A.B., Mount Holyoke College, Biology (1974)
Ph.D., Yale University, Cell Biology (1979)
T lymphocytes play a central role in the adaptive immune response. Understanding the biology of the response of T cells to immune challenge shoudl allow the development of new diagnostics and therapeutics to treat human disease. Research in my group is focuses on three areas:
1. Granulysin, a small molecule expressed by activated T cells and NK cells, lyses tumors and a variety of pathogens, including bacteria, fungi, and parasites. We have shown that synthetic peptides corresponding to linear regions of granulysin can recapitulate the lytic activity of the intact molecule. Substitution of critical residues resulted in mutant peptides that lyse pathogens but do not kill mammalian cells. We are currently developing granulysin derivatives as a new type of antibiotic as well as studying the mechanisms involved in granulysin induced cell death.
2. Tuberculosis kills more than 3,000,000 people each year. However, little is understood about the immune response to Mycobacterium tuberculosis, the bacterium that causes the disease. We are characterizing the immune response to individual antigens from Mycobacterium tuberculosis in lymphocytes from individuals with disease or who have been infected with Mycobacterium tuberculosis but do not develop tuberculosis. Correlates of protective immunity are critical to the development of an improved vaccine for tuberculosis.
3. Lymphotactin is a chemokine that is expressed in both activated and anergic T cells. Lymphocytes treated with lymphotactin are refractory to stimulation, suggesting that lymphotactin may be used to induce tolerance in vivo. Studies are ongoing to define the role of lymphotactin in the immune response in vitro and in vivo and to use this information to develop new tolerance inducing modalities.
Vaccination with Mycobacterium bovis bacille Calmette-Guérin (BCG) has variable efficacy in preventing tuberculosis. We hypothesized that some of this variation might be due to differences among BCG strains. To test this, neonates in Orizaba, Mexico, were vaccinated with one of three different BCG strains (BCG-Brazil [BBCG], BCG-Denmark [DBCG], or BCG-Japan [JBCG]). One year after vaccination, peripheral blood mononuclear cells (PBMC) were obtained and recall immune responses to culture filtrate proteins (CFP) of Mycobacterium tuberculosis were evaluated using quantitative real-time PCR. CFP-activated PBMC from BBCG- and DBCG-immunized children expressed high levels of cytokines characteristic of an adaptive immune response (gamma interferon, interleukin-2beta [IL-12beta], and IL-27), while those from children immunized with JBCG did not. In contrast, vaccination with JBCG resulted in significantly greater expression of cytokines characteristic of a proinflammatory immune response (IL-1alpha, IL-1beta, IL-6, and IL-24) in PBMC activated with CFP compared to PBMC from children vaccinated with BBCG or DBCG. Thus, different strains of BCG can activate different immune pathways, which may affect long-term vaccine efficacy.
View details for DOI 10.1128/IAI.00244-07
View details for Web of Science ID 000247707600048
View details for PubMedID 17502394
View details for PubMedCentralID PMC1932948
Activation of resting T lymphocytes initiates differentiation into mature effector cells over 3-7 days. The chemokine CCL5 (RANTES) and its major transcriptional regulator, Krüppel-like factor 13 (KLF13), are expressed late (3-5 days) after activation in T lymphocytes. Using yeast two-hybrid screening of a human thymus cDNA library, PRP4, a serine/threonine protein kinase, was identified as a KLF13-binding protein. Specific interaction of KLF13 and PRP4 was confirmed by reciprocal coimmunoprecipitation. PRP4 is expressed in PHA-stimulated human T lymphocytes from days 1 and 7 with a peak at day 3. Using an in vitro kinase assay, it was found that PRP4 phosphorylates KLF13. Furthermore, although phosphorylation of KLF13 by PRP4 results in lower binding affinity to the A/B site of the CCL5 promoter, coexpression of PRP4 and KLF13 increases nuclear localization of KLF13 and CCL5 transcription. Finally, knock-down of PRP4 by small interfering RNA markedly decreases CCL5 expression in T lymphocytes. Thus, PRP4-mediated phosphorylation of KLF13 plays a role in the regulation of CCL5 expression in T lymphocytes.
View details for Web of Science ID 000246896300049
View details for PubMedID 17513757
Differentiation of active from latent tuberculosis (TB) is a major challenge in the control of TB. In this study, PBMC from latent TB-infected subjects, TB patients, and tuberculin skin test-negative donors stimulated with the Mycobacterium tuberculosis (Mtb)-specific Ag, early secretory antigenic target 6, and mRNA for 45 immune-related genes was measured by quantitative real-time PCR. Univariate analysis showed significant differences in the expression of 10 genes (IFN-gamma, FOXP3, IL-1alpha, IL-1beta, IL-2, IL-6, IL-8, IL-12alpha, IL-12beta, and IL-24) in PBMC from TB patients vs latent TB-infected subjects (p < 0.01). Multivariate logistic regression and classification and regression tree analyses revealed that expression of three genes, IL-8, FOXP3, and IL-12beta, is predictive for TB vs latent Mtb infection. Thus, measurement of Ag-specific expression of these three genes may offer a specific and noninvasive means of differentiating between latent Mtb infection and TB.
View details for Web of Science ID 000244942400041
View details for PubMedID 17339466
View details for PubMedCentralID PMC2679258
The chemokine RANTES (regulated upon activation normal T cell expressed and secreted) is expressed "late" (3 to 5 days) after activation in T lymphocytes. In order to understand the molecular events that accompany changes in gene expression, a detailed analysis of the interplay between transcriptional machinery and chromatin on the RANTES promoter over time was undertaken. Krüppel-like factor 13 (KLF13), a sequence-specific DNA binding transcription factor, orchestrates the induction of RANTES expression in T lymphocytes by ordered recruitment of effector molecules, including Nemo-like kinase, p300/cyclic AMP response element binding protein (CBP), p300/CBP-associated factor, and Brahma-related gene 1, that initiate sequential changes in phosphorylation and acetylation of histones and ATP-dependent chromatin remodeling near the TATA box of the RANTES promoter. These events recruit RNA polymerase II to the RANTES promoter and are responsible for late expression of RANTES in T lymphocytes. Therefore, KLF13 is a key regulator of late RANTES expression in T lymphocytes.
View details for DOI 10.1128/MCB.01071-06
View details for Web of Science ID 000243136800021
View details for PubMedID 17074812
Granulysin (GNLY) is a cytolytic molecule expressed by human CTL and NK cells with activity against a variety of tumors and microbes, including Mycobacterium tuberculosis. Although the molecular mechanism of GNLY-induced apoptosis of Jurkat T cells is well defined in vitro, no direct evidence for its in vivo effects has been demonstrated. Because there is no murine homologue of GNLY, we generated mice expressing GNLY using a bacterial artificial chromosome containing the human GNLY gene and its 5' and 3' flanking regions. GNLY is expressed in leukocytes from transgenic mice with similar kinetics as in PBMC from humans: GNLY is constitutively expressed in NK cells and, following stimulation through the TCR, appears in T lymphocytes 8-10 days after activation. Both forms of GNLY (9 and 15 kDa) are produced by activated T cells, whereas the 15-kDa form predominates in freshly isolated NK cells from transgenic animals. GNLY mRNA is highest in spleen, with detectable expression in thymus and lungs, and minimal expression in heart, kidney, liver, muscle, intestine, and brain. Allospecific cell lines generated from GNLY transgenic animals showed enhanced killing of target cells. In vivo effects of GNLY were evaluated using the syngeneic T lymphoma tumor C6VL. GNLY transgenic mice survived significantly longer than nontransgenic littermates in response to a lethal tumor challenge. These findings demonstrate for the first time an in vivo effect of GNLY and suggest that GNLY may prove a useful therapeutic modality for the treatment of cancer.
View details for Web of Science ID 000243120900009
View details for PubMedID 17182542
Granulysin is a novel cationic molecule present in the granules of cytotoxic T lymphocytes and natural killer cells. Cytotoxic T lymphocytes have long been associated with graft destruction in transplant rejection. Recent studies implicate granulysin in cell-mediated cytotoxicity, chemoattraction, immune activation and as a potential diagnostic biomarker for transplant rejection.
View details for DOI 10.1111/j.1600-6143.2005.00970.x
View details for Web of Science ID 000230291500003
View details for PubMedID 15996224
Chemokines are a family of small, secreted chemoattractant cytokines that regulate distribution and function of leukocytes during immune responses. While most chemokines are members of the CC or CXC subgroups, XCL1, also known as lymphotactin, is the sole member of the C subgroup. XCL1 is produced by activated CD8(+) T cells, NK cells, gammadelta T cells, and mast cells. XCL1 differs from other chemokines in that it contains only a single disulfide bond and a mucin-like domain at its carboxy terminus that is glycosylated. Understanding the biologic functions of chemokines has largely depended upon expression of these recombinant molecules in E. coli. To examine the effects of glycosylation on the biologic activity of XCL1, we designed constructs for expression of human XCL1 in insect S2 cells. Comparison of this material with that expressed in E. coli reveals that glycosylation significantly increases the biologic activity of XCL1.
View details for DOI 10.1016/j.jim.2005.05.008
View details for Web of Science ID 000231490600013
View details for PubMedID 15992811
Granulysin, a cationic protein produced by activated human CTL and NK cells, is cytolytic against microbial and tumor targets. In this study we show that granulysin also functions as a chemoattractant and activates monocytes to produce cytokines/chemokines. Although granulysin-mediated cytotoxicity occurs at micromolar concentrations, chemoattraction occurs in the nanomolar range, and immune activation occurs over a wide range of concentrations (nanomolar to micromolar). Granulysin causes a 2- to 7-fold increase in chemotaxis of monocytes, CD4(+), and CD8(+) memory (CD45RO) but not naive (CD45RA) T cells, NK cells, and mature, but not immature, monocyte-derived dendritic cells. Pertussis toxin treatment abrogates chemoattraction by granulysin, indicating involvement of G-protein-coupled receptor(s). At low concentrations (10 nM), granulysin promotes a 3- to 10-fold increase in MCP-1 and RANTES produced by monocytes and U937 cells, while a 2-fold increase in TNF-alpha production by LPS-stimulated monocytes requires higher concentrations of granulysin (micromolar). Taken together, these data indicate that the local concentration of granulysin is critical for the biologic activity, with high concentrations resulting in cytotoxicity while lower concentrations, presumably further from the site of granulysin release, actively recruit immune cells to sites of inflammation.
View details for Web of Science ID 000228630800011
View details for PubMedID 15843520
Delivery of biologically active peptides into cells may help elucidate intracellular signal transduction pathways, identify additional in vivo functions, and develop new therapeutics. Although p21 was first identified as a major regulator of cell cycle progression, it is now clear that p21 subserves multiple functions. The amino terminus of p21 interacts with cyclins and cyclin-dependent kinases, while the carboxyl terminus interacts with proliferating cell nuclear antigen (PCNA), growth arrest and DNA damage-inducible gene 45 (GADD45), calmodulin, SET, and CCAAT/enhancer binding protein-alpha (C/EBP-alpha). A chimeric peptide, p21-IRS, consisting of the carboxyl terminal domain of p21 conjugated to a pentapeptide (RYIRS) rapidly enters lymphoid cells and activates apoptosis. In the present study, we investigate the molecular events involved in p21-activated apoptosis. Comparison of p21-IRS with other known proapoptotic agents demonstrates that p21-IRS activates a novel apoptotic pathway: mitochondria are central to the process, but caspases and a decrease in Deltapsi(m) are not involved. Targeting the p21 peptide to specific cell populations may allow development of novel therapies to eliminate aberrant cells in human diseases.
View details for DOI 10.1182/blood-2004-06-2188
View details for Web of Science ID 000226596700048
View details for PubMedID 15466931
Granulysin, a 9-kDa protein localized in human cytolytic T lymphocytes and natural killer cell granules, is cytolytic against tumors and microbes but not against red blood cells. Synthetic peptides corresponding to the central region of granulysin recapitulate the lytic activity of the intact molecule, and some peptides cause hemolysis of red blood cells. Peptides in which cysteine residues were replaced by serine maintain their activity against microbes but lose activity against human cells, suggesting their potential as antibiotics. Studies were undertaken to determine the mechanism of resistance of red blood cells to granulysin and sensitivity to a subset of granulysin-derived peptides. Granulysin lyses immature reticulocytes, which have mitochondria, but not red blood cells. Granulysin lyses U937 cells but not U937 cells lacking mitochondrial DNA and a functional respiratory chain (U937rho(o) degrees cells), further demonstrating the requirement of intact mitochondria for granulysin-mediated death. Peptide G8, which corresponds to helix 2/loop 2/helix 3, lyses red blood cells, while peptide G9, which is identical except that the cysteine residues were replaced by serine, does not lyse red blood cells. Granulysin peptide-induced hemolysis is markedly inhibited by an anion transporter inhibitor and by Na(+), K(+), and Ca(2+) channel blockers but not by Na(+)/K(+) pump, cotransport, or Cl(-) channel blockers. Although recombinant granulysin and G9 peptide do not induce hemolysis, they both competitively inhibit G8-induced hemolysis. The finding that some derivatives of granulysin are hemolytic may have important implications for the design of granulysin-based antimicrobial therapeutics.
View details for DOI 10.1128/AAC.49.1.388-397.2005
View details for Web of Science ID 000226035300050
View details for PubMedID 15616319
DQ 65-79, a peptide derived from residues 65-79 of the alpha-chain HLA class II molecule DQA03011, blocks T cell proliferation and induces T cell apoptosis. Using a yeast two-hybrid assay, we previously identified proliferating cell nuclear Ag (PCNA) as an intracellular ligand for DQ 65-79. In this study, we show that three regions of PCNA, residues 81-100, 121-140, and 241-261, interact with DQ 65-79. Residues 241-261 of PCNA also interact with the C terminus (residues 139-160) of the cell cycle regulator, p21, suggesting that DQ 65-79 and p21 might function similarly. We show here that DQ 65-79 competitively inhibits binding of p21 to PCNA and that both DQ 65-79 and p21 139-160 induce T cell apoptosis, suggesting that DQ 65-79 and p21 act similarly to inhibit cell growth.
View details for Web of Science ID 000186643300017
View details for PubMedID 14607903
Granulysin, a molecule expressed by human natural killer cells and activated T lymphocytes, exhibits cytolytic activity against a variety of microbes and tumors. Progress in understanding the structure, function and clinical relevance of granulysin over the past year encompasses three main areas: first, the solution of its crystal structure, providing new insights into its potential mechanism of target cell damage; second, inhibition of its function with small interfering RNA, indicating its relevance in microbial immunity; and third, association of granulysin expression in natural killer cells with good outcomes in cancer, indicating its potential utility as a diagnostic and suggesting its relevance to human disease.
View details for DOI 10.1016/S0952-7915(03)00097-9
View details for Web of Science ID 000185731800014
View details for PubMedID 14499265
Granulysin, a molecule present in the granules of CTL and NK cells, is cytolytic against microbes and tumors. Granulysin induces apoptosis of mammalian cells by damaging mitochondria and causing the release of cytochrome c and apoptosis-inducing factor, resulting in DNA fragmentation. Here we show that Ca2+ and K+ channels as well as reactive oxygen species are involved in granulysin-mediated Jurkat cell death. The Ca2+ channel blockers, nickel and econazole, and the K+ channel blockers, tetraethylammonium chloride, apamin, and charybdotoxin, inhibit the granulysin-induced increase in intracellular Ca2+ ([Ca2+](i)), the decrease in intracellular K+, and apoptosis. Thapsigargin, which releases Ca2+ from the endoplasmic reticulum, prevents a subsequent granulysin-induced increase in [Ca2+](i) in Jurkat cells, indicating that the initial increase in [Ca2+](i) is from intracellular stores. The rise in [Ca2+](i) precedes a decrease in intracellular K+, and elevated extracellular K+ prevents granulysin-mediated cell death. In granulysin-treated cells, electron transport is uncoupled, and reactive oxygen species are generated. Finally, an increase in intracellular glutathione protects target cells from granulysin-induced lysis, indicating the importance of the redox state in granulysin-mediated cell death.
View details for Web of Science ID 000184970900047
View details for PubMedID 12928406
In the present study, we have characterized the Xenopus Akt expressed in oocytes from the African clawed frog Xenopus laevis and tested whether its activity is required for the insulin- and progesterone-stimulated resumption of meiosis. A cDNA encoding the Xenopus Akt was isolated and sequenced, and its expression in the Xenopus oocyte was confirmed by reverse transcription PCR and Northern blotting. Using phosphospecific antibodies and enzyme assays, a large and rapid activation of the Xenopus Akt was observed upon insulin stimulation of the oocytes. In contrast, progesterone caused a modest activation of this kinase with a slower time course. To test whether the activation of Akt was required in the stimulation of the resumption of meiosis, we have utilized two independent approaches: a functional dominant negative Akt mutant and an inhibitory monoclonal antibody. Both the mutant Akt, as well as the inhibitory monoclonal antibody, completely blocked the insulin-stimulated resumption of meiosis. In contrast, both treatments only partially inhibited (by approx. 30%) the progesterone-stimulated resumption of meiosis when submaximal doses of this hormone were utilized. These data demonstrate a crucial role for Akt in the insulin-stimulated cell cycle progression of Xenopus oocytes, whereas Akt may have an ancillary function in progesterone signalling.
View details for Web of Science ID 000180715900003
View details for PubMedID 12374568
RFLAT-1/KLF13, a member of the Krüppel-like family of transcription factors, was identified as a transcription factor expressed 3-5 days after T lymphocyte activation. It binds to the promoter of the chemokine gene RANTES (regulated on activation normal T cell expressed and secreted) and regulates its "late" expression in activated T-cells. In this study, a series of experiments to define the functional domains of RFLAT-1/KLF13 were undertaken to further advance the understanding of the molecular mechanisms underlying transcriptional regulation by this factor. Using the GAL4 fusion system, distinct transcriptional activation and repression domains were identified. The RFLAT-1 minimum activation domain is localized to amino acids 1-35, whereas the repression domain resides in amino acids 67-168. Deletion analysis on the RFLAT-1 protein further supports these domain functions. The RFLAT-1 activation domain is similar to that of its closest family member, basic transcription element-binding protein 1. This domain is highly hydrophobic, and site-directed mutagenesis demonstrated that both negatively charged and hydrophobic residues are important for transactivation. The nuclear localization signal of RFLAT-1 was also identified using the RFLAT-1/green fluorescence protein fusion approach. RFLAT-1 contains two potent, independent nuclear localization signals; one is immediately upstream of the zinc finger DNA-binding domain, and the other is within the zinc fingers. Using mutational analysis, we also determined that the critical binding sequence of RFLAT-1 is CTCCC. The intact CTCCC box on the RANTES promoter is necessary for RFLAT-1-mediated RANTES transcription and is also required for the synergy between RFLAT-1 and NF-kappaB proteins.
View details for DOI 10.1074/jbc.M204278200
View details for Web of Science ID 000177509300088
View details for PubMedID 12050170
Activation of T lymphocytes by specific antigen triggers a 3- to 7-day maturation process. Terminal differentiation begins late after T cell activation and involves expression of effector genes, including the chemokine RANTES and its major transcriptional regulator, RANTES factor of late-activated T lymphocytes-1 (RFLAT-1). In this article we demonstrate that RFLAT-1 expression is translationally regulated through its 5'-UTR and in a cell type-specific manner. Overexpression of the translation initiation factor eIF4E increases RFLAT-1 protein, while inhibition of Mnk1, which phosphorylates eIF4E, reduces RFLAT-1 production, indicating cap-dependent translational regulation. These events are regulated by ERK-1/2 and p38 MAP kinases and allow T cells to rapidly adjust RANTES expression in response to changes in the cellular environment, such as stress and/or growth factors. These findings provide a molecular mechanism for a rheostat effect of increasing or decreasing RANTES expression at sites of inflammation. Memory T cells, already poised to make RANTES, are finely regulated by translational control of the major transcription factor regulating RANTES expression. This is the first example of such a mechanism regulating a chemokine, but it seems likely that this will prove to be a general way for cells to rapidly respond to stress, cytokines, and other proinflammatory factors in their local environment.
View details for DOI 10.1172/JCI200215336
View details for Web of Science ID 000176665100016
View details for PubMedID 12093895
A synthetic peptide corresponding to residues 65-79 of the alpha helix of the alpha-chain of the class II HLA molecule DQA03011 (DQ 65-79) inhibits the proliferation of human T lymphocytes in an allele nonrestricted manner. By using microarray technology, we found that expression of 29 genes was increased or decreased in a human CTL cell line after treatment with DQ 65-79. This study focuses on one of these genes, IkappaB-alpha, whose expression is increased by DQ 65-79. IkappaB proteins, including IkappaB-alpha and IkappaB-beta, are increased in T cells treated with DQ 65-79. Nuclear translocation of the NF-kappaB subunits p65 and p50 is decreased in T cells after treatment with DQ 65-79, while elevated levels of p65 and p50 are present in cytosol. DQ 65-79 inhibits the degradation of IkappaB-alpha mRNA and inhibits the activity of IkappaB kinase. These findings indicate that the DQ 65-79 peptide increases the level of IkappaB proteins, thereby preventing nuclear translocation of the transcription factor, NF-kappaB, and inhibiting T cell proliferation.
View details for Web of Science ID 000174566400029
View details for PubMedID 11907089
Granulysin is an antimicrobial and tumoricidal molecule expressed in granules of CTL and NK cells. In this study, we show that granulysin damages cell membranes based upon negative charge, disrupts the transmembrane potential (Deltapsi) in mitochondria, and causes release of cytochrome c. Granulysin-induced apoptosis is blocked in cells overexpressing Bcl-2. Despite the release of cytochrome c, procaspase 9 is not processed. Nevertheless, activation of caspase 3 is observed in granulysin-treated cells, suggesting that granulysin activates a novel pathway of CTL- and NK cell-mediated death distinct from granzyme- and death receptor-induced apoptosis.
View details for Web of Science ID 000170949100048
View details for PubMedID 11418670
Chemokines like RANTES appear to play a role in organ transplant rejection. Because RANTES is a potent agonist for the chemokine receptor CCR1, we examined whether the CCR1 receptor antagonist BX471 is efficacious in a rat heterotopic heart transplant rejection model. Treatment of animals with BX471 and a subtherapeutic dose of cyclosporin (2.5 mg/kg), which is by itself ineffective in prolonging transplant rejection, is much more efficacious in prolonging transplantation rejection than animals treated with either cyclosporin or BX471 alone. We have examined the mechanism of action of the CCR1 antagonist in in vitro flow assays over microvascular endothelium and have discovered that the antagonist blocks the firm adhesion of monocytes triggered by RANTES on inflamed endothelium. Together, these data demonstrate a significant role for CCR1 in allograft rejection.
View details for Web of Science ID 000166921200063
View details for PubMedID 11054419
Granulysin is a novel lytic molecule produced by human cytolytic T-lymphocytes (CTLs) and natural killer (NK) cells. It is active against a broad range of microbes, including Gram-positive and -negative bacteria, parasites and Mycobacterium tuberculosis. It is functionally related to other antibacterial peptides, like defensins and magainins, but is structurally distinct. It has structural similarity to porcine NK-lysin and to amoebapores made by Entamoeba histolytica. Synthetic peptides derived from granulysin have differential activity against eukaryotic cells and bacteria. Selective bactericidal peptides may have therapeutic roles as novel antibiotics.
View details for Web of Science ID 000166783200010
View details for PubMedID 11178344
Granulysin, a lytic protein present in cytolytic granules of human natural killer and cytotoxic T cells, entered cells infected with varicella-zoster virus (VZV). Exposure to granulysin accelerated death of infected cells as assessed by apoptosis markers. The functional domain of granulysin that mediated its antiviral effects was amino acid 23-51; this domain also mediates the additional antitumor cell effects of granulysin. Because granulysin is a product of natural killer cells and T lymphocytes, it is possible that its antiviral activity may act as a mediator of innate and adaptive immune mechanisms.
View details for Web of Science ID 000168993100005
View details for PubMedID 11398808
Differentiating etiologies of transplant dysfunction without biopsy and optimizing therapy for acute rejection by predicting steroid resistance will reduce patient morbidity. Granulysin is a cytolytic molecule released by CTL and NK cells and coexpressed with effectors of acute allograft rejection, like perforin and granzymes. Granulysin mRNA and protein expression were studied in peripheral blood lymphocytes (PBL; n = 61 total, n = 10 with intercurrent infections) and biopsy tissue from adult and children renal transplant recipients (n = 97) by competitive quantitative-reverse transcriptase-PCR (QC-RT-PCR) and immunohistochemistry. Differences in cell phenotypes were studied in steroid sensitive and resistant acute rejection biopsies. Granulysin was studied in phytohemagglutinin (PHA) stimulated cell lines (donor PBL and CD45RO(+) T cells) by FACS, Western blotting, and RT-PCR after pretreating with cyclosporine A (CSA), azathioprine, mycophenolic acid, and steroids. Granulysin mRNA was significantly increased in patient PBL and transplant biopsies during acute rejection (p < 0.0001) and infection (p < 0.001). Rejecting biopsies alone (n = 53) had mononuclear cell granulysin staining. Steroid resistant biopsies (n = 25) had denser granulysin staining (>2 cells/high power field) and CD45RO(+) lymphocytes, when compared with steroid sensitive (n = 28) rejecting tissue. Granulysin levels were unchanged after azathioprine and mycophenolic acid treatment, decreased after treating activated PBL with steroids and cyclosporine A (CSA), and paradoxically, increased (p < 0.05) after treating CD45RO(+) CTL with CSA. Elevated PBL granulysin is a peripheral marker for acute rejection and infection and dense granulysin staining a tissue marker for steroid resistance. Memory CTL abound in steroid resistant grafts and may have a markedly different response to CSA immunotherapy, suggesting a possible mechanism for steroid resistance.
View details for Web of Science ID 000167037400003
View details for PubMedID 11165712
Granulysin, a protein located in the acidic granules of human NK cells and cytotoxic T cells, has antimicrobial activity against a broad spectrum of microbial pathogens. A predicted model generated from the nuclear magnetic resonance structure of a related protein, NK lysin, suggested that granulysin contains a four alpha helical bundle motif, with the alpha helices enriched for positively charged amino acids, including arginine and lysine residues. Denaturation of the polypeptide reduced the alpha helical content from 49 to 18% resulted in complete inhibition of antimicrobial activity. Chemical modification of the arginine, but not the lysine, residues also blocked the antimicrobial activity and interfered with the ability of granulysin to adhere to Escherichia coli and Mycobacterium tuberculosis. Granulysin increased the permeability of bacterial membranes, as judged by its ability to allow access of cytosolic ss-galactosidase to its impermeant substrate. By electron microscopy, granulysin triggered fluid accumulation in the periplasm of M. tuberculosis, consistent with osmotic perturbation. These data suggest that the ability of granulysin to kill microbial pathogens is dependent on direct interaction with the microbial cell wall and/or membrane, leading to increased permeability and lysis.
View details for Web of Science ID 000165790500058
View details for PubMedID 11120840
A synthetic peptide corresponding to residues 75-84 of HLA-B2702 modulates immune responses in rodents and humans both in vitro and in vivo.We used a yeast two-hybrid screening, an in vitro biochemical method, and an in vivo animal model.Two cellular receptors for this novel immunomodulatory peptide were identified using a yeast two-hybrid screen: immunoglobulin binding protein (BiP), a member of the heat shock protein 70 family, and vascular cell adhesion molecule (VCAM)-1. Identification of BiP as a ligand for this peptide confirms earlier biochemical findings, while the interaction with VCAM-1 suggests an alternative mechanism of action. Binding to the B2702 peptide but not to closely related variants was confirmed by ligand Western blot analysis and correlated with immunomodulatory activity of each peptide. In mice, an ovalbumin-induced allergic pulmonary response was blocked by in vivo administration of either the B2702 peptide or anti-VLA-4 antibody.We propose that the immunomodulatory effect of the B2702 peptide is caused, in part, by binding to VCAM-1, which then prevents the normal interaction of VCAM-1 with VLA-4.
View details for Web of Science ID 000088986100021
View details for PubMedID 10972226
Granulysin, a 9-kDa protein localized to human CTL and NK cell granules, is cytolytic against tumor cells and microbes. Molecular modeling predicts that granulysin is composed of five alpha-helices separated by short loop regions. In this report, synthetic peptides corresponding to the linear granulysin sequence were characterized for lytic activity. Peptides corresponding to the central region of granulysin lyse bacteria, human cells, and synthetic liposomes, while peptides corresponding to the amino or carboxyl regions are not lytic. Peptides corresponding to either helix 2 or helix 3 lyse bacteria, while lysis of human cells and liposomes is dependent on the helix 3 sequence. Peptides in which positively charged arginine residues are substituted with neutral glutamine exhibit reduced lysis of all three targets. While reduction of recombinant 9-kDa granulysin increases lysis of Jurkat cells, reduction of cysteine-containing granulysin peptides decreases lysis of Jurkat cells. In contrast, lysis of bacteria by recombinant granulysin or by cysteine-containing granulysin peptides is unaffected by reducing conditions. Jurkat cells transfected with either CrmA or Bcl-2 are protected from lysis by recombinant granulysin or the peptides. Differential activity of granulysin peptides against tumor cells and bacteria may be exploited to develop specific antibiotics without toxicity for mammalian cells.
View details for Web of Science ID 000088340600042
View details for PubMedID 10903754
Synthetic peptides corresponding to structural regions of HLA molecules are novel immunosuppressive agents. A peptide corresponding to residues 65-79 of the alpha-chain of HLA-DQA03011 (DQ65-79) blocks cell cycle progression from early G1 to the G1 restriction point, which inhibits cyclin-dependent kinase-2 activity and phosphorylation of the retinoblastoma protein. A yeast two-hybrid screen identified proliferating cell nuclear Ag (PCNA) as a cellular ligand for this peptide, whose interaction with PCNA was further confirmed by in vitro biochemistry. Electron microscopy demonstrates that the DQ65-79 peptide enters the cell and colocalizes with PCNA in the T cell nucleus in vivo. Binding of the DQ65-79 peptide to PCNA did not block polymerase delta (pol delta)-dependent DNA replication in vitro. These findings support a key role for PCNA as a sensor of cell cycle progression and reveal an unanticipated function for conserved regions of HLA molecules.
View details for Web of Science ID 000087508500014
View details for PubMedID 10843669
MHC molecules bind antigenic peptides and present them to T cells. There is a growing body of evidence that MHC molecules also serve other functions. We and others have described synthetic peptides derived from regions of MHC molecules that inhibit T-cell proliferation or cytotoxicity in an allele-nonspecific manner that is independent of interaction with the T-cell receptor. In this report, we describe the mechanism of action of a synthetic MHC class II-derived peptide that blocks T-cell activation induced by IL-2. Both this peptide, corresponding to residues 65-79 of DQA*03011 (DQ 65-79), and rapamycin inhibit p70 S6 kinase activity, but only DQ 65-79 blocks Akt kinase activity, placing the effects of DQ 65-79 upstream of mTOR, a PI kinase family member. DQ 65-79, but not rapamycin, inhibits phosphatidylinositol 3-kinase (PI 3-kinase) activity in vitro. The peptide is taken up by cells, as demonstrated by confocal microscopy. These findings indicate that DQ 65-79 acts as an antagonist with PI 3-kinase, repressing downstream signaling events and inhibiting proliferation. Understanding the mechanism of action of immunomodulatory peptides may provide new insights into T-cell activation and allow the development of novel immunosuppressive agents.
View details for Web of Science ID 000087075300016
View details for PubMedID 10811852
An assay based on two-color flow cytometry has been developed to measure CTL and NK cell-mediated cytotoxicity. After effector/target cells are incubated together, CTL or NK populations are stained with an effector cell specific PE-conjugated mAb. Subsequently, annexin V-FITC binds to cells expressing phosphatidylserine (an early marker of apoptosis) on the cell surface. Target cells are gated upon as PE-negative and quantified with respect to their annexin V positivity. The shift from annexin Vneg to annexin Vhi is a discrete event such that all target cells fall within discernible populations with respect to annexin V. There is a strong correlation between cytotoxicity measured with our assay and a standard 51Cr release assay (r2 = 0.989). The PE/annexin V assay shows increased sensitivity at early timepoints after target/effector cell mixing. In addition, this method allows for analysis of target cells at the single cell level. Therefore, we have described a promising new technique to measure in vitro cell-mediated cytotoxicity. It avoids the potential difficulties of working with radioactive isotopes, and offers increased sensitivity and versatility.
View details for Web of Science ID 000080227300001
View details for PubMedID 10357200
A synthetic peptide corresponding to a region of the alpha1 alpha-helix of DQA03011 (DQ 65-79) inhibits the proliferation of human PBL and T cells in an allele-nonspecific manner. It blocks proliferation stimulated by anti-CD3 mAb, PHA-P, and alloantigen, but not by PMA and ionomycin. Substitution of each amino acid with serine shows that residues 66, 68, 69, 71-73, and 75-79 are critical for function. Inhibition of proliferation is long lasting and is not reversible with exogenous IL-2. The peptide can be added 24 to 48 h after stimulation and still block proliferation. The DQ 65-79 peptide does not affect expression of IL-2 or IL-2R; however, IL-2-stimulated proliferation is inhibited. Cell cycle progression is blocked at the G1/S transition, and the activity of cdk2 (cyclin-dependent kinase 2) kinase is impaired by the continued presence of p27. Although these results suggest a mechanism similar to that of rapamycin, the peptide inhibition is not reversed with FK-506, which indicates a distinct mechanism.
View details for Web of Science ID 000072115700024
View details for PubMedID 9498760
Functional properties of the B*4801 allotype were investigated using HLA class I-deficient 221 cells transfected with B*4801 cDNA. From pool sequence analysis of endogenously bound peptides, B*4801 was shown to select for nonamer peptides having glutamine or lysine at position 2 and leucine at the carboxyl-terminus. In an in vitro cell-cell binding assay, B*4801 binds CD8 alpha homodimers weakly due to the presence of a threonine residue at position 245 in the alpha 3 domain. A mutant B*4801 molecule in which alanine replaces threonine 245, binds CD8 alpha homodimers at levels comparable to those of other HLA class I allotypes. Despite the low affinity of B*4801 for CD8 alpha, alloreactive T-cells that recognize B*4801 molecules expressed by the 221 transfectant are inhibited by anti-CD8 monoclonal antibodies. Analysis of 25 B*48-expressing individuals from various populations showed threonine 245 was encoded by every B*48 allele.
View details for Web of Science ID A1997XX09000005
View details for PubMedID 9331948
Over the past decade the use of synthetic peptides corresponding to linear sequences of HLA molecules has progressed from a concept to a reality. These peptides are currently being evaluated in clinical trials. In animal models these peptides, given over 1 week with cyclosporin alone, induced long-term immunological tolerance (Figure 3). They may similarly induce tolerance in humans. The next major hurdle for such tolerogenic drugs, however, is to prove efficacy in clinical circumstances. Many of the drugs used to treat transplant patients today (steroids, cyclosporin, azathioprine, mycophenolate mofetil) may actually inhibit the 'active' processes of induction and maintenance of immunological tolerance. Demonstration that new drugs induce tolerance will require efficacy studies in other immune-mediated diseases in which monotherapy is feasible (such as psoriasis), before further advances can be made in the induction of transplant tolerance. In addition, rapid assays of rejection must be developed in order to reverse tolerance induction failures without graft damage or loss. Lastly, it will require heroic physicians, surgeons, and patients to make immunological tolerance a reproducible clinical reality.
View details for Web of Science ID A1997WZ68100001
View details for PubMedID 9175033
Synthetic peptides corresponding to linear sequences of HLA class I molecules have profound immunomodulatory effects in vitro and in vivo. Recent clinical trials confirm their potential as the therapeutics for transplantation and for a variety of immune-mediated diseases. These peptides also inhibit NK responses in vivo in humans. The importance of the carboxy end of the alpha 1 alpha helix in negative signaling to both T cells and NK cells focuses attention on new targets for immunotherapy.
View details for Web of Science ID A1996VW99900006
View details for PubMedID 8962174
In summary, synthetic peptides corresponding to linear sequences of HLA class I molecules can inhibit T-cell responses in vitro and in vivo. These peptides induce immunologic tolerance by binding to hsp-70 family members, causing an increase in intracellular calcium, and down-regulating the nuclear factor of activated T cells, NF-AT. We suggest that heat shock proteins may function as novel immunophilins (Fig 2). Like cyclophilins and FK 506 binding proteins, heat shock proteins are ubiquitous, are involved in protein folding and trafficking, and bind exogenous drugs. Cyclosporine and FK 506 exert immunosuppressive effects by binding immunophilins, which as a result interrupt the phosphatase activity of calcineurin. Although the precise pathways involved in the synthetic HLA peptide effects are not as well worked out, it seems likely that peptide binding to heat shock protein is disrupting normal events in T-cell activation, giving rise to an apparently permanent state of anergy.
View details for Web of Science ID A1996VD46300022
View details for PubMedID 8769160
Patients with non-Hodgkin's B-cell lymphoma who received an antitumor vaccine of idiotypic ig protein showed humoral and proliferative immune responses. Because immunity to some antigens, including tumor antigens and human pathogenic viruses, may be better correlated with the cytolytic cellular immune response, we evaluated 16 non-Hodgkin's lymphoma patients immunized with autologous idiotypic ig molecules for changes in tumor-specific cytotoxic T-lymphocyte precursor (CTLp) frequency using limiting dilution analysis. Eleven patients had a significant increase in tumor-specific CTLp. Eight of these 11 patients remain without evidence of disease or with stable minimal disease. In contrast, all five patients who did not have a significant change in tumor-specific CTLp have developed progressive disease. Patient vaccination with tumor associated protein antigens can increase tumor-specific CTLp frequencies. The correlation of increased tumor specific CTLp with freedom from progression is significant at P = .002. This study indicates that measurement of CTLp frequencies are relevant to the clinical evaluation of human tumor vaccines and suggests that cell-mediated cytolytic immune responses may be an important determinant of vaccine efficacy.
View details for Web of Science ID A1996UW48800023
View details for PubMedID 8695806
Synthetic peptides corresponding to sequences of HLA class I molecules have inhibitory effects on T cell function. The peptides investigated in this study have sequences corresponding to the relatively conserved region of the alpha 1 helix of HLA class I molecules that overlaps the "public epitope" Bw4/Bw6. These HLA-derived peptides exhibit inhibitory effects on T lymphocytes and have beneficial effects on the survival of allogenic organ transplants in mice and rats. Peptides corresponding to the Bw4a epitope appear most potent as they inhibit the differentiation of T cell precursors into mature cytotoxic T lymphocytes (CTL) and target cell lysis by established CTL lines and clones. To elucidate the mechanism through which these peptides mediate their inhibitory effect on T lymphocytes, peptide binding proteins were isolated from T cell lysates. We show that the inhibitory Bw4a peptide binds two members of the heat-shock protein (HSP) 70 family, constitutively expressed HSC70 and heat-inducible HSP70. Peptide binding to HSC/HSP70 is sequence specific and follows the rules defined by the HSC70 binding motif. Most intriguing, however, is the strict correlation of peptide binding to HSC/HSP70 and the functional effects such that only inhibitory peptides bind to HSC70 and HSP70 whereas noninhibitory peptides do not bind. This correlation suggests that small molecular weight HLA-derived peptides may modulate T cell responses by directly interacting with HSPs. In contrast to numerous reports of HSP70 expression at the surface of antigen-presenting cells and some tumor cells, we find no evidence that HSC/HSP70 are expressed at the surface of the affected T cells. Therefore, we believe that the peptides' immunodulatory effects are not mediated through a signaling event initiated by interaction of peptide with surface HSP, but favor a model similar to the action of other immunomodulatory compounds, FK506 and cyclosporin A, with a role for HSC/HSP70 similar to that for immunophilins, FKBPs and CyP40.
View details for Web of Science ID A1996TW10900001
View details for PubMedID 8627147
Elucidation of the structure of MHC molecules has provided profound new insights into their function in antigen presentation. In addition, structural studies have implicated certain regions of MHC molecules in specific functions. Although much of MHC biology has concentrated on the extensive polymorphism among these molecules, there is also evolutionary pressure to maintain the relatively monomorphic portions of these molecules. Drs. Krensky and Clayberger have found that synthetic peptides corresponding to linear sequences of HLA molecules have immunomodulatory effects both in vitro and in vivo. In this paper, they review the structure of HLA molecules and their studies of HLA derived peptides as novel immunotherapeutics. Members of the heat shock protein 70 family are implicated in the HLA derived peptide immunosuppressive pathway.
View details for PubMedID 8782740
Studies to determine if Toxoplasma gondii-specific human T cells lyse parasite-infected cells have yielded conflicting results. Furthermore, attempts to obtain human cytotoxic CD8+ T lymphocytes have been difficult because of the lack of a reproducible system for their generation. By using paraformaldehyde-fixed, T. gondii-infected peripheral blood mononuclear cells as antigen-presenting cells, we developed a method whereby T. gondii-specific T-cell lines can be reproducibly generated. Six T. gondii-specific T-cell lines were generated from an individual chronically infected with T. gondii. Cytofluorometric analysis of these lines revealed > 99% CD3+, 85 to 95% CD3+ alpha beta T-cell-receptor-positive (TCR+), 5 to 9% CD3+ gamma delta TCR+, 50 to 70% CD4+, and 20 to 40% CD8+ cells when cells were examined during the first 3 weeks of stimulation and >99% CD3+, >99% CD3+ alpha beta TCR+, < 1% CD3+ gamma delta TCR+, 20 to 40% CD4+, and 60 to 80% CD8+ cells when cells were examined between 5 and 11 weeks. Both CD4+ and CD8+ T cells had remarkable cytotoxic activity against T. gondii-infected target cells (30 to 50% specific Cr release at an effector-to-target ratio of 30:1) but not against uninfected target cells ( < 10% at an effector-to-target ratio of 30:1). Cytotoxic activity by the whole T-cell lines was not T. gondii strain specific. Whole T-cell lines were cytotoxic for target cells infected with the C56 and ME49 strains and the RH strain (which was used to infect peripheral blood mononuclear cells). T. gondii-specific T-cell lines displayed the predominant expression of V beta 7 TCR. The CDR3 regions of the V beta 7 TCRs of these T-cell lines showed a striking degree of sequence identity (oligoclonality). T-cell lines obtained by the method reporter here can be used to characterize functional activity of T-lymphocyte subsets in humans infected with T. gondii.
View details for Web of Science ID A1996TM71800025
View details for PubMedID 8557337
The induction of tolerance is a long-standing goal in transplantation. Intact MHC molecules, or fragments of them, are being used to render T cells unresponsive both in vitro and in vivo. Elucidation of the mechanisms underlying the effects of these treatments should aid the design of novel therapies for transplantation.
View details for Web of Science ID A1995TD86600008
View details for PubMedID 8573307
Cellular mechanisms responsible for maintenance of peripheral transplant tolerance in a rodent model were evaluated. Donor-specific tolerance was established in ACI rats given a vascularized heterotopic cardiac allograft followed by a 10-day course of cyclosporine. Tolerance was associated with a reduction in donor-specific cytotoxic T lymphocyte precursors and the presence within the spleen of cells capable of transferring suppression in adoptive transfer assays. Experiments using thymectomized animals revealed that the establishment and maintenance of tolerance occurred peripherally, independently of the thymus. Adoptive transfer experiments demonstrated that ongoing graft tolerance was mediated by suppressor cells that were antigen-restricted, radiosensitive, and capable of preventing allograft rejection by naive as well as sensitized cells in vivo. Studies designed to disrupt tolerance demonstrated a remarkable durability of graft protection once established, and give insight into the identity and mechanism of action of suppressor cells generated in this model.
View details for Web of Science ID A1995RB42900015
View details for PubMedID 7539554
Soluble forms of HLA may be natural immunoregulatory molecules and may explain the "transfusion effect" seen in transplant patients in the precyclosporine era. Synthetic peptides corresponding to short linear sequences of HLA molecules have inhibitory effects on human T lymphocytes in vitro and cause tolerance induction in a rat heterotopic heart transplant model. Recent studies indicate that a subset of these peptides causes an increase in intracellular calcium which appears to account for T cell unresponsiveness and tolerance induction. The peptides bind to members of the heat-shock protein 70 family in a specific manner, depending upon peptide sequence. These studies provide a mechanism to explain the biologic activities of these new immunotherapeutic reagents.
View details for Web of Science ID A1995QT37900002
View details for PubMedID 7704968
Synthetic peptides corresponding to linear sequences of HLA class I and class II molecules can potently inhibit T lymphocytes responses both in vitro and in vivo. The class I and class II peptides studied to date seem to function by different mechanisms. Nevertheless, several different peptides have been shown to potently induce T cell anergy. Opelz and Terasaki first demonstrated that blood transfusions improved graft survival in transplant patients (19). Data from several sources indicate that blood transfusions are immunosuppressive in: 1) increasing infections in trauma patients who have received transfusions (20), 2) increasing metastases and/or relapses in cancer patients who have been transfused (21), and 3) remissions of autoimmune diseases associated with pregnancy and/or transfusion (22). It is likely that the active constituent of blood transfusions is soluble HLA molecule (23,24). Liver transplants, which are profoundly immunosuppressive in themselves, produce large amounts of soluble HLA (25). Both B and T lymphocytes in culture secrete soluble HLA (26). We hypothesize that soluble HLA is a natural immunoregulatory molecule involved in dampening of the immune response, but, even if this is not the case, synthetic peptides corresponding to HLA sequences have profound effects on T lymphocytes and may prove to be effective for the induction of clinical tolerance in transplant patients.
View details for Web of Science ID A1995TF49700010
View details for PubMedID 8630749
Allospecific T lymphocytes mediate graft rejection through specific, direct or indirect, recognition of processed determinants of foreign MHC class I molecules. Small synthetic peptides derived from highly conserved sequences of the alpha 1 helix of the first domain of certain MHC class I molecules have been shown to inhibit CTL responses in vitro and to prolong graft survival in rats when combined with subtherapeutic doses of cyclosporine. Here, we report that the survival of LEW.1W heart allografts was significantly prolonged when transplanted into congenic LEW.1A recipients treated only with a peptide corresponding to residues 75-84 of the human HLA-B7-01 molecule (B7.75-84) before transplantation. The experimental value for mean survival time (+/- SD) in untreated recipients was 13 +/- 6 days and in peptide-treated recipients was 42 +/- 27 days (P < 0.002). A total of 64% of treated recipients had a functioning graft at 30 days, while grafts were rejected in all rats belonging to the control group within this time. Within graft-infiltrating leukocytes (GIL) in B7.75-84-treated animals, the proportion of T cells was significantly lower and that of CD5-/TCR alpha beta-/CD16-/CD8+ and MHC class II+ cells concomitantly increased, as compared with nontreated animals. GIL from B7.75-84-treated animals also exhibited a dramatic decrease (approximately 70%) of allospecific and spontaneous (NK) cytotoxic activity, whereas their proliferation and IL-2 production were similar in both experimental groups. The IFN-gamma, IL-2, and IL-10 mRNA levels from GIL from peptide-treated recipients were similar to levels of controls, reflecting a state of activation of GIL. Perforin and granzyme A mRNA, the level of which may be modulated parallel to impaired cytotoxic functions, were at similar levels in both experimental groups. These data demonstrate that B7.75-84 significantly prolongs graft survival in LEW.1A rats when given as a single agent and suggests that a specifically decreased cytotoxic response (allospecific and spontaneous) plays a major role.
View details for Web of Science ID A1995QM65600003
View details for PubMedID 7886788
Recently, Clayberger et al. demonstrated that ALLOTRAP, small synthetic peptides derived from a conserved region of the alpha 1 helix of certain HLA class I molecules, inhibited human CTL responses in vitro. In rats, ALLOTRAP 07 therapy combined with a subtherapeutic dose of cyclosporine led to the permanent acceptance of heart allografts. In the present study, the effect of ALLOTRAP on the survival of skin allografts in mice was studied. The tail skin of male C57B1/6 (H-2b) mice was grafted on the back of male CBA (H-2k) recipients. In untreated animals, the skin graft was rejected after 11.6 +/- 1.13 days (MST +/- SD). Cyclosporine administered orally for 5 days after transplantation prolonged graft survival to 13.1 +/- 2.13 days. ALLOTRAP 2702 prolonged graft survival to 16.57 +/- 2.15 days when administered orally for five days posttransplantation and to 18.86 +/- 0.38 when administered intraperitoneally until rejection. Thus, ALLOTRAP peptides derived from human MHC class I sequences, in addition to inhibiting human T cell responses in vitro, also prolong allograft survival in rats and mice.
View details for Web of Science ID A1995QK22500001
View details for PubMedID 7878745
Although gamma delta T cells have been postulated to act as a surveillance mechanism that eliminates transformed or otherwise damaged cells, little is known about tumor recognition by gamma delta T cells, including the Ags that are recognized and the molecules that present them. Previously, we described human gamma delta CTL that recognize the autologous B cell lymphoma. Here we report that these gamma delta CTL lyse heterologous cells transfected with the tumor Ig lambda chain gene. Furthermore, the lambda chain is recognized as processed peptide in an Id-specific manner. T cell recognition does not involve classical MHC molecules, but it could be blocked by Abs directed against the heat shock protein grp75. These findings show a specific gamma delta T cell response to a highly polymorphic Ag such as tumor Id and implicate heat shock protein as a molecule required for recognition.
View details for Web of Science ID A1995QG20800010
View details for PubMedID 7836746
Polymorphism among class I molecules of the major histocompatibility complex (MHC) confers allotypic specificity on the peptides that these molecules bind and present to cytotoxic T lymphocytes. Evolution of new human HLA class I alleles usually involves gene recombination events that replace a segment of one allele with the homologous region of another. In this study, the impact of these evolutionary changes has been assessed by comparison of the peptide-binding specificities of six related HLA-B allotypes.Endogenous peptides bound by HLA-B*5401, HLA-B*5501, HLA-B*5502, HLA-B*5601, HLA-B*6701 and HLA-B*0702 were characterized. Despite differing by 1-9 of the amino-acid residues comprising their peptide-binding sites, all these allotypes share a dominant preference for peptides that have proline at position 2. Polymorphism results in differing selection of carboxy-terminal and secondary anchor residues, but the peptide-binding specificities are sufficiently similar that there is overlap in the repertoires of peptides bound by these allotypes. Complete sequence determination of individual peptides revealed four that could be isolated from two or more allotypes. Members of the closely related HLA-B22 family--HLA-B*5401, HLA-B*5501, HLA-B*5502 and HLA-B*5601--show only minor differences in their peptide-binding specificities. This marked similarity is reflected at the functional level, as alloreactive cytotoxic T lymphocytes generated against HLA-B*5401 and HLA-B*5501 exhibited cross-reactive recognition.The isolation of identical endogenously bound peptides from six HLA-B allotypes demonstrates overlap in the repertoires of peptides bound in vivo by different allotypes. We speculate that the shared preference for binding peptides with proline at position 2 reflects a selective pressure to retain this specificity, which may be based upon peptide availability in vivo. Characterization of the overlap between the repertoires of peptides bound by HLA-B allotypes could simplify the development of peptide-based vaccines that are targeted to cytotoxic T cells, as single peptides would be effective for humans of different HLA types.
View details for Web of Science ID A1995QG89200018
View details for PubMedID 7743181
Tumor specific cytotoxic T lymphocytes (CTL) recognize antigen via the T-cell receptor (TCR). In addition, recognition requires accessory molecules involved in adhesion and signal transduction. The authors previously have characterized an autologous, Burkitt's lymphoma specific CTL line that uses the gamma-delta TCR to recognize antigen in a nonclassical context. The current study was undertaken to identify novel accessory molecules involved in this unusual TCR-tumor cell interaction.A panel of monoclonal antibodies was generated against a Burkitt's lymphoma cell line and was screened for inhibition of autologous, tumor specific, cytolysis by a gamma-delta CTL line. Proteins identified by these monoclonal antibodies were further characterized by fluorescent-activated cell sorter analysis, Western blot and immunoprecipitation.Three known (CD5, CD43, and CD11a/CD18) and three novel (BAM-1, BAM-2, and BAM-3) cell surface molecules involved in the gamma-delta CTL-Burkitt's lymphoma interaction were identified and characterized.This study identifies and provides a preliminary characterization of three novel Burkitt's lymphoma-associated molecules involved in the gamma-delta CTL-tumor cell interaction and demonstrates that CD5, CD43, and CD11a/CD18 are involved in this interaction. It is likely that other unidentified accessory molecules are also involved in this and other effector cell-tumor interactions. Identification of such molecules may be useful in the design of new immunotherapeutic approaches.
View details for Web of Science ID A1995QC44900020
View details for PubMedID 7530169
Bronchoalveolar lavage (BAL) cells and peripheral blood leucocytes (PBL) from 24 lung transplant recipients were analysed for leucocyte subsets and expression of cell surface antigens. Total and differential white cell counts were performed on BAL, and lymphocyte subsets were evaluated in both BAL and peripheral blood. Measurement of immunofluorescence by flow cytometry was used to assess the percentage of: T cells (CD3+); T-helper cells (CD4+); T-cytotoxic/suppressor cells (CD8+); and activated lymphocytes (HLA-DR+). Lymphocyte subsets in BAL demonstrated marked differences to those in blood. A lower percentage of CD3+ and CD4+ lymphocytes were found in BAL, whereas CD8+ cells were more prevalent in BAL than in PBL. The mean CD4:CD8 ratio was significantly lower in BAL (1:1) than in blood (2.1:1). In the absence of pulmonary infection, there was a trend for a lower CD4:CD8 ratio in BAL associated with acute rejection (1.1:1) and obliterative bronchiolitis (1:1), when compared to the group with no evidence of rejection (1.4:1). In the absence of pulmonary rejection, pulmonary infection was associated with a marginally lower CD4:CD8 ratio in BAL (0.7:1), than when infection was absent (1.4:1). This difference was more evident in cases of cytomegalovirus (CMV) infection with a mean CD4:CD8 ratio of 0.3:1, compared to 1.5:1 in the absence of CMV disease (P < 0.05).
View details for Web of Science ID A1995QB91800006
View details for PubMedID 7708976
Immunological tolerance is the ultimate goal of transplantation immunobiology. Current therapies involve nonspecific immunosuppression with concomitant risks for infection, malignancy, and drug-specific side effects. By inducing specific immune unresponsiveness to the graft it should be possible to maintain transplants without the need for chronic drug administration and without the risk of nonspecific immunosuppression. This review highlights recent progress in the understanding of immunological tolerance, with special attention to the long-term prospects for successful induction of tolerance in renal transplant patients.
View details for Web of Science ID A1994PW73600030
View details for PubMedID 7696124
The identification of immunomodulatory approaches that allow the induction of antigen-specific unresponsiveness is required for long-term graft survival without the complications of chronic immunosuppression. Recent novel strategies based upon treatment with synthetic peptides corresponding to linear sequences of MHC class I and II molecules reproducibly induce tolerance to alloantigens. Although the mechanisms involved are still not completely understood, the phenomenology reported makes these approaches promising for evaluation in clinical trials.
View details for Web of Science ID A1994PM73500017
View details for PubMedID 7826536
The long-term success of heart transplantation for end-stage heart disease has been hindered by the problems associated with acute and chronic graft rejection, opportunistic infections and potentially fatal complications of intensive immunosuppression. A more complete understanding of the biology of transplant rejection should provide the basis for the development of improved methods for controlling and monitoring rejection. Cytokines, the soluble factors which regulate the immune response, are central to the rejection process. The objective of this study was to analyse cytokine mRNA transcripts in 99 biopsy samples and 89 blood samples from 65 and 35 Stanford Medical Center cardiac transplant recipients, respectively, gathered between January 1990 and January 1992. Following RNA extraction and conversion to cDNA, samples were amplified with cytokine-specific primers for interleukins (IL) 1 to 8, TNF-beta (tumour necrosis factor-beta) and IFN-gamma (interferon-gamma) and were analysed by gel electrophoresis and Southern blot hybridization. Our results demonstrate that despite chronic immunosuppressive therapy, the peripheral blood of transplant recipients expressed a higher combined percentage of different cytokine transcripts than did peripheral blood obtained from normal volunteers. In transplant patients, detection of cytokine transcripts for IL-1 alpha, IL-1 beta and IL-2 increased with time after transplantation. Intragraft IL-7 gene expression was significantly increased in biopsies diagnosed with mild (grade 1) rejection when compared to those with no evidence of rejection or with moderate to severe rejection. Implications of these results in light of possible mechanisms of rejection and of new approaches to immunotherapy are discussed.
View details for PubMedID 8000848
A major function of the immune response is the discrimination of self from nonself. It is this response that must be overcome in transplant rejection. Progress in understanding these basic immune mechanisms has helped to improve clinical outcome and lays the foundation for a new generation of therapies.
View details for Web of Science ID A1994PA77100012
View details for PubMedID 8047371
CD4 and CD8 are cell surface glycoproteins that serve as co-receptors for Ag with the TCR. Recent studies have shown that both CD4 and CD8 interact with conserved regions of MHC class II and class I, respectively. To investigate further the roles of CD4 and CD8 in the immune response, we prepared synthetic peptides corresponding to the HLA sequences with which CD4 and CD8 are thought to interact. The peptide corresponding to residues 222 to 235 of the HLA class I heavy chain blocked the differentiation of human CTL precursors into active effect cells but affected neither the ability of PBLs to proliferate in response to mitogen nor the cytotoxic activity of established CTLs. In contrast, the peptide corresponding to residues 134 to 152 of the HLA-DR beta-chain inhibited the differentiation of CTL precursors, the proliferative response of freshly isolated PBL, and the proliferation of an established alloreactive CD4+ T cell clone to Ag. The inhibitory effect of the DR.134-152 peptide on CTL differentiation could be overcome by addition of exogenous IL-2 to the limiting dilution cultures, whereas the effect of the HLA-1.222-235 peptide was unaffected by exogenous IL-2. These results directly demonstrate a functional role for these regions of MHC molecules and underscore the central role of both CD4 and CD8 in the effective initiation of a CTL response.
View details for Web of Science ID A1994NY34200004
View details for PubMedID 8027565
With the increasing frequency of transplantation of two or more organs into a single recipient, it has become evident that different organs are rejected with different kinetics. In this study the kinetics of skin, lung, and heart allograft rejection were compared in a rodent model. To study the influence of different allografts on the recipient's immune system, simultaneous or sequential skin, lung, or heart transplants were performed in various combinations, using DA rats as recipients for PVG allografts. Recipients receiving primary allografts were treated postoperatively with ten doses of cyclosporine (CsA) or preoperatively with 4 doses of rabbit antirat thymocyte globulin (ATG). Subsequent transplants were performed a minimum of 40 days later without additional immunosuppression. All primary skin allografts and 60% of primary lung allografts were rejected, while 100% of the heart allografts were accepted indefinitely. Recipients of primary skin allografts rejected subsequent skin, lung, or heart allografts with accelerated kinetics. Recipients of primary heart allografts accepted subsequent skin, lung, and heart allografts indefinitely without further immunosuppression. Surprisingly, animals that had rejected a primary lung allograft accepted subsequent skin or heart allografts indefinitely. Simultaneously transplanted skin and lung allografts were concordantly rejected. However, these animals accepted a subsequent heart allograft indefinitely, suggesting a strong tolerizing effect of lung allografts. Our results indicate that tissue-specific differences are critical, not only in determining acceptance or rejection of a primary allograft but also in determining the fate of subsequent allografts.
View details for Web of Science ID A1994NV56800019
View details for PubMedID 8016886
T cell recognition of MHC molecules initiates a cascade of events resulting in allograft rejection. CTLs damage the graft by targeting nonself-MHC class I molecules. We and others have previously shown that small synthetic peptides corresponding to regions of certain MHC class I molecules can inhibit the CTL response against MHC class I alloantigens in vitro. Here we report that rat heart allografts survived survived indefinitely when transplanted into recipients treated with a synthetic peptide corresponding to residues 75-84 of (B7.75-84) in combination with a subtherapeutic dose of cyclosporine A. Furthermore, this treatment induced long-term donor-specific tolerance that was mediated by anergic cells, indicating that such peptides may have potential as therapeutics for human organ transplantation.
View details for Web of Science ID A1994NF01800009
View details for PubMedID 8144948
This study examined whether posttransplant anti-T cell monoclonal or polyclonal antibody therapy could provide a window of treatment to allow posttransplant total lymphoid irradiation (TLI) to induce tolerance. These experiments were conducted in a high responder strain combination of an ACI cardiac allograft into a Lewis rat. In this situation, treatment with antibody or posttransplant TLI alone is insufficient to induce tolerance, while similar treatments alone have been shown to induce tolerance in low responder strains. The affects of three anti-T cell therapies were compared: anti-CD4 mAb therapy, anti-CD3 mAb, and rabbit antithymocyte globulin (RATG). None of these antibody therapies alone prolonged graft survival indefinitely. Combining anti-CD4 therapy with posttransplant TLI markedly delayed rejection but failed to induce long-term graft survival. Tolerance could be induced by a combination of anti-pan T cell antibody (anti-CD3) and TLI, and, all grafts survived beyond 100 days. RATG failed to prevent graft rejection when used alone or in combination with TLI. However, posttransplant therapy with a combination of RATG, TLI, and single-donor blood transfusion resulted in graft survival beyond 100 days. Recipients bearing long-term donor grafts rejected third-party (PVG) grafts within 2 weeks. Low density donor bone marrow cells used instead of a blood transfusion did not facilitate tolerance. The results indicate that monoclonal or polyclonal anti-pan T cell antibodies, TLI, and a donor blood cell infusion function synergistically in facilitating tolerance to allografts in the posttransplant period.
View details for Web of Science ID A1993MQ05600031
View details for PubMedID 8279017
Understanding the nature of allorecognition is fundamental to the design of antigen-specific therapies for transplantation. As recently as 10 years ago it was generally believed that recognition of major histocompatibility complex (MHC) molecules by T lymphocytes was direct and represented the "simplest" kind of T-lymphocyte interaction. It is now clear that the nature of allorecognition is complex, involving a variety of different forms of MHC antigens with or without peptides contained in their antigen-binding groove. In addition, there is renewed interest in alternative forms of allorecognition, including so-called indirect allorecognition, in which donor alloantigens are recognized as peptides in the context of recipient self. Lastly, it appears that cells other than T lymphocytes, eg, natural killer cells, are capable of antigen-specific recognition and may be responsible for heretofore underappreciated mechanisms of transplant rejection.
View details for PubMedID 7922230
The polymerase chain reaction was used to evaluate cytokine gene expression in bronchoalveolar lavage (BAL) cells and peripheral blood leukocytes in 31 human lung transplant recipients. All patients were maintained on a triple immunosuppression regimen consisting of CsA, AZA, and prednisone. Posttransplant survival ranged from 0.5 to 100.5 months (mean = 16.3 months). Cytokines IL-1 alpha, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, TNF-beta, and IFN-gamma were studied. In BAL, transcripts for IL-1 alpha, IL-7, IL-8, and TNF-beta were found in over 60% of samples and those for IL-5, IL-6, and IFN-gamma in 40-50%, while IL-2 and IL-4 mRNA were rarely found (< 20%). Considerable variation in the frequency of cytokine gene expression between BAL and peripheral blood was observed. When analyzed for the presence of acute pulmonary allograft rejection (without infection), transcripts for IL-4 and IL-6 in BAL demonstrated the greatest increase in frequency compared with nil rejection (P = 0.07 and P = 0.17, respectively). Pulmonary infection (without rejection) was associated with a modest increase in the expression of genes for IL-1 alpha and IFN-gamma (> 10%). Transcripts for IL-4 were not found in association with pulmonary infection, suggesting that this cytokine may be useful as a discriminatory rejection marker.
View details for Web of Science ID A1993MD80500034
View details for PubMedID 7692639
As the simultaneous transplantation of two or more organs into a single recipient has become increasingly common, asynchronous allograft rejection has become an important clinical problem. To investigate the cellular and molecular mechanisms underlying differential organ rejection, we developed a rat model in which heart and lung allografts were transplanted sequentially. Heterotopic heart allografts transplanted into DA recipients from PVG donors survived indefinitely if the recipients were given a short course of rabbit antirat thymocyte globulin or cyclosporine at the time of transplantation. In contrast, orthotopic left lungs transplanted under the same conditions were rejected in ATG-treated recipients and accepted in most CsA-treated recipients. These animals were then given a second organ allograft from the same strain or a third party to assess whether they exhibited donor specific tolerance and whether the acceptance or rejection of the first allograft would influence the survival of the second transplant. Animals tolerized to a heart allograft with ATG rejected an orthotopic lung transplant from the same strain as the original allograft, whereas recipients treated with CsA at the time of their heart transplant accepted a subsequent lung graft. Surprisingly, animals treated with either ATG or CsA that had rejected a lung allograft accepted a subsequent heart transplant. Using limiting dilution analysis and adoptive transfer studies, we found that some recipients had developed suppressor cells while others demonstrated anergy. We conclude that major histocompatibility complex antigens as well as other antigens are involved in the differential rejection of heart and lung allografts.
View details for Web of Science ID A1993KT96100035
View details for PubMedID 8456486
The CD8 coreceptor interacts with MHC class I molecules through an acidic loop in the MHC alpha 3 domain. Mutations in this region reduced binding between cells expressing mutant HLA molecules and CHO cells transfected with CD8 alpha chain, with mutations at residue 227 having the greatest effects. This study was undertaken to examine the role of the CD8-HLA interaction in the generation of primary and long-term CTLs. HLA-A*0201 genes (wild type or mutated at residue 227) were transfected into a cell line that lacked expression of HLA-A or B molecules but expressed HLA-Cw4. These cells were used as stimulators for PBLs from a normal donor. Cultures were tested for cytotoxicity at various times thereafter. Transfectants expressing the HLA-A*0201 mutant gene were poor stimulators of primary HLA-A2-specific CTLs. In long-term culture, HLA-Cw4-specific CTLs predominated, indicating that continuous expansion of allogeneic CTLs depends upon an efficient CD8-MHC class I interaction.
View details for Web of Science ID A1993LC17900003
View details for PubMedID 8320133
The three-dimensional structure of the HLA class I molecules has highlighted the importance of the "groove" formed by the helices. We used site-directed mutagenesis to construct a series of HLA-B27 mutants with different substitutions at the sites of the conserved amino acid residues of HLA-B27 subtypes, specifically residue 77 which is thought to be critical to the binding site of the molecule, and a residue at the CD8 binding site. We formed an anti-B27 CTL line and derived six anti-B27 clones. Each of the six clones showed a different pattern of reaction, reflecting the diversity of the epitopes recognized. All nine mutants were effective in altering allorecognition by HLA-B27 specific CTL, although positions 45 and 77 caused the most drastic effect. The residue in position 77 is also the last amino acid of the peptide sequence shared with Klebsiella. Our results highlight the importance of certain epitopes in allorecognition that may have important implications for the immunotherapy of autoimmune diseases.
View details for Web of Science ID A1992KD27100006
View details for PubMedID 1282853
Previously, autologous Burkitt lymphoma-specific cytotoxic T-lymphocytes (CTL) were found to express the gamma and delta T-cell receptor and recognize tumor idiotype in a major histocompatibility complex (MHC) unrestricted fashion.In this study, the authors established autologous CTL lines and clones specific for a B-cell follicular lymphoma.These CTL are tumor specific and inhibited by antiimmunoglobulin monoclonal antibodies, but unlike the Burkitt lymphoma-specific CTL, they are MHC restricted and express the alpha and beta T-cell receptor.These studies suggest that different B-cell lymphomas can induce CTL of different phenotypes and MHC restriction.
View details for Web of Science ID A1992JT85900027
View details for PubMedID 1394049
Heart transplantation is now a viable therapeutic option for patients with certain end-stage cardiac diseases. However, episodes of rejection, opportunistic infection, and life-threatening side effects of generalized immunosuppression remain very real problems for these patients. A better understanding of the molecular mechanisms underlying rejection may provide the basis for the development of more specific, less toxic immunosuppressive therapies. While cytokines have long been implicated in the pathogenesis of rejection, the precise role of each cytokine in this process has yet to be defined. We report here the application of the polymerase chain reaction (PCR) to the detection of cytokine mRNA in biopsies obtained from heterotopic abdominal cardiac allografts in cynomolgus monkeys. With the exception of IL-6 and IL-8, cytokine transcripts were undetectable in samples obtained from the donor heart pretransplant. In contrast, IFN-gamma transcripts were detected in all transplants two days after surgery before evidence of rejection was demonstrable by histopathologic analysis. IL-1 beta, IL-2, and IL-6 transcripts were detected when minimal rejection was noted. At later times, IL-1 alpha, IL-1 beta, IL-2, IL-6, IL-8, TNF-beta, and IFN-gamma transcripts were detectable. Further characterization of the spectrum of cytokines expressed at various stages of rejection may lead to insights into the biology of transplant rejection and to the development of more specific and potent reagents to diagnose and/or treat rejection.
View details for Web of Science ID A1992JJ75300025
View details for PubMedID 1496544
We have identified and cloned cDNA for a novel cell-surface protein that we have named Tactile for T cell activation, increased late expression. It is expressed on normal T cell lines and clones, and some transformed T cells, but no other cultured cell lines tested. It is expressed at low levels on peripheral T cells and is strongly up-regulated after activation, peaking 6 to 9 days after the activating stimulus. It is also up-regulated on NK cells activated in allogeneic cultures. It is not found on peripheral B cells but is expressed at very low levels on activated B cells. Tactile-specific mAb immunoprecipitates a band of 160 kDa when reduced and bands of 240, 180, and 160 kDa nonreduced. Using an antiserum produced with affinity-purified Tactile protein to screen a lambda gt11 library, we have identified Tactile cDNA. Northern blot analysis shows an expression pattern similar to that of the protein and transfection of COS cells with the full-length 5.2-kb cDNA results in cell-surface expression. Comparison with the sequence databanks show that Tactile is a member of the immunoglobulin gene superfamily, with similarity to Drosophila amalgam, the melanoma Ag MUC-18, members of the carcinoembryonic Ag family, the poliovirus receptor, and the neural cell adhesion molecule. The deduced primary sequence encodes a protein with three Ig domains, a long serine/threonine/proline-rich region typical of an extensively O-glycosylated domain, a transmembrane domain, and a 45 residue cytoplasmic domain. These data suggest that Tactile may be involved in adhesive interactions of activated T and NK cells during the late phase of the immune response.
View details for Web of Science ID A1992HN74000041
View details for PubMedID 1313846
Correlation between the effective refractory period of the conduction system and the histopathologic grade of the ventricle was examined in the rat cardiac allograft during acute rejection. Lewis rats were recipients of intraabdominal heart grafts from brown Norway rats (allogeneic group, n = 42) or Lewis rats (syngeneic group, n = 15). No immunosuppressant was given. The effective refractory period of the conduction system was measured by the programmed atrial extrastimulus method (basic cycle length, 150 msec) just before the time of death. Specimens of the transplanted hearts were examined histopathologically and the histopathologic grade of rejection was scored according to the standardized grading system of the International Society for Heart and Lung Transplantation. The effective refractory period of the allogeneic heart was significantly longer starting on the third day after transplantation (p less than 0.01). The effective refractory period of the allogeneic heart was more prolonged as the severity of rejection increased. The correlation between the effective refractory period (Y) and the histopathologic grade (X) of the allogeneic group was statistically significant (r = 0.955; Y = 10.3X + 81.0; p less than 0.01). The effective refractory period of all syngeneic hearts and allogeneic hearts of postoperative day 1 and 2 were statistically equivalent to the period of native rat hearts (81.0 +/- 2.0 msec; n = 6). The histopathologic grade of the conduction system was the same as that of the ventricle. We conclude that the effective refractory period of the conduction system could be a useful measure to predict the histopathologic grade of cardiac allograft rejection.
View details for Web of Science ID A1992HN91000008
View details for PubMedID 1576134
Dissection of the peptide binding grooves of seven subtypes of human histocompatibility leukocyte antigen (HLA)-B27 into the six specificity pockets defined by the 2.6-A structure of HLA-A*0201 revealed just one pocket, the B ("45") pocket, that is conserved among all the HLA-B27 subtypes. Functional studies of mutant HLA-B*2705 molecules with point substitutions in residues of the B pocket show that this structure, and the glutamine residue at position 45 in particular, plays a critical role in cell surface expression, peptide binding, and in the presentation of both exogenous and endogenous peptides by HLA-B*2705. We predict that the B pocket of HLA-B*2705 interacts with an amino acid side chain that anchors peptides in the binding groove, and that this peptide motif is present in most endogenously processed peptides that bind to all seven subtypes of HLA-B27.
View details for Web of Science ID A1992HF64000019
View details for PubMedID 1371304
More than one-half of adults with non-Hodgkin's B cell lymphomas present with low-grade follicular lymphomas. These tumor cells are found in close association with follicular T lymphocytes and dendritic cells, suggesting that the surrounding cells may play a role in the support of follicular tumors. Supernatants from activated human peripheral blood lymphocytes were found to promote the in vitro proliferation of follicular tumor cells. This effect was entirely due to interleukin 3 (IL-3), a factor generally thought to cause the growth and differentiation of immature hematopoietic cells. IL-3 receptors were detected on fresh isolates of all primary follicular cell tumors examined. These findings suggest that follicular cell tumors may be dependent in vivo on IL-3 and that therapies directed against IL-3, its receptor, or the T cells that produce it may be effective treatment for follicular lymphoma.
View details for Web of Science ID A1992HB06100007
View details for PubMedID 1732410
Functional subsets of human T cells were delineated by analyzing patterns of lymphokines produced by clones from individuals with leprosy and by T cell clones of known function. CD4 clones from individuals with strong cell-mediated immunity produced predominantly interferon-gamma, whereas those clones that enhanced antibody formation produced interleukin-4. CD8 cytotoxic T cells secreted interferon-gamma. Interleukin-4 was produced by CD8 T suppressor clones from immunologically unresponsive individuals with leprosy and was found to be necessary for suppression in vitro. Both the classic reciprocal relation between antibody formation and cell-mediated immunity and resistance or susceptibility to certain infections may be explained by T cell subsets differing in patterns of lymphokine production.
View details for Web of Science ID A1991GJ64200037
View details for PubMedID 1681588
One approach to the diagnosis and therapy of T cell-mediated diseases is to develop reagents specific for T cell receptor (TcR) variable (V) regions. To date, however, TcR expressed on the surface of antigen-specific T lymphocytes have proven to be poorly immunogenic. As a result, few monoclonal antibodies (mAb) recognizing human variable regions are available. In this report, we have used the "phosphatidylinositol linkage" strategy to generate soluble forms of two human allogeneic TcR derived from human cytotoxic T lymphocytes (CTL) known to be specific for HLA-A2 and HLA-Aw68/HLA-Aw69, respectively. Monomeric TcR alpha and beta chains from the HLA-A2-specific CTL were purified in large quantities from CHO cells and each was used to immunize mice to generate mAb. In particular, the anti-beta chain mAb, denoted anti-V beta 13, stain a significant (approximately 5%) fraction of human peripheral blood alpha/beta T lymphocytes, immunoprecipitate native anti-A2 TcR molecules, and activate T cells transfected with the relevant alpha and beta chain cDNA. Anti-alpha chain mAb were also obtained against a constant region determinant which can immunoprecipitate detergent-solubilized polypeptides. In general, we find that immunizations with soluble protein are far superior to those with cells bearing TcR chimeras or in combination with the purified protein.
View details for Web of Science ID A1991GF65500019
View details for PubMedID 1832385
View details for PubMedID 1879125
The complementary adhesion molecules LFA-1 (CD11a, 18)/ICAM-1 (CD54) and LFA-2 (CD2)/LFA-3 (CD58) have been shown to be important in T cell interaction with lymphoid target cells. The role of these ligand pairs in cytotoxicity against somatic cells is less well established. While LFA-3 is expressed by all cells in the kidney, ICAM-1 expression is low in normal kidneys but is increased in allograft rejection. An in vitro cytotoxicity assay was used to examine the relative importance of the two adhesion ligands in immune damage against kidney cells in rejection. HLA-A2 specific cytotoxic T lymphocyte (CTL) recognition of cultured human kidney cells (HKC), of predominantly renal tubular cell origin, was studied. Immunofluorescence studies showed that both induced and uninduced HKC target cells expressed ICAM-1, MHC class I and LFA-3, but only MHC class I and class II antigens and ICAM-1 were significantly upregulated by cytokine induction. Effector cells expressed LFA-1 and LFA-2 but little or no ICAM-1 and LFA-3. Cytokine induction of ICAM-1 expression on HKC target cells increased their susceptibility to lysis. Monoclonal antibody against ICAM-1 or LFA-1 produced the greatest inhibition of HKC lysis, and their effects were not additive. Antibody against LFA-2 (CD2) or LFA-3 also produced significant inhibition, but to a lesser degree, and no additive effect was found.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1991FA89000012
View details for PubMedID 1706002
The human class I HLA molecules are composed of both polymorphic, or private, and conserved, or public, regions. The private regions are recognized by alloreactive T cells and also serve as restriction elements for peptide presentation to autologous cells. Although the ability of public determinants to elicit antibody responses is well documented, little is known of their role in T cell function. In this study we examine the ability of one of these public HLA determinants, designated Bw4, to serve as a target Ag for CTL. We show that for some individuals the HLA-Bw4 epitope can function as a restriction element for CTL. This finding has important implications for organ transplantation.
View details for Web of Science ID A1990DF84400013
View details for PubMedID 2341716
Adhesion measurements between CD8 and 48 point mutants of HLA-A2.1 show that the CD8 alpha-chain binds to the alpha 3 domain of HLA-A2.1. Three clusters of alpha 3 residues contribute to the binding, with an exposed, negatively charged loop (residues 223-229) playing a dominant role. CD8 binding correlates with cytotoxic T-cell recognition and sensitivity to inhibition by anti-CD8 antibodies. Impaired alloreactive T-cell recognition of an HLA-A2.1 mutant with reduced affinity for CD8 is not restored by functional CD8 binding sites on an antigenically irrelevant class I molecule. Therefore, complexes of CD8 and the T-cell receptor bound to the same class I major histocompatibility complex molecule seem to be necessary for T-cell activation.
View details for Web of Science ID A1990DB95700049
View details for PubMedID 2109837
We report here the localization of the gene for a human T-cell-specific molecule, designated RANTES, to human chromosome region 17q11.2-q12 by in situ hybridization and analysis of somatic cell hybrids using a cDNA probe to the gene. We have recently shown that this gene, which encodes a small, secreted, putative lymphokine, is a member of a larger gene family some of whose members reside on chromosome 4 but most of whose members have not to date been mapped. A secondary hybridization peak was noted on the region of human chromosome 5q31-q34, which may represent the location of other members of the gene family. Interestingly, this latter region overlaps with the location of an extended linked cluster of growth factor and receptor genes, some of which may be coregulated with members of the RANTES gene family.
View details for Web of Science ID A1990CQ97700017
View details for PubMedID 1691736
In situ hybridization was used to localize the lymphocyte activation gene 519 (D2S69E) on human metaphase chromosomes. A significant proportion of the grains were situated over chromosome 2p12----q11, a region which contains other genes involved in immunologic functions.
View details for Web of Science ID A1990ED25500013
View details for PubMedID 2209093
Human cytolytic T lymphocytes (CTL) were generated in the presence and absence of histamine in order to define the role of this autacoid in immune regulation. Histamine (10(-8)-10(-4) M) suppressed the generation of class I specific CTL but, at 10(-4) M, actually increased class II specific cytolysis. Histamine acted at the level of CTL generation; histamine was not present in the cytolytic assay. When histamine was added to the cytolytic assay with CTL grown without histamine, the lytic ability of the effector cells was similar to that of controls. Histamine-induced suppression of class I specific cytolysis was blocked by continuous culture with the H2 antagonist ranitidine but not with the H1 antagonist pyrilamine. These data suggest that suppression was mediated by the H2 receptor. Continuous culture with histamine had no effect on T cell proliferation or the expression of cell surface molecules. Histamine-induced suppression of class I specific cytolysis was reversed by the addition of PHA to the cytotoxicity assay, showing that the cytolytic machinery was intact. These data provide evidence that histamine is involved in regulation of cytolytic T cells.
View details for Web of Science ID A1989U778900005
View details for PubMedID 2541932
CTL are thought to play a role in the elimination of transformed cells in vivo. The effectiveness of such CTL is in part dependent on recognition of tumor specific antigens. Among the best characterized tumor-specific antigens are the unique or idiotypic determinants on the Ig of B cell lymphomas. Here we describe the generation and properties of human CTL specific for the idiotype on autologous B cell tumors. These cells are CD3+,CD4-,CD8- and express the delta chain of the TCR. Such cells may prove useful in tumor-specific adoptive therapy.
View details for Web of Science ID A1989U366600004
View details for PubMedID 2541219
Cytotoxic T lymphocytes (CTL) expressing the CD8 glycoprotein recognize peptide antigens presented by class I major histocompatibility complex (MHC) molecules. This correlation and the absence of CD8 polymorphism led to the hypothesis that CD8 binds to a conserved site of class I MHC molecules. Using a cell-cell binding assay we previously demonstrated specific interaction between human class I MHC (HLA-A,B,C) molecules and CD8. Subsequent analysis of the products of 17 HLA-A,B alleles revealed a natural polymorphism for CD8 binding in the human population. Two molecules, HLA-Aw68.1 and HLA-Aw68.2, which do not bind CD8, have a valine residue at position 245 whereas all other HLA-A,B,C molecules have alanine. Site-directed mutagenesis shows that this single substitution in the alpha 3 domain is responsible for the CD8 binding phenotype and also affects recognition by alloreactive and influenza-specific CTL. Our results indicate that CD8 binds to the alpha 3 domain of class I MHC molecules.
View details for Web of Science ID A1989T800800061
View details for PubMedID 2784196
HLA-A2.1 and HLA-A2.3, which differ from one another at residues 149, 152, and 156, can be distinguished by the mAb CR11-351 and many allogeneic and xenogeneic CTL. Site-directed mutagenesis was used to incorporate several different amino acid substitutions at each of these positions in HLA-A2.1 to evaluate their relative importance to serologic and CTL-defined epitopes. Recognition by mAb CR11-351 was completely lost when Thr but not Pro was substituted for Ala149. A model to explain this result based on the 3-dimensional structure of HLA-A2.1 is presented. In screening eight other mAb, only the substitutions of Pro for Val152 or Gly for Leu156 led to the loss of mAb binding. Because other non-conservative substitutions at these same positions had no effect, these results suggest that the loss of serologic epitopes is in many cases due to a more indirect effect on molecular conformation. Specificity analysis using 28 HLA-A2.1-specific alloreactive and xenoreactive CTL clones showed 19 distinct patterns of recognition. The epitopes recognized by alloreactive CTL clones demonstrated a pronounced effect by all substitutions at residue 152, including the very conservation substitution of Ala for Val. Overall, the most disruptive substitution at amino acid residue 152 was Pro, followed by Glu, Gln, and then Ala. In contrast, substitutions at 156 had little or no effect on allogeneic CTL recognition, and most clones tolerated either Gly, Ser, or Trp at this position. Similar results were seen using a panel of murine HLA-A2.1-specific CTL clones, except that substitutions at position 156 had a greater effect. The most disruptive substitution was Trp, followed by Ser and then Gly. In addition, when assessed on the entire panel of CTL, the effects of Glu and Gln substitutions at position 152 demonstrated that the introduction of a charge difference is no more disruptive than a comparable change in side chain structure that does not alter charge. Taken together, these results indicate that the effect of amino acid replacements at positions 152 and 156 on CTL-defined epitopes depends strongly on the nature of the substitution. Thus, considerable caution must be exercised in evaluating the significance of particular positions on the basis of single mutations. Nonetheless, the more extensive analysis conducted here indicates that there are differences among residues in the class I Ag "binding pocket," with residue 152 playing a relatively more important role in formation of allogeneic CTL-defined epitopes than residue 156.
View details for Web of Science ID A1989T588500048
View details for PubMedID 2466083
Lymphocyte function-associated antigen 1 (LFA-1) is a glycoprotein involved in virtually all aspects of the immune response requiring direct cell to cell contact. It has been suggested that lack of LFA-1 expression in lymphomas may represent a mechanism of escape from immunologic surveillance. We investigated the expression of LFA-1 in a series of more than 250 lymphoid neoplasms and reactive lymphoid proliferations using a frozen section immunoperoxidase technique. LFA-1 was expressed by all lymphoid populations in the reactive cases. In contrast, absence of LFA-1 alpha or beta chains was found in 44% of non-Hodgkin's lymphomas, including 50% of B-cell lymphomas. These findings suggest that loss of LFA-1 expression may be of great use in the differential diagnosis of benign versus malignant lymphoproliferations. Eighty percent of initial biopsy specimens of low-grade lymphoma exhibited LFA-1 expression, whereas only 8% of recurrent specimens retained expression of both LFA-1 subunits. However, we found no correlation between LFA-1 expression and clinical course in a series of 64 patients with diffuse large cell lymphomas.
View details for Web of Science ID A1989R775500008
View details for PubMedID 2642732
Hemi-exon shuffling and site-directed mutagenesis have been used to determine which amino acid differences between HLA-A2.1 and HLA-A2.2 alter the CTL-defined epitopes on these two molecules. Two genes were constructed that encode novel molecules in which the effect of amino acid differences at residues 9, 43, and 95, or at residue 156 could be separately evaluated. Using both human and murine CTL that were specific for either HLA-A2.1 or HLA-A2.2, four types of epitopes were identified: 1) epitopes that were insensitive to substitutions at either residues 9, 43, and 95, or residue 156 but were lost when all four positions were changed; 2) epitopes that were dependent on the residues 9, 43, 95, but not residue 156; 3) epitopes that were dependent on residue 156, but not amino acid residues 9, 43, and 95; and 4) epitopes that were dependent on residues 9, 43, and 95, as well as amino acid residue 156. Overall, there was a roughly equal distribution of clones recognizing each of these types of epitopes. Additional molecules were constructed by hemi-exon shuffling between the HLA-A2.2 and HLA-A2.3 genes, and by site-directed mutagenesis, to analyze the epitopes recognized by two HLA-A2.2/A2.1 cross-reactive murine CTL that do not recognize HLA-A2.3. Although the epitopes recognized by these CTL were unaffected by changes occurring at residues 9, 43, and 95, or at residues 149, 152, and 156 alone, simultaneous changes in both of these regions acted in concert to destroy the epitopes. Both of the CTL recognized epitopes that were lost when substitutions were made at residues 9, 43, 95, 149, and 152. The epitope recognized by one of the CTL was also destroyed by the substitution of residues 9, 43, 95, 152, and 156. Overall, these results indicate that residues 9, 43, and 95, as well as residues in the alpha-helical region of the molecule, are all capable of contributing to the definition of the epitopes recognized by HLA-A2.1- and HLA-A2.2-specific CTL. They further indicate that some epitopes can be mapped to a particular region of the molecule, whereas other epitopes are formed through a complex interaction of residues in distant regions of the molecule.
View details for Web of Science ID A1988R004700046
View details for PubMedID 2460555
Site-directed mutagenesis of HLA-A2.1 has been used to identify the amino acid substitutions in HLA-A2.3 that are responsible for the lack of recognition of the latter molecule by the HLA-A2/A28 specific antibody, CR11-351, and by HLA-A2.1 specific CTL. Three genes were constructed that encoded HLA-A2 derivatives containing one of the amino acids known to occur in HLA-A2.3: Thr for Ala149, Glu for Val152, and Trp for Leu156. Three additional genes were constructed that encoded the different possible combinations of two amino acid substitutions at these residues. Finally, a gene encoding all three substitutions and equivalent to HLA-A2.3 was constructed. These genes were transfected into the class I negative, human cell line Hmy2.C1R. Analysis of this panel of cells revealed that recognition by the antibody CR11-351 was completely lost when Thr was substituted for Ala149, whereas substitutions at amino acids 152 and 156, either singly or in combination, had no effect on the binding of this antibody. The epitopes recognized by the allogeneic and xenogeneic HLA-A2.1 specific CTL clones used in this study were all affected by either one or two amino acid substitutions. Of those epitopes sensitive to single amino acid changes, none were affected by the substitution of Thr for Ala149, whereas all of them were affected by at least one of the substitutions of Glu for Val 152 or Trp for Leu156. Overall, amino acid residue 152 exerted a stronger effect on the epitopes recognized by HLA-A2.1 specific CTL than did residue 156. Of those epitopes affected only by multiple amino acid substitutions, double substitutions at residues 149 and 152 or at 152 and 156 resulted in a loss of recognition, whereas a mutant with substitutions at residues 149 and 156 was recognized normally. This reemphasizes the importance of residue 152 and indicates that residue 149 can affect epitope formation in conjunction with another amino acid substitution. These results are discussed in the context of current models for the recognition of alloantigens and in light of the recently published three-dimensional structure of the HLA-A2.1 molecule.
View details for Web of Science ID A1988Q240000052
View details for PubMedID 2459215
We have used a cDNA library enriched for T cell-specific sequences to isolate genes expressed by T cells but not by other cell types. We report here one such gene, designated RANTES, which encodes a novel T cell-specific molecule. The RANTES gene product is predicted to be 10 kDa and, after cleavage of the signal peptide, approximately 8 kDa. Of the 68 residues, 4 are cysteines, and there are no sites for N-linked glycosylation. RANTES is expressed by cultured T cell lines that are Ag specific and growth factor dependent. RANTES expression is inducible in PBL by Ag or mitogen. In CTL, expression of RANTES decreases after stimulation with Ag and growth factors. Interestingly, RANTES was not expressed by any T cell tumor line tested. There is significant homology between the RANTES sequence and several other T cell genes, suggesting that they comprise a previously undescribed family of small T cell molecules.
View details for Web of Science ID A1988P361900046
View details for PubMedID 2456327
The Daudi cell line is a B-lymphoblastoid line derived from a Burkitt lymphoma. Daudi cells lack cell surface expression of class I HLA molecules despite the presence of intracellular class I heavy chains. They have a defect in the gene encoding beta 2-microglobulin (beta 2m), resulting in lack of translatable mRNA for this protein. It has been thought that this deficiency is responsible for the lack of cell surface class I expression. However, data have recently been presented demonstrating that at least one mouse class I heavy chain can be expressed on the cell surface in the absence of beta 2m. These results raised the questions of whether the lack of beta 2m is the only defect in Daudi and whether transfer of this single gene could restore surface class I expression. We found that transfection of the mouse beta 2m gene into Daudi indeed rescued cell surface expression of class I HLA molecules, and that these molecules could be recognized both by monomorphic and allospecific mAbs. CTL clones specific for HLA-B17 or a determinant shared by HLA-B17 and HLA-A2 killed the Daudi cells transfected with the beta 2m gene, but not untransfected Daudi or Daudi transfected with vector alone. Mouse beta 2m on the transfected Daudi cells could exchange with human beta 2m when the cells were incubated in human serum. This exchange did not alter the ability of the cells to be killed by the specific CTLs. These results demonstrate that the lack of beta 2m is the sole reason for lack of surface class I molecules in Daudi cells, and that beta 2m is required for cell surface expression of the specific class I heavy chains of Daudi.
View details for Web of Science ID A1988M473200004
View details for PubMedID 3279151
Because keratinocytes (KCs) express HLA-DR in a wide variety of skin diseases in which mononuclear leukocytes are observed in close apposition to KCs (i.e., graft-versus-host disease), and since gamma interferon (IFN-gamma) induces HLA-DR expression on KCs, we asked whether IFN-gamma treatment of KCs would influence the adherence of mononuclear leukocytes. When allogeneic peripheral blood mononuclear leukocytes (PBML) and a Leu-3+ T cell clone were coincubated with IFN-gamma-treated KCs (300 U/ml, 3 days), there was a marked increase in binding compared with nontreated KCs. Similar binding results were obtained using a cutaneous squamous carcinoma cell line (SCL-1) after IFN-gamma treatment. The IFN effect was relatively specific for IFN-gamma, as neither IFN-alpha nor -beta had any effect. Tumor necrosis factor exposure (500 U/ml, 3 days) increased the binding of the Leu-3+ T cell clone to both KCs and SCL-1 cells. Neutrophils displayed a less marked (but statistically significant) increase in binding to IFN-gamma-treated KCs. Using the Leu-3+ cell clone and SCL-1 cells, detailed kinetic analysis of the effect of IFN-gamma on binding was performed. The increased adherence between the cells began to appear after only 7 hours of treatment with r-IFN-gamma (300 U/ml) and reached a plateau at 48 hours, with significantly enhanced binding continuing for at least 48 hours after removal of IFN-gamma. The mechanism of binding was explored by preincubation of the PBML/Leu-3+ T cells with anti-LFA-1 (lymphocyte function-associated antigen) antibody (0.6-6.0 micrograms/ml), which totally inhibited the binding with no effect by anti-LFA-2 or -3 or class I or II antibodies despite documented binding of these antibodies to the cells. These results suggest that, after exposure to IFN-gamma, the ability of KCs to bind mononuclear leukocytes is strongly enhanced, and this adherence may be important in leukocyte trafficking in the skin as well as contributing to altered KC-leukocyte interaction, which may be of fundamental importance in a variety of skin disease.
View details for Web of Science ID A1988L481700004
View details for PubMedID 2447190
The class-I and class-II molecules encoded by the major histocompatibility complex (MHC) are homologous proteins which allow cytotoxic and helper T cells to recognize foreign antigens. Recent studies have shown that the form of the antigen recognized by T cells is generally not a native protein but rather a short peptide fragment and that class-II molecules specifically bind antigenic peptides. Furthermore, the three-dimensional structure of the human MHC class-I molecule, HLA-A2, is consistent with a peptide-binding function for MHC class-I molecules. An outstanding question concerns the molecular nature and involvement of MHC-bound peptides in antigens recognized by alloreactive T cells. In this study the effects of peptides derived from HLA-A2 on cytolysis of alloreactive cytotoxic T cells (TC) cells are presented. Peptides can inhibit lysis by binding to the T cell or sensitize to lysis by binding an HLA-A2-related class-I molecule (HLA-Aw69) on the target cell. Thus, allospecific TC cells can recognize HLA-derived peptides in the context of the MHC.
View details for Web of Science ID A1987L431600073
View details for PubMedID 3501071
During studies of T-cell recognition of autologous tumour cells, a number of tumour cell lines derived from patients with lymphoma proved to be poor stimulators of both autologous and allogeneic T-cell responses. Analysis of the tumour cell surface molecules indicated that expression of the lymphocyte-function-associated antigen, LFA-1, was lacking, whereas normal leucocytes from these patients expressed normal levels of LFA-1. Examination of other lymphoid tumours revealed that most high grade lymphomas, but not most low or intermediate grade lymphomas, do not express the LFA-1 molecule. Furthermore, in an initial survey, the tumours from 5 of 7 patients with non-relapsing large cell lymphomas expressed LFA-1 whereas only 3 of 18 patients with relapsing lymphomas had tumours that did so. These findings suggest that tumour cells lacking surface LFA-1 cannot initiate an effective immune response in vivo. This lack of immunogenicity might contribute to escape from immunosurveillance.
View details for Web of Science ID A1987J846800004
View details for PubMedID 2887833
We have studied the interaction of HLA class I antigens with alloreactive cytotoxic T lymphocytes and monoclonal antibodies using site-directed mutagenesis and expression of an HLA-Aw68.1 gene. Two mutants containing distinct substitutions at polymorphic residues near the NH2-terminal end of the alpha 2 domain were made. One mutant with substitutions at positions 95 and 97 corresponding to residues found in HLA-A2.1 showed no alterations in binding of HLA-Aw68- or HLA-A2-specific monoclonal antibodies, but was reactive with some HLA-A2-specific CTL clones. A second mutant, in which glycine at position 107 was replaced with tryptophan found at that position in HLA-A2.1, was recognized by HLA-A2-specific CTL clones and HLA-A2, Aw69-specific monoclonal antibodies. Thus, substitution of a single amino acid residue at position 107 of the HLA-Aw68.1 molecule generates an allospecific determinant shared with HLA-A2.1 and recognized by both B and T lymphocytes.
View details for Web of Science ID A1987H960000025
View details for PubMedID 2439636
The Leu-19 (NKH-1) antigen is expressed on human peripheral blood NK cells and a subset of peripheral blood cytotoxic T lymphocytes that kill "NK-sensitive" tumor cell targets without major histocompatibility complex restriction. In the present study, we demonstrate that the Leu-19 (NKH-1) antigen is also expressed on most interleukin 2 (IL 2) dependent T cell lines and clones that have been maintained in long term culture. The Leu-19 (NKH-1) antigen expressed on an antigen-specific, class I directed cytotoxic T lymphocyte cell line was an approximately 200,000 to 220,000 dalton protein, similar to Leu-19 (NKH-1) protein expressed on natural killer cells and KG1a, an immature stem cell leukemia cell line. Furthermore, Leu-19 (NKH-1) was expressed on both CD4+ and CD8+ IL 2 dependent T cell clones, and was present on both cytotoxic and non-cytotoxic T cell clones. Thus expression of Leu-19 (NKH-1) antigen on cultured cell lines does not directly correlate with cytotoxic function, antigenic specificity, or cell lineage.
View details for Web of Science ID A1987G571600002
View details for PubMedID 2951430
A variety of molecules are involved in the interaction of human allospecific cytolytic T lymphocytes (CTL) with target cells. Monoclonal antibodies specific for these molecules inhibit CTL-target conjugate formation and/or lysis. To further study recognition and lysis of targets by human CTL, we used a murine mastocytoma cell line transfected with the histocompatibility leukocyte antigen (HLA)-A2 gene (P815-A2+) as a target for human HLA-A2-specific CTL. We find that only a subset of human HLA-A2-specific CTL can lyse murine P815-A2+ cells, suggesting that the murine cells may lack one or more accessory molecules needed for CTL recognition and lysis.
View details for Web of Science ID A1987G571600005
View details for PubMedID 3549894
We describe the function and cell distribution of two novel cell surface antigens, L24 and L25. These antigens are broadly distributed on human lymphocytes. Monoclonal antibodies specific for these molecules block lysis by Class I- and II-specific cytotoxic T lymphocytes, but do not affect any other T cell functions tested. Anti-L24 antibody immunoprecipitates a molecule composed of two disulfide-linked monomers of 140 kd each. Anti-L25 antibody immunoprecipitates three proteins of 150, 85, and 75 kd. The study of these and other function associated molecules may provide insight into mechanisms of cytotoxic T lymphocyte recognition and/or function.
View details for Web of Science ID A1987G268700031
View details for PubMedID 3492556
Using a subtractive hybridization procedure we have constructed a cDNA library enriched for sequences present in functional human T cell lines, but not in human EBV-transformed B cell lines. We have isolated a cDNA clone, AH2-519, representing a novel gene, designated 519. This novel gene is expressed in functional human cytolytic and Th cell lines but not in a variety of other cell lines, including several long-term human T cell tumor lines. The expression of gene 519 is inducible in cultures of normal human PBL using antigenic or mitogenic stimulation. Neither the DNA sequence determined from a full-length cDNA clone overlapping with clone AH2-519 nor the amino acid sequence of its predicted protein product has significant homology to published sequences in the GenBank or NBRF databases. The restricted expression of gene 519 suggests that its gene product is involved in the growth and/or differentiation of normal T cells. The data also show that normal, nontransformed, functional T cells express gene products that can not be readily identified in long-term tumor lines of the same cell lineage.
View details for Web of Science ID A1987G292200002
View details for PubMedID 2434598
Class I major histocompatibility complex (MHC) molecules function in the recognition of antigens by cytotoxic T lymphocytes (CTL). Although this biological role is firmly established and much has been learnt about their structure and polymorphic variation, little is known of the regions of class I molecules that are involved in functional interactions with components of the T-cell surface. Here we show that peptides derived from residues 98-113 of the alpha 2 domain of HLA-A2 specifically inhibit the recognition of target cells by many HLA-A2-specific CTL. In addition to identifying a region that is probably involved in binding the T-cell receptor these results raise the possibility that alloreactive CTL may recognize degraded fragments of class I histocompatibility antigens.
View details for Web of Science ID A1987F973500058
View details for PubMedID 2433598
Natural killer (NK) cell have been implicated in immune responses to tumor and viral antigens. We describe here a monoclonal antibody, anti-KC-1, that blocks lysis of NK targets by fresh but not activated NK cells. Anti-KC-1 has no effect on cytotoxic T lymphocyte activity or on antibody-dependent cellular cytotoxicity. This antibody may be useful in the analysis of NK cell activation and the mechanism of lysis.
View details for Web of Science ID A1986A163400002
View details for PubMedID 3081628
In earlier studies we showed that hapten-specific inducer T cell clones specifically induce B cells from immunized donors to secrete IgM antibodies. However, IgG responses were not observed, suggesting that an additional signal(s) was required. In this report, we show that an autoreactive T cell clone produces a factor(s) that collaborates with antigen-specific inducer T cells to promote specific IgG responses. This factor is not restricted by antigen or MHC determinants and promotes IgG production both in vivo and in vitro. These findings suggest that autoreactive cells may play an important role in the regulation of isotype expression.
View details for Web of Science ID A1985AAL9200007
View details for PubMedID 2578161
We described a cloned dendritic cell, clone Den-1, that is a potent accessory cell for some B cell responses. Clone Den-1 produces a novel lymphokine that is distinct from previously described factors produced by T cells. In the present study, we compare the role of nonspecific helper factors produced by Den-1 (Den-1 SN) or the T cell thymoma EL4 (EL4-SN) in promotion of B cell plaque-forming cell (PFC) responses to a variety of antigens. We find that the antigen in culture determines the B cell requirement for dendritic and/or T cell factors. B cell PFC responses to TNP-Brucella abortus (BA) and TNP-lipopolysaccharide (LPS) are greatly increased by EL4-SN but show little, if any, enhancement with Den-1 SN. Responses to TNP-polyacrylamide are reconstituted by either Den-1 SN or EL4-SN, whereas responses to TNP-Ficoll, TNP-dextran and TNP-levan are reconstituted by Den-1 SN and are much less sensitive to factors present in EL4-SN. Responses to SRBC require the presence of both Den-1 SN and EL4-SN. We also show that the time at which Den-1 SN must be provided to the B cell is dependent on the antigen in culture. Our findings are discussed in terms of present classification of antigens based on their ability to stimulate various B cell subpopulations.
View details for Web of Science ID A1985AFX6000009
View details for PubMedID 2580002
Mice expressing the X-linked recessive CBA/N genetic defect xid lack a subpopulation of B cells which appears late in normal B cell ontogeny and is characterized by expression of the cell surface antigens Lyb3, Lyb5, and Lyb7. In adult mice with the xid defect, responses to Type 2 antigens such as TNP-ficoll are entirely absent and responses to Type 1 antigens such as TNP-LPS are somewhat reduced. Primary in vitro responses to the T dependent antigen sheep red blood cells (SRBC) are defective but secondary responses are normal. These and other findings have led to the hypothesis that one effect of the xid defect is the inability of a subset of B cells to respond to nonspecific signals from T cells or accessory cells. The cellular basis of the xid defect is not well understood. The simplest explanation is that it reflects a primary lesion in the development of a subpopulation of B cells responsible for the types of immune responses described above. An alternative notion is that the xid defect is expressed primarily in a non-B cell population (e.g., T cells or accessory cells) which are necessary for the development and/or function of this B cell subset. A direct approach to this problem depends on the availability of homogeneous populations of B cells, T cells, or accessory cells. Recently we have described a cloned dendritic cell, Den-1, which is a potent stimulator of some B and T cell responses.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1985AND7900002
View details for PubMedID 3916931
Efficient B cell responses to most polysaccharide antigens such as TNP-PAA or TNP-Ficoll require factors produced by activated T cells. However, the mechanism of T cell activation during such responses has not been established, because these antigens do not activate T cells, either directly or in conjunction with I-A gene products. We used a panel of antigen-specific monoclonal helper T cells to study T cell activation during the course of such responses. We show that activated I-A-identical B cells directly stimulate these monoclonal T cells, and that this stimulation is in the absence of nominal antigen. The high frequency of inducer cells that are stimulated by activated B cells suggests a major biologic role for this novel pathway of T cell activation.
View details for Web of Science ID A1985TX32500015
View details for PubMedID 3155473
We have described a trinitrophenyl (TNP)-specific inducer clone, clone Ly-1-T1, which responds to a variety of different stimuli, including a) soluble TNP-protein conjugates plus syngeneic (H-2d) spleen cells, b) TNP directly coupled to syngeneic or allogeneic spleen cells, and c) activated I-A identical B cells in the absence of nominal antigen. In the present study we used a panel of antibodies to investigate the recognition structures involved in the activation of clone Ly-1-T1 by these different stimuli. We show that allogeneic spleen cells must be conjugated by using relatively high concentrations of TNBS to be efficient stimulators of the clone. In contrast, syngeneic spleen cells conjugated by using a much wider range of concentrations will activate the clone. The response of the clone to TNP-coupled allogeneic spleen cells is inhibited by anti-L3T4 and anti-Ia antibodies. In contrast, stimulation of the clone with syngeneic spleen cells coupled by using the same concentrations of TNBS is not inhibited with either anti-Ia or anti-L3T4 antibody. The inhibition pattern observed with anti-Ia and anti-L3T4 antibodies was also determined by the nature of the accessory population used to present soluble TNP-protein conjugates. Anti-I-Ad antibodies blocked the activation of clone Ly-1-T1 by TNP-protein plus splenic adherent cells, indicating the involvement of polymorphic I-A determinants in this response. Anti-L3T4 antibody had little or no effect on this response, suggesting that a significant L3T4-Ia interaction is not required. Finally, the response of the clone to activated B cells in the presence or absence of TNP-protein is exquisitely sensitive to inhibition by anti-L3T4 as well as anti-I-A antibodies. The data suggest that the requirement for an L3T4-I interaction depends on the combination of antigen and accessory cell type used to stimulate the clone.
View details for Web of Science ID A1985AQY7500002
View details for PubMedID 3897372
To function efficiently in vivo, lymphocytes must circulate from the blood into lymphoid tissues and other sites of immune reaction. Herein, we show that human cytotoxic and helper T cell clones and lines, maintained in vitro with IL-2, express the functional capacity to recognize and bind to high endothelial venules (HEV), a capacity essential for lymphocyte exit from the blood, and hence for normal lymphocyte trafficking. The expression of functional homing receptors distinguishes human T cell clones from their murine counterparts, which uniformly lack receptors for HEV and are unable to migrate normally from the blood in vivo. The results raise the possibility that human T cell clones may be more effective in mediating in vivo immune responses than is suggested by murine models.
View details for Web of Science ID A1985AQH0300021
View details for PubMedID 3875680
We describe a cloned dendritic cell, clone Den-1, which is a potent accessory cell for some B-cell responses. Clone Den-1 produces a unique lymphokine that induces polyclonal B-cell proliferation in the absence of other costimulators. This clone or factors produced by it also stimulate purified B cells to develop plaque-forming cell responses to type 2 antigens. The effect of this factor(s) on various B-cell populations and its relationship to previously described B-cell-stimulating factors is discussed.
View details for Web of Science ID A1985AAV3800039
View details for PubMedID 3871522
Two long-term cytolytic T lymphocyte (CTL) lines derived from the peripheral blood lymphocytes (PBL) of a single donor were analyzed for target specificity and involvement of cell surface molecules in CTL-target interactions. One line, AH2, was generated after stimulation with B lymphoblastoid cells. Cytolysis by these cells was restricted to targets expressing the appropriate HLA-A2 specificity and was blocked by mAb recognizing CD2, CD3, CD8, LFA-1, and LFA-3. The second line, AE1, was generated after stimulation with cultured endothelial cells derived from human newborn preputial microvessels. These CTL lysed all human target cells tested, except autologous cells and the Class I negative cell line Daudi. In addition, mAb specific for CD2, CD3, and CD8 did not affect cytolysis. Anti-LFA-1 and -LFA-3 mAb blocked cytolysis of B lymphoblastoid targets but not endothelial targets. These results indicate that some CTL utilize as yet uncharacterized cell surface structures for CTL-target interactions.
View details for Web of Science ID A1985AKS7900005
View details for PubMedID 2582029
The effect of heritable LFA-1 deficiency on T lymphocyte function was measured. After primary mixed lymphocyte stimulation, all six patients studied showed diminished allospecific T lymphocyte cytolytic and NK activity as compared with kindred and normal controls. MLR and mitogen-induced proliferative responses were consistently depressed. LFA-1-deficient, EBV-transformed B cell lines were poor stimulators of T cell responses. Primary cytolytic responses by lymphocytes from severely LFA-1-deficient patients (less than 0.2% of normal surface expression) were consistently more profoundly depressed than those by lymphocytes from moderately deficient patients (about 5% of normal surface expression). These results demonstrate the importance of LFA-1 in lymphocyte function. After repeated MLR restimulation, proliferative and cytolytic capacity improved and CTL lines could be established from all patients. Cytolysis by lines from one but not a second severe patient, and by four of four moderate patients, was inhibited by anti-LFA-1 MAb, and at 10-fold lower concentrations than required for inhibition of killing by control CTL lines. The locus of inhibition was on the target cell for the severely deficient CTL line, and on both the target and effector cells for moderately deficient CTL lines. In contrast, the locus of inhibition for normal CTL is on the effector cell. These findings show that LFA-1 can participate bidirectionally in cell interactions. The in vitro results are discussed in terms of the clinical findings in patients.
View details for Web of Science ID A1985ASW4100028
View details for PubMedID 3900204
The major histocompatibility complex class I HLA molecules are the primary determinants recognized by allogeneic cytotoxic T lymphocytes (CTL), and serve as restricting elements for CTL recognition of viral, chemical, or minor histocompatibility antigens. HLA-Aw69 is a naturally occurring hybrid class I molecule that we have used to investigate the regions of class I antigens involved in human CTL recognition. HLA-Aw69 appears to have resulted from an exon shuffle between two closely related class I genes: the alpha 1 domain of HLA-Aw69 is identical to that of HLA-Aw68, while the alpha 2 and alpha 3 domains are identical to HLA-A2. The determinants recognized by human allogeneic CTL clones specific for HLA-A2, -Aw68, and/or -Aw69 fall into three patterns: (a) CTL determinants are located on both the alpha 1 and alpha 2 domains; (b) interaction of the alpha 1 and alpha 2 domains results in new combinatorial determinants; (c) interaction of the alpha 1 and alpha 2 domains in the hybrid molecule results in the loss of CTL determinants that are present on both parental molecules. Thus, using human CTL clones, target cells, and HLA molecules, we show that the interaction of the alpha 1 and alpha 2 domains alters CTL determinants in ways not directly predictable from primary structure.
View details for Web of Science ID A1985ATL1600021
View details for PubMedID 2414389
We have studied the interactions of peripheral blood T lymphocytes with cultured human vascular endothelial cells, focusing upon endothelial cell surface antigens important for T cell recognition. Under standard culture conditions endothelial cells express class I but not class II major histocompatibility complex (MHC) antigens. However, class II antigens may be induced by activated T cells or T cell products, including the lymphokine immune interferon. Immune interferon concomitantly increases class I antigen expression and causes a change in cell shape. In addition to vascular endothelial cells, we have found that vascular smooth muscle cells and human dermal fibroblasts may also be induced by immune interferon to express class II antigens. All known human class II antigens are induced (i.e. HLA-DR, DC and SB) as is the associated invariant chain. Induced antigen expression in these cells is stable over several days, although mRNA levels decline rapidly upon withdrawal of interferon. Vascular and stromal cell class II antigens are functional, in that they can be recognized by cytolytic and helper T cell clones. Several non-MHC antigens are also involved in the recognition of endothelial and stromal cells by T cells. We propose a model for the role of inducible class II molecules on endothelium and stromal cells in vivo: The induction of class II MHC antigens on endothelial cells, locally mediated by activated T cells, enables endothelium to present an immunogenic cell surface structure, comprised of antigen plus self class II polymorphic determinants, which in turn, serves to recruit additional antigen-specific T cells from the circulation into the site of a developing cell mediated immune response. Class II molecules on stromal cells, also induced locally at the site of a developing response, confers immune accessory function on these cells and may serve to augment and sustain a T cell response.
View details for Web of Science ID A1984AAK9500030
View details for PubMedID 6397428
We have derived a Ly-1+, 2-3- T cell clone that is specific for autologous I-A on activated but not on resting cells. After activation, this clone produces factors that induce purified (B + adherent) cells to secrete antibody in response to sheep red blood cells and type 2 T-independent antigens. Regulation of plaque-forming cell responses by this clone is dose dependent: low numbers enhance the plaque-forming cell response, whereas high numbers suppress the response. The inhibition observed with high doses is associated with cytolysis of I-A+ cells, and this can be blocked by the addition of anti-I-A antibodies. The physiologic significance of this novel cell type in regulating immune responses is discussed.
View details for Web of Science ID A1984SN37200015
View details for PubMedID 6201537
Monoclonal antibodies (mAb) against cell surface structures have been used to identify several molecules involved in the interaction of human cytolytic T lymphocytes (CTL) with lymphoid and other bone marrow-derived targets. In allograft rejection or in graft-vs-host disease, however, major cellular targets are vascular and stromal cells, especially endothelium. Yet little is known about whether the same cell surface molecules are involved in the interactions of CTL with these cell types. We assessed the ability of mAb against effector or target cell structures to inhibit cytolysis of susceptible, cultured human vascular endothelium or dermal fibroblasts by a cloned human CTL line. Using mAb reactive with T3, T4, LFA-1, LFA-2, LFA-3, and HLA-DR, we found a qualitatively similar but quantitatively different pattern of inhibition of cytolysis as previously established for lymphoid targets by using the same CTL clone. These results have two implications: 1) the target cell structures recognized by CTL molecules such as T4, LFA-1 and LFA-2 are present on diverse cell types; and 2) the relative importance of such interactions may vary with target cell type. Furthermore, our studies provide several insights into the mechanisms of the interacting molecules. Our model system, and the use of pathophysiologically important target cells, may be useful for further analysis of CTL-mediated immune injury.
View details for Web of Science ID A1984TJ51800031
View details for PubMedID 6432903
We have described a cloned dendritic cell, Clone Den-1, which is a potent accessory cell for some T-dependent immune responses. Clone Den-1 activates T cells in both autologous and allogeneic mixed lymphocyte reactions, but cannot present soluble antigen to T cells that co-recognize nominal antigen plus I-Ab gene products. In addition, Clone Den-1 or factors produced by it can reconstitute plaque-forming cell responses by accessory cell-depleted B plus T cells to the T-dependent antigen sheep red blood cells. The relationship of this clone to heterogeneous populations of dendritic cells and other accessory cells is discussed.
View details for Web of Science ID A1984TF41100016
View details for PubMedID 6379045
Hapten-reactive inducer T cell clones can be divided into two groups based on their activation specificity. The first and largest group is conjugate specific. These clones are activated only by hapten coupled to the same carrier protein used for in vitro selection. The second group, which is quite rare, is hapten specific. Clones of this type are activated by hapten coupled to all foreign and autologus proteins tested. Both types of clones corecognize soluble antigen in association with products of the I-A locus. The hapten-specific cells were used to analyze the molecular basis of I-A vs. I-E gene control. The physiologic significance of hapten- and carrier-specific inducer T cells in the response to foreign antigens and autoantigens is discussed.
View details for Web of Science ID A1983QV71000015
View details for PubMedID 6602202
A human cytotoxic T lymphocyte (CTL) line, A9, was generated by limiting dilution and was selected because of its apparent DC specificity. A9 is 100% OKT3+, 90% OKT4+, and 10% OKT8+, but by negative selection the CTL present are entirely OKT8+. These OKT8+ CTL are totally inhibitable by Genox 3.53, an anti-DC1 monoclonal antibody (mAb), and Leu-10, an anti-DC subgroup mAb, but are not inhibitable by a panel of anti-HLA-DR mAb. These CTL are also inhibitable by anti-OKT3 and anti-LFA-2 but not by OKT4 or OKT8 mAb. These findings extend previous studies that showed that OKT8+ CTL recognize HLA-A,B antigens, whereas OKT4+ CTL recognize HLA-DR and SB antigens. It is possible that an as yet undefined T cell surface molecule is involved in DC recognition.
View details for Web of Science ID A1983RT38500031
View details for PubMedID 6605989
T-lymphocyte-mediated responses to the cellular components of blood vessels are important in rejection of allografts. The induction of cytolytic T lymphocytes (CTLs) depends on recognition of foreign class II major histocompatibility complex antigens (human HLA-DR, DC/DS, SB and others, collectively referred to as Ia) on the target cells whereas killing by CTLs usually depends on recognition of foreign class I antigens (HLA-A, B), although some alloreactive CTLs recognize foreign Ia instead of HLA-A, B (refs 5-8). The expression of Ia antigens has traditionally been regarded as restricted to immunological cell types, and the presence of class II antigen-bearing 'passenger' leukocytes in rodent organ grafts appears necessary for graft rejection. Recently, Ia antigens have been observed by immunofluorescence microscopy on human renal and dermal capillary endothelium. We have previously shown that human umbilical vein endothelial (HUVE) cells in standard culture conditions do not bear Ia antigens, but may be induced to do so by products of lectin- or alloantigen-activated T lymphocytes. Furthermore, we found that recombinant immune interferon (IFN-gamma), free of other lymphokines, is a potent inducer of Ia expression in HUVE cells. Here we report that IFN-gamma also induces Ia expression on human foreskin capillary endothelial (HFCE) cells, HUVE cells transformed by Simian virus 40 viral DNA (SV-HUVE cells) and human dermal fibroblast (HDF) cells in culture. Further, we present evidence that Ia present on HUVE cells and HDF cells can be functionally recognized by human T cells, resulting in a two-way interaction between T cells and mesenchymal cells that may be important in allograft rejection.
View details for Web of Science ID A1983RM23900055
View details for PubMedID 6415484
We have prepared a cDNA probe that detects genes that are rapidly and abundantly expressed after exposure of inducer T-lymphocyte clones to antigen or mitogen. All inducer cells tested express a characteristic set of new mRNA, and these mRNAs are not expressed after activation of other lymphocytes. This initial burst of mRNA synthesis is paralleled by synthesis and secretion of a family of polypeptides that mediate inducer cell activity, including T- and B-cell growth factors, interferon, and molecules that bind to antigen. Expression of this initial genetic program precedes mitosis and is replaced within 74 hr by a different genetic program, which may control further cell division. The action of these sequential sets of genetic programs defines two stages of the cell's differentiation and accounts for altered expression of the cell's immunological functions.
View details for Web of Science ID A1983QW61000046
View details for PubMedID 6602984
The authors review their recent research involving the generation of cytotoxic T lymphocytes (CTL) directed against HLA-DR antigens. A mouse anti-human xenogeneic system first suggested that HLA-DR antigens could be recognized by CTL. Human allogeneic CTL specific for HLA-DR6 were generated and found to be OKT4+. The fact that these CTL were OKT4+ while anti HLA-A,B CTL were OKT8+ suggested that these T cell surface antigens may be involved in MHC antigen recognition; ie, they may be part of the T cell receptor. These OKT4+, HLA-DR specific CTL were further used to generate monoclonal antibodies (1) which block cytolysis and define novel antigens involved in the CTL-target interaction and (2) which define an antigenic complex on alloantigen activated T cells.
View details for PubMedID 6238749
The biologic activity of molecules synthesized and secreted by hapten-specific inducer T cells was examined. After activation, a single inducer clone secretes both antigen-specific inducer peptides as well as nonspecific factors. The nonspecific factors augment the in vitro response of B cells to sheep erythrocytes (SRBC) and Type 2 T-independent antigens. The antigen-specific molecules (ABM) induce plaque-forming cell (PFC) responses in cultures containing ABM, B cells, and antigen that links the epitope recognized by ABM with the B cell epitope. Induction of B cells by ABM is limited to B cells expressing the same I-A allele as the source of the ABM and this reflects binding by ABM to I-A products on B lymphocytes. The data reported here strongly support the view that inducer cells can activate at least some B cells by secretion of a modified form of the T cell surface receptor.
View details for Web of Science ID A1983RV00200007
View details for PubMedID 6196433
OKT4+, HLA-DR-specific CTL were cloned by limiting dilution, and two clones were evaluated. One clone, B8, specifically recognized DR6 antigens, whereas another cloned, C6, recognized an Ia-like determinant on some DR 3, 5, and 6 target cells. Both clones were OKT3+, OKT4+, and OKT8-, and their cytolysis could be blocked by OKT3 and OKT4, but not OKT8, antibodies. A panel of monoclonal antibodies that recognize DR molecules blocked target cell recognition by these OKT+ CTL. Although cloned B8 recognized a DR6-specific determinant, clone C6 appeared to recognize a supratypic determinant that may be common to some DR molecules (e.g., MT2) or possibly another human Ia-like antigen (e.g., SB). The availability of OKT+ CTL clones should help to dissect the Ia-like antigens recognized by human T cells.
View details for Web of Science ID A1982PM14700036
View details for PubMedID 6181153
The regulation of cyclic AMP metabolism in the rat erythrocyte has been investigated during chronic exposure to the beta agonist isoproterenol. A triphasic response is observed: 1) an acute increase in cyclic AMP to levels four- to fivefold greater than basal, maximal by 1 minute (Phase I); 2) a gradual decline in cAMP content to levels near basal during the next 15-20 minutes (Phase II) and a second sustained rise in cAMP, maximal by 60 minutes, to a concentration greater than that observed during the first minute (Phase III). Extensively washed Phase II and Phase III cells are refractory to a second challenge by isoproterenol. In phosphodiesterase-inhibited intact Phase II and III cells adenylate cyclase activity is maximally activated. Isoproterenol has no effect on soluble phosphodiesterase activity but increases membrane-bound phosphodiesterase activity 3- and 2.2-fold in Phase II and Phase III cells, respectively. The activation of this membrane-bound enzyme activity appears to be mediated by the calcium-dependent regulatory protein, calmodulin, because 1) the amount of exogenous calmodulin required to achieve half-maximal activation of membrane-bound phosphodiesterase is 3.7, 2.0, and 1.2 micrograms in control, Phase III and Phase II membranes, respectively; and 2) there is less calmodulin in membrane-free lysates prepared from Phase II cells than control cells. These data support the idea that the major mechanism regulating cAMP content in the rat erythrocyte during chronic isoproterenol stimulation is the membrane-bound phosphodiesterase and that there is a translocation of calmodulin from the cytoplasm to the membrane during hormone stimulation.
View details for Web of Science ID A1981LK73900004
View details for PubMedID 6260952
View details for PubMedID 42982
View details for PubMedID 218310