A., Herzenberg, L., Gronert, K., Moss, R. B. WILEY-BLACKWELL. 2010: 243-243
  • Activation of critical, host-induced, metabolic and stress pathways marks neutrophil entry into cystic fibrosis lungs PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Makam, M., Diaz, D., Laval, J., Gernez, Y., Conrad, C. K., Dunn, C. E., Davies, Z. A., Moss, R. B., Herzenberg, L. A., Herzenberg, L. A., Tirouvanziam, R. 2009; 106 (14): 5779-5783


    Cystic fibrosis (CF) patients undergo progressive airway destruction caused in part by chronic neutrophilic inflammation. While opportunistic pathogens infecting CF airways can cause inflammation, we hypothesized that host-derived metabolic and stress signals would also play a role in this process. We show that neutrophils that have entered CF airways have increased phosphorylation of the eukaryotic initiation factor 4E and its partner the 4E-binding protein 1; 2 key effectors in the growth factor- and amino acid-regulated mammalian target of rapamycin (mTOR) pathway. Furthermore CF airway neutrophils display increased phosphorylation of the cAMP response element binding protein (CREB), a major transcriptional coactivator in stress signaling cascades. These active intracellular pathways are associated with increased surface expression of critical adaptor molecules, including the growth factor receptor CD114 and the receptor for advanced glycation end-products (RAGE), a CREB inducer and sensor for host-derived damage-associated molecular patterns (DAMPs). Most CF airway fluids lack any detectable soluble RAGE, an inhibitory decoy receptor for DAMPs. Concomitantly, CF airway fluids displayed high and consequently unopposed levels of S100A12; a potent mucosa- and neutrophil-derived DAMP. CF airway neutrophils also show increased surface levels of 2 critical CREB targets, the purine-recycling enzyme CD39 and the multifunctional, mTOR-inducing CXCR4 receptor. This coordinated set of events occurs in all patients, even in the context of minimal airway inflammation and well-preserved lung function. Taken together, our data demonstrate an early and sustained activation of host-responsive metabolic and stress pathways upon neutrophil entry into CF airways, suggesting potential targets for therapeutic modulation.

    View details for DOI 10.1073/pnas.0813410106

    View details for Web of Science ID 000264967500059

    View details for PubMedID 19293384

  • Antigen- specific B-1a antibodies induced by Francisella tularensis LPS provide long-term protection against F. tularensis LVS challenge PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Cole, L. E., Yang, Y., Elkins, K. L., Fernandez, E. T., Qureshi, N., Shlomchik, M. J., Herzenberg, L. A., Herzenberg, L. A., Vogel, S. N. 2009; 106 (11): 4343-4348


    Francisella tularensis (Ft), a gram-negative intracellular bacterium, is the etiologic agent of tularemia. Infection of mice with <10 Ft Live Vaccine Strain (Ft LVS) organisms i.p. causes a lethal infection that resembles human tularemia. Here, we show that immunization with as little as 0.1 ng Ft LVS lipopolysaccharide (Ft-LPS), but not Ft lipid A, generates a rapid antibody response that protects wild-type (WT) mice against lethal Ft LVS challenge. Protection is not induced in Ft-LPS-immunized B cell-deficient mice (muMT or JhD), male xid mice, or Ig transgenic mice that produce a single IgH (not reactive with Ft-LPS). Focusing on the cellular mechanisms that underlie this protective response, we show that Ft-LPS specifically stimulates proliferation of B-1a lymphocytes that bind fluorochrome-labeled Ft-LPS and the differentiation of these cells to plasma cells that secrete antibodies specific for Ft-LPS. This exclusively B-1a antibody response is equivalent in WT, T-deficient (TCRalphabeta(-/-), TCRgammadelta(-/-)), and Toll-like receptor 4 (TLR4)-deficient (TLR4(-/-)) mice and thus is not dependent on T cells or typical inflammatory processes. Serum antibody levels peak approximately 5 days after Ft-LPS immunization and persist at low levels for months. Thus, immunization with Ft-LPS activates a rare population of antigen-specific B-1a cells to produce a persistent T-independent antibody response that provides long-term protection against lethal Ft LVS infection. These data support the possibility of creating effective, minimally invasive vaccines that can provide effective protection against pathogen invasion.

    View details for DOI 10.1073/pnas.0813411106

    View details for Web of Science ID 000264278800053

    View details for PubMedID 19251656

  • Inherited disorders affecting mitochondrial function are associated with glutathione deficiency and hypocitrullinemia PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Atkuri, K. R., Cowan, T. M., Kwan, T., Ng, A., Herzenberg, L. A., Herzenberg, L. A., Enns, G. M. 2009; 106 (10): 3941-3945


    Disorders affecting mitochondria, including those that directly affect the respiratory chain function or result from abnormalities in branched amino acid metabolism (organic acidemias), have been shown to be associated with impaired redox balance. Almost all of the evidence underlying this conclusion has been obtained from studies on patient biopsies or animal models. Since the glutathione (iGSH) system provides the main protection against oxidative damage, we hypothesized that untreated oxidative stress in individuals with mitochondrial dysfunction would result in chronic iGSH deficiency. We confirm this hypothesis here in studies using high-dimensional flow cytometry (Hi-D FACS) and biochemical analysis of freshly obtained blood samples from patients with mitochondrial disorders or organic acidemias. T lymphocyte subsets, monocytes and neutrophils from organic acidemia and mitochondrial patients who were not on antioxidant supplements showed low iGSH levels, whereas similar subjects on antioxidant supplements showed normal iGSH. Measures of iROS levels in blood were insufficient to reveal the chronic oxidative stress in untreated patients. Patients with organic acidemias showed elevated plasma protein carbonyls, while plasma samples from all patients tested showed hypocitrullinemia. These findings indicate that measurements of iGSH in leukocytes may be a particularly useful biomarker to detect redox imbalance in mitochondrial disorders and organic acidemias, thus providing a relatively non-invasive means to monitor disease status and response to therapies. Furthermore, studies here suggest that antioxidant therapy may be useful for relieving the chronic oxidative stress that otherwise occurs in patients with mitochondrial dysfunction.

    View details for DOI 10.1073/pnas.0813409106

    View details for Web of Science ID 000264036900054

    View details for PubMedID 19223582

  • Automatic clustering of flow cytometry data with density-based merging. Advances in bioinformatics Walther, G., Zimmerman, N., Moore, W., Parks, D., Meehan, S., Belitskaya, I., Pan, J., Herzenberg, L. 2009: 686759-?


    The ability of flow cytometry to allow fast single cell interrogation of a large number of cells has made this technology ubiquitous and indispensable in the clinical and laboratory setting. A current limit to the potential of this technology is the lack of automated tools for analyzing the resulting data. We describe methodology and software to automatically identify cell populations in flow cytometry data. Our approach advances the paradigm of manually gating sequential two-dimensional projections of the data to a procedure that automatically produces gates based on statistical theory. Our approach is nonparametric and can reproduce nonconvex subpopulations that are known to occur in flow cytometry samples, but which cannot be produced with current parametric model-based approaches. We illustrate the methodology with a sample of mouse spleen and peritoneal cavity cells.

    View details for DOI 10.1155/2009/686759

    View details for PubMedID 20069107

  • FR255734, a humanized, Fc-silent, anti-CD28 antibody, improves psoriasis in the SCID mouse-psoriasis xenograft model JOURNAL OF INVESTIGATIVE DERMATOLOGY Raychaudhuri, S. P., Kundu-Raychaudhuri, S., Tamura, K., Masunaga, T., Kubo, K., Hanaoka, K., Jiang, W., Herzenberg, L. A., Herzenberg, L. A. 2008; 128 (8): 1969-1976


    In psoriasis, CD28/B7 costimulatory molecules are well characterized. Here, using the severe combined immunodeficient (SCID) mouse-psoriasis xenograft model, we report therapeutic efficacy of a humanized anti-CD28 monoclonal antibody (FR255734; Astellas Pharmaceuticals Inc., Tokyo, Japan). Transplanted psoriasis plaques on the SCID mouse were treated weekly for 4 weeks with intraperitoneal injections of FR255734 at 10, 3, and 1-mg kg(-1) doses. Groups treated with doses of 10 and 3 mg kg(-1) had significant thinning of the epidermis and reduced HLA-DR-positive lymphocytic infiltrates. The length of the rete pegs changed from 415.2+/-59.6 to 231.4+/-40.4 microm (P<0.005) in the 10-mg kg(-1) group, and from 323.4+/-69.6 to 237.5+/-73.6 microm in the 3-mg kg(-1) group (P=0.002). Positive controls treated with CTLA4-Ig and cyclosporine had significant histological improvement, whereas plaques treated with saline and isotype controls (human and mouse IgG2) remained unchanged. In vitro studies have shown that FR255734 effectively blocked T-cell proliferation and proinflammatory cytokine production. These observations warrant studies to evaluate the efficacy of FR255734 in human autoimmune diseases.

    View details for DOI 10.1038/jid.2008.38

    View details for Web of Science ID 000258031700016

    View details for PubMedID 18337836

  • CD11b expression distinguishes sequential stages of peritoneal B-1 development PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ghosn, E. E., Yang, Y., Tung, J., Herzenberg, L. A., Herzenberg, L. A. 2008; 105 (13): 5195-5200


    Peritoneal cavity (PerC) B-1 cells have long been known to express CD11b, which is coexpressed with CD18 to form the Mac-1/CR3 complement receptor and adhesion molecule. However, although all PerC B-1 cells are commonly believed to express CD11b, we show here that nearly half of the cells in each of the PerC B-1 subsets (B-1a and B-1b) do not express this surface receptor. The CD11b(+) cells in each B-1 subset are larger and more granular and express higher levels of surface IgM than the CD11b(-) B-1 cells. In addition, the CD11b(+) B-1 cells initiate the formation of tightly associated doublets that are present at high frequency in adult PerC. Finally, and most importantly from a developmental standpoint, the CD11b(+) B-1 cells have a limited reconstitution capability: when sorted and transferred into congenic recipients, they reconstitute their own (CD11b(+)) B-1 subset but do not reconstitute the CD11b(-) B-1 subset. In contrast, CD11b(-) B-1 cells transferred under the same conditions efficiently replenish all components of the PerC B-1 population in appropriate proportions. During ontogeny, CD11b(-) B-1 cells appear before CD11b(+) B-1 cells. However, the clear phenotypic differences between the neonatal and adult CD11b B-1 subsets argue that although CD11b(-) B-1 give rise to CD11b(+) B-1 in both cases different forces may regulate this transition.

    View details for DOI 10.1073/pnas.0712350105

    View details for Web of Science ID 000254723700044

    View details for PubMedID 18375763

  • Antigen-induced B-1 class switch and persistent B-1 memory Yang, Y., Cole, L. E., Vogel, S. N., Wang, D., Herzenberg, L. A., Herzenberg, L. A. FEDERATION AMER SOC EXP BIOL. 2008
  • Culturing of human peripheral blood cells reveals unsuspected lymphocyte responses relevant to HIV disease PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sahaf, B., Atkuri, K., Heydari, K., Malipatlolla, M., Rappaport, J., Regulier, E., Herzenberg, L. A., Herzenberg, L. A. 2008; 105 (13): 5111-5116


    Recombinant HIV-Tat (Tat) induces extensive apoptosis in peripheral blood mononuclear cells (PBMCs) cultured in typical CO2 incubators, which are equilibrated with air (21% O2). However, as we show here, Tat apoptosis induction fails in PBMCs cultured at physiological oxygen levels (5% O2). Under these conditions, Tat induces PBMCs to divide, efficiently primes them for HIV infection, and supports virus production by the infected cells. Furthermore, Tat takes only 2 h to prime PBMCs under these conditions. In contrast, PHA/IL-2, which is widely used to prime cells for HIV infection, takes 2-3 days. These findings strongly recommend culturing primary cells at physiological oxygen levels. In addition, they suggest HIV-Tat as a key regulator of HIV disease progression.

    View details for DOI 10.1073/pnas.0712363105

    View details for Web of Science ID 000254723700029

    View details for PubMedID 18364393

  • Profound functional and signaling changes in viable inflammatory neutrophils homing to cystic fibrosis airways PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Tirouvanziam, R., Gernez, Y., Conrad, C. K., Moss, R. B., Schrijver, I., Dunn, C. E., Davies, Z. A., Herzenberg, L. A., Herzenberg, L. A. 2008; 105 (11): 4335-4339


    Blood neutrophils recruited to cystic fibrosis (CF) airways are believed to be rapidly killed by resident bacteria and to passively release elastase and other toxic by-products that promote disease progression. By single-cell analysis, we demonstrate that profound functional and signaling changes readily occur within viable neutrophils recruited to CF airways, compared with their blood counterparts. Airway neutrophils have undergone conventional activation, as shown by decreased intracellular glutathione, increased lipid raft assembly, surface mobilization of CD11b+ and CD66b+ granules, and increased levels of the cytoskeleton-associated phospho-Syk kinase. Unexpectedly, they also mobilize to the surface CD63+ elastase-rich granules, usually confined intracellularly, and lose surface expression of CD16 and CD14, both key receptors in phagocytosis. Furthermore, they express CD80, major histocompatibility complex type II, and the prostaglandin D2 receptor CD294, all normally associated with other lineages, which reflects functional reprogramming. This notion is reinforced by their decreased total phosphotyrosine levels, mirroring a postactivated stage, and increased levels of the phospho-S6 ribosomal protein, a key anabolic switch. Thus, we identified a subset of neutrophils within CF airways with a viable but dysfunctional phenotype. This subset provides a possible therapeutic target and indicates a need to revisit current paradigms of CF airway disease.

    View details for DOI 10.1073/pnas.0712386105

    View details for Web of Science ID 000254263300048

    View details for PubMedID 18334635

  • B cell lineage contributions to antiviral host responses SPECIALIZATION AND COMPLEMENTATION OF HUMORAL IMMUNE RESPONSES TO INFECTION Baumgarth, N., Choi, Y. S., Rothaeusler, K., Yang, Y., Herzenberg, L. A. 2008; 319: 41-61


    B cell responses are a major immune protective mechanism induced against a large variety of pathogens. Technical advances over the last decade, particularly in the isolation and characterization of B cell subsets by multicolor flow cytometry, have demonstrated the multifaceted nature of pathogen-induced B cell responses. In addition to participation by the major follicular B cell population, three B cell subsets are now recognized as key contributors to pathogen-induced host defenses: marginal zone (MZ) B cells, B-1a and B-1b cells. Each of these subsets seems to require unique activation signals and to react with distinct response patterns. Here we provide a brief review of the main developmental and functional features of these B cell subsets. Furthermore, we outline our current understanding of how each subset contributes to the humoral response to influenza virus infection and what regulates their differential responses. Understanding of the multilayered nature of the humoral responses to infectious agents and the complex innate immune signals that shape pathogen-specific humoral responses are likely at the heart of enhancing our ability to induce appropriate and long-lasting humoral responses for prophylaxis and therapy.

    View details for Web of Science ID 000252396900003

    View details for PubMedID 18080414

  • Phospho-FACS: a powerful tool for exploring intracellular transduction cascades REVUE DES MALADIES RESPIRATOIRES Gernez, Y., Herzenberg, L. A., Herzenberg, L. A., Tirouvanziam, R. 2007; 24 (8): 955-964


    FACS (fluorescence-activated cell sorting), or flow cytometry, was developed in 1971 by Leonard Herzenberg's team at Stanford University. Under continuous development, this technology enables single-cell multiparametric analysis and sorting, based on physical properties of cells and/or their relative expression levels of specific glycoproteic epitopes and metabolites.Recently, the use of fluorescent antibodies specific for phosphorylated epitopes - or "phospho-epitopes" - within proteins of interest has further extended the range of FACS analyses. This new application, dubbed "phospho-FACS", has quickly become a tool of choice for delineating intracellular phosphorylation cascades.In both basic research and clinical research, the application of phospho-FACS to cellular subsets from blood or the periphery, whether frequent or rare, enables the discovery of pathological biomarkers and therapeutic innovation.Thanks to its rapid implementation and its ability to generate single-cell data, the phospho-FACS technique features numerous advantages compared to preexisting analytical methods for intracellular phosphorylation cascades.

    View details for DOI 10.1019/20072001118

    View details for Web of Science ID 000251260100004

    View details for PubMedID 18033184

  • Modern flow cytometry: A practical approach CLINICS IN LABORATORY MEDICINE Tung, J. W., Heydari, K., Tirouvanziam, R., Parks, D. R., Herzenberg, L. A., Herzenberg, L. A. 2007; 27 (3): 453-?


    The demonstration that CD T-cell counts can be used to monitor HIV disease progression opened the way to the first clinical application for fluorescence activated cell sorting (FACS) technology. Modern FACS methodologies such multicolor staining and sorting has opened the way to new and constructive therapeutic and clinical applications. This article outlines approaches in which current users can use to improve the quality of their FACS work without undue effort. FACS technology development and the emergence of new software support for this technology are cooperating in this effort.

    View details for DOI 10.1016/j.cII.2007.05.001

    View details for Web of Science ID 000249231600002

    View details for PubMedID 17658402

  • N-Acetylcysteine - a safe antidote for cysteine/glutathione deficiency CURRENT OPINION IN PHARMACOLOGY Atkuri, K. R., Mantovani, J. J., Herzenberg, L. A., Herzenberg, L. A. 2007; 7 (4): 355-359


    Glutathione (GSH) deficiency is associated with numerous pathological conditions. Administration of N-acetylcysteine (NAC), a cysteine prodrug, replenishes intracellular GSH levels. NAC, best known for its ability to counter acetaminophen toxicity, is a safe, well-tolerated antidote for cysteine/GSH deficiency. NAC has been used successfully to treat GSH deficiency in a wide range of infections, genetic defects and metabolic disorders, including HIV infection and COPD. Over two-thirds of 46 placebo-controlled clinical trials with orally administered NAC have indicated beneficial effects of NAC measured either as trial endpoints or as general measures of improvement in quality of life and well-being of the patients.

    View details for DOI 10.1016/j.coph.2007.04.005

    View details for Web of Science ID 000249682800002

    View details for PubMedID 17602868

  • B-cell development fails in the absence of the Pbx1 proto-oncogene BLOOD Sanyal, M., Tung, J. W., Karsunky, H., Zeng, H., Selleri, L., Weissman, I. L., Herzenberg, L. A., Cleary, M. L. 2007; 109 (10): 4191-4199


    Pbx1, a homeodomain transcription factor that was originally identified as the product of a proto-oncogene in acute pre-B-cell leukemia, is a global regulator of embryonic development. However, embryonic lethality in its absence has prevented an assessment of its role in B-cell development. Here, using Rag1-deficient blastocyst complementation assays, we demonstrate that Pbx1 null embryonic stem (ES) cells fail to generate common lymphoid progenitors (CLPs) resulting in a complete lack of B and NK cells, and a partial impairment of T-cell development in chimeric mice. A critical role for Pbx1 was confirmed by rescue of B-cell development from CLPs following restoration of its expression in Pbx1-deficient ES cells. In adoptive transfer experiments, B-cell development from Pbx1-deficient fetal liver cells was also severely compromised, but not erased, since transient B lymphopoiesis was detected in Rag-deficient recipients. Conditional inactivation of Pbx1 in pro-B (CD19(+)) cells and thereafter revealed that Pbx1 is not necessary for B-cell development to proceed from the pro-B-cell stage. Thus, Pbx1 critically functions at a stage between hematopoietic stem cell development and B-cell commitment and, therefore, is one of the earliest-acting transcription factors that regulate de novo B-lineage lymphopoiesis.

    View details for DOI 10.1182/blood-2006-10-054213

    View details for Web of Science ID 000246609100023

    View details for PubMedID 17244677

  • Unraveling B-1 progenitors CURRENT OPINION IN IMMUNOLOGY Tung, J. W., Herzenberg, L. A. 2007; 19 (2): 150-155


    B-1 cells comprise a small percentage of the B lymphocytes that reside in multiple tissues in the mouse, including the peritoneal and pleural cavities. Functionally, B-1 cells participate in innate immunity by producing the majority of the natural IgM in serum, which protects against invading pathogens before the onset of the adaptive immune response. B-1 cells arise from fetal and neonatal progenitors and are distinct from the adult bone marrow progenitors that give rise to follicular and marginal zone B-2 cells. Recent studies have attempted to delineate the progenitors of B-1 cells from those of B-2 cells. Notably, the identification of CD45R(-/lo)CD19(+) B-1 progenitors and expression of two surface determinants, CD138 and major histocompatibility class II antigens, distinguish developing B-1 cells from B-2 cells.

    View details for DOI 10.1016/j.coi.2007.02.012

    View details for Web of Science ID 000245355400007

    View details for PubMedID 17303402

  • Division and differentiation of natural antibody-producing cells in mouse spleen PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Yang, Y., Tung, J. W., Ghosn, E. E., Herzenberg, L. A., Herzenberg, L. A. 2007; 104 (11): 4542-4546


    B-1a cells reside in both the peritoneal cavity and the spleen. LPS stimulates splenic B-1a to differentiate to plasma cells producing natural IgM specific for microbial and self antigens. However, there are conflicting views as to whether the B-1a cells divide before this differentiation occurs, and hence how the resident B-1a population is maintained in the spleen. Studies here resolve this dispute in favor of both sides: we show that (some or all) B-1a cells resident in the spleen respond to LPS by differentiating to plasma cells immediately, without dividing; however, we also show that additional B-1a cells immigrate into the spleen after LPS stimulation and divide at least once before differentiating. Importantly, the studies we presently describe reveal the complex cell migration and differentiation events that collectively underlie the rapid production of natural antibodies in response to in vivo LPS stimulation. Thus, the studies present a different view of the roles that B-1a cells play in the early phases of the innate immune response.

    View details for DOI 10.1073/pnas.0700001104

    View details for Web of Science ID 000244972700054

    View details for PubMedID 17360560

  • Importance of culturing primary lymphocytes at physiological oxygen levels PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Atkuri, K. R., Herzenberg, L. A., Niemi, A., Cowan, T., Herzenberg, L. A. 2007; 104 (11): 4547-4552


    Although studies with primary lymphocytes are almost always conducted in CO(2) incubators maintained at atmospheric oxygen levels (atmosO(2); 20%), the physiological oxygen levels (physO(2); 5%) that cells encounter in vivo are 2-4 times lower. We show here that culturing primary T cells at atmosO(2) significantly alters the intracellular redox state (decreases intracellular glutathione, increases oxidized intracellular glutathione), whereas culturing at physO(2) maintains the intracellular redox environment (intracellular glutathione/oxidized intracellular glutathione) close to its in vivo status. Furthermore, we show that CD3/CD28-induced T cell proliferation (based on proliferation index and cell yield) is higher at atmosO(2) than at physO(2). This apparently paradoxical finding, we suggest, may be explained by two additional findings with CD3/CD28-stimulated T cells: (i) the intracellular NO (iNO) levels are higher at physO(2) than at atmosO(2); and (ii) the peak expression of CD69 is significantly delayed and more sustained at physO(2) that at atmosO(2). Because high levels of intracellular NO and sustained CD69 tend to down-regulate T cell responses in vivo, the lower proliferative T cell responses at physO(2) likely reflect the in vitro operation of the natural in vivo regulatory mechanisms. Thus, we suggest caution in culturing primary lymphocytes at atmosO(2) because the requisite adaptation to nonphysiological oxygen levels may seriously skew T cell responses, particularly after several days in culture.

    View details for DOI 10.1073/pnas.0611732104

    View details for Web of Science ID 000244972700055

    View details for PubMedID 17360561

  • Photogenerated glycan arrays identify immunogenic sugar moieties of Bacillus anthracis exosporium PROTEOMICS Wang, D., Carroll, G. T., Turro, N. J., Koberstein, J. T., Kovac, P., Saksena, R., Adamo, R., Herzenberg, L. A., Herzenberg, L. A., Steinman, L. 2007; 7 (2): 180-184


    Using photogenerated glycan arrays, we characterized a large panel of synthetic carbohydrates for their antigenic reactivities with pathogen-specific antibodies. We discovered that rabbit IgG antibodies elicited by Bacillus anthracis spores specifically recognize a tetrasaccharide chain that decorates the outermost surfaces of the B. anthracis exosporium. Since this sugar moiety is highly specific for the spores of B. anthracis, it appears to be a key biomarker for detection of B. anthracis spores and development of novel vaccines that target anthrax spores.

    View details for DOI 10.1002/pmic.200600478

    View details for Web of Science ID 000243957700003

    View details for PubMedID 17205603

  • Knowledge acquisition and visualization for biomedical research. AMIA ... Annual Symposium proceedings / AMIA Symposium. AMIA Symposium Zimmerman, N., Meehan, S., Herzenberg, L. A., Das, A. K. 2007: 1177-?


    The TreeTableWidget is a user interface component that allows instances in a knowledge-base to be sorted on the value of one or more of its attributes. Any combination of attributes can be sorted simultaneously by assigning a precedence to each sort. This results in a hierarchy of attributes such that instances can be grouped and visualized according to shared attribute values in a tree view. The resulting tree provides a mechanism to navigate and edit a large number of instances based on attribute values. The component is available as a Protg slot-widget plug-in.

    View details for PubMedID 18694273

  • FR255734, a humanized CD28 antibody is therapeutically effective in psoriasis: An in vivo study using the SCID mouse-psoriasis xenograft model. Raychaudhuri, S. P., Raychaudhuril, S. K., Tamura, K., Masunaga, T., Kubo, K., Jiang, W. Y., Herzenberg, L. A., Herzenberg, L. A. WILEY-BLACKWELL. 2006: 4090-4090
  • Utilization of host SR protein kinases and RNA-splicing machinery during viral replication PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Fukuhara, T., Hosoya, T., Shimizu, S., Sumi, K., Oshiro, T., Yoshinaka, Y., Suzuki, M., Yamamoto, N., Herzenberg, L. A., Herzenberg, L. A., Hagiwara, M. 2006; 103 (30): 11329-11333


    Although the viral genome is often quite small, it encodes a broad series of proteins. The virus takes advantage of the host-RNA-processing machinery to provide the alternative splicing capability necessary for the expression of this proteomic diversity. Serine-arginine-rich (SR) proteins and the kinases that activate them are central to this alternative splicing machinery. In studies reported here, we use the HIV genome as a model. We show that HIV expression decreases overall SR protein/activity. However, we also show that HIV expression is significantly increased (20-fold) when one of the SR proteins, SRp75 is phosphorylated by SR protein kinase (SRPK)2. Thus, inhibitors of SRPK2 and perhaps of functionally related kinases, such as SRPK1, could be useful antiviral agents. Here, we develop this hypothesis and show that HIV expression down-regulates SR proteins in Flp-In293 cells, resulting in only low-level HIV expression in these cells. However, increasing SRPK2 function up-regulates HIV expression. In addition, we introduce SR protein phosphorylation inhibitor 340 (SRPIN340), which preferentially inhibits SRPK1 and SRPK2 and down-regulates SRp75. Although an isonicotinamide compound, SPRIN340 (or its derivatives) remain to be optimized for better specificity and lower cytotoxicity, we show here that SRPIN340 suppresses propagation of Sindbis virus in plaque assay and variably suppresses HIV production. Thus, we show that SRPK, a well known kinase in the cellular RNA-processing machinery, is used by at least some viruses for propagation and hence suggest that SRPIN340 or its derivatives may be useful for curbing viral diseases.

    View details for DOI 10.1073/pnas.0604616103

    View details for Web of Science ID 000239353900041

    View details for PubMedID 16840555

  • MYC can induce DNA breaks in vivo and in vitro independent of reactive oxygen species CANCER RESEARCH Ray, S., Atkuri, K. R., Deb-Basu, D., Adler, A. S., Chang, H. Y., Herzenberg, L. A., Felsher, D. W. 2006; 66 (13): 6598-6605


    MYC overexpression is thought to initiate tumorigenesis by inducing cellular proliferation and growth and to be restrained from causing tumorigenesis by inducing cell cycle arrest, cellular senescence, and/or apoptosis. Here we show that MYC can induce DNA breaks both in vitro and in vivo independent of increased production of reactive oxygen species (ROS). We provide an insight into the specific circumstances under which MYC generates ROS in vitro and propose a possible mechanism. We found that MYC induces DNA double-strand breaks (DSBs) independent of ROS production in murine lymphocytes in vivo as well as in normal human foreskin fibroblasts (NHFs) in vitro in normal (10%) serum, as measured by gammaH2AX staining. However, NHFs cultured in vitro in low serum (0.05%) and/or ambient oxygen saturation resulted in ROS-associated oxidative damage and DNA single-strand breaks (SSBs), as measured by Ape-1 staining. In NHFs cultured in low versus normal serum, MYC induced increased expression of CYP2C9, a gene product well known to be associated with ROS production. Specific inhibition of CYP2C9 by small interfering RNA was shown to partially inhibit MYC-induced ROS production. Hence, MYC overexpression can induce ROS and SSBs under some conditions, but generally induces widespread DSBs in vivo and in vitro independent of ROS production.

    View details for DOI 10.1158/0008-5472.CAN-05-3115

    View details for Web of Science ID 000238825800021

    View details for PubMedID 16818632

  • Phenotypically distinct B cell development pathways map to the three B cell lineages in the mouse PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Tung, J. W., Mrazek, M. D., Yang, Y., Herzenberg, L. A., Herzenberg, L. A. 2006; 103 (16): 6293-6298


    A recent article by Montecino-Rodriguez et al. [Montecino-Rodriguez, E., Leathers, H. & Dorshkind, K. (2006) Nat. Immunol.7, 293-301] has distinguished the early progenitors for B-1 cells, which principally develop in neonates, from early progenitors for B-2 cells, which principally develop in adult bone marrow. Here we introduce syndecan-1 (CD138) and MHC class II (I-A) as markers of early B cell development [Hardy, R. R., Carmack, C. E., Shinton, S. A., Kemp, J. D. & Hayakawa, K. (1991) J. Exp. Med. 173, 1213-1225; Hardy fractions B-D] and show that the expression of these markers distinguishes the predominant B cell development pathway in neonates from the corresponding predominant pathway in adults (both progenitors are present but differently represented in each case). We show that pre-B cells (Hardy fraction D) in the predominant adult pathway express high levels of CD138 and intermediate levels of I-A, whereas the corresponding pre-B cells in the pathway that predominates in neonates do not express either of these markers. As expected, because most of the pre-B cells in adults express CD138, we find that sorted CD138+ adult pre-B cells differentiate to IgM+ B cells in vitro. Sorted CD138- pre-B cells from neonates, the majority subset at this age, also mature to IgM+ cells (without passing through a CD138+ stage). Importantly, our studies here confirm the differential representation of adult and neonatal progenitor populations and further demonstrate that CD138 expression subdivides the adult CD19+, B220-6B2-/low population shown to contain B-1 progenitors in a way consistent with the predominance of B-1b progenitors in adults. Thus, CD138 expression provides a key route to distinguishing early B cell development pathway for what now are clearly three B cell lineages.

    View details for DOI 10.1073/pnas.0511305103

    View details for Web of Science ID 000236999000043

    View details for PubMedID 16606838

  • High-dose oral N-acetylcysteine, a glutathione prodrug, modulates inflammation in cystic fibrosis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Tirouvanziam, R., Conrad, C. K., Bottiglieri, T., Herzenberg, L. A., Moss, R. B., Herzenberg, L. A. 2006; 103 (12): 4628-4633


    Neutrophilic airway inflammation is a hallmark of cystic fibrosis (CF). As high oxidant producers, airway neutrophils contribute largely to the systemic redox imbalance seen in CF. In turn, this chronic and profound imbalance can impact circulating neutrophils before their migration into airways. Indeed, in 18 CF patients with stable disease, blood neutrophils were readily deficient in the pivotal antioxidant glutathione (P = 0.003, compared with 9 healthy controls). In a phase 1 study, this deficiency was improved (P = 0.025) by the glutathione prodrug N-acetylcysteine, given orally in high doses (0.6 to 1.0 g three times daily, for 4 weeks). This treatment was safe and markedly decreased sputum elastase activity (P = 0.006), the strongest predictor of CF pulmonary function. Consistently, neutrophil burden in CF airways was decreased upon treatment (P = 0.003), as was the number of airway neutrophils actively releasing elastase-rich granules (P = 0.005), as measured by flow cytometry. Pulmonary function measures were not improved, as expected with short-term treatment. After excluding data from subjects without baseline airway inflammation, positive treatment effects were more pronounced and included decreased sputum IL-8 levels (P = 0.032). Thus, high-dose oral N-acetylcysteine has the potential to counter the intertwined redox and inflammatory imbalances in CF.

    View details for DOI 10.1073/pnas.0511304103

    View details for Web of Science ID 000236362600055

    View details for PubMedID 16537378

  • B cell lineages: documented at last! NATURE IMMUNOLOGY Herzenberg, L. A., Tung, J. W. 2006; 7 (3): 225-226

    View details for Web of Science ID 000235360400003

    View details for PubMedID 16482166

  • Wnt signaling in the thymus is regulated by differential expression of intracellular signaling molecules PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Weerkamp, F., Baert, M. R., Naber, B. A., Koster, E. E., de Haas, E. F., Atkuri, K. R., van Dongen, J. J., Herzenberg, L. A., Staal, F. J. 2006; 103 (9): 3322-3326


    Wnt signaling is essential for T cell development in the thymus, but the stages in which it occurs and the molecular mechanisms underlying Wnt responsiveness have remained elusive. Here we examined Wnt signaling activity in both human and murine thymocyte populations by determining beta-catenin levels, Tcf-reporter activation and expression of Wnt-target genes. We demonstrate that Wnt signaling occurs in all thymocyte subsets, including the more mature populations, but most prominently in the double negative (DN) subsets. This differential sensitivity to Wnt signaling was not caused by differences in the presence of Wnts or Wnt receptors, as these appeared to be expressed at comparable levels in all thymocyte subsets. Rather, it can be explained by high expression of activating signaling molecules in DN cells, e.g., beta-catenin, plakoglobin, and long forms of Tcf-1, and by low levels of inhibitory molecules. By blocking Wnt signaling from the earliest stage onwards using overexpression of Dickkopf, we show that inhibition of the canonical Wnt pathway blocks development at the most immature DN1 stage. Thus, responsiveness to developmental signals can be regulated by differential expression of intracellular mediators rather than by abundance of receptors or ligands.

    View details for DOI 10.1073/pnas.0511299103

    View details for Web of Science ID 000235780700058

    View details for PubMedID 16492759

  • Haploidentical non-myeloablative allogeneic hematopoietic cell transplantation (HCT) using total lymphoid irradiation (TLI) and anti-thymocyte globulin (ATG) conditioning protects against acute graft versus host disease (GVHD) Lowsky, R., Heydari, K., Sahaf, B., Shizuru, J., Laport, G., Johnston, L., Stockerl-Goldstein, K., Arai, S., Miklos, D., Blume, K., Herzenberg, L., Negrin, R., Strober, S. AMER SOC HEMATOLOGY. 2005: 814A-815A
  • Molecular imaging using labeled donor tissues reveals patterns of engraftment, rejection, and survival in transplantation TRANSPLANTATION Cao, Y. A., Bachmann, M. H., Beilhack, A., Yang, Y., Tanaka, M., Swijnenburg, R. J., Reeves, R., Taylor-Edwards, C., Schulz, S., Doyle, T. C., Fathman, C. G., Robbins, R. C., Herzenberg, L. A., Negrin, R. S., Contag, C. H. 2005; 80 (1): 134-139


    Tissue regeneration and transplantation of solid organs involve complex processes that can only be studied in the context of the living organism, and methods of analyzing these processes in vivo are essential for development of effective transplantation and regeneration procedures. We utilized in vivo bioluminescence imaging (BLI) to noninvasively visualize engraftment, survival, and rejection of transplanted tissues from a transgenic donor mouse that constitutively expresses luciferase. Dynamic early events of hematopoietic reconstitution were accessible and engraftment from as few as 200 transplanted whole bone marrow (BM) cells resulted in bioluminescent foci in lethally irradiated, syngeneic recipients. The transplantation of autologous pancreatic Langerhans islets and of allogeneic heart revealed the tempo of transplant degeneration or immune rejection over time. This imaging approach is sensitive and reproducible, permits study of the dynamic range of the entire process of transplantation, and will greatly enhance studies across various disciplines involving transplantation.

    View details for DOI 10.1097/01.TP.0000164347.50559.A3

    View details for Web of Science ID 000230473800023

    View details for PubMedID 16003245

  • Culturing at atmospheric oxygen levels impacts lymphocyte function PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Atkuri, K. R., Herzenberg, L. A., Herzenberg, L. A. 2005; 102 (10): 3756-3759


    To determine whether culturing peripheral blood mononuclear cells at atmospheric oxygen levels skews responses in comparison with culturing lymphocytes at physiologic oxygen levels, we cultured peripheral blood mononuclear cells at 5%, 10%, and atmospheric (20%) gas-phase oxygen for 5 days. We found that incubator oxygen levels influenced lymphocyte proliferation stimulated by two commonly used stimuli: Con A and antibodies that crosslink surface CD3 and CD28 to mimic antigen presentation. In both cases, proliferation increased as gas-phase oxygen levels increased. In contrast, oxygen levels did not influence proliferation stimulated by phytohemagglutinin, another commonly used mitogen. Similarly, oxygen levels did not impact cell viability in unstimulated cultures. Thus, we conclude that the influence of oxygen levels on proliferation depends on the stimulus, and, most importantly from the standpoint of immune responses, culturing cells at atmospheric rather than physiologic oxygen levels results in significantly increased proliferation responses to the CD3/CD28 crosslinking, a proliferation stimulus commonly used to mimic T cell antigen receptor signaling.

    View details for DOI 10.1073/pnas.0409910102

    View details for Web of Science ID 000227533100043

    View details for PubMedID 15738407

  • Inherent specificities in natural antibodies: a key to immune defense against pathogen invasion SPRINGER SEMINARS IN IMMUNOPATHOLOGY Baumgarth, N., Tung, J. W., Herzenberg, L. A. 2005; 26 (4): 347-362


    Natural antibodies are produced at tightly regulated levels in the complete absence of external antigenic stimulation. They provide immediate, early and broad protection against pathogens, making them a crucial non-redundant component of the humoral immune system. These antibodies are produced mainly, if not exclusively, by a subset of long-lived, self-replenishing B cells termed B-1 cells. We argue here that the unique developmental pattern of these B-1 cells, which rests on positive selection by self antigens, ensures production of natural antibodies expressing evolutionarily important specificities that are required for the initial defense against invading pathogens. Positive selection for reactivity with self antigens could also result in the production of detrimental anti-self antibodies. However, B-1 cells have evolved a unique response pattern that minimizes the risk of autoimmunity. Although these cells respond rapidly and strongly to host-derived innate signals, such as cytokines, and to pathogen-encoded signals, such as lipopolysaccharide and phosphorylcholine, they respond very poorly to receptor-mediated activation. In addition, they rarely enter germinal centers and undergo affinity maturation. Thus, their potential for producing high-affinity antibodies with harmful anti-self specificity is highly restricted. The positive selection of B-1 cells occurs during the neonatal period, during which the long-lived self-renewing B-1 population is constituted. Many of these cells (B-1a) express CD5, although a smaller subset (B-1b) does not express this surface marker. Importantly, B-1a cells should not be confused with short-lived anergic B-2 cells, which originate in the bone marrow in adults and initiate CD5 expression and programmed cell death following self-antigen recognition. In summary, we argue here that the mechanisms that enable natural antibody production by B-1 cells reflect the humoral immune system, which has evolved in layers whose distinct developmental mechanisms generate complementary repertoires that collectively operate to maximize flexibility in responses to invading pathogens. B-2 cells, present in what may be the most highly evolved layer(s), express a repertoire that is explicitly selected against self recognition and directed towards the generation of high-affinity antibody response to external antigenic stimuli. B-1 cells, whose repertoire is selected by recognition of self antigen, belong to what may be earlier layer(s) and inherently maintain production of evolutionarily important antibody specificities that respond to pathogen-related, rather then antigen-specific signals.

    View details for DOI 10.1007/s00281-004-0182-2

    View details for Web of Science ID 000228526100002

    View details for PubMedID 15633017

  • The extracellular microenvironment plays a key role in regulating the redox status of cell surface proteins in HIV-infected subjects ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Sahaf, B., Heydari, K., Herzenberg, L. A., Herzenberg, L. A. 2005; 434 (1): 26-32


    There is an overwhelming interest in the study of the redox status of the cell surface affecting redox signaling in the cells and also predicting the total redox status of the cells. Measuring the total surface thiols (cell surface molecule thiols, csm-SH) we have shown that the overall level of surface thiols is tightly controlled. In vitro, the total concentration of intracellular glutathione (iGSH) seems to play a regulatory role in determination of the amounts of reduced proteins on cells. In addition, short term exposure of the cell surface to glutathione disulfide (GSSG, oxidized GSH) seems to reduce the overall levels of csm-SH suggesting that the function of some cysteine containing proteins on the cell surface may be regulated by the amount of GSSG secreted from the cells or the GSSG available in the extracellular environment. Examination of peripheral blood mononuclear cells (PBMCs) from healthy or HIV-infected subjects failed to reveal a similar correlation between the intra- and extracellular thiol status of cells. Although there is a relatively wide variation between individuals in both csm-SH and iGSH there is no correlation between the iGSH and csm-SH levels measured for healthy and HIV-infected individuals. There are many reports suggesting different redox active proteins on the cell surface to be the key players in the total cell surface redox regulation. However, we suggest that the redox status of the cells is regulated through a complex and tightly regulated mechanism that needs further investigation. In the mean time, overall surface thiol measurements together with case specific protein determinations may offer the most informative approach. In this review, we discuss our own results as well as results from other laboratories to argue that the overall levels of surface thiols on the exofacial membrane are regulated primarily by redox status of the cell surface microenvironment.

    View details for DOI 10.1016/j.jabb.2004.11.015

    View details for Web of Science ID 000226457700005

    View details for PubMedID 15629105

  • Functional evolution of the vertebrate Myb gene family: B-Myb, but neither A-Myb nor C-Myb, complements drosophila Myb in hemocytes GENETICS Davidson, C. J., Tirouvanziam, R., Herzenberg, L. A., Lipsick, J. S. 2005; 169 (1): 215-229


    The duplication of genes and genomes is believed to be a major force in the evolution of eukaryotic organisms. However, different models have been presented about how duplicated genes are preserved from elimination by purifying selection. Preservation of one of the gene copies due to rare mutational events that result in a new gene function (neofunctionalization) necessitates that the other gene copy retain its ancestral function. Alternatively, preservation of both gene copies due to rapid divergence of coding and noncoding regions such that neither retains the complete function of the ancestral gene (subfunctionalization) may result in a requirement for both gene copies for organismal survival. The duplication and divergence of the tandemly arrayed homeotic clusters have been studied in considerable detail and have provided evidence in support of the subfunctionalization model. However, the vast majority of duplicated genes are not clustered tandemly, but instead are dispersed in syntenic regions on different chromosomes, most likely as a result of genome-wide duplications and rearrangements. The Myb oncogene family provides an interesting opportunity to study a dispersed multigene family because invertebrates possess a single Myb gene, whereas all vertebrate genomes examined thus far contain three different Myb genes (A-Myb, B-Myb, and c-Myb). A-Myb and c-Myb appear to have arisen by a second round of gene duplication, which was preceded by the acquisition of a transcriptional activation domain in the ancestral A-Myb/c-Myb gene generated from the initial duplication of an ancestral B-Myb-like gene. B-Myb appears to be essential in all dividing cells, whereas A-Myb and c-Myb display tissue-specific requirements during spermatogenesis and hematopoiesis, respectively. We now report that the absence of Drosophila Myb (Dm-Myb) causes a failure of larval hemocyte proliferation and lymph gland development, while Dm-Myb(-/-) hemocytes from mosaic larvae reveal a phagocytosis defect. In addition, we show that vertebrate B-Myb, but neither vertebrate A-Myb nor c-Myb, can complement these hemocyte proliferation defects in Drosophila. Indeed, vertebrate A-Myb and c-Myb cause lethality in the presence or absence of endogenous Dm-Myb. These results are consistent with a neomorphic origin of an ancestral A-Myb/c-Myb gene from a duplicated B-Myb-like gene. In addition, our results suggest that B-Myb and Dm-Myb share essential conserved functions that are required for cell proliferation. Finally, these experiments demonstrate the utility of genetic complementation in Drosophila to explore the functional evolution of duplicated genes in vertebrates.

    View details for DOI 10.1534/genetics.104.034132

    View details for Web of Science ID 000227039900018

    View details for PubMedID 15489525

  • The E47 transcription factor negatively regulates CD5 expression during thymocyte development PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Yang, Y., Contag, C. H., Felsher, D., Shachaf, C. M., Cao, Y., Herzenberg, L. A., Herzenberg, L. A., Tung, J. W. 2004; 101 (11): 3898-3902


    The expression of CD5 increases progressively as thymocytes mature. We have shown that CD5 expression is controlled by a tissue-specific regulatory promoter located upstream of the CD5 translation start sites. Deletion of this regulatory promoter, which contains three potential transcription factor binding sites (CCAAT, kappa E2, and ets) reduces the promoter activity to basal level. Of these sites, only ets proved essential for CD5 expression in T cell lines. Here, we introduce a role for the E47 transcription factor and the CD5 promoter kappa E2 site in regulating CD5 expression during thymocyte development. Using T cell lines, we show that (i) mutation of the kappa E2 site in the CD5 regulatory promoter results in a significant elevation of CD5 promoter activity; (ii) the E47 transcription factor binds to the kappa E2 site; and (iii) overexpression of E47 inhibits CD5 expression. We then show, in high-dimensional fluorescence-activated cell sorting studies with primary thymocytes at successive developmental stages, that (i) intracellular E47 levels decrease as surface CD5 expression increases; (ii) E47 expression is down-regulated and CD5 expression is correspondingly up-regulated in DN3 thymocytes in RAG-2-deficient mice injected with anti-CD3 to mimic pre-T cell receptor stimulation; and (iii) E47 expression is down-regulated and CD5 expression is up-regulated when double positive thymocytes are stimulated in vitro with anti-CD3. Based on these data, we propose that E47 negatively regulates CD5 expression by interacting with the kappa E2 site in the CD5 regulatory promoter and that decreases in E47 in response to developmental signals are critical to the progressive increase in CD5 expression as thymocytes mature.

    View details for DOI 10.1073/pnas.0308764101

    View details for Web of Science ID 000220314500034

    View details for PubMedID 15001710

  • Fluorescence-activated cell sorting (FACS) of Drosophila hemocytes reveals important functional similarities to mammalian leukocytes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Tirouvanziam, R., Davidson, C. J., Lipsick, J. S., Herzenberg, L. A. 2004; 101 (9): 2912-2917


    Drosophila is a powerful model for molecular studies of hematopoiesis and innate immunity. However, its use for functional cellular studies remains hampered by the lack of single-cell assays for hemocytes (blood cells). Here we introduce a generic method combining fluorescence-activated cell sorting and nonantibody probes that enables the selective gating of live Drosophila hemocytes from the lymph glands (larval hematopoietic organ) or hemolymph (blood equivalent). Gated live hemocytes are analyzed and sorted at will based on precise quantitation of fluorescence levels originating from metabolic indicators, lectins, reporters (GFP and beta-galactosidase) and antibodies. With this approach, we discriminate and sort plasmatocytes, the major hemocyte subset, from lamellocytes, an activated subset present in gain-of-function mutants of the Janus kinase and Toll pathways. We also illustrate how important, evolutionarily conserved, blood-cell-regulatory molecules, such as calcium and glutathione, can be studied functionally within hemocytes. Finally, we report an in vivo transfer of sorted live hemocytes and their successful reanalysis on retrieval from single hosts. This generic and versatile fluorescence-activated cell sorting approach for hemocyte detection, analysis, and sorting, which is efficient down to one animal, should critically enhance in vivo and ex vivo hemocyte studies in Drosophila and other species, notably mosquitoes.

    View details for DOI 10.1073/pnas.0308734101

    View details for Web of Science ID 000220065300049

    View details for PubMedID 14976247

  • New approaches to fluorescence compensation and visualization of FACS data CLINICAL IMMUNOLOGY Tung, J. W., Parks, D. R., Moore, W. A., Herzenberg, L. A., Herzenberg, L. A. 2004; 110 (3): 277-283


    The Fluorescence Activated Cell Sorter (FACS) is an invaluable tool for clinicians and researchers alike in phenotyping and sorting individual cells. With the advances in FACS methodology, notably intracellular staining for cytokines, transcription factors and phosphoproteins, and with increases in the number of fluorescence detection channels, researchers now have the opportunity to study individual cells in far greater detail than previously possible. In this chapter, we discuss High-Definition (Hi-D) FACS methods that can improve analysis of lymphocyte subsets in mouse and man. We focus on the reasons why fluorescence compensation, which is necessary to correct for spectral overlap between two or more fluorochromes used in the same staining combination, is best done as a computed transformation rather than using the analog circuitry available on many flow cytometers. In addition, we introduce a new data visualization method that scales the axes on histograms and two-dimensional contour (or dot) plots to enable visualization of signals from all cells, including those that have minimal fluorescence values and are not properly represented with traditional logarithmic axes. This "Logicle" visualization method, we show, provides superior representations of compensated data and makes correctly compensated data look correct. Finally, we discuss controls that facilitate recognition of boundaries between positive and negative subsets.

    View details for DOI 10.1016/j.clim.2003.11.016

    View details for Web of Science ID 000220831800009

    View details for PubMedID 15047205

  • B lineage-specific regulation of V(D)J recombinase activity is established in common lymphoid progenitors JOURNAL OF EXPERIMENTAL MEDICINE BORGHESI, L., Hsu, L. Y., Miller, J. P., Anderson, M., Herzenberg, L., Herzenberg, L., Schlissel, M. S., Allman, D., Gerstein, R. M. 2004; 199 (4): 491-502


    Expression of V(D)J recombinase activity in developing lymphocytes is absolutely required for initiation of V(D)J recombination at antigen receptor loci. However, little is known about when during hematopoietic development the V(D)J recombinase is first active, nor is it known what elements activate the recombinase in multipotent hematopoietic progenitors. Using mice that express a fluorescent transgenic V(D)J recombination reporter, we show that the V(D)J recombinase is active as early as common lymphoid progenitors (CLPs) but not in the upstream progenitors that retain myeloid lineage potential. Evidence of this recombinase activity is detectable in all four progeny lineages (B, T, and NK, and DC), and rag2 levels are the highest in progenitor subsets immediately downstream of the CLP. By single cell PCR, we demonstrate that V(D)J rearrangements are detectable at IgH loci in approximately 5% of splenic natural killer cells. Finally, we show that recombinase activity in CLPs is largely controlled by the Erag enhancer. As activity of the Erag enhancer is restricted to the B cell lineage, this provides the first molecular evidence for establishment of a lineage-specific transcription program in multipotent progenitors.

    View details for DOI 10.1084/jem.20031800

    View details for Web of Science ID 000189185600006

    View details for PubMedID 14769852

  • Ontogeny of gamma delta T cells in humans JOURNAL OF IMMUNOLOGY De Rosa, S. C., Andrus, J. P., Perfetto, S. P., Mantovani, J. J., Herzenberg, L. A., Herzenberg, L. A., Roederer, M. 2004; 172 (3): 1637-1645


    T cell receptors consist either of an alpha-chain combined with a beta-chain or a gamma-chain combined with a delta-chain. alphabeta T cells constitute the majority of T cells in human blood throughout life. Flow cytometric analyses presented in this study, which focus on the representation of the developmental (naive and memory) subsets of gammadelta T cells, show by function and phenotype that this lineage contains both naive and memory cells. In addition, we show that the representation of naive T cells is higher among alphabeta than gammadelta T cells in adults and that the low frequency of naive gammadelta T cells in adults reflects ontological differences between the two major gammadelta subsets, which are distinguished by expression of Vdelta1 vs Vdelta2 delta-chains. Vdelta1 cells, which mirror alphabeta cells with respect to naive representation, predominate during fetal and early life, but represent the minority of gammadelta cells in healthy adults. In contrast, Vdelta2 cells, which constitute the majority of adult gammadelta cells, show lower frequencies of naive cells than Vdelta1 early in life and show vanishingly small naive frequencies in adults. In essence, nearly all naive Vdelta2 cells disappear from blood by 1 year of life. Importantly, even in children less than 1 year old, most of the nonnaive Vdelta2 cells stain for perforin and produce IFN-gamma after short-term in vitro stimulation. This represents the earliest immunological maturation of any lymphocyte compartment in humans and most likely indicates the importance of these cells in controlling pathology due to common environmental challenges.

    View details for Web of Science ID 000188378700039

    View details for PubMedID 14734745

  • Identification of B-cell subsets: an exposition of 11-color (Hi-D) FACS methods. Methods in molecular biology (Clifton, N.J.) Tung, J. W., Parks, D. R., Moore, W. A., Herzenberg, L. A., Herzenberg, L. A. 2004; 271: 37-58


    In the last few years, the effectiveness of developmental and functional studies of individual subsets of cells has increased dramatically owing to the identification of additional subset markers and the extension of fluorescence-activated cell sorter (FACS) capabilities to simultaneously measure the expression of more markers on individual cells. For example, introduction of a 6-8 multiparameter FACS instrument resulted in significant advances in understanding B-cell development. In this chapter, we describe 11-color high-dimensional (Hi-D) FACS staining and data analysis methods that provide greater clarity in identifying the B-cell subsets in bone marrow, spleen, and peritoneal cavity. Further, we show how a single Hi-D FACS antibody reagent combination is sufficient to unambiguously identify most of the currently defined B-cell developmental subsets in the bone marrow (Hardy fractions A-F) and the functional B-cell subsets (B-1a, B-1b, B-2, and marginal zone [MZ] B cells) in the periphery. Although we focus on murine B-cell subsets, the methods we discuss are relevant to FACS studies conducted with all types of cells and other FACS instruments. We introduce a new method for scaling axes for histograms or contour plots of FACS data. This method, which we refer to as Logicle visualization, is particularly useful in promoting correct interpretations of fluorescence-compensated FACS data and visual confirmation of correct compensation values. In addition, it facilitates discrimination of valid subsets. Application of Logicle visualization tools in the Hi-D FACS studies discussed here creates a strong new base for in-depth analysis of B-cell development and function.

    View details for PubMedID 15146111

  • Non-myeloablative conditioning with total lymphoid irradiation (TLI) and anti-thymocyte globulin (ATG) for allogeneic hematopoietic cell transplantation (HCT) results in high levels of regulatory natural killer T cells and low incidences of acute GVHD and tumor relapse. Lowsky, R., Jones, S. D., Mitra, S., Shizuru, J. A., Laport, G. G., Stockerl-Goldstein, K., JOHNSTON, L. J., Stuart, M. J., Herzenberg, L. A., Hoppe, R. T., Blume, K. G., Negrin, R. S., Strober, S. AMER SOC HEMATOLOGY. 2003: 152A-153A
  • Leukocyte functional antigen 1 lowers T cell activation thresholds and signaling through cytohesin-1 and Jun-activating binding protein 1 NATURE IMMUNOLOGY Perez, O. D., Mitchell, D., Jager, G. C., South, S., Murriel, C., McBride, J., Herzenberg, L. A., Kinoshita, S., Nolan, G. P. 2003; 4 (11): 1083-1092


    Leukocyte functional antigen 1 (LFA-1), with intercellular adhesion molecule ligands, mediates T cell adhesion, but the signaling pathways and functional effects imparted by LFA-1 are unclear. Here, intracellular phosphoprotein staining with 13-dimensional flow cytometry showed that LFA-1 activation induced phosphorylation of the beta(2) integrin chain and release of Jun-activating binding protein 1 (JAB-1), and mediated signaling of kinase Erk1/2 through cytohesin-1. Dominant negatives of both JAB-1 and cytohesin-1 inhibited interleukin 2 production and impaired T helper type 1 differentiation. LFA-1 stimulation lowered the threshold of T cell activation. Thus, LFA-1 signaling contributes to T cell activation and effects T cell differentiation.

    View details for DOI 10.1038/ni984

    View details for Web of Science ID 000186241100012

    View details for PubMedID 14528303

  • Lymphocyte surface thiol levels PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sahaf, B., Heydari, K., Herzenberg, L. A., Herzenberg, L. A. 2003; 100 (7): 4001-4005


    Recent studies have implicated reduced thiols (cysteine -SH) in the function of individual cell surface proteins. Studies presented here demonstrate that the overall level of reduced thiols on cell surface molecules differs on individual subsets of peripheral blood mononuclear cells and that these levels can be manipulated in vitro by altering the level of intracellular glutathione (iGSH). To quantitate cell surface thiols, we have developed a Hi-D (11-color) fluorescence-activated cell sorter method in which we covalently couple a fluorescent molecule, Alexa-maleimide, to free (reduced) -SH groups on proteins or other molecules exposed on the cell surface (exofacial membrane). In addition, to reveal changes in cell surface thiol levels in response to various in vitro treatments, we used a pair of fluorescent Alexa dyes with distinct excitation and emission spectra to stain the cells before and after treatments. These in vitro studies demonstrate that decreasing iGSH, by specifically inhibiting its synthesis, decreases cell surface molecule thiols (csm-SH) and that preventing loss of iGSH also prevents loss of csm-SH. However, examination of peripheral blood mononuclear cell subsets tested immediately after isolation from healthy or HIV-infected subjects failed to reveal a similar relationship between internal iGSH and csm-SH. Although there is a relatively wide variation between individuals in both csm-SH and iGSH, there is no correlation between median iGSH and csm-SH compared for 22 healthy and 36 HIV-infected subjects. Collectively, our findings indicate that local environment plays a greater role in determining the redox status of cell surface molecules than the internal redox status of the cells.

    View details for DOI 10.1073/pnas.2628032100

    View details for Web of Science ID 000182058400085

    View details for PubMedID 12642656

  • B cell-dependent T cell responses: IgM antibodies are required to elicit contact sensitivity JOURNAL OF EXPERIMENTAL MEDICINE Tsuji, R. F., Szczepanik, M., Kawikova, I., Paliwal, V., Campos, R. A., Itakura, A., Akahira-Azuma, M., Baumgarth, N., Herzenberg, L. A., Askenase, P. W. 2002; 196 (10): 1277-1290


    Contact sensitivity (CS) is a classic example of in vivo T cell immunity in which skin sensitization with reactive hapten leads to immunized T cells, which are then recruited locally to mediate antigen-specific inflammation after subsequent skin challenge. We have previously shown that T cell recruitment in CS is triggered by local activation of complement, which generates C5a that triggers C5a receptors most likely on mast cells. Here, we show that B-1 cell-derived antihapten IgM antibodies generated within 1 day (d) of immunization combine with local challenge antigen to activate complement to recruit the T cells. These findings overturn three widely accepted immune response paradigms by showing that (a) specific IgM antibodies are required to initiate CS, which is a classical model of T cell immunity thought exclusively due to T cells, (b) CS priming induces production of specific IgM antibodies within 1 d, although primary antibody responses typically begin by day 4, and (c) B-1 cells produce the 1-d IgM response to CS priming, although these cells generally are thought to be nonresponsive to antigenic stimulation. Coupled with previous evidence, our findings indicate that the elicitation of CS is initiated by rapidly formed IgM antibodies. The IgM and challenge antigen likely form local complexes that activate complement, generating C5a, leading to local vascular activation to recruit the antigen-primed effector T cells that mediate the CS response.

    View details for DOI 10.1084/jem.20020649

    View details for Web of Science ID 000179412600002

    View details for PubMedID 12438420

  • The history and future of the fluorescence activated cell sorter and flow cytometry: A view from Stanford CLINICAL CHEMISTRY Herzenberg, L. A., Parks, D., Sahaf, B., Perez, O., Roederer, M., Herzenberg, L. A. 2002; 48 (10): 1819-1827


    The Fluorescence Activated Cell Sorter (FACS) was invented in the late 1960s by Bonner, Sweet, Hulett, Herzenberg, and others to do flow cytometry and cell sorting of viable cells. Becton Dickinson Immunocytometry Systems introduced the commercial machines in the early 1970s, using the Stanford patent and expertise supplied by the Herzenberg Laboratory and a Becton Dickinson engineering group under Bernie Shoor. Over the years, we have increased the number of measured FACS dimensions (parameters) and the speed of sorting to where we now simultaneously measure 12 fluorescent colors plus 2 scatter parameters. In this history, I illustrate the great utility of this state-of-the-art instrument, which allows us to simultaneously stain, analyze, and then sort cells from small samples of human blood cells from AIDS patients, infants, stem cell transplant patients, and others. I also illustrate analysis and sorting of multiple subpopulations of lymphocytes by use of 8-12 colors. In addition, I review single cell sorting used to clone and analyze hybridomas and discuss other applications of FACS developed over the past 30 years, as well as give our ideas on the future of FACS. These ideas are currently being implemented in new programs using the internet for data storage and analysis as well as developing new fluorochromes, e.g., green fluorescent protein and tandem dyes, with applications in such areas as apoptosis, gene expression, cytokine expression, cell biochemistry, redox regulation, and AIDS. Finally, I describe new FACS methods for measuring activated kinases and phosphatases and redox active enzymes in individual cells simultaneously with cell surface phenotyping. Thus, key functions can be studied in various subsets of cells without the need for prior sorting.

    View details for Web of Science ID 000178238100033

    View details for PubMedID 12324512

  • Identification of a germ-line pro-B cell subset that distinguishes the fetal/neonatal from the adult B cell development pathway PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lu, L. S., Tung, J., Baumgarth, N., Herman, O., Gleimer, M., Herzenberg, L. A., Herzenberg, L. A. 2002; 99 (5): 3007-3012


    Studies presented here show that the expression of CD4, MHC class II (Ia,) and B220 cleanly resolves a major and a minor subset within the earliest pro-B cell population (germ-line pro-B) in adult bone marrow (BM). The major subset expresses intermediate B220 and low CD4 levels. The minor subset, which constitutes roughly 20% of the adult germ-line pro-B, expresses very low B220 levels and does not express CD4. Ia is clearly detectable at low levels on the major germ-line pro-B subset, both in wild-type adult mice and in gene-targeted mice (RAG2-/- and microMT), in which B cell development terminates before the pre-B cell stage. A small proportion of cells in the more mature pro-B cell subsets (Hardy Fractions B and C) also express Ia at this level. In contrast, Ia levels on the minor subset are barely above (or equal to) background. Surprisingly, the major germ-line pro-B cell subset found in adults is missing in fetal and neonatal animals. All of the germ-line pro-B in these immature animals express a phenotype (very low B220, no CD4, or Ia) similar to that of the minor pro-B cell subset in adult BM. Because B cell development in fetal/neonatal animals principally results in B-1 cells, these findings demonstrate that the B-1 development pathway does not include the major germ-line pro-B subset found in adult BM and hence identify a very early difference between the B-1 and -2 development pathways.

    View details for DOI 10.1073/pnas.052715399

    View details for Web of Science ID 000174284600075

    View details for PubMedID 11867763

  • Motexafin gadolinium (Gd-Tex) selectively induces apoptosis in HIV-1 infected CD4+T helper cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Perez, O. D., Nolan, G. P., Magda, D., MILLER, R. A., Herzenberg, L. A., Herzenberg, L. A. 2002; 99 (4): 2270-2274


    Here, we show that motexafin gadolinium (Gd-Tex), a compound that promotes intracellular oxidative stress, selectively induces apoptosis in HIV-1-infected CD4(+) T cells in IL-2-stimulated cultures of peripheral blood mononuclear cells infected in vitro with HIV-1. This selective induction of apoptosis, which we detect by FACS analysis of intracellular HIV/p24 and concomitant surface and apoptosis marker expression, is abrogated by the glutathione precursor, N-acetyl-l-cysteine. Importantly, it occurs at Gd-Tex concentrations that are not cytotoxic to uninfected cells in the culture. These findings suggest that Gd-Tex may have therapeutic utility as an anti-HIV agent capable of selectively targeting and removing HIV-infected cells in an infected host.

    View details for DOI 10.1073/pnas.261711499

    View details for Web of Science ID 000174031100093

    View details for PubMedID 11854523

  • Protein glutathionylation: coupling and uncoupling of glutathione to protein thiol groups in lymphocytes under oxidative stress and HIV infection MOLECULAR IMMUNOLOGY Ghezzi, P., Romines, B., Fratelli, M., Eberini, I., Gianazza, E., Casagrande, S., Laragione, T., Mengozzi, M., Herzenberg, L. A., Herzenberg, L. A. 2002; 38 (10): 773-780


    We show here that exposure to oxidative stress induces glutathione (GSH) modification of protein cysteinyl residues (glutathionylation) in T cell blasts. Treating the cells with the oxidant diamide induces thiolation of a series of proteins that can be detected by 2D electrophoresis when 35S-cysteine is used to label the intracellular GSH pool. This thiolation is reversible, proteins are rapidly dethiolated and GSH is released from proteins once the oxidants are washed and the cells are allowed to recover. Dethiolation is dependent on the availability of GSH and thiols, since it is inhibited by GSH-depleting agents and improved by N-acetyl-L-cysteine (NAC). The capacity of these agents to reverse glutathionylation is diminished in T cell blasts infected in vitro with HIV, which is known to cause oxidative stress. Consistent with these findings, the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme known to be inhibited by glutathionylation, is inhibited in diamide-treated cells and recovers rapidly when cells are allowed to dethiolate. Further, GAPDH activity is diminished by GSH-depleting agents and augmented by NAC. Thus, reversible glutathionylation of proteins can rapidly shift the activity of a key metabolic enzyme and thereby result in dramatic, reversible changes in cellular metabolism.

    View details for Web of Science ID 000174234700008

    View details for PubMedID 11841837

  • Circulating thioredoxin suppresses lipopolysaccharide-induced neutrophil chemotaxis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Nakamura, H., Herzenberg, L. A., Bai, J., ARAYA, S., Kondo, N., Nishinaka, Y., Herzenberg, L. A., Yodoi, J. 2001; 98 (26): 15143-15148


    Thioredoxin (Trx), a redox enzyme with a conserved active site (Cys-32-Gly-Pro-Cys-35), is induced and secreted into circulation in response to inflammation. Studies here demonstrate that elevating Trx levels in circulation either by i.v. injection of recombinant Trx or stimulating Trx release in Trx-transgenic mice dramatically blocks lipopolysaccharide (LPS)-stimulated neutrophil migration in the murine air pouch chemotaxis model. Furthermore, we show that leukocyte recruitment induced by the murine chemokines KC/GROalpha, RANTES (regulated upon activation, normal T cell expressed and secreted), and monocyte chemoattractant protein-1 (MCP-1) is suppressed also in Trx-transgenic mice. Addressing the mechanism responsible for this suppression, we show that circulating Trx blocks (i) the LPS-stimulated in vitro activation of neutrophil p38 mitogen-activated protein kinase, (ii) the normal down-regulation of CD62L on neutrophils migrating into the LPS-stimulated air pouch, and (iii) the in vitro adhesion of LPS-activated neutrophils on endothelial cells. However, as we also show, Trx does not alter the expression of endothelial cell adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1, CD62P, and CD62E) within 3 h. Collectively, these findings indicate that elevated levels of circulating Trx interfere with chemotaxis by acting directly on neutrophils. We discuss these findings in the context of recent studies reporting beneficial effects of acutely elevated Trx in ischemic injury and negative effects associated with chronically elevated Trx in HIV disease.

    View details for Web of Science ID 000172848800073

    View details for PubMedID 11742067

  • Dynamics of cytokine and perforin expression in T cells after allogeneic bone marrow transplantation: Possible predictors of GVHD. Watanabe, N., DeRosa, S. C., Chao, N. J., Herzenberg, L. A., Herzenberg, L. A., Roederer, M. AMER SOC HEMATOLOGY. 2001: 661A-661A
  • V delta and V delta 2 gamma delta T cells express distinct surface markers and might be developmentally distinct lineages JOURNAL OF LEUKOCYTE BIOLOGY De Rosa, S. C., Mitra, D. K., Watanabe, N., Herzenberg, L. A., Herzenberg, L. A., Roederer, M. 2001; 70 (4): 518-526


    We report here that the two major types of gammadelta T cells found in human blood, Vdelta1 and Vdelta2, were found to have markedly different phenotypes. Vdelta2 cells had a phenotype typical of most alphabeta T cells in blood; i.e., they were CD5(+), CD28(+), and CD57(-). In contrast, Vdelta1 cells tended to be CD5(-/dull), CD28(-), and CD57(+). Furthermore, although Vdelta1 T cells appeared to be "naive" in that they were CD45RA(+), they were CD62L(-) and on stimulation uniformly produced interferon-gamma, indicating that they are in fact memory/effector cells. This phenotype for Vdelta1 cells was similar to that of intestinal intraepithelial lymphocytes, a subset that can develop in the absence of the thymus. We suggest that the Vdelta1 and Vdelta2 T cell subsets represent distinct lineages with different developmental pathways. The disruption of the supply of normal, thymus-derived T cells in HIV-infected individuals might be responsible for the shift in the Vdelta2/Vdelta1 ratio that occurs in the blood of individuals with HIV disease.

    View details for Web of Science ID 000171467500005

    View details for PubMedID 11590187

  • Naive CD4 T cells inhibit CD28-costimulated R5 HIV replication in memory CD4 T cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Mengozzi, M., Malipatlolla, M., De Rosa, S. C., Herzenberg, L. A., Herzenberg, L. A., Roederer, M. 2001; 98 (20): 11644-11649


    Stimulation with antibodies to CD3 and CD28 coimmobilized on beads can be used to significantly expand T cells ex vivo. With CD4 T cells from HIV-infected patients, this expansion usually is accompanied by complete suppression of viral replication, presumed to be caused by down-regulation of the viral coreceptor CCR5 and up-regulation of CCR5 ligands. Here we show that this suppression occurs in total CD4 T cells acutely infected with R5 HIV, but not in purified CD62L(-) memory CD4 T cells. The lack of complete suppression in these memory cells, typically comprising 10-40% of total CD4 T cells, occurs despite high levels of CCR5 ligand secretion and down-regulation of CCR5. Significantly, adding back naive or CD62L(+) memory CD4 T cells inhibits the viral replication in the CD62L(-) cells, with the naive cells capable of completely repressing the virus. Although this inhibition was previously thought to be specific to bead-bound anti-CD3/CD28 stimulation, we show that the same suppression is obtained with sufficiently strong anti-CD3/B7.1 stimulation. Our results show that inhibitory mechanisms, expressed predominantly by strongly stimulated naive CD4 T cells and mediated independently of CCR5-binding chemokines, play a role in the inhibition of R5 HIV replication in CD4 T cells upon CD28 costimulation.

    View details for Web of Science ID 000171237100118

    View details for PubMedID 11562498

  • Probability binning comparison: A metric for quantitating univariate distribution differences CYTOMETRY Roederer, M., Treister, A., Moore, W., Herzenberg, L. A. 2001; 45 (1): 37-46


    Comparing distributions of data is an important goal in many applications. For example, determining whether two samples (e.g., a control and test sample) are statistically significantly different is useful to detect a response, or to provide feedback regarding instrument stability by detecting when collected data varies significantly over time.We apply a variant of the chi-squared statistic to comparing univariate distributions. In this variant, a control distribution is divided such that an equal number of events fall into each of the divisions, or bins. This approach is thereby a mini-max algorithm, in that it minimizes the maximum expected variance for the control distribution. The control-derived bins are then applied to test sample distributions, and a normalized chi-squared value is computed. We term this algorithm Probability Binning.Using a Monte-Carlo simulation, we determined the distribution of chi-squared values obtained by comparing sets of events derived from the same distribution. Based on this distribution, we derive a conversion of any given chi-squared value into a metric that is analogous to a t-score, i.e., it can be used to estimate the probability that a test distribution is different from a control distribution. We demonstrate that this metric scales with the difference between two distributions, and can be used to rank samples according to similarity to a control. Finally, we demonstrate the applicability of this metric to ranking immunophenotyping distributions to suggest that it indeed can be used to objectively determine the relative distance of distributions compared to a single control.Probability Binning, as shown here, provides a useful metric for determining the probability that two or more flow cytometric data distributions are different. This metric can also be used to rank distributions to identify which are most similar or dissimilar. In addition, the algorithm can be used to quantitate contamination of even highly-overlapping populations. Finally, as demonstrated in an accompanying paper, Probability Binning can be used to gate on events that represent significantly different subsets from a control sample. Published 2001 Wiley-Liss, Inc.

    View details for Web of Science ID 000171015000005

    View details for PubMedID 11598945

  • Probability binning comparison: A metric for quantitating multivariate distribution differences CYTOMETRY Roederer, M., Moore, W., Treister, A., Hardy, R. R., Herzenberg, L. A. 2001; 45 (1): 47-55


    While several algorithms for the comparison of univariate distributions arising from flow cytometric analyses have been developed and studied for many years, algorithms for comparing multivariate distributions remain elusive. Such algorithms could be useful for comparing differences between samples based on several independent measurements, rather than differences based on any single measurement. It is conceivable that distributions could be completely distinct in multivariate space, but unresolvable in any combination of univariate histograms. Multivariate comparisons could also be useful for providing feedback about instrument stability, when only subtle changes in measurements are occurring.We apply a variant of Probability Binning, described in the accompanying article, to multidimensional data. In this approach, hyper-rectangles of n dimensions (where n is the number of measurements being compared) comprise the bins used for the chi-squared statistic. These hyper-dimensional bins are constructed such that the control sample has the same number of events in each bin; the bins are then applied to the test samples for chi-squared calculations.Using a Monte-Carlo simulation, we determined the distribution of chi-squared values obtained by comparing sets of events from the same distribution; this distribution of chi-squared values was identical as for the univariate algorithm. Hence, the same formulae can be used to construct a metric, analogous to a t-score, that estimates the probability with which distributions are distinct. As for univariate comparisons, this metric scales with the difference between two distributions, and can be used to rank samples according to similarity to a control. We apply the algorithm to multivariate immunophenotyping data, and demonstrate that it can be used to discriminate distinct samples and to rank samples according to a biologically-meaningful difference.Probability binning, as shown here, provides a useful metric for determining the probability with which two or more multivariate distributions represent distinct sets of data. The metric can be used to identify the similarity or dissimilarity of samples. Finally, as demonstrated in the accompanying paper, the algorithm can be used to gate on events in one sample that are different from a control sample, even if those events cannot be distinguished on the basis of any combination of univariate or bivariate displays. Published 2001 Wiley-Liss, Inc.

    View details for Web of Science ID 000171015000006

    View details for PubMedID 11598946

  • Isoelectric focusing and enzyme overlay membrane analysis of caspase 3 activation ANALYTICAL BIOCHEMISTRY Breithaupt, T. B., Shires, A. L., Voehringer, D. W., Herzenberg, L. A., Herzenberg, L. A. 2001; 292 (2): 313-316

    View details for Web of Science ID 000168902300023

    View details for PubMedID 11355870

  • Phycoerythrin-allophycocyanin: A resonance energy transfer fluorochrome for immunofluorescence CYTOMETRY Tjioe, I., Legerton, T., Wegstein, J., Herzenberg, L. A., Roederer, M. 2001; 44 (1): 24-29


    As immunofluorescence experiments become more complex, the demand for new dyes with different properties increases. Fluorescent dyes with large Stoke's shifts that are very bright and have low background binding to cells are especially desirable. We report on the properties of the resonance energy tandems of phycoerythrin and allophycocyanin (PE-APC). PE-APC is the original fluorescence resonance energy tandem dye described in the literature, but it has not been utilized because of the difficulty of synthesizing and preparing a consistent product.PE-APC complexes comprising different ratios of the two phycobiliproteins conjugated to streptavidin were synthesized using standard protein-protein conjugation chemistry. The PE-APC streptavidins were evaluated for flow cytometric analysis. They were compared directly to Cy5PE conjugates because Cy5PE is the fluorophore that is spectrally most like the PE-APC.PE-APC complexes showed the expected fluorescence spectral properties of a tandem: excitation was excellent at 488 nm (and best at the PE excitation maximum) and emission was greatest at the APC emission maximum at about 660 nm. The efficiency of transfer of energy from PE to APC was about 90%.PE-APC can be considered an excellent substitute for Cy5PE. Compared with Cy5PE, PE-APC has similar brightness (in staining experiments), slightly greater compensation requirements with PE but much lower compensation with Cy5.5PE or Cy5.5PerCP, and lower nonspecific background binding. PE-APC is a useful alternative to Cy5PE, especially in applications in which the use of Cy5 is impractical. Cytometry 44:24-29, 2001. Published 2001 Wiley-Liss, Inc.

    View details for Web of Science ID 000168346600004

    View details for PubMedID 11309805

  • MHC class II expression is initiated at the earliest stage of B cell development in murine bone marrow Lu, L. S., Baumgarth, N., Herzenberg, L. A. FEDERATION AMER SOC EXP BIOL. 2001: A320-A320
  • Chronic elevation of plasma thioredoxin: Inhibition of chemotaxis and curtailment of life expectancy in AIDS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Nakamura, H., De Ros, S. C., Yodoi, J. J., Holmgren, A., Ghezzi, P., Herzenberg, L. A., Herzenberg, L. A. 2001; 98 (5): 2688-2693


    Thioredoxin (Trx) is an intracellular redox protein with extracellular cytokine-like and chemokine-like activities. We show here that, although plasma Trx levels are unrelated to survival of HIV-infected individuals with CD4 cell counts above 200/microl blood, survival is significantly impaired (P = 0.003) when plasma Trx is chronically elevated in HIV-infected subjects with CD4 T cell counts below this level (i.e., with Centers for Disease Control (CDC)-defined AIDS). Relevant to the mechanism potentially underlying this finding, we also present data from experimental studies in mice showing that elevated plasma Trx efficiently blocks lipopolysaccharide (LPS)-induced chemotaxis, an innate immune mechanism that is particularly crucial when adaptive immunity is compromised. Thus, we propose that elevated plasma Trx in HIV-infected individuals with low CD4 T cell counts directly impairs survival by blocking pathogen-induced chemotaxis, effectively eliminating the last (innate) barrier against establishment of opportunistic and other infections in these immunodeficient individuals.

    View details for Web of Science ID 000167258900104

    View details for PubMedID 11226300

  • 11-color, 13-parameter flow cytometry: Identification of human naive T cells by phenotype, function, and T-cell receptor diversity NATURE MEDICINE De Rosa, S. C., Herzenberg, L. A., Herzenberg, L. A., Roederer, M. 2001; 7 (2): 245-248

    View details for Web of Science ID 000166756300044

    View details for PubMedID 11175858

  • The regulation of CD5 expression in murine T cells. BMC molecular biology Tung, J. W., Kunnavatana, S. S., Herzenberg, L. A., Herzenberg, L. A. 2001; 2: 5-?


    CD5 is a pan-T cell surface marker that is also present on a subset of B cells, B-1a cells. Functional and developmental subsets of T cells express characteristic CD5 levels that vary over roughly a 30-fold range. Previous investigators have cloned a 1.7 Kb fragment containing the CD5 promoter and showed that it can confer similar lymphocyte-specific expression pattern as observed for endogenous CD5 expression.We further characterize the CD5 promoter and identify minimal and regulatory regions on the CD5 promoter. Using a luciferase reporter system, we show that a 43 bp region on the CD5 promoter regulates CD5 expression in resting mouse thymoma EL4 T cells and that an Ets binding site within the 43 bp region mediates the CD5 expression. In addition, we show that Ets-1, a member of the Ets family of transcription factors, recognizes the Ets binding site in the electrophoretic mobility shift assay (EMSA). This Ets binding site is directly responsible for the increase in reporter activity when co-transfected with increasing amounts of Ets-1 expression plasmid.We also identify two additional evolutionarily-conserved regions in the CD5 promoter (CD5X and CD5Y) and demonstrate the respective roles of the each region in the regulation of CD5 transcription.Our studies define a minimal and regulatory promoter for CD5 and show that the CD5 expression level in T cells is at least partially dependent on the level of Ets-1 protein. Based on the findings in this report, we propose a model of CD5 transcriptional regulation in T cells.

    View details for PubMedID 11389772

  • Increased production of IL-7 accompanies HIV-l-mediated T-cell depletion: implications for T-cell homeostasis NATURE MEDICINE Napolitano, L. A., Grant, R. M., Deeks, S. G., Schmidt, D., De Rosa, S. C., Herzenberg, L. A., Herndier, B. G., Andersson, J., McCune, J. M. 2001; 7 (1): 73-79


    We hypothesized that HIV-1-mediated T-cell loss might induce the production of factors that are capable of stimulating lymphocyte development and expansion. Here we perform cross-sectional (n = 168) and longitudinal (n = 11) analyses showing that increased circulating levels of interleukin (IL)-7 are strongly associated with CD4+ T lymphopenia in HIV-1 disease. Using immunohistochemistry with quantitative image analysis, we demonstrate that IL-7 is produced by dendritic-like cells within peripheral lymphoid tissues and that IL-7 production by these cells is greatly increased in lymphocyte-depleted tissues. We propose that IL-7 production increases as part of a homeostatic response to T-cell depletion.

    View details for Web of Science ID 000166243100038

    View details for PubMedID 11135619

  • Apoptolidin, a selective cytotoxic agent, is an inhibitor of F0F1-ATPase CHEMISTRY & BIOLOGY Salomon, A. R., Voehringer, D. W., Herzenberg, L. A., Khosla, C. 2001; 8 (1): 71-80


    Apoptolidin is a macrolide originally identified on the basis of its ability to selectively kill E1A and E1A/E1B19K transformed rat glial cells while not killing untransformed glial cells. The goal of this study was to identify the molecular target of this newly discovered natural product.Our approach to uncovering the mechanism of action of apoptolidin utilized a combination of molecular and cell-based pharmacological assays as well as structural comparisons between apoptolidin and other macrocyclic polyketides with known mechanism of action. Cell killing induced by apoptolidin was independent of p53 status, inhibited by BCL-2, and dependent on the action of caspase-9. PARP was completely cleaved in the presence of 1 microM apoptolidin within 6 h in a mouse lymphoma cell line. Together these results suggested that apoptolidin might target a mitochondrial protein. Structural comparisons between apoptolidin and other macrolides revealed significant similarity between the apoptolidin aglycone and oligomycin, a known inhibitor of mitochondrial F0F1-ATP synthase. The relevance of this similarity was established by demonstrating that apoptolidin is a potent inhibitor of the F0F1-ATPase activity in intact yeast mitochondria as well as Triton X-100-solubilized ATPase preparations. The K(i) for apoptolidin was 4-5 microM. The selectivity of apoptolidin in the NCI-60 cell line panel was found to correlate well with that of several known anti-fungal natural products that inhibit the eukaryotic mitochondrial F0F1-ATP synthase.Although the anti-fungal activities of macrolide inhibitors of the mitochondrial F0F1-ATP synthase such as oligomycin, ossamycin and cytovaricin are well-documented, their unusual selectivity toward certain cell types is not widely appreciated. The recent discovery of apoptolidin, followed by the demonstration that it is an inhibitor of the mitochondrial F0F1-ATP synthase, highlights the potential relevance of these natural products as small molecules to modulate apoptotic pathways. The mechanistic basis for selective cytotoxicity of mitochondrial ATP synthase inhibitors is discussed.

    View details for Web of Science ID 000167275800008

    View details for PubMedID 11182320

  • Understanding and exploiting the mechanistic basis for selectivity of polyketide inhibitors of F0F1-ATPase PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Salomon, A. R., Voehringer, D. W., Herzenberg, L. A., Khosla, C. 2000; 97 (26): 14766-14771


    Recently, a family of polyketide inhibitors of F(0)F(1)-ATPase, including apoptolidin, ossamycin, and oligomycin, were shown to be among the top 0.1% most cell line selective cytotoxic agents of 37, 000 molecules tested against the 60 human cancer cell lines of the National Cancer Institute. Many cancer cells maintain a high level of anaerobic carbon metabolism even in the presence of oxygen, a phenomenon that is historically known as the Warburg effect. A mechanism-based strategy to sensitize such cells to this class of potent small molecule cytotoxic agents is presented. These natural products inhibit oxidative phosphorylation by targeting the mitochondrial F(0)F(1) ATP synthase. Evaluation of gene expression profiles in a panel of leukemias revealed a strong correlation between the expression level of the gene encoding subunit 6 of the mitochondrial F(0)F(1) ATP synthase (known to be the binding site of members of this class of macrolides) and their sensitivity to these natural products. Within the same set of leukemia cell lines, comparably strong drug-gene correlations were also observed for the genes encoding two key enzymes involved in central carbon metabolism, pyruvate kinase, and aspartate aminotransferase. We propose a simple model in which the mitochondrial apoptotic pathway is activated in response to a shift in balance between aerobic and anaerobic ATP biosynthesis. Inhibitors of both lactate formation and carbon flux through the Embden-Meyerhof pathway significantly sensitized apoptolidin-resistant tumors to this drug. Nine different cell lines derived from human leukemias and melanomas, and colon, renal, central nervous system, and ovarian tumors are also sensitized to killing by apoptolidin.

    View details for Web of Science ID 000165993700138

    View details for PubMedID 11121076

  • An early oxygen-dependent step is required for dexamethasone-induced apoptosis of immature mouse thymocytes JOURNAL OF IMMUNOLOGY Torres-Roca, J. F., Tung, J. W., Greenwald, D. R., Brown, J. M., Herzenberg, L. A., Herzenberg, L. A., Katsikis, P. D. 2000; 165 (9): 4822-4830


    The roles of oxygen and reactive oxygen intermediates in apoptosis are unclear at present. Although oxygen and reactive oxygen intermediates are not required for the execution of apoptosis, oxygen may be involved in at least some forms of apoptosis. In this study we show that dexamethasone (Dex)-induced apoptosis of immature mouse thymocytes is completely inhibited by hypoxic culture. In contrast, anti-CD95 thymocyte apoptosis is unaffected by hypoxia, indicating the existence of two forms of thymocyte apoptosis: an oxygen-dependent pathway (Dex induced) and an oxygen-independent pathway (anti-CD95 induced). Furthermore, hypoxia inhibited mitochondrial permeability transition (PT) in Dex-treated, but not in anti-CD95-treated, thymocytes, suggesting that the oxygen-sensitive step is upstream of mitochondria. Both Dex- and anti-CD95-induced PT and apoptosis were dependent on activation of IL-converting enzyme-like protease, as PT and apoptosis were inhibited by preincubation with Cbz-Val-Ala-Asp-fluoromethyl ketone, an irreversible inhibitor of IL-converting enzyme-like proteases. In addition, hypoxia inhibited the activation by Dex of caspase-3 (CPP32)-like proteases. Our data show that the private signaling pathways of Dex (oxygen dependent) and anti-CD95 (oxygen independent) both converge upstream of mitochondrial changes. The oxygen-dependent step in Dex-induced apoptosis lies upstream of caspase-3-like protease activation. Our observations support a model of apoptosis signaling in which independent pathways (oxygen dependent and oxygen independent) particular to each stimuli converge at a central point in the apoptotic cascade.

    View details for Web of Science ID 000090076000009

    View details for PubMedID 11046005

  • N-acetylcysteine replenishes glutathione in HIV infection EUROPEAN JOURNAL OF CLINICAL INVESTIGATION De Rosa, S. C., Zaretsky, M. D., Dubs, J. G., Roederer, M., Anderson, M., Green, A., Mitra, D., Watanabe, N., Nakamura, H., Tjioe, I., Deresinski, S. C., Moore, W. A., Ela, S. W., Parks, D., Herzenberg, L. A., Herzenberg, L. A. 2000; 30 (10): 915-929


    Glutathione (GSH) deficiency is common in HIV-infected individuals and is associated with impaired T cell function and impaired survival. N-acetylcysteine (NAC) is used to replenish GSH that has been depleted by acetaminophen overdose. Studies here test oral administration of NAC for safe and effective GSH replenishment in HIV infection.Oral NAC administration in a randomized, 8-week double-blind, placebo-controlled trial followed by optional open-label drug for up to 24 weeks.HIV-infected, low GSH, CD4 T cells < 500 micro L(-1), no active opportunistic infections or other debilitation; n = 81. Study conducted prior to introduction of protease inhibitors.Whole blood GSH levels in NAC arm subjects significantly increased from 0.88 mM to 0.98 mM, bringing GSH levels in NAC-treated subjects to 89% of uninfected controls (P = 0.03). Baseline GSH levels in the placebo group (0.91) remained essentially the same during the 8 week placebo-controlled trial. T cell GSH, adjusted for CD4 T cell count and beta2-microglobulin levels, also increased in the NAC-treated subjects (P = 0.04). Adverse effects were minimal and not significantly associated with NAC ingestion.NAC treatment for 8 weeks safely replenishes whole blood GSH and T cell GSH in HIV-infected individuals. Thus, NAC offers useful adjunct therapy to increase protection against oxidative stress, improve immune system function and increase detoxification of acetaminophen and other drugs. These findings suggest that NAC therapy could be valuable in other clinical situations in which GSH deficiency or oxidative stress plays a role in disease pathology, e.g. rheumatoid arthritis, Parkinson's disease, hepatitis, liver cirrhosis, septic shock and diabetes.

    View details for Web of Science ID 000090133200012

    View details for PubMedID 11029607

  • Monoclonal antibodies and the FACS: complementary tools for immunobiology and medicine IMMUNOLOGY TODAY Herzenberg, L. A., De Rosa, S. C., Herzenberg, L. A. 2000; 21 (8): 383-390


    The histories of monoclonal antibodies and the fluorescence activated cell sorter (FACS) are as closely intertwined as their current uses in biology and medicine. Here, Leonore Herzenberg, Stephen De Rosa and Leonard Herzenberg recount the meeting and the mating of these two technologies, whose offspring now populate clinical and research laboratories throughout the world.

    View details for Web of Science ID 000088680900007

    View details for PubMedID 10916141

  • B-1 and B-2 cell-derived immunoglobulin M antibodies are nonredundant components of the protective response to influenza virus infection JOURNAL OF EXPERIMENTAL MEDICINE Baumgarth, N., Herman, O. C., Jager, G. C., Brown, L. E., Herzenberg, L. A., Chen, J. Z. 2000; 192 (2): 271-280


    We have studied the role of secreted immunoglobulin (Ig)M in protection from infection with influenza virus and delineated the relative contributions of B-1 versus B-2 cell-derived IgM in this process. Mice deficient in secreted IgM but capable of expressing surface IgM and secreting other Ig classes show significantly reduced virus clearance and survival rates compared with wild-type controls. Irradiation chimeras in which only either B-1 or B-2 cells lack the ability to secrete IgM show mortality rates similar to those of mice in which neither B-1 nor B-2 cells secrete IgM. Dependence on both sources of IgM for survival is partially explained by findings in allotype chimeras that broadly cross-reactive B-1 cell-derived natural IgM is present before infection, whereas virus strain-specific, B-2 cell-derived IgM appears only after infection. Furthermore, lack of IgM secreted from one or both sources significantly impairs the antiviral IgG response. Reconstitution of chimeras lacking B-1 cell-derived IgM only with IgM-containing serum from noninfected mice improved both survival rates and serum levels of virus-specific IgG. Thus, virus-induced IgM must be secreted in the presence of natural IgM for efficient induction of specific IgG and for immune protection, identifying B-1 and B-2 cell-derived IgM antibodies as nonredundant components of the antiviral response.

    View details for Web of Science ID 000088261100013

    View details for PubMedID 10899913

  • B-1 cells: the lineage question revisited IMMUNOLOGICAL REVIEWS Herzenberg, L. A. 2000; 175: 9-22


    The origins and functions of B-1 cells have sparked a good deal of controversy, largely centered on whether these B cells are developmentally distinct from the principal B cell populations (B-2) found in peripheral lymphoid organs. However, the prime criteria for assigning B-1 and B-2 cells to separate developmental lineages are satisfied by studies published some time ago that 1) identify distinct sources of progenitors for B-1 and B-2 cells; 2) show that these progenitors express their inherent commitment developing under the same conditions in co-transfer recipients; and, 3) have distinctive developmental patterns revealed by analysis of cells at various stages along the B-cell development pathway. I review these developmental studies here both to clarify the issue and to set the stage for presentation of evidence from more recent studies, which further define the functional differences between B-1 and B-2 cells and reveal intriguing complexities in the selective and other mechanisms that control the V(H) composition of the B-1 antibody repertoire.

    View details for Web of Science ID 000088202800002

    View details for PubMedID 10933587

  • CD4(+) T cells derived from B cell-deficient mice inhibit the establishment of peripheral B cell pools PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Baumgarth, N., Jager, G. C., Herman, O. C., Herzenberg, L. A., Herzenberg, L. A. 2000; 97 (9): 4766-4771


    We demonstrate that adoptive transfer of peritoneal cavity B cells fails to replenish the peripheral B-1 cells in adult B cell-deficient (mu(-/-)) mice but does replenish adult RAG-1(-/-) mice. We show that this lack of self-replenishment in mu(-/-) mice is mediated by strongly inhibitory, radiation-sensitive CD4(+) T cells that also function in cotransfer studies to block the reconstitution of B-1 cells and inhibit accumulation of bone marrow-derived B-2 cells in the periphery in irradiated recipients. CD8(+) T cells from mu(-/-) do not mediate this inhibition. The inhibitory CD4(+) T cells develop early in life, because B-1 cell replenishment occurs normally when B-1 cells are transferred into mu(-/-) neonates. Thus, we conclude that the presence of B cells in the neonate conditions the CD4(+) T-cell population to permit the establishment and maintenance of normal B cell pools throughout life.

    View details for Web of Science ID 000086703000068

    View details for PubMedID 10781082

  • Gene microarray identification of redox and mitochondrial elements that control resistance or sensitivity to apoptosis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Voehringer, D. W., Hirschberg, D. L., Xiao, J., Lu, Q., Roederer, M., Lock, C. B., Herzenberg, L. A., Steinman, L., Herzenberg, L. A. 2000; 97 (6): 2680-2685


    Multigenic programs controlling susceptibility to apoptosis in response to ionizing radiation have not yet been defined. Here, using DNA microarrays, we show gene expression patterns in an apoptosis-sensitive and apoptosis-resistant murine B cell lymphoma model system both before and after irradiation. From the 11,000 genes interrogated by the arrays, two major patterns emerged. First, before radiation exposure the radioresistant LYar cells expressed significantly greater levels of message for several genes involved in regulating intracellular redox potential. Compared with LYas cells, LYar cells express 20- to 50-fold more mRNA for the tetraspanin CD53 and for fructose-1,6-bisphosphatase. Expression of both of these genes can lead to the increase of total cellular glutathione, which is the principle intracellular antioxidant and has been shown to inhibit many forms of apoptosis. A second pattern emerged after radiation, when the apoptosis-sensitive LYas cells induced rapid expression of a unique cluster of genes characterized by their involvement in mitochondrial electron transport. Some of these genes have been previously recognized as proapoptotic; however others, such as uncoupling protein 2, were not previously known to be apoptotic regulatory proteins. From these observations we propose that a multigenic program for sensitivity to apoptosis involves induction of transcripts for genes participating in mitochondrial uncoupling and loss of membrane potential. This program triggers mitochondrial release of apoptogenic factors and induces the "caspase cascade." Conversely, cells resistant to apoptosis down-regulate these biochemical pathways, while activating pathways for establishment and maintenance of high intracellular redox potential by means of elevated glutathione.

    View details for Web of Science ID 000085941400051

    View details for PubMedID 10716996

  • The role of B-1 and B-2 cells in immune protection from influenza virus infection Baumgarth, N., Chen, J., Herman, O. C., Jager, G. C., Herzenberg, L. A. SPRINGER-VERLAG BERLIN. 2000: 163-169

    View details for Web of Science ID 000167097400017

    View details for PubMedID 11125473

  • B-1 cell origins and V-H repertoire determination Herzenberg, L. A., Baumgarth, N., Wilshire, A. SPRINGER-VERLAG BERLIN. 2000: 3-13

    View details for Web of Science ID 000167097400001

    View details for PubMedID 11125487

  • Differential representations of memory T cell subsets are characteristic of polarized immunity in leprosy and atopic diseases INTERNATIONAL IMMUNOLOGY Mitra, D. K., De Rosa, S. C., Luke, A., Balamurugan, A., Khaitan, B. K., Tung, J., Mehra, N. K., Terr, A. I., O'Garra, A., Herzenberg, L. A., Herzenberg, L. A., Roederer, M. 1999; 11 (11): 1801-1810


    We identified functionally polarized subsets of CD4 memory T cells on the basis of the expression of CD11a, CD45RA and CD62L. Within the several phenotypically distinct subsets of CD4 memory cells are two that, upon stimulation, produce primarily IL-4 (MT(2), CD45RA(-)CD62L(+)CD11a(dim)) or primarily IFN-gamma (MT(1), CD45RA(-)CD62L(-)CD11a(bright)). In addition, four other phenotypically distinct subsets of CD4 cells have unique cytokine profiles. To determine the clinical relevance of the representation of these cell types, we analyzed blood from patients with the chronic diseases leprosy and atopy. These diseases are characterized as immunologically polarized, since T cell responses in affected individuals are often strongly biased towards T(h)1 (dominated by IFN-gamma production) or T(h)2 (IL-4 production). We show here that this polarization reflects homeostatic or differentiation mechanisms affecting the representation of the functionally distinct subsets of memory CD4 T cells, MT(1) and MT(2). Significantly, the representation of the MT(1) and MT(2) subsets differs dramatically between subjects with tuberculoid leprosy (a T(h)1 disease), or lepromatous leprosy or atopic disease (T(h)2 diseases). However, there was no difference in the cytokine profiles of these or any of the other finely resolved CD4 subsets, when compared between individuals across all disease states. Thus, it is the representation of these subsets in peripheral blood that is diagnostic of the polarized state of the immune system.

    View details for Web of Science ID 000083797800009

    View details for PubMedID 10545484

  • An improved monobromobimane assay for glutathione utilizing tris(2-carboxyethyl)phosphine as the reductant ANALYTICAL BIOCHEMISTRY Anderson, M. T., Trudell, J. R., Voehringer, D. W., Tjioe, I. M., Herzenberg, L. A., Herzenberg, L. A. 1999; 272 (1): 107-109

    View details for Web of Science ID 000081616600015

    View details for PubMedID 10405300

  • Thioredoxin, a redox enzyme released in infection and inflammation, is a unique chemoattractant for neutrophils, monocytes, and T cells JOURNAL OF EXPERIMENTAL MEDICINE BERTINI, R., Howard, O. M., Dong, H. F., Oppenheim, J. J., Bizzarri, C., Sergi, R., Caselli, G., Pagliei, S., Romines, B., Wilshire, J. A., Mengozzi, M., Nakamura, H., Yodoi, J., Pekkari, K., Gurunath, R., Holmgren, A., Herzenberg, L. A., Herzenberg, L. A., Ghezzi, P. 1999; 189 (11): 1783-1789


    Thioredoxin (Trx) is a ubiquitous intracellular protein disulfide oxidoreductase with a CXXC active site that can be released by various cell types upon activation. We show here that Trx is chemotactic for monocytes, polymorphonuclear leukocytes, and T lymphocytes, both in vitro in the standard micro Boyden chamber migration assay and in vivo in the mouse air pouch model. The potency of the chemotactic action of Trx for all leukocyte populations is in the nanomolar range, comparable with that of known chemokines. However, Trx does not increase intracellular Ca2+ and its activity is not inhibited by pertussis toxin. Thus, the chemotactic action of Trx differs from that of known chemokines in that it is G protein independent. Mutation of the active site cysteines resulted in loss of chemotactic activity, suggesting that the latter is mediated by the enzyme activity of Trx. Trx also accounted for part of the chemotactic activity released by human T lymphotropic virus (HTLV)-1-infected cells, which was inhibited by incubation with anti-Trx antibody. Since Trx production is induced by oxidants, it represents a link between oxidative stress and inflammation that is of particular interest because circulating Trx levels are elevated in inflammatory diseases and HIV infection.

    View details for Web of Science ID 000080855200012

    View details for PubMedID 10359582

  • Nine color eleven parameter immunophenotyping using three laser flow cytometry CYTOMETRY Bigos, A., Baumgarth, N., Jager, G. C., Herman, O. C., Nozaki, T., Stovel, R. T., Parks, D. R., Herzenberg, L. A. 1999; 36 (1): 36-45


    This study describes a three laser flow cytometer, reagents, and software used to simultaneously evaluate nine distinct fluorescent parameters on one cell sample. We compare the quality of data obtained with (1) full software compensation and (2) the use of partial spectral compensation of selected pairs of parameters in analog hardware, in combination with final software compensation. An application characterizing low frequency murine B cell subpopulations is given.The fluorochromes used are: fluorescein (FITC), phycoerythrin (PE), Cy5PE and Cy7PE, excited at 488 nm by an argon laser; Texas Red (TR), allophycocyanin (APC), and Cy7APC excited at 595 nm by a pumped dye laser; and cascade blue (CB) and cascade yellow (CY) excited at 407 nm by a violet-enhanced krypton laser. Custom additions to commercial electronics and an extended optical bench allow the measurement of these nine parameters plus forward and side scatter light signals.We find the use of partial analog compensation reduces the variation in the background staining levels introduced by the compensation process. Novel B cell populations with frequencies below 1% are characterized.Nine color flow cytometry is capable of providing measurements with high information content. The choice of reagent-dye combinations and the ability to compensate in multi-parameter measurement space are crucial to obtaining satisfactory results.

    View details for Web of Science ID 000079988800005

    View details for PubMedID 10331625

  • Predominant V-H genes expressed in innate antibodies are associated with distinctive antigen-binding sites PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Seidl, K. J., Wilshire, J. A., MacKenzie, J. D., Kantor, A. B., Herzenberg, L. A., Herzenberg, L. A. 1999; 96 (5): 2262-2267


    Antibodies to phosphatidylcholine (PtC), a common constituent of mammalian and bacterial cell membranes, represent a large proportion of the natural antibody repertoire in mice. Previous studies of several mouse strains (e.g., C57BL/6) have shown that anti-PtC antibodies are mainly encoded by the VH11 and VH12 immunoglobulin heavy chain variable region gene families. We show here, however, that VH11 and VH12 encode only a small proportion of the anti-PtC antibodies in BALB/c mice. Instead, VHQ52-encoded antibodies predominate in this strain. In addition, two-thirds of the cells expressing VHQ52 family genes use a single gene (which, interestingly, has been previously shown to predominate in the anti-oxazolone response). We also show here that in anti-PtC antibodies from all strains, the distinctive antigen-binding sites associated with VHQ52 differ substantially from those associated with VH11 and VH12. That is, VHQ52-containing transcripts preferentially use the joining region JH4 rather than JH1 and exhibit more diverse complementarity-determining region 3 (CDR3) junctions with more N-region nucleotide additions at the gene segment junctions. Thus, the VH gene family that predominates in the anti-PtC repertoire differs among mouse strains, whereas the distinctive VHDJH rearrangements (CDR3, JH) associated with each VH gene family are similar in all strains. We discuss these findings in the context of a recent hypothesis suggesting that CDR3 structure, independent of VH framework, is sufficient to define the specificity of an antibody.

    View details for Web of Science ID 000078956600082

    View details for PubMedID 10051629

  • Innate and acquired humoral immunities to influenza virus are mediated by distinct arms of the immune system PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Baumgarth, N., Herman, O. C., Jager, G. C., Brown, L., Herzenberg, L. A., Herzenberg, L. A. 1999; 96 (5): 2250-2255


    "Natural" Igs, mainly IgM, comprise part of the innate immune system present in healthy individuals, including antigen-free mice. These Igs are thought to delay pathogenicity of infecting agents until antigen-induced high affinity Igs of all isotypes are produced. Previous studies suggested that the acquired humoral response arises directly from the innate response, i.e., that B cells expressing natural IgM, upon antigen encounter, differentiate to give rise both to cells that secrete high amounts of IgM and to cells that undergo affinity maturation and isotype switching. However, by using a murine model of influenza virus infection, we demonstrate here that the B cells that produce natural antiviral IgM neither increase their IgM production nor undergo isotype switching to IgG2a in response to the infection. These cells are distinct from the B cells that produce the antiviral response after encounter with the pathogen. Our data therefore demonstrate that the innate and the acquired humoral immunities to influenza virus are separate effector arms of the immune system and that antigen exposure per se is not sufficient to increase natural antibody production.

    View details for Web of Science ID 000078956600080

    View details for PubMedID 10051627

  • Single cell analysis and selection of living retrovirus vector-corrected mucopolysaccharidosis VII cells using a fluorescence-activated cell sorting-based assay for mammalian beta-glucuronidase enzymatic activity JOURNAL OF BIOLOGICAL CHEMISTRY Lorincz, M. C., Parente, M. K., Roederer, M., Nolan, G. P., Diwu, Z. J., Martin, D. I., Herzenberg, L. A., Wolfe, J. H. 1999; 274 (2): 657-665


    Mutations in the acid beta-glucuronidase gene lead to systemic accumulation of undegraded glycosaminoglycans in lysosomes and ultimately to clinical manifestations of mucopolysaccharidosis VII (Sly disease). Gene transfer by retrovirus vectors into murine mucopolysaccharidosis VII hematopoietic stem cells or fibroblasts ameliorates glycosaminoglycan accumulation in some affected tissues. The efficacy of gene therapy for mucopolysaccharidosis VII depends on the levels of beta-glucuronidase secreted by gene-corrected cells; therefore, enrichment of transduced cells expressing high levels of enzyme prior to transplantation is desirable. We describe the development of a fluorescence-activated cell sorter-based assay for the quantitative analysis of beta-glucuronidase activity in viable cells. Murine mucopolysaccharidosis VII cells transduced with a beta-glucuronidase retroviral vector can be isolated by cell sorting on the basis of beta-glucuronidase activity and cultured for further use. In vitro analysis revealed that sorted cells have elevated levels of beta-glucuronidase activity and secrete higher levels of cross-correcting enzyme than the population from which they were sorted. Transduced fibroblasts stably expressing beta-glucuronidase after subcutaneous passage in the mucopolysaccharidosis VII mouse can be isolated by cell sorting and expanded ex vivo. A relatively high percentage of these cells maintain stable expression after secondary transplantation, yielding significantly higher levels of enzymatic activity than that generated in the primary transplant.

    View details for Web of Science ID 000077968500014

    View details for PubMedID 9872999

  • Flow cytometric analysis and FACS sorting of cells based on GFP accumulation METHODS IN CELL BIOLOGY, VOL 58 Galbraith, D. W., Anderson, M. T., Herzenberg, L. A. 1999; 58: 315-341

    View details for Web of Science ID 000165166500019

    View details for PubMedID 9891389

  • Flow cytometric analysis of transgene expression in higher plants: Green fluorescent protein GREEN FLUORESCENT PROTEIN Galbraith, D. W., Herzenberg, L. A., Anderson, M. T. 1999; 302: 296-315

    View details for Web of Science ID 000087254200026

    View details for PubMedID 12876781

  • Pairs of violet-light-excited fluorochromes for flow cytometric analysis CYTOMETRY Anderson, M. T., Baumgarth, N., Haugland, R. P., Gerstein, R. M., Tjioe, T., Herzenberg, L. A., Herzenberg, L. A. 1998; 33 (4): 435-444


    We describe pairs of fluorochromes for use with the 407-nm line of a violet-light-enhanced krypton ion laser. These fluorochromes and a previously described violet-light-excited reporter variant, GFP-Vex, fall into two emission classes: blue for Cascade Blue, and green/yellow for Cascade Yellow, Lucifer Yellow, and GFP-Vex. Cascade Yellow is a new fluorochrome that we have synthesized and is used for the first time in the present study. The two emission classes are sufficiently different that Cascade Blue can be paired with Cascade Yellow, Lucifer Yellow, or GFP-Vex in flow cytometric analysis. Furthermore, with proper detection filters, these fluorochromes can be combined with all of the currently used fluorochromes in a three-laser FACS system. With these data, the total number of fluorochromes that can be used as antibody labels for simultaneous detection in combined FACS analysis increases to nine. This study demonstrates the sensitivity and power of the combined use of these reagents in a single eight-color analysis by identifying murine T-lymphocyte subsets that could not otherwise be readily distinguished.

    View details for Web of Science ID 000077135400007

    View details for PubMedID 9845438

  • CD1 expression defines subsets of follicular and marginal zone B cells in the spleen: beta(2)-microglobulin-dependent and independent forms JOURNAL OF IMMUNOLOGY Amano, M., Baumgarth, N., Dick, M. D., Brossay, L., Kronenberg, M., Herzenberg, L. A., Strober, S. 1998; 161 (4): 1710-1717


    We have used multicolor FACS analysis, immunohistology, and functional assays to study the expression of CD1 on B cell subsets from normal and beta 2m-/- mice. Two B cell subpopulations were identified that express high levels of CD1 in normal mice: splenic marginal zone B cells (IgMhigh IgDlow CD21high CD24intermediate CD23- CD43-) and a newly identified subpopulation of follicular B cells. The latter cells are unusual, because they are IgDhigh CD23+, like follicular B cells, but express high levels of CD21 and IgM, an expression pattern that is associated with marginal zone B cells. Therefore, the high-level expression of CD1 and CD21 was found to be closely associated on splenic B cells. Immunohistology confirmed the expression of CD1 on marginal zone B cells and on clusters of B cells in splenic follicles. Both the high-level CD1 expression by these cells and the low-level CD1 expression by subpopulations of B cells in the spleen, lymph node, peritoneal cavity, and bone marrow were markedly reduced in beta 2m-/- mice. Despite this, a CD1-restricted T cell clone proliferated vigorously in response to LPS-activated spleen cells that had been obtained from both beta 2m-/- and wild-type mice. This response was inhibited by the 3C11 anti-CD1 mAb. These results show the heterogeneity of B cell subsets in their expression of the beta 2m-dependent form of CD1. They further suggest that a beta 2m-independent form of CD1 is expressed on B cells that can stimulate T cells; however, this form is not easily visualized with the anti-CD1 mAb used here.

    View details for Web of Science ID 000075345400018

    View details for PubMedID 9712035

  • Neutrophil CD11b expression as a diagnostic marker for early-onset neonatal infection JOURNAL OF PEDIATRICS Weirich, E., Rabin, R. L., Maldonado, Y., Benitz, W., Modler, S., Herzenberg, L. A., Herzenberg, L. A. 1998; 132 (3): 445-451


    To determine whether neutrophil surface expression of CD11b predicts early-onset infection or suspected infection in at-risk infants.CD11b expression on peripheral blood neutrophils was determined by flow cytometry of whole blood samples. Blood (0.1 ml) was obtained from a convenience sample of at-risk infants admitted to the neonatal intensive care unit, stained with antibodies detecting CD11b and CD15, chilled, and analyzed within 8 hours. Blood for culture, blood counts, and C-reactive protein (CRP) determination was obtained simultaneously. Subjects were grouped on the basis of culture results and clinical signs, and investigators were blinded to CD11b level.Of 106 subjects, seven had positive bacterial or viral cultures ("confirmed infection"), 17 had clinical signs of infection but negative cultures ("suspected infection"), and 82 had negative cultures and no clinical signs ("no infection"). Neutrophil CD11b was elevated in all infants with confirmed infection, 94% with suspected infection, and none with no infection. The negative and positive predictive values, sensitivity, and specificity were 100%, 99%, 96%, and 100%, respectively, for diagnosis of neonatal infection at initial evaluation. CD11b levels correlated with peak CRP (r2 = 0.76, p < 0.0001); however, CD11b was elevated at the time of admission in all five infants with proven bacterial infection, whereas CRP was normal until the second day in the neonatal intensive care unit in three of these five. Both infants with positive viral cultures had elevated CD11b, but the CRP levels remained within normal limits. The negative predictive value of neutrophil CD11b for identifying suspected or confirmed infection was 99%.This assay for neutrophil CD11b is a promising test for exclusion of early-onset neonatal infection. If validated prospectively, this assay may reduce hospital and antibiotic use in the population of neonates at risk for early-onset infection.

    View details for Web of Science ID 000072877800018

    View details for PubMedID 9544899

  • Dynamics of fine T-cell subsets during HIV disease and after thymic ablation by mediastinal irradiation. Seminars in immunology Roederer, M., De Rosa, S. C., Watanabe, N., Herzenberg, L. A. 1997; 9 (6): 389-396


    The T-cell compartment is considerably more complex than just CD4 and CD8 T cells. Indeed, we can identify dozens of functionally and phenotypically distinct subsets within the peripheral blood of humans. These subsets are differentially affected in diseases which may underly some of the functional defects attributable to the disease. In HIV disease, all thymic-derived T-cell populations are gradually lost at identical rates during late-stage disease progression, while unusual, perhaps extrathymically-derived T cells expand. This expansion may reflect an attempt on the part of the immune system to compensate for the significant insult of HIV infection to the host: the abrogation of normal thymopoiesis and T-cell homeostasis.

    View details for PubMedID 9405268

  • 8 color, 10-parameter flow cytometry to elucidate complex leukocyte heterogeneity CYTOMETRY Roederer, M., DeRosa, S., Gerstein, R., Anderson, M., Bigos, M., Stovel, R., Nozaki, T., Parks, D., Herzenberg, L., Herzenberg, L. 1997; 29 (4): 328-339


    We developed the chemistry, instrumentation, and software technologies needed to measure, simultaneously and independently, eight different fluorescent molecules on individual cells. Conjugation of these fluorochromes to monoclonal antibodies is straightforward; all immunofluorescence staining is accomplished with direct stains only. We built a hybrid flow cytometer with eight fluorescence detectors and two light scatter channels, with excitation provided by three spatially separated laser beams emitting at 407 nm, 488 nm, and 595 nm. The fluorescence compensation required to make the data orthogonal is of sufficient complexity that it cannot be performed manually; thus, we use software to compensate the data post hoc, based on data collected from singly stained compensation control samples. In this report, we evaluate the 8 color staining technology. Of the seven fluorochromes other than fluorescein, six have a useful brightness at least as great as fluorescein. Three of the fluorochromes (phycoerythrin, allophycocyanin, and the Cy5 resonance energy tandem of phycoerythrin) are considerably brighter than fluorescein and are useful for detecting antigens expressed at low levels. Finally, we show the power and utility of the 8 color, 10-parameter technology using staining experiments on both human and murine immune systems.

    View details for Web of Science ID A1997YK15100009

    View details for PubMedID 9415416

  • Long-term depletion of naive T cells in patients treated for Hodgkin's disease BLOOD Watanabe, N., DeRosa, S. C., Cmelak, A., Hoppe, R., Herzenberg, L. A., Herzenberg, L. A., Roederer, M. 1997; 90 (9): 3662-3672


    We investigated the representation of T cells in patients who had been treated for Hodgkin's disease (HD). We found a marked depletion in both CD4 and CD8 naive T-cell counts that persists up to 30 years after completion of treatment. In contrast, CD4 and CD8 memory T-cell subsets recovered to normal or above normal levels by 5 years posttreatment. Thus, the previously-reported long-term deficit in total CD4 T-cell counts after treatment for HD is due to specific depletion of naive T cells. Similarly, total CD8 T-cell counts return to normal by 5 years only because CD8 memory T cells expand to higher than normal levels. These findings suggest that the treatment (mediastinal irradiation) results in a longterm dysregulation of T-cell subset homeostasis. The profound depletion of naive T cells may explain the altered T-cell function in treated patients, including the poor response to immunization after treatment for HD. Further, in some individuals, we identified expansions of unusual subsets expressing low levels of CD8. Eight-color fluorescence-activated cell sorting analyses showed that these cells largely express CD8alphaalpha homodimers and CD57, consistent with the phenotype of potentially extrathymically derived T cells. In addition, these cells, both CD4+ and CD4-, are probably cytotoxic lymphocytes, as they express high levels of intracellular perforin. In adults treated for HD, an increased activity of extrathymic T-cell differentiation may partially compensate for the loss of thymic-derived T cells.

    View details for Web of Science ID A1997YD10800043

    View details for PubMedID 9345051

  • Interleukin-1 beta converting enzyme-like protease involvement in Fas-induced and activation-induced peripheral blood T cell apoptosis in HIV infection. TNF-related apoptosis-inducing ligand can mediate activation-induced T cell death in HIV infection JOURNAL OF EXPERIMENTAL MEDICINE Katsikis, P. D., GarciaOjeda, M. E., TORRESROCA, J. F., Tijoe, I. M., Smith, C. A., Herzenberg, L. A., Herzenberg, L. A. 1997; 186 (8): 1365-1372


    Apoptosis of peripheral blood T cells has been suggested to play an important role in the pathogenesis of human immunodeficiency virus (HIV) infection. Spontaneous, Fas (CD95)-induced and activation-induced T cell apoptosis have all been described in peripheral blood mononuclear cell cultures of HIV-infected individuals. We have previously shown that activation-induced T cell apoptosis is Fas independent in peripheral blood T cells from HIV+ individuals. In this study, we extend and confirm these observations by using an inhibitor of interleukin-1 beta converting enzyme (ICE) homologues. We show that z-VAD-fmk, a tripeptide inhibitor of ICE homologues, can inhibit Fas-induced apoptosis of peripheral blood CD4(+) and CD8+ T cells from asymptomatic HIV+ individuals. z-VAD-fmk also inhibited activation (anti-CD3)- induced CD4+ and CD8+ T cell apoptosis (AICD) in some but not all asymptomatic HIV+ individuals. Apoptosis was measured by multiparameter flow cytometry. The z-VAD-fmk inhibitor also enhanced survival of T cells in anti-Fas or anti-CD3 antibody-treated cultures and inhibited DNA fragmentation. AICD that could be inhibited by z-VAD-fmk was Fas independent and could be inhibited with a blocking monoclonal antibody to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a recently described member of the TNF/nerve growth factor ligand family. The above findings show that Fas-induced T cell apoptosis is ICE dependent in HIV infection. AICD can be blocked by ICE inhibitors in some patients, and this AICD is mediated by TRAIL. These results show that TRAIL can be a mediator of AICD in T cells. These different mechanisms of peripheral blood T cell apoptosis may play different roles in the pathogenesis of HIV infection.

    View details for Web of Science ID A1997YC95000021

    View details for PubMedID 9334376

  • HIV type 1 Tat protein enhances activation- not Fas (CD95)-induced peripheral blood T cell apoptosis in healthy individuals INTERNATIONAL IMMUNOLOGY Katsikis, P. D., GarciaOjeda, M. E., TORRESROCA, J. F., Greenwald, D. R., Herzenberg, L. A., Herzenberg, L. A. 1997; 9 (6): 835-841


    T cell apoptosis may play an important role in the depletion and functional defects of T cells in HIV disease. A number of investigators have shown that peripheral blood T cells in HIV disease undergo spontaneous and activation-induced apoptosis. We found recently that peripheral blood T cells from HIV+ individuals undergo apoptosis when stimulated through Fas. Also, a number of investigators have shown that Tat protein from HIV-1 can increase spontaneous and activation-induced apoptosis. In the present study we examined the effect of HIV type 1 Tat protein on spontaneous, activation-induced and Fas-induced apoptosis of peripheral blood T cells from HIV- individuals. We find that Tat protein has no effect on spontaneous apoptosis but does enhance activation-induced apoptosis of both CD4+ and CD8+ T cells. Tat, however, failed to enhance Fas-induced apoptosis of CD4+ and CD8+ T cells. Examining the mechanisms by which Tat induces apoptosis, we found that inhibitors of reactive oxygen intermediate (ROI) generation or neutralizers of ROI, such as rotenone, a potent inhibitor of mitochondrial complex I of the respiratory chain, and 3,3,5,5-tetramethylpyrroline N-oxide (TMPO), an electron spin trap, could both enhance the spontaneous apoptosis induced by Tat. This enhancement of Tat-induced apoptosis by rotenone and TMPO was independent of ICE activation as it could not be inhibited by the tripeptide z-VAD-fmk, an irreversible inhibitor of ICE/ced-3 protease homologs. These findings suggest that Tat induced enhancement of activation-induced cell death may involve complex mechanisms, some of which are ROI independent. These results indicate that a HIV-specific mechanism other than Tat is responsible for the previously observed increased susceptibility of peripheral blood T cells from HIV-infected individuals to undergo apoptosis in response to Fas stimulation.

    View details for Web of Science ID A1997XE89100004

    View details for PubMedID 9199966

  • Detection and isolation of gene-corrected cells in Gaucher disease via a fluorescence-activated cell sorter assay for lysosomal glucocerebrosidase activity BLOOD Lorincz, M., Herzenberg, L. A., Diwu, Z. J., Barranger, J. A., Kerr, W. G. 1997; 89 (9): 3412-3420


    Gaucher disease type 1 results from the accumulation of glucocerebroside in macrophages of the reticuloendothelial system, as a consequence of a deficiency in glucocerebrosidase (GC) activity. Recent improvements in the methodologies for introducing foreign genes into bone marrow stem cells have prompted several groups to test the efficacy of gene transfer therapy as a curative treatment for Gaucher disease. Limitations of this approach include the potential for insufficient engraftment of gene-corrected cells and incomplete transduction of hematopoietic stem cells using retroviral gene transfer. Overcoming these obstacles may be critical in the case of treatment for Gaucher disease type 1, because GC transduced cells have not been shown to have a growth advantage over noncorrected cells. Here, we describe the development and application of a novel, fluorescence-activated cell sorter based assay that directly quantitates GC activity at the single cell level. In a test of this application, fibroblasts from a Gaucher patient were transduced, and high expressing cells sorted based on GC activity. Reanalysis of cultured sorted fibroblasts reveals that these cells maintain high levels of enzymatic activity, compared with the heterogeneous population from which they were sorted. The assay is sufficiently sensitive to distinguish GC activity found in Gaucher patient monocytes from that in normal controls. Furthermore, preliminary results indicate that increased GC activity can be detected in transduced, CD34+ enriched peripheral blood mononuclear cells isolated from a Gaucher patient. This method should be a useful addition to current gene therapy protocols as a means to quantitatively assess gene correction of relevant cell populations and potentially purify transduced cells for transplantation.

    View details for Web of Science ID A1997WX54200041

    View details for PubMedID 9129049

  • Frequent occurrence of identical heavy and light chain Ig rearrangements INTERNATIONAL IMMUNOLOGY Seidl, K. J., MacKenzie, J. D., Wang, D. N., Kantor, A. B., KABAT, E. A., Herzenberg, L. A., Herzenberg, L. A. 1997; 9 (5): 689-702


    Single-cell PCR analyses of expressed Ig H and L chain sequences presented here show that certain rearrangements occur repeatedly and account for a major segment of the well-studied repertoire of B-1 cell autoantibodies that mediate the lysis of bromelain-treated mouse erythrocytes, i.e. antibodies reactive with phosphatldyicholine (PtC). We repeatedly isolated at least 10 different types of VH region rearrangements, involving three distinct germline genes, among FACS-sorted PtC-binding B-1 cells from three strains of mice (C57BL/6J, BALB/c and C.B-17). The predominant rearrangement, VH11-DSP-JH1 (VH11 type 1), has been previously found in anti-PtC hybridomas in several studies. We show that within each of six mice from two strains (C57BL/6J and BALB/c), unique instances of IgH/IgL pairing arose either from different B cell progenitors prior to IgH rearrangement or from pre-B cells which expanded after IgH rearrangement but prior to IgL rearrangement. Together with other recurrent rearrangements described here, our findings demonstrate that clonal expansion of mature B cells cannot account for all repeated rearrangements. As suggested by initial studies of dominant idiotype expression, these findings confirm that clonal expansion is only one of the mechanisms contributing to the establishment of recurrent rearrangements.

    View details for Web of Science ID A1997XD24000006

    View details for PubMedID 9184914

  • Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zambrowicz, B. P., Imamoto, A., Fiering, S., Herzenberg, L. A., Kerr, W. G., Soriano, P. 1997; 94 (8): 3789-3794


    The ROSA beta geo26 (ROSA26) mouse strain was produced by random retroviral gene trapping in embryonic stem cells. Staining of ROSA26 tissues and fluorescence-activated cell sorter-Gal analysis of hematopoietic cells demonstrates ubiquitous expression of the proviral beta geo reporter gene, and bone marrow transfer experiments illustrate the general utility of this strain for chimera and transplantation studies. The gene trap vector has integrated into a region that produces three transcripts. Two transcripts, lost in ROSA26 homozygous animals, originate from a common promoter and share identical 5' ends, but neither contains a significant ORF. The third transcript, originating from the reverse strand, shares antisense sequences with one of the noncoding transcripts. This third transcript potentially encodes a novel protein of at least 505 amino acids that is conserved in humans and in Caenorhabditis elegans.

    View details for Web of Science ID A1997WW81000057

    View details for PubMedID 9108056

  • HIV does not replicate in naive CD4 T cells stimulated with CD3/CD28 JOURNAL OF CLINICAL INVESTIGATION Roederer, M., Raju, P. A., Mitra, D. K., Herzenberg, L. A., Herzenberg, L. A. 1997; 99 (7): 1555-1564


    In this report, we demonstrate that the T cell tropic strain of HIV, LAI, does not replicate in naive CD4 T cells stimulated by cross-linking CD3 and CD28. In contrast, LAI replicates well in memory CD4 T cells stimulated in the same way. Unlike this physiologically relevant stimulation, PHA stimulates productive LAI replication in both naive and memory T cells. These studies were conducted with highly purified (FACS-isolated) subsets of CD4 T cells identified by expression of both CD45RA and CD62L. Remixing of purified T cells showed that naive T cells do not suppress LAI replication in memory T cells and that memory T cells do not restore LAI expression in naive T cells. The suppression of productive LAI replication in naive T cells is not due to differential expression of viral coreceptors, nor is it due to inhibition of activation of the important HIV transcription factors, nuclear factor-kappaB and activator protein-1. The inherent resistance of naive T cells to productive HIV infection, coupled with their proliferative advantage as demonstrated here, provides a sound basis for proposed clinical therapies using ex vivo expansion and reinfusion of CD4 T cells from HIV-infected adults.

    View details for Web of Science ID A1997WT44400014

    View details for PubMedID 9119999

  • Glutathione deficiency is associated with impaired survival in HIV disease PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Herzenberg, L. A., DeRosa, S. C., Dubs, J. G., Roederer, M., Anderson, M. T., Ela, S. W., Deresinski, S. C., Herzenberg, L. A. 1997; 94 (5): 1967-1972


    Glutathione (GSH), a cysteine-containing tripeptide, is essential for the viability and function of virtually all cells. In vitro studies showing that low GSH levels both promote HIV expression and impair T cell function suggested a link between GSH depletion and HIV disease progression. Clinical studies presented here directly demonstrate that low GSH levels predict poor survival in otherwise indistinguishable HIV-infected subjects. Specifically, we show that GSH deficiency in CD4 T cells from such subjects is associated with markedly decreased survival 2-3 years after baseline data collection (Kaplan-Meier and logistic regression analyses, P < 0.0001 for both analyses). This finding, supported by evidence demonstrating that oral administration of the GSH prodrug N-acetylcysteine replenishes GSH in these subjects and suggesting that N-acetylcysteine administration can improve their survival, establishes GSH deficiency as a key determinant of survival in HIV disease. Further, it argues strongly that the unnecessary or excessive use of acetaminophen, alcohol, or other drugs known to deplete GSH should be avoided by HIV-infected individuals.

    View details for Web of Science ID A1997WM05900068

    View details for PubMedID 9050888

  • An unbiased analysis of V-H-D-J(H) sequences from B-1a, B-1b, and conventional B cells JOURNAL OF IMMUNOLOGY Kantor, A. B., Merrill, C. E., Herzenberg, L. A., Hillson, J. L. 1997; 158 (3): 1175-1186


    Previous studies conclude that the repertoire of B-1a (CD5+ B) cells is highly restricted. Studies here, which use FACS sorting and single-cell PCR methodology to develop an unbiased representation of the IgH repertoires of B-1a, B-1b, and conventional B cells from the peritoneal cavity, demonstrate that the B-1a cell repertoire is more diverse than previously thought. Furthermore, adult B-1a cells have significantly fewer noncoded nucleotide (N) insertions than conventional B cells. However, B-1a cells are not defined by the absence of these regions, since such insertions are present in two-thirds of B-1a cell transcripts. All three B cell populations use a wide spectrum of V(H), D, and J(H) elements and display considerable diversity in complementarity-determining region 3 (CDR3). However, characteristic differences in the repertoires of all three B cell populations also exist, suggesting different selective and/or developmental forces act to shape each repertoire.

    View details for Web of Science ID A1997WE02000019

    View details for PubMedID 9013957

  • Recurrent identical rearrangement and repeated expression of identical heavy and light chains in single anti-phosphatidylcholine B cells B LYMPHOCYTES AND AUTOIMMUNITY Seidl, K. J., MacKenzie, J. D., Wang, D. N., Kantor, A. B., KABAT, E. A., Herzenberg, L. A., Herzenberg, L. A. 1997; 815: 484-488

    View details for Web of Science ID A1997BH93X00071

    View details for PubMedID 9186705

  • NF-kappa B homodimer binding within the HIV-1 initiator region and interactions with TFII-I PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Montano, M. A., Kripke, K., Norina, C. D., Achacoso, P., Herzenberg, L. A., Roy, A. L., Nolan, G. P. 1996; 93 (22): 12376-12381


    We show that the binding of Rel p50 and p52 homodimers at sites within the transcriptional initiation region of HIV-1 provides for their ability to interact with other proteins that bind the initiator. The binding of one such protein, the initiator protein TFII-I, to the initiation region of HIV-1 is augmented in the presence of Rel p50 and Rel p52 homodimers. Consistent with this, in vitro Rel homodimers potentiate HIV-1 transcription in a manner dependent upon TFII-I. The findings suggest that Rel dimers may regulate HIV-1 transcription in two ways. First, through binding at the kappa B enhancer sites at (-104 to -80), NF-kappa B p50:p65 participates in classical transcriptional activation. Second, Rel dimers such as p50 or p52 might bind at initiator sequences to regulate the de novo binding of components of certain preinitiation complexes. These findings, and the existence of Rel binding sites at the initiators of other genes, suggest roles for Rel proteins in early events determining transcriptional control.

    View details for Web of Science ID A1996VP93700060

    View details for PubMedID 8901589

  • Simultaneous fluorescence-activated cell sorter analysis of two distinct transcriptional elements within a single cell using engineered green fluorescent proteins PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Anderson, M. T., Tjioe, I. M., Lorincz, M. C., Parks, D. R., Herzenberg, L. A., Nolan, G. P., Herzenberg, L. A. 1996; 93 (16): 8508-8511


    Green fluorescent protein (GFP) is widely used as a reporter gene in both prokaryotes and eukaryotes. However, the fluorescence levels of wild-type GFP (wtGFP) are not bright enough for fluorescence-activated cell sorting or flow cytometry. Several GFP variants were generated that are brighter or have altered excitation spectra when expressed in prokaryotic cells. We engineered two GFP genes with different combinations of these mutations, GFP(S65T,V163A) termed GFP-Bex1, and GFP(S202F,T203I,V163A) termed GFP-Vex1. Both show enhanced brightness and improved signal-to-noise ratios when expressed in mammalian cells and appropriately excited, compared with wtGFP. Each mutant retains only one of the two excitation peaks of the wild-type protein. GFP-Bex1 excites at 488 nm (blue) and GFP-Vex1 excites at 406 nm (violet), both of which are available laser lines. Excitation at these wavelengths allows for the independent analyses of these mutants by fluorescence-activated cell sorting, permitting simultaneous, quantitative detection of expression from two different genes within single mammalian cells.

    View details for Web of Science ID A1996VB32500065

    View details for PubMedID 8710900

  • Activation-induced peripheral blood T cell apoptosis is fas independent in HIV-infected individuals INTERNATIONAL IMMUNOLOGY Katsikis, P. D., GarciaOjeda, M. E., Wunderlich, E. S., Smith, C. A., Yagita, H., Okumura, K., Kayagaki, N., Alderson, M., Herzenberg, L. A., Herzenberg, L. A. 1996; 8 (8): 1311-1317


    T cell apoptosis has been proposed as an important contributor to the functional defects and depletion of T cells in HIV-infected individuals. However, the mechanisms involved in this apoptosis have not been elucidated. We recently showed that peripheral blood T cells from HIV-infected individuals are especially susceptible to Fas antigen-induced apoptosis. In this study we examine the role of Fas, CTLA-4, tumor necrosis factor (TNF) receptors (TNFR) and CD30, receptors known to be involved in T cell activation-induced cell death (AICD), in the spontaneous and activation (anti-CD3)-induced apoptosis of peripheral blood T cells from asymptomatic HIV-infected individuals. We report here that spontaneous and activation-induced T cell apoptosis cannot be inhibited by reagents that block interactions of Fas, CTLA-4, p55 and p75 TNFR and CD30 with their respective ligands. We also show that IL-12, IFN-gamma, IL-4 and IL-10 cannot modify spontaneous, activation- and anti-Fas-induced apoptosis. Anti-Fas preferentially induced CD4+ T cell apoptosis whereas AICD induced apoptosis equally in CD4+ and CD8+ T cells. We conclude that T cell AICD in HIV infection is not mediated by Fas, thus indicating that Fas-induced and activation-induced T cell apoptosis are independent mechanisms of apoptosis which may play different roles in the pathogenesis of HIV infection.

    View details for Web of Science ID A1996VE10900014

    View details for PubMedID 8918700

  • Enzyme-generated intracellular fluorescence for single-cell reporter gene analysis utilizing Escherichia coli beta-glucuronidase CYTOMETRY Lorincz, M., Roederer, M., Diwu, Z., Herzenberg, L. A., Nolan, G. P. 1996; 24 (4): 321-329


    We report the development of a new fluorescence-activated cell sorter (FACS)-based reporter gene system utilizing the enzymatic activity of the E. coli beta-glucuronidase (gus) gene. When loaded with the Gus substrate fluorescein-di-beta-D-glucuronide (FDGlcu), individual mammalian cells expressing and translating gus mRNA liberate sufficient levels of intracellular fluorescein for quantitative analysis by flow cytometry. This assay can be used to FACS sort viable cells based on Gus enzymatic activity, and the efficacy of the assay can be measured independently by using a fluorometric lysate assay. Furthermore, both the beta-glucuronidase and the previously described E. coli beta-galactosidase enzymes have high specificities for their cognate substrates, allowing each reporter gene to be measured by FACS independently.

    View details for Web of Science ID A1996VD78900004

    View details for PubMedID 8866216

  • Cy7PE and Cy7APC: Bright new probes for immunofluorescence CYTOMETRY Roederer, M., Kantor, A. B., Parks, D. R., Herzenberg, L. A. 1996; 24 (3): 191-197


    We demonstrate the utility of indotricarbocyanine (Cy7) conjugates of the phycobiliproteins phycoerythrin (PE) and allophycocyanin (APC) in flow cytometry. This is the first demonstration of the use of an APC tandem dye for fluorescence measurements. These resonance energy transfer tandem dyes can be excited by the phycobiliprotein-specific excitation wavelengths and fluoresce at wavelengths above 780 nm. The tandem dyes, when conjugated to antibodies, are suitable for flow cytometry and other immunofluorescence applications. These conjugates are easily detectable above the very low autofluorescence in this part of the spectrum. Indeed, the Cy7-conjugated PE tandem (Cy7PE) has a "brightness" (fluorescence signal over cellular autofluorescence) comparable to that of fluorescein, and the Cy7APC tandem has a "brightness" comparable to that of APC. These tandems are also easily distinguished from other commonly used fluorophores, making them suitable for high-order multiparametric analysis. We show an example of six-color immunofluorescence analysis by flow cytometry, simultaneously measuring fluorescences from fluorescein, PE, Cy5PE, Texas red, APC, and Cy7APC.

    View details for Web of Science ID A1996UU65300001

    View details for PubMedID 8800551

  • Analysis of lipopolysaccharide-response genes in B-lineage cells demonstrates that they can have differentiation stage-restricted expression and contain SH2 domains PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kerr, W. G., Heller, M., Herzenberg, L. A. 1996; 93 (9): 3947-3952


    Bacterial lipopolysaccharide (LPS) is a potent stimulator of B-cell activation, proliferation, and differentiation. We examined the genetic response of B-lineage cells to LPS via trapping of expressed genes with a gene-trap retrovirus. This analysis showed that expression of only a small fraction of genes is altered during LPS stimulation of B-lineage cells. Isolation of the cellular portion of the trapped LPS-response genes via 5' RACE (rapid amplification of cDNA ends) cloning identified novel genes for all the cloned loci. These novel LPS-response genes were also found to have differentiation stage-restricted expression within the B-lymphoid lineage. That LPS-response genes in B cells also have differentiation stage-restricted expression suggests that these genes may be involved in the control of B-cell function and differentiation, since the known members of this class of genes have frequently been found to play a role in the function and differentiation of B-lineage cells. The isolation of novel members of this class of genes, including a gene that contains a putative SH2 domain, will further increase our understanding of the molecular events involved in the control of B-cell differentiation and function.

    View details for Web of Science ID A1996UK55700039

    View details for PubMedID 8632995

  • Elevation of plasma thioredoxin levels in HIV-infected individuals INTERNATIONAL IMMUNOLOGY Nakamura, H., DeRosa, S., Roederer, M., Anderson, M. T., Dubs, J. G., Yodoi, J., Holmgren, A., Herzenberg, L. A., Herzenberg, L. A. 1996; 8 (4): 603-611


    Thioredoxin (Trx), a ubiquitous protein intimately involved in redox and protein disulfide reductions, has been shown to be released from cells and to have cytokine-like activities. In addition, Trx has been implicated in the redox regulation of immunological responses and shown to be deficient in tissues from AIDS patients. In studies presented here, plasma Trx levels were measured by ELISA in plasma samples from HIV-infected individuals (n = 136) and HIV-negative controls (n = 47). To account for the release of Trx into plasma due to hemolysis, the Trx measurements were corrected according to the level of hemoglobin in the plasma sample. Data presented show that, in contrast to tissue Trx levels, corrected plasma Trx levels are significantly higher in HIV-infected individuals than in controls (P < 0.0001). Furthermore, approximately 25% of the HIV-infected individuals studied have plasma Trx levels greater than the highest levels found in controls (37 ng/ml). Detailed multiparameter FACS analysis of peripheral blood mononuclear cells (PBMC) from the infected individuals demonstrates that those with higher plasma Trx levels (37 ng/ml or greater) tend to have lower overall CD4 counts. In addition, increases in plasma Trx levels correlate with decreases in monochlorobimane staining (indicative of lower intracellular glutathione levels in PBMC) and with changes in surface antigen expression (CD62L, CD38 and CD20) that occur in the later stages of HIV infection. These correlations suggest that elevation of plasma Trx levels may be an important component of advanced HIV disease, perhaps related to the oxidative stress that often occurs at this stage.

    View details for Web of Science ID A1996UG69100019

    View details for PubMedID 8671648

  • Changes in antigen densities on leukocyte subsets correlate with progression of HIV disease INTERNATIONAL IMMUNOLOGY Roederer, M., Herzenberg, L. A., Herzenberg, L. A. 1996; 8 (1): 1-11


    In a cross-sectional study of 154 HIV-infected and 33 uninfected healthy adults, we show that characteristic changes in the levels of expression of leukocyte surface antigens occur in the HIV-infected individuals. These changes, which collectively occur on virtually every leukocyte subset, are specific: a particular antigen may increase or decrease on one subset of PBMC but remain constant on another. Furthermore, within any particular subset, the levels of one or more antigens may change, while the levels of other surface antigens on the same cells remain constant. Some of these antigens density changes have been noted before, e.g. increased CD20 on B cells, and increased CD38 and HLA-DR on CD8 T cells. However, the multiparameter flow cytometry methodology used here reveals changes in a substantially larger number of surface markers, some of which are restricted to fine subsets of PBMC, such as naive or memory T cell subsets. For many of these antigens, the change in expression correlates with absolute CD4 counts >500/microl; others differ only in those with counts >100microl. The changes in antigen densities we observed on B and T cells are consistent with the observation of a persistent quasi-activated state of these cells in HIV-infected individuals. Similarly, the altered expression of the signal-transducing molecules CD7 and CD16 that we demonstrated for NK cells may correlate with the functional defects previously demonstrated in NK cells. Thus, measurements of antigen densities such as those demonstrated here may provide surrogate markers for the altered functional capacities of PBMC subsets in HIV-infected individuals, and may thereby provide a much simpler assay for immunocompetence than in vitro functional assays.

    View details for Web of Science ID A1996TT32900001

    View details for PubMedID 8671584



    Calcium flux measurements of different subpopulations of cells by flow cytometry are important in understanding complex interactions in the immune system. This paper discusses the use of the difference of Log signals as a preferred method for obtaining this information simultaneously with other immunofluorescence parameters. We describe simple modifications to a commercial instrument that enables the measurement of calcium flux in addition to three immunofluorescence parameters. Finally, we show an application of this technique to measuring calcium flux of T cell subsets in human blood. We show that different subsets of peripheral CD4 T cells have significantly different capabilities to flux calcium after CD3 stimulation. These differences are related to the functional capacities of the cells within these subsets.

    View details for Web of Science ID A1995TA35300010

    View details for PubMedID 8582239

  • DEFECTIVE B-CELL DEVELOPMENT AND FUNCTION IN BTK-DEFICIENT MICE IMMUNITY Khan, W. N., Alt, F. W., Gerstein, R. M., Malynn, B. A., Larsson, I., Rathbun, G., Davidson, L., Muller, S., Kantor, A. B., Herzenberg, L. A., Rosen, F. S., Sideras, P. 1995; 3 (3): 283-299


    Mutations in the Bruton's tyrosine kinase (Btk) gene have been linked to severe early B cell developmental blocks in human X-linked agammaglobulinemia (XLA), and to milder B cell activation deficiencies in murine X-linked immune deficiency (Xid). To elucidate unequivocally potential Btk functions in mice, we generated mutations in embryonic stem cells, which eliminated the ability to encode Btk pleckstrin homology or kinase domains, and assayed their effects by RAG2-deficient blastocyst complementation or introduction into the germline. Both mutations block expression of Btk protein and lead to reduced numbers of mature conventional B cells, severe B1 cell deficiency, serum IgM and IgG3 deficiency, and defective responses in vitro to various B cell activators and in vivo to immunization with thymus-independent type II antigens. These results prove that lack of Btk function results in an Xid phenotype and further suggest a differential requirement for Btk during the early stages of murine versus human B lymphocyte development.

    View details for Web of Science ID A1995RX64500004

    View details for PubMedID 7552994

  • ISOLATION OF MUTANT T-LYMPHOCYTES WITH DEFECTS IN CAPACITATIVE CALCIUM-ENTRY IMMUNITY SERAFINI, A. T., Lewis, R. S., Clipstone, N. A., Bram, R. J., Fanger, C., Fiering, S., Herzenberg, L. A., Crabtree, G. R. 1995; 3 (2): 239-250


    Calcium and calcium-binding proteins play important roles in the signaling cascade leading from the initial engagement of TCRs on T cells to the fully activated state. To undertake a molecular dissection of this cascade, we first isolated a Jurkat T cell line derivative containing the NF-AT promoter element driving transcription of the diphtheria toxin A chain gene (dipA), resulting in rapid cell death. Selecting viable cells that fail to activate NF-AT-dependent transcription, we isolated two independent cell lines possessing defects in capacitative Ca2+ entry. NF-AT-dependent transcription can be restored in these cells by expression of a constitutively active calcineurin, but not overexpression of the Ca2+ regulatory protein CAML, which can normally replace the Ca2+ signal. The defect in these cell lines probably lies between CAML and calcineurin in the T cell activation cascade.

    View details for Web of Science ID A1995RR33900009

    View details for PubMedID 7648396



    Apoptosis (programmed cell death) of T lymphocytes has been proposed as a mechanism which plays an important role in the pathogenesis of human immunodeficiency virus (HIV) disease. Activation of Fas (CD95) can either result in costimulation of proliferation and cytokine production or in the induction of apoptosis of T lymphocytes. This raises the possibility that Fas is involved in the observed T cell apoptosis during HIV disease. In this report we show that peripheral blood CD4+ and CD8+ T lymphocytes from HIV-infected individuals undergo apoptosis in vitro in response to antibody stimulation (cross-linking) of Fas at a much higher frequency than from uninfected controls. This anti-Fas-induced T cell apoptosis is markedly higher than spontaneous T cell apoptosis in HIV-infected individuals. Antibodies against other members of the tumor necrosis factor (TNF)/nerve growth factor receptor family such as CD27, CD30, CD40, 4-1BB, p55 TNF receptor, p75 TNF receptor, and TNF receptor-related protein did not result in any increase of T cell apoptosis above that spontaneously observed in HIV+ individuals. Anti-Fas-induced apoptosis was much higher in symptomatic HIV-infected individuals; and the magnitude of anti-Fas-induced CD4+ T cell apoptosis correlated inversely with peripheral blood CD4+ T cell absolute counts. Surface expression of Fas on T cells was also found to be higher in HIV-infected individuals. Resting and activated CD4+ and CD8+ T cells both underwent apoptosis in response to anti-Fas antibody. L-Selectin positive memory CD4+ T cells were especially susceptible to anti-Fas-induced apoptosis. These findings show that CD4+ and CD8+ T lymphocytes in HIV-infected individuals are primed in vivo to undergo apoptosis in response to Fas stimulation, suggesting that Fas signaling may be responsible for the T lymphocyte functional defects and depletion observed in HIV disease.

    View details for Web of Science ID A1995RA60500011

    View details for PubMedID 7539037

  • ALTERED REPRESENTATION OF NAIVE AND MEMORY CD8 T-CELL SUBSETS IN HIV-INFECTED CHILDREN JOURNAL OF CLINICAL INVESTIGATION Rabin, R. L., Roederer, M., Maldonado, Y., Petru, A., Herzenberg, L. A., Herzenberg, L. A. 1995; 95 (5): 2054-2060


    CD8 T cells are divided into naive and memory subsets according to both function and phenotype. In HIV-negative children, the naive subset is present at high frequencies, whereas memory cells are virtually absent. Previous studies have shown that the overall number of CD8 T cells does not decrease in HIV-infected children. In studies here, we use multiparameter flow cytometry to distinguish naive from memory CD8 T cells based on expression of CD11a, CD45RA, and CD62L. With this methodology, we show that within the CD8 T cell population, the naive subset decreases markedly (HIV+ vs. HIV-, 190 vs. 370 cells/microliter; P < or = 0.003), and that there is a reciprocal increase in memory cells, such that the total CD8 T cell counts remained unchanged (800 vs. 860 cells/microliter; P < or = 0.76). In addition, we show that for HIV-infected children, the naive CD8 T cell and total CD4 T cell counts correlate (chi 2 P < or = 0.001). This correlated loss suggests that the loss of naive CD8 T cells in HIV infection may contribute to the defects in cell-mediated immunity which become progressively worse as the HIV disease progresses and CD4 counts decrease.

    View details for Web of Science ID A1995QW20500014

    View details for PubMedID 7738172

  • CD8 NAIVE T-CELL COUNTS DECREASE PROGRESSIVELY IN HIV-INFECTED ADULTS JOURNAL OF CLINICAL INVESTIGATION Roederer, M., Dubs, J. G., Anderson, M. T., Raju, P. A., Herzenberg, L. A., Herzenberg, L. A. 1995; 95 (5): 2061-2066


    We show here that CD8 naive T cells are depleted during the asymptomatic stage of HIV infection. Although overall CD8 T cell numbers are increased during this stage, the naive CD8 T cells are progressively lost and fall in parallel with overall CD4 T cell counts. In addition, we show that naive CD4 T cells are preferentially lost as total CD4 cell counts fall. These findings, presented here for adults, and in the accompanying study for children, represent the first demonstration that HIV disease involves the loss of both CD4 T cells and CD8 T cells. Furthermore, they provide a new insight into the mechanisms underlying the immunodeficiency of HIV-infected individuals, since naive T cells are required for all new T cell-mediated immune responses. Studies presented here also show that the well-known increase in total CD8 counts in most HIV-infected individuals is primarily due to an expansion of memory cells. Thus, memory CD8 T cells comprise over 80% of the T cells in PBMC from individuals with < 200 CD4/microliter, whereas they comprise roughly 15% in uninfected individuals. Since the naive and memory subsets have very different functional activities, this altered naive/memory T cell representation has significant consequences for the interpretation of data from in vitro functional studies.

    View details for Web of Science ID A1995QW20500015

    View details for PubMedID 7738173

  • Development of the antibody repertoire as revealed by single-cell PCR of FACS-sorted B-cell subsets Kantor, A. B., Merrill, C. E., MacKenzie, J. D., Herzenberg, L. A., Hillson, J. L. NEW YORK ACAD SCIENCES. 1995: 224-227

    View details for Web of Science ID A1995BE39E00034

    View details for PubMedID 7486528


    View details for Web of Science ID A1995BE08C00017

    View details for PubMedID 7476350



    Pregnancy is a unique immunologic state where a natural homeostasis exists between antigenically different tissues. Several earlier studies have addressed the fluctuations in the number and/or function of lymphocytes, including B cells during pregnancy, but changes within the subsets of B lymphocytes, conventional (CD5-) and B-1 (CD5+), have not been addressed. Here we demonstrate that the frequency of B-1 cells decreases dramatically during pregnancy, whereas the frequency of conventional B cells remains relatively constant. The missing B-1 cells return to pre-pregnancy levels 8-10 weeks after parturition. The polyreactive autoantibodies secreted by B-1 cells have been implicated in autoimmunity and immune regulation. The possible role of B-1 cells during pregnancy will be discussed in that context.

    View details for Web of Science ID A1995QH79400005

    View details for PubMedID 7537825

  • DE-NOVO DEVELOPMENT AND SELF-REPLENISHMENT OF B-CELLS INTERNATIONAL IMMUNOLOGY Kantor, A. B., STALL, A. M., Adams, S., Watanabe, K., Herzenberg, L. A. 1995; 7 (1): 55-68


    Previous studies distinguished two murine B cell lineages: the conventional lineage, which comprises the majority of B cells, and the Ly-1 B lineage (B-1a), which represents a small percentage of total adult B cells. A third subset, B-1b cells, shares many properties with B-1a cells, including the characteristic ability to self-replenish, but does not express Ly-1 (CD5). Reconstitution studies presented here show that (i) although the B220- population in adult spleen and bone marrow contains very little progenitor activity for B-1a cells, it can reconstitute roughly half the normal number of B-1b cells; (ii) B-1 progenitors present in adult bone marrow and spleen function at low levels in adult animals; (iii) peritoneal B-1 cells (principally B-1b) that develop following bone marrow transfer, like B-1 cells from normal animals, are capable of substantial self-replenishment; and (iv) conventional B cells do not expand (self-replenish) in adoptive recipients, although they can persist for long periods. Collectively, these progenitor and self-replenishment characteristics provide a developmental base for distinguishing B-1a, B-1b and conventional B cells.

    View details for Web of Science ID A1995QD67400007

    View details for PubMedID 7536467



    Studies presented here show that overall NF-kappa B signal transduction begins with a parallel series of stimuli-specific pathways through which cytokines (tumor necrosis factor alpha), oxidants (hydrogen peroxide and mitomycin C), and phorbol ester (phorbol 12-myristate 13-acetate) individually initiate signaling. These initial pathways culminate in a common pathway through which all of the stimulating agents ultimately signal NF-kappa B activation. We distinguish the stimuli-specific pathways by showing that the oxidative stimuli trigger NF-kappa B activation in only one of two human T-cell lines (Wurzburg but not Jurkat), whereas tumor necrosis factor alpha and phorbol 12-myristate 13-acetate readily stimulate in both lines. We propose the common pathway as the simplest way of accounting for the common requirements and properties of the signaling pathway. We include a redox-regulatory mechanism(s) in this common pathway to account for the previously demonstrated redox regulation of NF-kappa B activation in Jurkat cells (in which oxidants don't activate NF-kappa B); we put tyrosine phosphorylation in the common pathway by showing that kinase activity (inhibitable by herbimycin A and tyrphostin 47) is required for NF-kappa B activation by all stimuli tested in both cell lines. Since internal sites of oxidant production have been shown to play a key role in the cytokine-stimulated activation of NF-kappa B, and since tyrosine kinase and phosphatase activities are known to be altered by oxidants, these findings suggest that intracellular redox status controls NF-kappa B activation by regulating tyrosine phosphorylation event(s) within the common step of the NF-kappa B signal transduction pathway.

    View details for Web of Science ID A1994PU28500050

    View details for PubMedID 7526398

  • PATTERN SORTING - A COMPUTER-CONTROLLED MULTIDIMENSIONAL SORTING METHOD USING K-D TREES CYTOMETRY Bigos, M., Parks, D. R., Moore, W. A., Herzenberg, L. A., Herzenberg, L. A. 1994; 16 (4): 357-363


    Multidimensional binary trees provide a memory efficient and general method for computing sorting decisions in real time for a flow cytometer. Their fundamental advantage over conventional lookup table sorting techniques is that sort criteria in the full N-dimensional data space which cannot be described by projections onto two-dimensional parameter planes can be effectively implemented. This becomes particularly relevant when multidimensional analysis methods such as principal components or clustering are employed. We describe a prototype implementation of this method and point out other possible implementations.

    View details for Web of Science ID A1994NY12100010

    View details for PubMedID 7988296



    N-Acetyl-L-cysteine (NAC) and L-2-oxothiazolidine 4-carboxylate (OTC) are pro-GSH drugs that been proposed for AIDS therapy. In this article we compare the antiviral activities of these compounds in various in vitro HIV infection models. Although both compounds blocked cytokine induction of HIV in acute and chronic infection models, and in HIV-LTR reporter cell systems, NAC was far more effective than OTC, even at suboptimal doses. To test whether this difference is due to GSH conversion efficacies of these compounds, we measured GSH restoration by NAC or OTC in GSH-depleted peripheral blood mononuclear cells (PBMCs), using flow cytometry. In isolated PBMCs, NAC fully replenishes depleted intracellular GSH whereas OTC only minimally replenishes GSH. This ability to replenish GSH in vitro and its ability to scavenge free radicals directly explain why NAC has more potent antiviral activities in vitro.

    View details for Web of Science ID A1994PE52200015

    View details for PubMedID 7811547



    We tested the hypothesis that different genes can have different abilities to be amplified after transfection under comparable selection conditions. DNA from human lymphoid or choriocarcinoma cell lines was transfected into L cells. Transfectants for CD5, CD8A, TROP1, and TROP2, genes expressed on lymphocytes or trophoblast and carcinomas, were selected by fluorescence-activated cell sorting. To select for amplification of the transfected gene we cloned twice by fluorescence-activated cell sorting the transfectants with the highest expression. We analyzed a total of 38 families (1768 clones) derived from the original transfectants. We then analyzed by Southern blotting the clones with the highest increase in surface expression and determined the copy number of each transfected gene. CD5, CD8A, and TROP2 were amplified with high frequency and progressively, whereas TROP1 essentially was not amplified at all. We examined the hypothesis that DNA methylation prevents the amplification of the TROP1 gene by treating JAR choriocarcinoma cells with 5-azacytidine to decrease DNA methylation. DNA extracted at different times after the treatment was used for transfection. When DNA that showed demethylation of the TROP1 gene was used, 16 Trop-1 transfectants were obtained and 6 of them were found to contain up to 40 copies of the TROP1 gene per haploid genome. Thus, we showed that transfectants obtained from a demethylated TROP1 gene were amplified efficiently and progressively. We propose that DNA methylation affects DNA amplification either by altering the recognition of methylated DNA sequences or by changing the conformation of the chromatin of methylated segments. We speculate that DNA methylation is a determinant of gene amplification in vivo, for example in tumor cells.

    View details for Web of Science ID A1994NT46100021

    View details for PubMedID 8016075

  • CONTRIBUTION OF A SINGLE HEAVY-CHAIN RESIDUE TO SPECIFICITY OF AN ANTIDIGOXIN MONOCLONAL-ANTIBODY PROTEIN SCIENCE Schildbach, J. F., Shaw, S. Y., Bruccoleri, R. E., Haber, E., Herzenberg, L. A., Jager, G. C., Jeffrey, P. D., Panka, D. J., Parks, D. R., Near, R. I., Novotny, J., Sheriff, S., Margolies, M. N. 1994; 3 (5): 737-749


    Two distinct spontaneous variants of the murine anti-digoxin hybridoma 26-10 were isolated by fluorescence-activated cell sorting for reduced affinity of surface antibody for antigen. Nucleotide and partial amino acid sequencing of the variant antibody variable regions revealed that 1 variant had a single amino acid substitution: Lys for Asn at heavy chain position 35. The second variant antibody had 2 heavy chain substitutions: Tyr for Asn at position 35, and Met for Arg at position 38. Mutagenesis experiments confirmed that the position 35 substitutions were solely responsible for the markedly reduced affinity of both variant antibodies. Several mutants with more conservative position 35 substitutions were engineered to ascertain the contribution of Asn 35 to the binding of digoxin to antibody 26-10. Replacement of Asn with Gln reduced affinity for digoxin 10-fold relative to the wild-type antibody, but maintained wild-type fine specificity for cardiac glycoside analogues. All other substitutions (Val, Thr, Leu, Ala, and Asp) reduced affinity by at least 90-fold and caused distinct shifts in fine specificity. The Ala mutant demonstrated greatly increased relative affinities for 16-acetylated haptens and haptens with a saturated lactone. The X-ray crystal structure of the 26-10 Fab in complex with digoxin (Jeffrey PD et al., 1993, Proc Natl Acad Sci USA 90:10310-10314) reveals that the position 35 Asn contacts hapten and forms hydrogen bonds with 2 other contact residues. The reductions in affinity of the position 35 mutants for digoxin are greater than expected based upon the small hapten contact area provided by the wild-type Asn. We therefore performed molecular modeling experiments which suggested that substitution of Gln or Asp can maintain these hydrogen bonds whereas the other substituted side chains cannot. The altered binding of the Asp mutant may be due to the introduction of a negative charge. The similarities in binding of the wild-type and Gln-mutant antibodies, however, suggest that these hydrogen bonds are important for maintaining the architecture of the binding site and therefore the affinity and specificity of this antibody. The Ala mutant eliminates the wild-type hydrogen bonding, and molecular modeling suggests that the reduced side-chain volume also provides space that can accommodate a congener with a 16-acetyl group or saturated lactone, accounting for the altered fine specificity of this antibody.

    View details for Web of Science ID A1994NH62300003

    View details for PubMedID 8061604



    Studies presented here show that altering the intracellular redox balance by decreasing glutathione levels profoundly affects early signal transduction events in human T cells. In a T-cell receptor (TCR) signaling model, short-term pretreatment with buthionine sulfoximine, which specifically decreases intracellular glutathione, essentially abrogates the stimulation of calcium influx by anti-CD3 antibodies without significantly impairing other aspects of TCR-initiated signal transduction, such as overall levels of TCR-stimulated tyrosine phosphorylation. In an inflammatory-cytokine signaling model, the failure of tumor necrosis factor alpha to stimulate more than minimal tyrosine phosphorylation in lymphocytes is overcome by buthionine sulfoximine pretreatment--i.e., tumor necrosis factor alpha stimulates extensive tyrosine phosphorylation in glutathione-depleted lymphocytes. These redox-dependent changes in T-cell responsiveness suggest that the glutathione deficiency that we and others have demonstrated in human immunodeficiency virus-infected individuals may contribute significantly to the immunodeficiency and the increased inflammatory reactions in these individuals.

    View details for Web of Science ID A1994NJ03400031

    View details for PubMedID 7513425



    Myxococcus xanthus strains containing transcriptional fusions to lacZ were analyzed and fractionated by differences in their levels of beta-galactosidase expression. The fluorogenic substrate for beta-galactosidase, fluorescein di-beta-galactopyranoside, was introduced into M. xanthus cells during a rapid decrease in osmolarity of the medium followed by a return to isoosmolarity. Fluorescein, the product of hydrolysis, was retained within the cells and their viability was preserved. Fluorescence increased linearly with time and was proportional to beta-galactosidase activity. beta-Galactosidase expression in most fusion strains, though beginning at different phases of growth or development, was distributed unimodally amongst cells. However, fusion strain Tn5 lac omega 4473 was shown to be heterogeneous at 9 hr of development. It was possible to separate physically cells that expressed beta-galactosidase at a high level from other, still viable, cells with no expression. The approach described here could be adapted to study differentiation in plants and animals as well, where transcriptional fusions and fluorogenic substrates for enzyme probes of gene expression also can be used.

    View details for Web of Science ID A1993LV64400060

    View details for PubMedID 8396263

  • ANTIOXIDANTS INHIBIT STIMULATION OF HIV TRANSCRIPTION AIDS RESEARCH AND HUMAN RETROVIRUSES Staal, F. J., Roederer, M., Raju, P. A., Anderson, M. T., Ela, S. W., Herzenberg, L. A., Herzenberg, L. A. 1993; 9 (4): 299-306


    In studies presented here, we demonstrate that antioxidants regulate NF-kappa B activation and signal transduction pathways leading to HIV expression. We show (1) that N-acetyl-L-cysteine (NAC), an antioxidant and an efficient glutathione (GSH) precursor, inhibits NF-kappa B activation and HIV expression under conditions in which GSH is depleted and NAC cannot be converted to GSH, (2) that the D-stereoisomer of NAC and a wide variety of chemically unrelated antioxidants also inhibit NF-kappa B activation and/or transcription directed by the HIV LTR, and (3) that depletion of GSH, the principal intracellular antioxidant, augments HIV production in an acute infection model. Taken together, these findings suggest direct antioxidant action as the mechanism for inhibition of HIV transcription by NAC. They also confirm that GSH, acting in its capacity as an antioxidant, regulates HIV expression and that exogenous antioxidants can potentiate this regulation.

    View details for Web of Science ID A1993KY80800002

    View details for PubMedID 8512745

  • N-ACETYLCYSTEINE - POTENTIAL FOR AIDS THERAPY PHARMACOLOGY Roederer, M., Staal, F. J., Ela, S. W., Herzenberg, L. A., Herzenberg, L. A. 1993; 46 (3): 121-129


    The observations that people infected with HIV suffer not only from an inflammatory stress but also from depleted glutathione levels have led to a general hypothesis that these two are causally related, and that treatment of AIDS should include thiol-replenishment therapy. In particular, inflammatory stimulations are dependent on intracellular thiol levels, as they are potentiated at low glutathione levels (oxidative stress) and inhibited at high glutathione levels. Inflammatory stress may itself lead to decreased levels of glutathione. HIV has taken advantage of inflammatory signals to regulate its own replication; thus, the HIV infection is exacerbated by low levels of glutathione. We have shown that N-acetylcysteine can inhibit inflammatory stimulations, including that of HIV replication. Since N-acetylcysteine can replenish depleted glutathione levels in vivo, we suggest that it be used as an adjunct in the treatment of AIDS.

    View details for Web of Science ID A1993KP55700001

    View details for PubMedID 8441760

  • B-CELL LINEAGES EXIST IN THE MOUSE IMMUNOLOGY TODAY Herzenberg, L. A., Kantor, A. B. 1993; 14 (2): 79-83

    View details for Web of Science ID A1993KL80100010

    View details for PubMedID 8447936

  • DISREGULATION OF LEUKOCYTE GLUTATHIONE IN AIDS CLINICAL FLOW CYTOMETRY Roederer, M., Staal, F. J., Anderson, M., Rabin, R., Raju, P. A., Herzenberg, L. A., Herzenberg, L. A. 1993; 677: 113-125
  • ORIGIN OF MURINE B-CELL LINEAGES ANNUAL REVIEW OF IMMUNOLOGY Kantor, A. B., Herzenberg, L. A. 1993; 11: 501-538


    Until recently, the hematopoietic stem cells (HSC) that appear early in ontogeny were thought to constitute a homogeneous, self-replenishing population whose developmental potential remains constant throughout the life of the animal. Studies reviewed here, however, demonstrated clear differences in the developmental potential of fetal and adult progenitor populations (including FACS-sorted HSC). These studies, which chart the ability of various progenitor sources to reconstitute functionally distinct B cell populations, define three B cell lineages: B-1a cells (CD5 B cells), derived from progenitors that are present in fetal omentum and fetal liver but are largely absent from adult bone marrow; B-1b cells ("sister" population), derived from progenitors that are present in fetal omentum, fetal liver, and also in adult bone marrow; and conventional B cells, whose progenitors are missing from fetal omentum but are found in fetal liver and adult bone marrow. B-1a and B-1b cells share many properties, including self-replenishment and feedback regulation of development. These B cell studies, in conjunction with evidence for a similar developmental switch for T cells and erythrocytes, suggest that evolution has created a "layered" immune system in which successive progenitors (HSC) reach predominance during development and give rise to differentiated cells (B, T, etc) responsible for progressively more complex immune functions.

    View details for Web of Science ID A1993KX30600018

    View details for PubMedID 8476571



    We report the cloning of a cDNA encoding the alpha-chain of the bovine CD8 (BoCD8 alpha). A bovine thymus cDNA library was hybridized at low stringency with a human CD8 alpha cDNA clone. The first round of screening of 5 x 10(4) independent colonies yielded 12 clones containing incomplete BoCD8 alpha genes. Two further rounds of colony hybridization were conducted, each using as a probe the 5' fragment from the longest BoCD8 alpha clone previously isolated. The final screening yielded a clone containing a 2 kilobase (kb) insert. We mapped and sequenced the 2 kb BoCD8 alpha clone and compared it with the published sequences of the genes encoding the human, mouse and rat CD8 alpha. Sequence analysis confirmed that the clone under study encoded the BoCD8 alpha. The overall similarity of the BoCD8 alpha coding region with the human CD8 alpha coding sequence is 74.7% at the nucleotide level and 62.1% at the protein level. Lower levels of similarity are found with the mouse and rat CD8 alpha. Interestingly, three separate highly homologous regions are clearly defined at the peptide level in bovine versus human and mouse versus rat comparisons. Two of the regions are highly conserved among all species analysed, while the most 5' region is not. We speculate that the latter region may contain the binding site of CD8 alpha to the alpha 3 domain of major histocompatibility complex (MHC) class I molecules. Sequence analysis showed that the 2 kb BoCD8 alpha clone contains an incomplete coding region, i.e. lacks six bases corresponding to the first two amino acids of the leader region. To allow efficient translation and processing of the BoCD8 alpha gene, we constructed a chimeric gene containing the coding sequence of the BoCD8 alpha clone and synthetic sequences corresponding to the first two amino acids of the human CD8 alpha leader sequence. The chimeric gene was subcloned in the pKSV10 expression vector. The pKSV10-BoCD8 alpha construct is efficiently expressed both transiently in COS cells and stably in L cells, as determined by Northern blot and by FACS analysis, using the ILA-51 monoclonal antibody to BoCD8 alpha. The latter result formally proves that the ILA-51 antibody does indeed recognize the product of the BoCD8 alpha gene, as previously suggested on serological grounds.

    View details for Web of Science ID A1992HV85200016

    View details for PubMedID 1628904



    Cell-transfer studies presented here distinguish three murine B cell lineages: conventional B cells, which develop late and are continually replenished from progenitors in adult bone marrow; Ly-1 B cells (B-1a), which develop early and maintain their numbers by self-replenishment; and Ly-1B "sister" (B-1b) cells, which share many of the properties of Ly-1 B cells, including self-replenishment and feedback regulation of development but can also readily develop from progenitors in adult bone marrow. The sequential emergence of these lineages, the time at which their progenitors function during ontogeny, and the distinctions among their repertoires and functions suggest that evolution has created a layered immune system in which the immune response potential of each successive lineage is adapted to its particular niche.

    View details for Web of Science ID A1992HP04300033

    View details for PubMedID 1565622

  • GLUTATHIONE DEFICIENCY AND HUMAN-IMMUNODEFICIENCY-VIRUS INFECTION LANCET Staal, F. J., Ela, S. W., Roederer, M., Anderson, M. T., Herzenberg, L. A., Herzenberg, L. A. 1992; 339 (8798): 909-912

    View details for Web of Science ID A1992HN48300013

    View details for PubMedID 1348307

  • INTRACELLULAR GLUTATHIONE LEVELS IN T-CELL SUBSETS DECREASE IN HIV-INFECTED INDIVIDUALS AIDS RESEARCH AND HUMAN RETROVIRUSES Staal, F. J., Roederer, M., Israelski, D. M., BUBP, J., Mole, L. A., McShane, D., Deresinski, S. C., Ross, W., Sussman, H., Raju, P. A., Anderson, M. T., Moore, W., Ela, S. W., Herzenberg, L. A., Herzenberg, L. A. 1992; 8 (2): 305-311


    The authors have shown previously that intracellular glutathione (GSH) plays an important role in the regulation of human immunodeficiency virus (HIV) transcription and replication in vitro, through modulation of signal transduction by inflammatory cytokines. Moreover, intracellular GSH levels are known to regulate T-lymphocyte function. In multiparameter FACS studies presented here, we show that relative GSH levels in CD4+ and CD8+ T cells from HIV+ individuals are significantly lower than in corresponding subsets from uninfected controls. These studies define the relative intracellular glutathione (GSH) levels in CD4+ T cells, CD8+ T cells, B cells, and monocytes from 134 HIV-infected individuals and 31 uninfected controls. The greatest decreases in intracellular GSH occur in subsets of T cells in individuals in the later stages of the HIV infection. In AIDS patients, GSH levels are 63% of normal in CD4+ T cells (p less than 0.0001) and are 62% of normal in CD8+ T cells (p less than 0.0001). Similarly, in AIDS-related complex (ARC) patients, GSH levels are 66% of normal in CD4+ T cells (p less than 0.003) and are 69% of normal in CD8+ T cells (p less than 0.003). These findings suggest that low intracellular GSH levels may be an important factor in HIV infection and in the resulting immunodeficiency.

    View details for Web of Science ID A1992HG48900025

    View details for PubMedID 1540417

  • N-ACETYLCYSTEINE - A NEW APPROACH TO ANTI-HIV THERAPY AIDS RESEARCH AND HUMAN RETROVIRUSES Roederer, M., Ela, S. W., Staal, F. J., Herzenberg, L. A., Herzenberg, L. A. 1992; 8 (2): 209-217


    Several investigators have implicated depletion of glutathione (GSH) and production of reactive oxygen intermediates (ROIs) in the regulation of the human immunodeficiency virus (HIV). We have shown directly that N-acetylcysteine (NAC) blocks HIV expression in chronic and acute infection models, and HIV replication in normal peripheral blood mononuclear cells. NAC is a cysteine prodrug which maintains intracellular thiol levels during oxidative stress and replenishes depleted GSH. The observed antiviral effect of NAC is due to inhibition of viral stimulation by ROIs, which are produced in response to inflammatory cytokines. We have also shown that HIV-infected individuals have decreased intracellular GSH levels in their circulating T cells. Since GSH is the major protection against the production of ROIs, we hypothesize that the observed decrease is due to a chronic oxidative stress induced by continual exposure to elevated levels of inflammatory cytokines. Together, these results provide a rationale for clinical trials testing the efficacy of GSH-replenishing drugs such as NAC in the treatment of AIDS. NAC is different than many other antiviral drugs in that it inhibits host-mediated stimulation of viral replication arising in normal immune responses, and may thereby extend latency. In addition, it inhibits the action of inflammatory cytokines which may mediate cachexia, thereby raising the possibility that it may alleviate the deleterious wasting that accompanies late stage AIDS.

    View details for Web of Science ID A1992HG48900012

    View details for PubMedID 1540408

  • THE ONTOGENY AND FUNCTIONAL-CHARACTERISTICS OF HUMAN B-1 (CD5+B) CELLS INTERNATIONAL IMMUNOLOGY Bhat, N. M., Kantor, A. B., Bieber, M. M., STALL, A. M., Herzenberg, L. A., Teng, N. N. 1992; 4 (2): 243-252


    We demonstrate that, on average, greater than 90% of B lymphocytes in fetal spleen express CD5 at gestational ages of 17-23 weeks. Similarly, CD5+ B cells (B-1 cells) are the major B cell subset in umbilical cord blood. These findings depend on the optimization of fluorochrome conjugated anti-CD5 reagents for multiparameter fluorescent-activated cell sorter (FACS) analysis. From infancy through childhood the percentage of B-1 cells gradually diminishes in both spleen and peripheral blood. Stable adult levels, 25-35% of the total B cell population, are reached in late adolescence. The decrease in the percentage of B-1 cells in spleen is accompanied by an increase in conventional (CD5-) B cells, keeping the percentage of total B cells per mononuclear cells relatively constant. In contrast, in peripheral blood, the concentration of both B-1 cells and total B cells decreases, while T cells increase. At the functional level, we show that polyreactive IgM autoantibodies are produced by FACS-sorted CD5high B cells, but not by CD5- B cells from adolescent spleen. In contrast, fetal splenic CD5high and CD5- B cells appear functionally uniform, both producing IgM autoantibodies that are typical of B-1 cells. The apparent level of CD5- B cells in fetal spleen, on average 10% of total B cells, may still result from limitations of our reagent. The prominence of B-1 cells in fetal spleen and cord blood, the gradual reduction of B-1 cells with increasing age, and its characteristic repertoire, all suggest a role for this cell type in immunologically immature hosts.

    View details for Web of Science ID A1992HF01700016

    View details for PubMedID 1377947



    In this paper we have outlined the evidence for two distinct branches of the B-1 cell lineage. The data show that phenotypically B-1a and B-1b cells are essentially identical, distinguished only by the presence or absence of the CD5 antigen. Functionally no differences between the two populations have yet been identified. Both produce anti-PtC antibodies, a specificity not observed in conventional B cells. Both produced high levels of IgM as measured in adoptive transfer experiments. Developmentally, B-1a and B-1b cells are indistinguishable with respect to generation from progenitors present in fetal liver and omentum, feedback regulation of new B-1a and B-1b cells from bone marrow, self-replenishment from Ig+ cells following adoptive transfer, and the generation of clonal populations. The major difference in the two populations is seen in the development of B-1a and B-1b cells from B220- progenitors in the adult bone marrow. Although B220- B-1a progenitors are rare in adult (greater than 6 weeks) bone marrow, the progenitors for B-1b cells persist well into adulthood. Our understanding of B-1b cell ontogeny is at a stage similar to that of B-1a cells five years ago. We have evidence from transfer experiments that strongly suggests the existence of two distinct progenitors for B-1a and B-1b, but we have yet to physically separate these progenitors as Solvansen et al. have done for B-1 and conventional B cells. Furthermore we must determine whether the B-1b cells that develop from fetal liver and bone marrow are functionally and developmentally equivalent to those that develop from adult bone marrow. As with B-1a cells, the role of B-1b cells in the immune system is unclear. Although we have not yet discerned functional differences between B-1a and B-1b, given the recent identification of CD72 (Lyb-2) as the ligand for CD5, it is tempting to speculate that B-1a cells are more involved in B-B cell interactions such as idiotype-anti-idiotype regulation of the early B-cell repertoire and that B-1b cells are more involved in B-T cell interactions. Whatever their function, it is clear that in trying to understand the role of the B-1 lineage it is important to consider both the B-1a and B-1b lineages.

    View details for Web of Science ID A1992BW41S00004

  • CD20 expression is increased on B lymphocytes from HIV-infected individuals. Journal of acquired immune deficiency syndromes Staal, F. J., Roederer, M., BUBP, J., Mole, L. A., Anderson, M. T., Raju, P. A., Israelski, D. M., Deresinski, S. C., Moore, W. A., Herzenberg, L. A. 1992; 5 (6): 627-632


    In studies presented here, we show that expression of the pan B cell marker CD20 is markedly increased on B lymphocytes from HIV-infected individuals and that this increase tends to be greater in individuals with more advanced disease. By using multiparameter FACS analyses to quantitate surface density of CD20 and intracellular glutathione (GSH) levels simultaneously, we further show that the distribution of intracellular glutathione (GSH) levels in B cells of HIV-infected individuals is more heterogeneous than in uninfected controls. Finally, we show that the intracellular GSH levels correlate with CD20 expression on a per-cell basis in all infected individuals. These findings suggest that CD20 expression, which can be precisely measured, may prove to be a useful surrogate marker for monitoring HIV infection.

    View details for PubMedID 1375291

  • ADOPTIVE TRANSFER OF MURINE B-CELL LINEAGES CD5 B CELLS IN DEVELOPMENT AND DISEASE Kantor, A. B., STALL, A. M., Adams, S., Herzenberg, L. A., Herzenberg, L. A. 1992; 651: 168-169


    Studies presented here show that heparin alters immunoglobulin expression by murine pre-B cell lines and normal Ly-1 (CD5+) B cells. Previous studies have shown that pre-B cell lines 70Z/3 and NFS-5.3 express mu heavy chains in the cytoplasm and a small amount on the cell surface. Both these cytoplasmic and surface mu are disulfide-linked to omega (lambda 5) surrogate light chains and are noncovalently associated with iota (Vpre-B) variable region-like proteins. We show that culturing 70Z/3 with heparin reduces the amount of the membrane-form mu (micron) on the cell surface. Culturing NFS-5.3 with heparin similarly decreases the membrane-form mu; however, it increases the surface level of a pentameric mu molecule containing secreted-form mu (microS) heavy chains, disulfide-linked omega (lambda 5) chains, and noncovalently associated proteins. Culturing peritoneal B cells with heparin also increases the production of the secreted-form microS, detectable in this case by the secretion of classical pentameric IgM. Similarly, injecting heparin intraperitoneally increases IgM secretion by peritoneal Ly-1 B cells. Thus heparin could influence pre-B cell and B cell differentiation and function.

    View details for Web of Science ID A1991GQ76300008

    View details for PubMedID 1722112



    Maintenance of intracellular glutathione (GSH) levels has been implicated in blocking cytokine-stimulated HIV replication in vitro, in both acute and latent infection models. We demonstrate here that subsets of human peripheral blood mononuclear cells differ substantially in mean GSH levels, as measured on a cell-by-cell basis with the fluorescence-activated cell sorter (FACS): B cells have the lowest GSH levels; T cells are intermediate; and monocytes and macrophages have the highest levels. Furthermore, GSH levels subdivide the CD4 and CD8 T cell subsets into two classes each: high- and low-GSH cells, which cannot be distinguished by cell size or by currently known surface markers. Significantly, the high-GSH T cells are selectively depleted early during the HIV infection, and are effectively missing in all ARC and AIDS patients.

    View details for Web of Science ID A1991GE61600010

    View details for PubMedID 1681892



    The progression of the human immunodeficiency virus (HIV) infection from its early latent (asymptomatic) stage to active, late-stage acquired immunodeficiency syndrome (AIDS) apparently begins with the production of inflammatory cytokines that stimulate the expression and replication of the latent virus. We have shown that N-acetylcysteine, a cysteine precursor that is converted intracellularly into glutathione, blocks cytokine-stimulated HIV replication in an acutely infected T-cell line and in acutely infected peripheral blood mononuclear cells from normal individuals. In this report, we show that N-acetylcysteine also inhibits stimulated HIV expression in chronically infected monocyte and T-cell lines which are used as models for latent infection in AIDS. Furthermore, we show that N-acetylcysteine blocks viral production in monocyte cell lines more effectively than it blocks viral production in T cells. Since monocytes are a major reservoir for HIV in infected individuals, these results suggest that N-acetylcysteine may slow the change from latency to the later stages of AIDS in HIV-infected individuals.

    View details for Web of Science ID A1991FU36100010

    View details for PubMedID 1931232

  • WHOLE ANIMAL-CELL SORTING OF DROSOPHILA EMBRYOS SCIENCE Krasnow, M. A., Cumberledge, S., Manning, G., Herzenberg, L. A., Nolan, G. P. 1991; 251 (4989): 81-85


    Use of primary culture cells has been limited by the inability to purify most types of cells, particularly cells from early developmental stages. In whole animal cell sorting (WACS), live cells derived from animals harboring a lacZ transgene are purified according to their level of beta-galactosidase expression with a fluorogenic beta-galactosidase substrate and fluorescence-activated cell sorting. With WACS, incipient posterior compartment cells that express the engrailed gene were purified from early Drosophila embryos. Neuronal precursor cells were also purified, and they differentiated into neurons with high efficiency in culture. Because there are many lacZ strains, it may be possible to purify most types of Drosophila cells. The same approach is also applicable to other organisms for which germ-line transformation is possible.

    View details for Web of Science ID A1991EQ60300032

    View details for PubMedID 1898782



    The previously reported FACS-Gal assay (Nolan et al., Proc Natl Acad Sci USA 85:2603-2607, 1988) measures E. coli lacZ-encoded beta-galactosidase activity in individual viable eukaryotic cells for a variety of molecular and cellular biological applications. Enzyme activity is measured by flow cytometry, using a fluorogenic substrate, which is hydrolyzed and retained intracellularly. In this system, lacZ serves both as a reporter gene to quantitate gene expression and as a selectable marker for the fluorescence-activated sorting of cells based on their lacZ expression level. This report details the following improvements of the original assay: 1) use of phenylethyl-beta-D-thiogalactoside, a competitive inhibitor, to inhibit beta-galactosidase activity; 2) reduction of false positives by two-color measurements; and 3) inhibition of interfering mammalian beta-galactosidases by the weak base chloroquine. We found an exponential relationship between fluorescence generated by beta-galactosidase in this assay and the intracellular concentration of beta-galactosidase molecules. Finally, we report conditions for optimal loading of the substrate (FDG) and retention of the product, fluorescein. Under these conditions, we found uniform loading of FDG in all cells of a clone in individual experiments. Together, these improvements make FACS-Gal an extremely powerful tool for investigation of gene expression in eukaryotic cells.

    View details for Web of Science ID A1991FK38600001

    View details for PubMedID 1905992



    We demonstrate that infection of an LPS-responsive pre-B cell line with transcriptionally-defective retroviruses containing a reporter gene (lacZ) can result in viral integrations where expression of lacZ is differentiation stage-dependent. Because expression of lacZ is dependent upon flanking cellular sequences these retroviral integrations represent in situ gene fusions with cellular enhancers (Enhsr1) and genes (Gensr1) which are either induced or repressed during LPS-stimulated differentiation. One of the well-documented effects of LPS upon pre-B cells is the induction of kappa light chain transcription via NF-kappa B. The identification of LPS-stimulated gene repression during B cell differentiation indicates that LPS has multiple effects upon gene expression during the pre-B to B cell transition. The identification of cellular enhancers and genes which are downregulated during the transition from the pre-B to the B cell stage indicates that other transcription factors, in addition to NF-kappa B, are required for this step in differentiation. Finally, we present some initial experiments which indicate the gene-search retroviruses can introduce expression of lacZ into normal hematopoietic cells in vitro and in vivo.

    View details for Web of Science ID A1991BT82R00021

    View details for PubMedID 1950769



    The activation of nuclear factor kappa B (NF-kappa B) has been implicated in the regulation of transcription of a variety of genes and has been shown to be essential for the expression of genes controlled by the long terminal repeat of human immunodeficiency virus (HIV LTR). We show here that intracellular thiol levels play a key role in regulating this process. That is, stimulation with tumor necrosis factor alpha and/or phorbol 12-myristate 13-acetate activates NF-kappa B and markedly decreases intracellular thiols; N-acetyl-L-cysteine, an efficient thiol source, prevents this thiol decrease and blocks the activation of NF-kappa B; and the lack of activated NF-kappa B prevents the activation of the HIV LTR and the transcription of genes under its control. These findings reveal a previously unrecognized genetic regulatory mechanism in which cytokine-induced shifts in intracellular thiol levels are crucial in the control of NF-kappa B activity and thereby influence the spectrum of genes expressed by cytokine-stimulated cells.

    View details for Web of Science ID A1990EN15900090

    View details for PubMedID 2263644

  • THE ACTIONS OF CYCLOSPORINE-A AND FK506 SUGGEST A NOVEL STEP IN THE ACTIVATION OF LYMPHOCYTES-T EMBO JOURNAL Mattila, P. S., Ullman, K. S., Fiering, S., EMMEL, E. A., McCutcheon, M., Crabtree, G. R., Herzenberg, L. A. 1990; 9 (13): 4425-4433


    Cyclosporin A and FK506 are immunosuppressive compounds that have similar inhibitory effects on the expression of several lymphokines produced by T lymphocytes. Despite their similar effects the drugs bind to two different cytosolic protein, cyclophilin and FKBP respectively, which raises the possibility that they have different modes of action. Using constructs in which mRNA production controlled by a specific transcription factor could be readily measured we found that both cyclosporin A and FK506 completely inhibited transcription activated by NF-AT, NFIL2 A, NFIL2 B and partially inhibited transcription activated by NF kappa B. Cyclosporin A and FK506 inhibited only transcriptional activation that was dependent on Ca2+ mobilization. However, cyclosporin A and FK506 did not inhibit Ca2+ mobilization dependent expression of c-fos mRNA indicating that only a subset of signalling pathways regulated by Ca2+ is sensitive to these drugs. Furthermore, we did not observe any qualitative differences between the effect of cyclosporin A and FK506 on six different transcription factors which suggests that these drugs may interfere with the activity of a novel Ca2+ dependent step that regulates several transcription factors.

    View details for Web of Science ID A1990EN92100026

    View details for PubMedID 1702384



    Stimulation of T lymphocytes through their antigen receptor leads to the appearance of several transcription factors, including NF-AT and NF-kappa B, which are involved in regulating genes required for immunologic activation. To investigate the activity of a single transcription factor in individual viable cells, we have applied an assay that uses the fluorescence-activated cell sorter to quantitate beta-galactosidase (beta-gal). We have analyzed the distribution of NF-AT transcriptional activity among T cells undergoing activation by using a construct in which three tandem copies of the NF-AT-binding site directs transcription of the lacZ gene. Unexpectedly, stimulation of cloned stably transfected Jurkat T cells leads to a bimodal pattern of beta-gal expression in which some cells express no beta-gal and others express high levels. This expression pattern cannot be accounted for by cell-cycle position or heritable variation. Further results, in which beta-gal activity is correlated with NF-AT-binding activity, indicate that the concentration of NF-AT must exceed a critical threshold before transcription initiates. This threshold likely reflects the NF-AT concentration-dependent assembly of transcription complexes at the promoter. Similar constructs controlled by NF-kappa B or the entire interleukin-2 enhancer show bimodal expression patterns during induction, suggesting that thresholds set by the concentration of transcription factors may be a common property of inducible genes.

    View details for Web of Science ID A1990EC63000016

    View details for PubMedID 2123468



    Two-color flow cytometrical (FCM) analysis of rat peripheral lymphoid organs shows two distinct IgM/IgD-defined B cell subpopulations, similar to those of the mouse: a major population of cells expressing little IgM and high levels of IgD (population I) and a minor population of cells expressing high levels of IgM but little IgD (population III). In peripheral lymphoid organs population III cells are mainly found in spleen where they represent about 25% of the B cells; population III cells are almost absent from lymph nodes and Peyer's patches. In adult bone marrow and in neonatal spleen the majority of IgM/IgD-defined B cells (greater than 70%) are population III cells, similar to what is observed in the mouse. In contrast with mice, only a low proportion of the cells (1%) recovered from the peritoneal cavity are B cells, and most of them belong to population I. Previously defined monoclonal antibodies (HIS22 and HIS24) to B cell forms of the leukocyte common antigen (CD45R) in combination with staining for surface IgM and surface IgD demonstrates a further heterogeneity of rat B cells by three-color FCM analyses. HIS22 labels most population I cells; population III cells and a small subset (about one third) of population I express only very low levels of the HIS22 determinant. HIS24 reacts with population I cells and subdivides population III into two subsets: about one third of splenic population III cells are brightly stained with this antibody whereas fluorescence of the remaining two-thirds is lower. The HIS24bright population III cells likely are newly formed B cells since cells with this phenotype are the predominant surface Ig population found in adult bone marrow and neonatal spleen. In tissue sections of lymphoid organs, HIS22- and HIS24-positive cells are mainly found in lymphoid follicles; splenic marginal zones are almost unstained. Combining immunohistological analysis with the FCM data, we therefore conclude that the small follicular B cells are in population I and marginal zone B cells are found in the HIS24dull population III. The in situ localization of HIS24bright population III cells and the HIS22dull population I cells is not clear.(ABSTRACT TRUNCATED AT 400 WORDS)

    View details for Web of Science ID A1990DV21200017

    View details for PubMedID 2143728



    Studies presented here demonstrate that IgM and IgD molecules on normal murine B lymphocytes exist in different, noncovalently associated molecular complexes containing distinct but potentially related glycoproteins. The glycoproteins in these complexes, particularly those associated with IgD, show striking differences in various lymphoid organs and in X-linked immunodeficient (Xid) mice. These differences are due in part to post-translational processing. They apparently reflect the differential expression of the Ig-associated glycoproteins in the various B cell subpopulations and lineages and the differential distribution of the subpopulations and lineages in the various lymphoid organs. In addition, they reflect structural differences in the IgM and IgD complexes which, we suggest, permit differential signal transduction by IgM and IgD on the same B cell.

    View details for Web of Science ID A1990DL08100012

    View details for PubMedID 2357960



    Studies presented here demonstrate that heparin inhibits EcoRI endonuclease cleavage of DNA whereas related proteoglycans show no effect. The inhibition occurs at particular EcoRI sites that are near or overlap with palindromic sequences in the murine lambda 5 and Lyt-2 genes. Endogenous heparin from peritoneal mast cells co-isolates with DNA and inhibits digestion of peritoneal cell DNA at the inhibitable sites. Digestion of spleen DNA is inhibited at the same sites when commercial heparin is added prior to digestion. In both cases, the inhibition is abolished by pre-treating the DNA with heparinase. Thus, potential artifacts in restriction fragment length analyses could occur with DNA isolated either from cells that are naturally rich in heparin or from cells to which heparin has been added, e.g., as an anticoagulant.

    View details for Web of Science ID A1990DJ27100016

    View details for PubMedID 2356119



    We show that the stimulation of human immunodeficiency virus (HIV) brought about by tumor necrosis factor alpha and phorbol 12-myristate 13-acetate can be inhibited by adding N-acetyl-L-cysteine (NAC). NAC, which replenishes intracellular glutathione, effectively inhibits the tumor necrosis factor alpha- or phorbol ester-stimulated replication of HIV in acutely infected cell cultures. NAC also inhibits the cytokine-enhanced HIV long terminal repeat-directed expression of beta-galactosidase in in vitro HIV model systems. These results show that intracellular thiol levels influence HIV production. Furthermore, because NAC reverses tumor necrosis factor alpha toxicity both in cells and in animals and is a well-known drug that can be administered orally without known toxicity in humans, these results suggest that NAC is a possible therapeutic agent in AIDS.

    View details for Web of Science ID A1990DK27300100

    View details for PubMedID 2112750



    The treatment of female (New Zealand black x New Zealand white)F1 mice with total lymphoid irradiation resulted in a prolonged remission of autoimmune disease activity. Total lymphoid irradiation-treated mice also showed a marked reduction of Ly-1 B cells, which lasted up to 3 months. The subsequent return of Ly-1 B cells to preirradiation levels was not associated with a simultaneous return of disease when measured by parameters such as IgG anti-DNA antibodies and spontaneous secretion of IgG by splenic cells. In cell sorting experiments, most of the cells spontaneously secreting IgG were found within the Ly-1- (CD5-) splenic B cell population.

    View details for Web of Science ID A1990CZ97400013

    View details for PubMedID 2328033



    Using flow cytometry technology and multiparameter analyses, we report early and characteristic alterations in lymphoid cell profile in spleen and lymph nodes due to LP-BM5 retrovirus disease (murine AIDS (MAIDS)) and the effect of azido dideoxythymidine, a nucleoside inhibitor, on these changes. MAIDS has been characterized by rapid and profound lymphoproliferation accompanied by hypergammaglobulinemia and immunosuppression. As early as 2 wk postinfection, there is a selective depletion of CD8+ cells whereas the total number of CD4+ cells increases throughout the first 8 wk of infection although the frequency is relatively stable. These population changes were partially delayed by oral AZT therapy for 6 wk postinfection. Ly-6C (AL-21) is expressed on roughly 50% of CD4+ and CD8+ cells in C57BL/6 mice. In MAIDS, the residual population of CD8+ cells is primarily Ly-6C+. The CD4+ cells have a transient increase in ratio of Ly-6C+/Ly-6C- cells at 2 wk postinfection but by 6 wk are primarily Ly-6C-. There was an increase in both the total number and percentage of Mac 1+ cells and a selective depletion of certain splenic B cell subpopulations. Azido dideoxythymidine delays these early population changes.

    View details for Web of Science ID A1990CR42900022

    View details for PubMedID 1968486

  • TOWARD A LAYERED IMMUNE-SYSTEM CELL Herzenberg, L. A., Herzenberg, L. A. 1989; 59 (6): 953-954

    View details for Web of Science ID A1989CF97500002

    View details for PubMedID 2688900



    Although Ly-1 B cells produce autoantibodies and are found at elevated frequencies in certain autoimmune strains, very little is known about the role of these cells, if any, in autoimmune disease. In this publication, we summarize some recent findings relevant to Ly-1 B-cell development, clonal expansion and antibody production. We then consider the idea that Ly-1 B cells constitute the most primitive B-cell lineage in mammals and that the evolutionary niche occupied by these cells requires that they produce a basic set of autoantibodies that cross-react with common bacterial antigens.

    View details for Web of Science ID A1989AE29900024

    View details for PubMedID 2673274



    Studies presented here demonstrate that paternal allotype Ly-1 B cells are permanently depleted following neonatal treatment with antibodies to the paternal IgM allotype. Paternal allotype conventional B cells, in contrast, are temporarily depleted by treatment with either anti-IgM or anti-IgD allotype antibodies and return rapidly to normal frequencies once the antibody treatment disappears. These differences are explained by basic developmental differences between Ly-1 B and conventional lineage B cells. That is, the conventional B cell population is replenished from Ig- precursors throughout life and, therefore, is only temporarily affected when depleted in neonates. The Ly-1 B cell population, in contrast, develops from Ig- progenitors during the prenatal and neonatal life but survives because it is exclusively self-replenishing in adults. Therefore, elimination of a population of Ly-1 B cells from neonates is tantamount to removing it forever. These findings suggest that while conventional B cells turn over rapidly and have an effectively unlimited repertoire, Ly-1 B cells express a repertoire whose composition is strongly influenced by neonatal conditions that favor or select against the retention of cells producing certain antibody molecules. Thus, Ly-1 B cells play a unique role in the immune system in that they retain indefinitely the history of the neonatal animal's immunological experience.

    View details for Web of Science ID A1989U629900013

    View details for PubMedID 2785045



    Using a newly developed FACS method for quantifying the expression of the Escherischia coli lacZ reporter gene in viable mammalian cells, we have obtained cloned cell lines in which the expression of lacZ is under the control of native endogenous transcription elements. We infected the murine pre-B cell 70Z/3 with transcriptionally disabled retroviruses containing lacZ and employed the FACS-FDG technique to detect and sort rare lacZ+ cells in which we expect integration is near such endogenous transcription elements. After two rounds of enrichment we obtained a population of cells that was 80-90% positive for lacZ activity. Clones derived from the lacZ+ pool differ from each other with respect to their overall level of lacZ activity as well as in the pattern of lacZ expression among cells within an individual clone. Treatment of these lacZ+ 70Z/3 clones with lipopolysaccharide (LPS; which is known to stimulate differentiation of 70Z/3 from a pre-B cell to an IgM-expressing B cell) greatly decreased lacZ expression in one clone, 7e17. lacZ expression in this clone was 50-100 times lower within 24 hr of LPS addition and coincided with the acquisition of IgM kappa on the surface of 7e17. This suggests that a transcriptionally active domain of chromatin that harbors the lacZ construct is down-regulated during the transition induced by LPS stimulation.

    View details for Web of Science ID A1989AR92700015

    View details for PubMedID 2807403


    View details for Web of Science ID A1989JX74600019

    View details for PubMedID 2518010


    View details for Web of Science ID A1989JX74500025

    View details for PubMedID 2517920

  • Many of the IgA producing plasma cells in murine gut are derived from self-replenishing precursors in the peritoneal cavity INTERNATIONAL IMMUNOLOGY Kroese, F. G., Butcher, E. C., Stall, A. M., Lalor, P. A., Adams, S., Herzenberg, L. A. 1989; 1 (1): 75-84


    Long term B lineage chimeras are used here to study the origin of plasma cells in the mouse. Chimeric mice are constructed by reconstituting lethally irradiated mice with peritoneal cells (PerC) and bone marrow cells from congenic pairs of mice differing in Igh-C allotype. All conventional B cells in these mice express the allotype of the bone marrow donor and nearly all Ly-1 B lineage cells express the allotype of the PerC donor. FACS analysis and immunohistology of these mice shows that virtually all (sig+) B cells in peripheral lymphoid organs are derived from the bone marrow donor. However, despite this overwhelming number of bone marrow-derived B cells in these animals, immunohistological staining of lymphoid organs and gut shows that nearly half of the IgM, IgG, and IgA plasma cells derive from the PerC donor. These data demonstrate that the peritoneal cavity contains a major reservoir of self-replenishing cells that play a significant role in the mucosal immune response. The possibility that these are B cells that belong to the Ly-1 B lineage is discussed.

    View details for DOI 10.1093/intimm/1.1.75

    View details for Web of Science ID 000208919900010



    Studies presented examine the origin of IgA plasma cells in B lineage chimeric mice constructed by reconstituting lethally irradiated mice with a mixture of syngeneic bone marrow cells and peritoneal cells from Ig heavy chain allotype congenic donors. In these mice, essentially all B cells in spleen and Peyer's patches are derived from the bone marrow donor; however Ly-1 B lineage cells which have been mainly detected in the peritoneum are derived from the peritoneal cell donor. Surprisingly, roughly half of the IgA plasma cells in the lamina propria of the gut are also derived from the peritoneal cell donor, suggesting an important role for peritoneally-derived B cells in the mucosal immune response.

    View details for Web of Science ID A1989U349300008

    View details for PubMedID 2786500


    View details for Web of Science ID A1989AC35900017

    View details for PubMedID 2474188



    The usually small Ly-1 B cell population is markedly increased in older mice by expansion of certain clones. This results in a cellular picture very similar to human B chronic lymphocytic leukemia. Here we report a molecular analysis of the immunoglobulin gene rearrangements of the Ly-1 B cell populations in (NZB x NZW)F1 females. We find that (i) the number of clones found in the peritoneum (a major tissue source of Ly-1 B cells) decreases with age till mono- or biclonality is common by approximately 6 months, (ii) many clones from different mice show the same size rearrangements at both the Ig heavy and light chain loci and (iii) the IgH rearrangements found in a clone isolated from the spleen of one mouse are a subset of those found in the peritoneum of the same mouse, implying migration occurs from the peritoneum to the spleen. Molecular cloning and sequencing of the IgH rearrangements from the peritoneal clones of one B/W mouse revealed that all productive rearrangements used the identical unmutated VH and D elements joined to different JHS. Indeed, two VDJH4 rearrangements were recovered which were identical but for six junctional (N region) nucleotides. The conservation of VH and D segment usage in the rearrangements of these Ly-1 B cell clones could indicate some strong selective pressure for clonal expansion (for example antigen selection) operates via the immunoglobulin molecules of these cells. Southern analyses of other (NZB x NZW)F1 mice with this cloned VH and the usage of the same or similar VH genes among a number of Ly-1 B origin tumors in other mouse strains indicate the generality of this repetitive VH gene usage in individual mice.

    View details for Web of Science ID A1988Q990600010

    View details for PubMedID 2463165



    Using a new Ly-6C-specific antibody (Monts-1) we show that this class of antigens are differentially expressed on monocytes/macrophages and endothelial cells. Recently elicited peritoneal exudate Mac-1+ mononuclear cells, as well as Mac-1+ mononuclear cells in the bone marrow and in the peripheral blood, express high levels of Ly-6C. Ly-6C+ mononuclear Mac-1+ cells are absent in normal uninflamed skin, but are present in high numbers in skin lesions 3 days after the s.c. injection of lipopolysaccharide, concanavalin A or complete Freund's adjuvant. In addition, large Ly-6C+ mononuclear cells are predominant in chronic granulomas induced by complete Freund's adjuvant. Resident macrophages in a variety of tissues express low levels or in many cases do not express Ly-6C. Two out of three monocyte-like cell lines are Ly-6C+, whereas macrophage-like cell lines are negative. Ly-6C+ monocytes/macrophages lose the Ly-6C antigen within 24 h after in vitro culture. Ly-6C- cultured monocytes and Ly-6C- monocyte-like cell lines, but not fully differentiated macrophages and macrophage-like cell lines, can be induced to express the Ly-6C antigen by interferon-gamma. A population of small vessel endothelial cells in diverse tissues also express high levels of Ly-6C. The present findings suggest that the Ly-6C antigen family, shown by others to be involved in T cell activation, may have more general importance in immune responses and cellular differentiation than previously appreciated.

    View details for Web of Science ID A1988R780600024

    View details for PubMedID 2849552



    Sperm and trophoblast are among the few nucleated human cells that do not express HLA class I antigens. DNA methylation, which is proposed to be a tight mechanism of regulation, may be necessary to turn off these genes. We have investigated the transfectability of HLA class I genes and of the genes for the T-cell differentiation antigens Leu-1 (CD5) and Leu-2 (CD8) in mouse L cells by using human sperm cells and choriocarcinoma cell lines, tumors of trophoblastic origin, as sources of DNA. It was found that DNA from one choriocarcinoma line (JAR) does not transfect genes for HLA, Leu-1, or Leu-2 and that DNA from two other choriocarcinoma lines (BeWo and Ima) transfects only some of the surface markers. Sperm DNA transfects genes for all the surface antigens tested except Leu-1. DNA from control cells and from the line SCH transfects all the markers studied. Southern blots show that all cell types contain apparently intact genes encoding HLA, Leu-1, and Leu-2 and reveal differences in the DNA methylation patterns of genes from different sources of DNA. We treated JAR (the cell line with the lowest transfecting ability) with 5-azacytidine and obtained demethylation of its DNA. This demethylated DNA transfects genes for both HLA class I antigens and Leu-2. Further culture of JAR cells in the absence of 5-azacytidine results in remethylation of their DNA and decreased ability to transfect these surface antigens. These findings indicate that DNA methylation affects the efficiency of transfection of surface antigen genes in L cells.

    View details for Web of Science ID A1988Q990500005

    View details for PubMedID 3054885



    Studies presented here demonstrate that individually expanded clones of murine Ly-1 B cells, perhaps analogous to the expanded neoplastic Leu-1 B-cell clones in human chronic lymphocytic leukemias, are universally detectable in young New Zealand Black (NZB)-related autoimmune mice and in senescent normal mice (greater than 18 months old). These clones are visible as phenotypically homogeneous cell populations in multiparameter fluorescence-activated cell sorter analyses of peritoneal and splenic B cells; they show unique immunoglobulin heavy- and light-chain gene rearrangements in Southern gel analyses of peritoneal and splenic DNA; and, like the self-replenishing Ly-1 B-cell population from which they are drawn, they tend to grow readily in irradiated or unirradiated syngeneic or allotype congenic hosts. Furthermore, they develop and generalize in primary and secondary hosts in a characteristic pattern (peritoneum much greater than spleen greater than lymph node greater than bone marrow) that suggests that their initial growth is controlled by the mechanisms that normally control Ly-1 B-cell distribution in lymphoid organs. The universal emergence of these clones within the Ly-1 B-cell lineage may be explained by the substantially greater opportunity for hyperplastic and neoplastic transformation events in this long-lived self-replenishing Ly-1 B-cell population, which must divide relatively frequently to maintain its normal size throughout adulthood. Repeated exposure to internal or environmental antigens (with which Ly-1 B cells are known to react) may also play a role in driving the development of these clones.

    View details for Web of Science ID A1988Q358500058

    View details for PubMedID 3262873



    We generated a family of chimeric immunoglobulin G (IgG) molecules having identical antigen-combining sites for the dansyl (DNS) hapten, in conjunction with nine heavy chain constant (CH) regions. This family of antibody molecules allows comparison of CH dependent properties independent of possible variable region contributions to IgG function. The segmental flexibility and complement fixation activity were measured of six genetically engineered molecules (the four human IgG isotypes, mouse IgG3 and rabbit IgG) and the remaining three mouse IgG isotypes, (IgG1, IgG2a and IgG2b), isolated previously by somatic cell genetic techniques. These properties of antibody molecules each correlate with the length of the immunoglobulin hinge region which separate the first and second CH (CH1 and CH2) domains. These results attribute a structural basis for two critical properties of antibody molecules.

    View details for Web of Science ID A1988P074100009

    View details for PubMedID 3138110



    Transgenic mice carrying immunoglobulin genes coding for mu heavy chain and kappa light chain have been used to study the mechanisms involved in allelic and isotypic exclusion. We report here that individual cells from transgenic mice carrying a functionally rearranged mu heavy chain gene (capable of generating both membrane and secreted forms of IgM) can rearrange an endogenous mu heavy chain gene and simultaneously produce both transgenic and endogenous IgM. These "double-producing" cells express both endogenous and transgenic IgM in the cytoplasm (detected by immunohistology) and on the cell surface (detected by multiparameter fluorescence-activated cell sorter analysis). In addition, they secrete mixed IgM molecules containing both transgenic and endogenous mu heavy chains (detected in serum by radioimmune assay). The transgenic mice studied also have relatively large numbers of cells that produce endogenous immunoglobulin in the absence of detectable transgenic immunoglobulin ("endogenous-only cells"). The mechanisms that generate double-producing cells and endogenous-only cells appear to be under genetic control because the frequencies of these B-cell populations are characteristic for a given transgenic line. Thus, our findings indicate that more is involved in triggering allelic exclusion than the simple presence or absence of membrane mu heavy chains (as has been previously postulated).

    View details for Web of Science ID A1988N475300062

    View details for PubMedID 3130629



    We demonstrate that individual cells infected with and expressing a recombinant retrovirus carrying the Escherichia coli beta-galactosidase gene (lacZ) can be viably stained, analyzed, sorted, and cloned by fluorescence-activated cell sorting based on the levels of lacZ expressed. To accomplish this we have devised a method to enzymatically generate and maintain fluorescence in live mammalian cells. Accumulation of fluorescent products in cells is linear with time, with a direct correlation of fluorescence to enzymatic activity. This technology for beta-galactosidase detection is more sensitive than other available cytochemical or biochemical methods. We have used this procedure to show that the expression of psi-2-MMuLVSVnlsLacZ in the T-cell lymphoma BW5147 and the B-cell hybridoma SP2/0 is not completely stable and that subclones selected by the fluorescence-activated cell sorter for low lacZ activity demonstrate distinctly lower average expression of LacZ. These findings indicate the utility of beta-galactosidase as a reporter molecule at the single-cell level for studies of gene regulation, including studies of promoter efficacy, enhancer activity, trans-acting factors, and other regulatory elements.

    View details for Web of Science ID A1988N023400040

    View details for PubMedID 3128790



    The work conducted so far in this laboratory has demonstrated the application of the use of genes encoding lymphocyte differentiation molecules, in the isolation of homologous genes from other mammalian species, by the technique of cross-species DNA hybridization. The studies have also highlighted the use of transfection as a means of obtaining expression of genes, either from total genomic DNA or cloned in plasmids, which encode lymphocyte antigens. Preliminary work presented in this paper demonstrates the application of these technologies in the isolation and expression of genes for lymphocyte antigens from species in which the gene products have not been fully defined. We favour this approach because it may allow isolation and definition of important immunological molecules independently of the existence of specific antibodies. It therefore seems the most direct way to avoid the frustrating randomness in production of anti lymphocyte subset-specific monoclonal antibodies, and to shorten the time and effort needed to define the specificities of such reagents. Furthermore, the cDNA clones isolated from alternate species (in this case the bovine) have a use in classical immunological studies apart from the application of antibodies made to their products in veterinary immunology. That is, comparisons of the DNA sequences of lymphocyte differentiation antigens from different species provide much important information about structural or functional elements of evolutionarily conserved proteins involved in generation of immune responses.

    View details for Web of Science ID A1987L817900028

    View details for PubMedID 3324465



    Experimental allergic encephalomyelitis (EAE) is an autoimmune disease mediated by CD4+ T cells. Prior studies have established that monoclonal anti-CD4 antibodies can reverse EAE. To determine whether immunoglobulin isotype plays a role in the therapy of EAE with anti-CD4 antibody, an isotype switch variant family of the mouse IgG1 anti-rat CD4 antibody W3/25 was isolated with the fluorescence-activated cell sorter. The IgG1, IgG2b, and IgG2a W3/25 isotype variants all had identical binding capacities for rat CD4+ T cells. Although all three W3/25 isotypes showed some beneficial effects in the amelioration of EAE, the IgG1 and IgG2a W3/25 antibodies were superior to the IgG2b W3/25 in the treatment of EAE. Multiparameter fluorescence-activated cell sorter analysis of T cell subpopulations from treated rats showed that none of the antibodies of the W3/25 isotype switch variant family substantially depleted CD4+ target cells in vivo. These experiments demonstrate that immunoglobulin isotype is important in the monoclonal antibody therapy of autoimmune disease. They indicate that therapy of EAE may be successful without a major depletion of CD4+ lymphocytes. Immunotherapy may be optimized by selecting an appropriate isotype of a monoclonal antibody.

    View details for Web of Science ID A1987L166500017

    View details for PubMedID 3500226

  • PRODUCTION OF IMMUNOGLOBULIN ISOTYPES BY LY-1+ B-CELLS IN VIABLE MOTH-EATEN AND NORMAL MICE SCIENCE Sidman, C. L., Shultz, L. D., Hardy, R. R., Hayakawa, K., Herzenberg, L. A. 1986; 232 (4756): 1423-1425


    Almost all B cells in autoimmune mice with the viable motheaten (mev) mutation express the Ly-1 cell surface antigen, which marks a minor population of B cells constituting a separate lineage in normal mice. Immunoglobulins primarily of the M and G3 classes, which in both normal and mev mice contain high levels of lambda light chain, are produced in excess in mev mice. These and other observations suggest that the development of B cells that express Ly-1 is regulated independently from the development of B cells that do not express Ly-1. B cells bearing the Ly-1 surface antigen may play specialized roles in the normal immune system and in autoimmunity by regulating other B cells via lymphokines, by producing antibodies to self and certain foreign antigens, and by preferentially secreting immunoglobulin M and immunoglobulin G3.

    View details for Web of Science ID A1986C602400041

    View details for PubMedID 3487115



    Previous studies demonstrate that Ly-1 B cells and their progenitors are clearly detectable in peritoneum in normal mice. In this publication, we show (a) that peritoneal Ly-1 B cells resemble splenic Ly-1 B cells with respect to surface marker expression and functional activity (autoantibody production); (b) that Ly-1 B frequencies in peritoneum are considerably higher than in spleen; and (c) that genetic mechanisms reduce peritoneal Ly-1 B frequencies to minimal levels in SJL-related mice and to below detectability in CBA/N and other mice with the X-linked immunodeficiency (Xid). In addition, we show that that peritoneal (and perhaps splenic) Ly-1 B populations demonstrate an unique bias in immunoglobulin commitment. That is, they are selectively enriched for cells that express IgM heavy chains in association with lambda light chains. Thus, as a whole, evidence presented here defines the peritoneum as a tightly regulated lymphocyte compartment that normally houses a large population of mature Ly-1 B cells with distinctive functional properties.

    View details for Web of Science ID A1986C242800022

    View details for PubMedID 3084283



    We investigated whether spontaneous isotype switching in monoclonal antibody-producing hybridomas always occurs with genes on the same chromosome. Spleen cells of (BAB/ 25 X AKR/J) F1 mice, immunized with dansyl-keyhole limpet hemocyanin (DNS-KLH), were hybridized with NS-1 to generate hybridomas producing monoclonal anti-DNS antibodies of either the b or d haplotype of the BAB/25 or AKR/J parent, respectively. We selected isotype switch variants of such hybridomas using the fluorescence-activated cell sorter (FACS). Although in most cases the allotypic haplotype expressed by the parent and switch-variant hybridomas are the same, in one family of variants we noted a switch in haplotype along with the switch in isotype. This was noted in the selection of IgG2a switch variants from an IgG1 switch variant originally derived from an IgG3-producing parent. Biochemical and molecular studies confirm that the allotype switch variant expresses the same heavy-chain variable region gene complex as its parent hybridomas. As such, the allotype switch represents an example of spontaneous mitotic recombination between immunoglobulin heavy-chain genes, generating a single actively transcribed gene from loci previously positioned on different chromosomes.

    View details for Web of Science ID A1986A347600008

    View details for PubMedID 3519208



    Data from previous multiparameter fluorescence-activated cell sorter (FACS) analysis and sorting studies define a subset of murine B cells that expresses the Ly-1 surface determinant in conjunction with IgM, IgD, Ia, and other typical B cell markers. These Ly-1 B cells are physically and functionally distinct. They express more IgM and less IgD than most other B cells; they are not normally found in lymph node or bone marrow; they are always present at low frequencies (1-5%) in normal spleens, and, as we show here, they comprise about half of the B cells (10-20% of total cells) recovered from the peritoneal cavity in normal mice. Furthermore, most of the commonly studied IgM autoantibodies in normal and autoimmune mice are produced by these Ly-1 B cells, even though they seldom produce antibodies to exogenous antigens such as trinitrophenyl-Ficoll or trinitrophenyl-keyhole limpet hemocyanin. Cell transfer studies presented here demonstrate that the progenitors of Ly-1 B cells are different from the progenitors of the predominant B cell populations in spleen and lymph node. In these studies, we used FACS analysis and functional assays to characterize donor-derived (allotype-marked) B cells present in lethally irradiated recipients 1-2 mo after transfer. Surprisingly, adult bone marrow cells typically used to reconstitute B cells in irradiated recipients selectively failed to reconstitute the Ly-1 B subset. Liver, spleen, and bone marrow cells from young mice, in contrast, reconstituted all B cells (including Ly-1 B), and peritoneal "washout" cells (PerC) from adult mice uniquely reconstituted Ly-1 B. Bone marrow did not block Ly-1 B development, since PerC and newborn liver still gave rise to Ly-1 B when jointly transferred with marrow. These findings tentatively assign Ly-1 B to a distinct developmental lineage originating from progenitors that inhabit the same locations as other B cell progenitors in young animals, but move to unique location(s) in adults.

    View details for Web of Science ID A1985AKB7800021

    View details for PubMedID 3874257

  • LY-1 AS A DIFFERENTIATION ANTIGEN ON NORMAL AND NEOPLASTIC-B CELLS CURRENT TOPICS IN MICROBIOLOGY AND IMMUNOLOGY Hardy, R. R., Hayakawa, K., Herzenberg, L. A., Morse, H. C., Davidson, W. F., Herzenberg, L. A. 1984; 113: 231-236

    View details for Web of Science ID A1984TH34500039

    View details for PubMedID 6332718



    Studies presented here introduce another perspective on the mechanisms responsible for IgM autoantibody production. A unique subpopulation of B lymphocytes (Ly-1 B) that concomitantly expresses IgM, IgD, Ia, and Ly-1 membrane glycoproteins is present at higher frequencies in NZB and NZB-related mice. The Ly-1 B subpopulation in these autoimmune animals is responsible for the "spontaneous" IgM secretion demonstrated with cultured NZB spleen cells and contains the cells that secrete typical NZB IgM autoantibodies to single-stranded DNA and to thymocytes. In addition, the Ly-1 B population in normal mouse strains (and in NZB) contains virtually all of the spleen cells that secrete IgM autoantibodies reactive with bromelain-treated mouse erythrocytes. Since a different B-cell subpopulation (IgM+, IgD-, Ly-1) secretes most of the IgM antibodies produced in responses to exogenous antigens, we conclude that Ly-1 B cells constitute a functionally distinct B-cell population important in certain kinds of autoimmunity.

    View details for Web of Science ID A1984SR28800048

    View details for PubMedID 6609363


    View details for Web of Science ID A1982NX16800001

    View details for PubMedID 6811662


    View details for Web of Science ID A1982PY68800009

    View details for PubMedID 6984600

  • B-CELL SUB-POPULATIONS IDENTIFIED BY 2-COLOR FLUORESCENCE ANALYSIS NATURE Hardy, R. R., Hayakawa, K., Haaijman, J., Herzenberg, L. A. 1982; 297 (5867): 589-591

    View details for Web of Science ID A1982NT76200041

    View details for PubMedID 7045679



    We have used highly specific, directly fluorescein-conjugated heterologous (conventional) and monoclonal antibodies directed against mouse immunoglobulin isotypes in conjunction with the fluorescence activated cell sorter (FACS) to enrich and clone hybridoma cells producing new immunoglobulin heavy chain constant regions. Each variant retains the parental heavy chain variable region and the parental immunoglobulin light chain; thereby each variant binds the same dansyl (DNS) hapten. These isotype switch variants occur at frequencies of approximately 10-5 to 10-6. We were able to isolate the variants by first sorting for an approximate 1000-fold enrichment of the desired immunoglobulin-producing cells, growing these cells for five to nine days, followed by a second 1000-fold enrichment and direct cell cloning into 96 well culture trays. Clones were screened only 3-5 weeks after the original selection for secretion of dansyl-binding immunoglobulin of the selected isotype. Judicious combination of existing methods permits improved analytical techniques using the cell sorter. These include: first, "red" fluorescence staining of dead cells with ethidium bromide or propidium iodide and using the red fluorescence measurement to exclude dead cells from the green fluorescence selection; and second, the use logarithmic amplification of fluorescence signals, allowing for more succinct selection of fluorescence parameters for sorting.

    View details for Web of Science ID A1982NQ30900006

    View details for PubMedID 6804196

  • Immunoglobulin isoantigens (allotypes) in the mouse : V. Characterization of IgM allotypes. Immunogenetics Black, S. J., Goding, J. W., GUTMAN, G. A., Herzenberg, L. A., Loken, M. R., Osborne, B. A., van der Loo, W., Warner, N. L. 1978; 7 (1): 213-230


    Murine antisera raised against allogeneic lymphoid cells often contain antibodies to IgM allotypes. Rarely, allotypic antibodies to IgM have been found after immunization withB. pertussis anti-B. pertussis conjugates. Using both types of antibodies, we have defined a new constant-region locus for both secreted and membrane-bound ? chains. This locus,Ig-6, is closely linked to the previously described H-chain constant-region loci (Ig-1 throughIg-5) and is subject to allelic exclusion. We have identified three alleles and four antigenic specificities ofIg-6.

    View details for DOI 10.1007/BF01844009

    View details for PubMedID 21302076


    View details for Web of Science ID A19639818B00032

    View details for PubMedID 16591071

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