All Publications

Altered signaling in systemic juvenile idiopathic arthritis monocytes CLINICAL IMMUNOLOGY Macaubas, C., Wong, E., Zhang, Y., Nguyen, K. D., Lee, J., Milojevic, D., Shenoi, S., Stevens, A. M., Ilowite, N., Saper, V., Lee, T., Mellins, E. D. 2016; 163: 66-74
Abstract

Systemic juvenile idiopathic arthritis (sJIA) is characterized by systemic inflammation and arthritis. Monocytes are implicated in sJIA pathogenesis, but their role in disease is unclear. The response of sJIA monocytes to IFN may be dysregulated. We examined intracellular signaling in response to IFN type I (IFN?) and type II (IFN?) in monocytes during sJIA activity and quiescence, in 2 patient groups. Independent of disease activity, monocytes from Group 1 (collected between 2002 and 2009) showed defective STAT1 phosphorylation downstream of IFNs, and expressed higher transcript levels of SOCS1, an inhibitor of IFN signaling. In the Group 2 (collected between 2011 and 2014), monocytes of patients with recent disease onset were IFN? hyporesponsive, but in treated, quiescent subjects, monocytes were hyperresponsive to IFN?. Recent changes in medication in sJIA may alter the IFN hyporesponsiveness. Impaired IFN/pSTAT1 signaling is consistent with skewing of sJIA monocytes away from an M1 phenotype and may contribute to disease pathology.

View details for DOI 10.1016/j.clim.2015.12.011

View details for Web of Science ID 000370585600010

View details for PubMedID 26747737
HLA-DRB1*11 and variants of the MHC class II locus are strong risk factors for systemic juvenile idiopathic arthritis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ombrello, M. J., Remmers, E. F., Tachmazidou, I., Grom, A., Foell, D., Haas, J., Martini, A., Gattorno, M., Ozen, S., Prahalad, S., Zeft, A. S., Bohnsack, J. F., Mellins, E. D., Ilowite, N. T., Russo, R., Len, C., Hilario, M. O., Oliveira, S., Yeung, R. S., Rosenberg, A., Wedderburn, L. R., Anton, J., Schwarz, T., Hinks, A., Bilginer, Y., Park, J., Cobb, J., Satorius, C. L., Han, B., Baskin, E., Signa, S., Duerr, R. H., Achkar, J. P., Kamboh, M. I., Kaufman, K. M., Kottyan, L. C., Pinto, D., Scherer, S. W., Alarcon-Riquelme, M. E., Docampo, E., Estivill, X., Guel, A., de Bakker, P. I., Raychaudhuri, S., Langefeld, C. D., Thompson, S., Zeggini, E., Thomson, W., Kastner, D. L., Woo, P. 2015; 112 (52): 15970-15975
View details for DOI 10.1073/pnas.1520779112

View details for Web of Science ID 000367234700064
Tmem178 acts in a novel negative feedback loop targeting NFATc1 to regulate bone mass PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Decker, C. E., Yang, Z., Rimer, R., Park-Min, K., Macaubas, C., Mellins, E. D., Novack, D. V., Faccio, R. 2015; 112 (51): 15654-15659
View details for DOI 10.1073/pnas.1511285112

View details for Web of Science ID 000366916000047
pH-susceptibility of HLA-DO tunes DO/DM ratios to regulate HLA-DM catalytic activity SCIENTIFIC REPORTS Jiang, W., Strohman, M. J., Somasundaram, S., Ayyangar, S., Hou, T., Wang, N., Mellins, E. D. 2015; 5
View details for DOI 10.1038/srep17333

View details for Web of Science ID 000365399900001
Comparison of biomarkers for systemic juvenile idiopathic arthritis PEDIATRIC RESEARCH Shenoi, S., Ou, J., Ni, C., Macaubass, C., Gersuk, V. H., Wallace, C. A., Mellins, E. D., Stevens, A. M. 2015; 78 (5): 554-559
View details for DOI 10.1038/pr.2015.144

View details for Web of Science ID 000363601700012
Synthesis of Phospholipid-Protein Conjugates as New Antigens for Autoimmune Antibodies MOLECULES Maity, A., Macaubas, C., Mellins, E., Astakhova, K. 2015; 20 (6): 10253-10263
View details for DOI 10.3390/molecules200610253

View details for Web of Science ID 000357992700048
Immunological Basis for Rapid Progression of Diabetes in Older NOD Mouse Recipients Post BM-HSC Transplantation PLOS ONE Wang, N., Rajasekaran, N., Hou, T., Macaubas, C., Mellins, E. D. 2015; 10 (5)
View details for DOI 10.1371/journal.pone.0128494

View details for Web of Science ID 000355187300117
pH-susceptibility of HLA-DO tunes DO/DM ratios to regulate HLA-DM catalytic activity. Scientific reports Jiang, W., Strohman, M. J., Somasundaram, S., Ayyangar, S., Hou, T., Wang, N., Mellins, E. D. 2015; 5: 17333-?
Abstract

The peptide-exchange catalyst, HLA-DM, and its inhibitor, HLA-DO control endosomal generation of peptide/class II major histocompatibility protein (MHC-II) complexes; these complexes traffic to the cell surface for inspection by CD4+ T cells. Some evidence suggests that pH influences DO regulation of DM function, but pH also affects the stability of polymorphic MHC-II proteins, spontaneous peptide loading, DM/MHC-II interactions and DM catalytic activity, imposing challenges on approaches to determine pH effects on DM-DO function and their mechanistic basis. Using optimized biochemical methods, we dissected pH-dependence of spontaneous and DM-DO-mediated class II peptide exchange and identified an MHC-II allele-independent relationship between pH, DO/DM ratio and efficient peptide exchange. We demonstrate that active, free DM is generated from DM-DO complexes at late endosomal/lysosomal pH due to irreversible, acid-promoted DO destruction rather than DO/DM molecular dissociation. Any soluble DM that remains in complex with DO stays inert. pH-exposure of DM-DO in cell lysates corroborates such a pH-regulated mechanism, suggesting acid-activated generation of functional DM in DO-expressing cells.

View details for DOI 10.1038/srep17333

View details for PubMedID 26610428
The MHC Class II Cofactor HLA-DM Interacts with Ig in B Cells JOURNAL OF IMMUNOLOGY Macmillan, H., Strohman, M. J., Ayyangar, S., Jiang, W., Rajasekaran, N., Spura, A., Hessell, A. J., Madec, A., Mellins, E. D. 2014; 193 (6): 2641-2650
Abstract

B cells internalize extracellular Ag into endosomes using the Ig component of the BCR. In endosomes, Ag-derived peptides are loaded onto MHC class II proteins. How these pathways intersect remains unclear. We find that HLA-DM (DM), a catalyst for MHC class II peptide loading, coprecipitates with Ig in lysates from human tonsillar B cells and B cell lines. The molecules in the Ig/DM complexes have mature glycans, and the complexes colocalize with endosomal markers in intact cells. A larger fraction of Ig precipitates with DM after BCR crosslinking, implying that complexes can form when DM meets endocytosed Ig. In vitro, in the endosomal pH range, soluble DM directly binds the Ig Fab domain and increases levels of free Ag released from immune complexes. Taken together, these results argue that DM and Ig intersect in the endocytic pathway of B cells with potential functional consequences.

View details for DOI 10.4049/jimmunol.1400075

View details for Web of Science ID 000341859700004
Susceptibility to HLA-DM Protein Is Determined by a Dynamic Conformation of Major Histocompatibility Complex Class II Molecule Bound with Peptide JOURNAL OF BIOLOGICAL CHEMISTRY Yin, L., Trenh, P., Guce, A., Wieczorek, M., Lange, S., Sticht, J., Jiang, W., Bylsma, M., Mellins, E. D., Freund, C., Stern, L. J. 2014; 289 (34): 23449-23464
Abstract

HLA-DM mediates the exchange of peptides loaded onto MHCII molecules during antigen presentation by a mechanism that remains unclear and controversial. Here, we investigated the sequence and structural determinants of HLA-DM interaction. Peptides interacting nonoptimally in the P1 pocket exhibited low MHCII binding affinity and kinetic instability and were highly susceptible to HLA-DM-mediated peptide exchange. These changes were accompanied by conformational alterations detected by surface plasmon resonance, SDS resistance assay, antibody binding assay, gel filtration, dynamic light scattering, small angle x-ray scattering, and NMR spectroscopy. Surprisingly, all of those changes could be reversed by substitution of the P9 pocket anchor residue. Moreover, MHCII mutations outside the P1 pocket and the HLA-DM interaction site increased HLA-DM susceptibility. These results indicate that a dynamic MHCII conformational determinant rather than P1 pocket occupancy is the key factor determining susceptibility to HLA-DM-mediated peptide exchange and provide a molecular mechanism for HLA-DM to efficiently target unstable MHCII-peptide complexes for editing and exchange those for more stable ones.

View details for DOI 10.1074/jbc.M114.585539

View details for Web of Science ID 000341505000019
Assembly and architecture of the EBV B cell entry triggering complex. PLoS pathogens Sathiyamoorthy, K., Jiang, J., Hu, Y. X., Rowe, C. L., M�hl, B. S., Chen, J., Jiang, W., Mellins, E. D., Longnecker, R., Zhou, Z. H., Jardetzky, T. S. 2014; 10 (8)
Abstract

Epstein-Barr Virus (EBV) is an enveloped double-stranded DNA virus of the gammaherpesvirinae sub-family that predominantly infects humans through epithelial cells and B cells. Three EBV glycoproteins, gH, gL and gp42, form a complex that targets EBV infection of B cells. Human leukocyte antigen (HLA) class II molecules expressed on B cells serve as the receptor for gp42, triggering membrane fusion and virus entry. The mechanistic role of gHgL in herpesvirus entry has been largely unresolved, but it is thought to regulate the activation of the virally-encoded gB protein, which acts as the primary fusogen. Here we study the assembly and function of the reconstituted B cell entry complex comprised of gHgL, gp42 and HLA class II. The structure from negative-stain electron microscopy provides a detailed snapshot of an intermediate state in EBV entry and highlights the potential for the triggering complex to bring the two membrane bilayers into proximity. Furthermore, gHgL interacts with a previously identified, functionally important hydrophobic pocket on gp42, defining the overall architecture of the complex and playing a critical role in membrane fusion activation. We propose a macroscopic model of the initiating events in EBV B cell fusion centered on the formation of the triggering complex in the context of both viral and host membranes. This model suggests how the triggering complex may bridge the two membrane bilayers, orienting critical regions of the N- and C- terminal ends of gHgL to promote the activation of gB and efficient membrane fusion.

View details for DOI 10.1371/journal.ppat.1004309

View details for PubMedID 25144748
Assembly and Architecture of the EBV B Cell Entry Triggering Complex PLOS PATHOGENS Sathiyamoorthy, K., Jiang, J., Hu, Y. X., Rowe, C. L., Moehl, B. S., Chen, J., Jiang, W., Mellins, E. D., Longnecker, R., Zhou, Z. H., Jardetzky, T. S. 2014; 10 (8)
View details for DOI 10.1371/journal.ppat.1004309

View details for Web of Science ID 000341576300031
Serum amyloid A induces mitogenic signals in regulatory T cells via monocyte activation MOLECULAR IMMUNOLOGY Nguyen, K. D., Macaubas, C., Phi Truong, P., Wang, N., Hou, T., Yoon, T., Mellins, E. D. 2014; 59 (2): 172-179
Abstract

Serum amyloid A (SAA) has recently been identified by our group as a mitogen for regulatory T cells (Treg). However, the molecular mechanism by which SAA induces Treg proliferation is unknown. Here we provide evidence that IL-1? and IL-6 are directly involved in the SAA-mediated proliferation of Treg. By engaging its several cognate receptors, SAA induces IL-1? and IL-6 secretion by monocytes and drives them toward an HLA-DR(hi) HVEM(lo) phenotype resembling immature dendritic cells, which have been implicated in tolerance generation. This monocyte-derived cytokine milieu is required for Treg expansion, as inhibition of IL-1? and IL-6 abrogate the ability of SAA to induce Treg proliferation. Furthermore, both IL-1? and IL-6 are required for ERK1/2 and AKT signaling in proliferating Treg. Collectively, these results point to a novel mechanism, by which SAA initiates a monocyte-dependent process that drives mitogenic signals in Treg.

View details for DOI 10.1016/j.molimm.2014.02.011

View details for Web of Science ID 000334988800007
A160: role of interleukin-1 in abnormal monocyte phenotype in systemic onset juvenile idiopathic arthritis. Arthritis & rheumatology Zhang, Y., Macaubas, C., Gupta, S., Klein, C., pascual, V., Hay, A., Thompson, S. D., Sandborg, C. I., Ilowite, N. T., Mellins, E. D. 2014; 66: S207-8
Abstract

Monocytes phenotype changes in different microenvironments: the proinflammatory M1, regulatory M2, and M2-like phenotypes are each regulated by specific transcriptional factors (TFs). We have observed altered phenotypes in blood monocytes in systemic onset juvenile idiopathic arthritis (sJIA), including a decreased M1 cells, an increased mixed M1/M2 cells, and reduced secretion of IL-1, despite an increased IL-1b response to LPS (PMID 22281427). Here, we investigate whether these monocyte phenotypes are affected by IL-1 blockade. We analyzed monocytes from RAPPORT (RAndomized Placebo Phase study Of Rilonacept in the Treatment of sJIA) patients to determine levels of TFs involved in monocyte polarization and expression of genes related to IL-1 secretion, before and after treatment with Rilonacept, an IL-1 trap.Subjects on the Rilonacept arm received active drug from week 0 for a total of 24 weeks; subjects on the placebo arm received placebo for 4 weeks, then Rilonacept for 20 weeks. Blood samples were obtained at week 0, 2, 4, 14 and 24. We used real time PCR to measure M1 associated genes: Interferon Regulatory factor (IRF) family IRF5, STAT1; M2 associated genes IRF4, STAT6, Kruppel-Like Factor 4 (KLF4) and peroxisome proliferator-activated receptor-? (PPAR-?); IL-1 secretion related genes: RAB39, RAB27A, RAB27B, P2RX7, and IL-1?. All TFs levels were normalized by the average levels of 3 housekeeping genes.30 non-paired RNA samples from 15 subjects were tested. Samples from subjects treated with Rilonacept for ?10 weeks (Late RAPPORT) showed decreased expression of M2 genes, especially KLF4, compared to those untreated or treated for <10 weeks (Early RAPPORT) (Fig ), except for PPAR-?. Samples collected when there was clinical improvement also showed reduced KLF4 (Fig .). The expression of TFs following IL-1 inhibition in sJIA patients is distinct from normal controls. Levels of IL-1 secretion related genes, except for RAB27B, also were reduced in "Late RAPPORT" samples (Fig .). [Figure: see text] [Figure: see text] [Figure: see text]IL-1 blockade in sJIA is likely associated with changes in the activation profile and expression of IL-1 secretion related genes in circulating monocytes. Monocytes phenotypes in treated subjects are not "normal" and likely reflect changes associated with compensated inflammation.

View details for DOI 10.1002/art.38586

View details for PubMedID 24677915
HLA-DM and HLA-DO, key regulators of MHC-II processing and presentation CURRENT OPINION IN IMMUNOLOGY Mellins, E. D., Stern, L. J. 2014; 26: 115-122
Abstract

Peptide loading of class II MHC molecules in endosomal compartments is regulated by HLA-DM. HLA-DO modulates HLA-DM function, with consequences for the spectrum of MHC-bound epitopes presented at the cell surface for interaction with T cells. Here, we summarize and discuss recent progress in investigating the molecular mechanisms of action of HLA-DM and HLA-DO and in understanding their roles in immune responses. Key findings are the long-awaited structures of HLA-DM in complex with its class II substrate and with HLA-DO, and observation of a novel phenotype - autoimmunity combined with immunodeficiency - in mice lacking HLA-DO. We also highlight several areas where gaps persist in our knowledge about this pair of proteins and their molecular biology and immunobiology.

View details for DOI 10.1016/j.coi.2013.11.005

View details for Web of Science ID 000333001600016

View details for PubMedID 24463216
The IL-23/IL-17 axis in psoriatic arthritis. Autoimmunity reviews Suzuki, E., Mellins, E. D., Gershwin, M. E., Nestle, F. O., Adamopoulos, I. E. 2014
Abstract

Psoriatic arthritis (PsA) is an immune-mediated chronic inflammatory disease, affecting both the skin and joints. Disease progression is associated with aberrant cytokine expression, and TNF blockade is the most successful therapy to date. However, not all patients are responsive to anti-TNF treatment, highlighting the need to better understand the cellular and molecular mechanisms that govern the disease. PsA associations with single nucleotide polymorphisms in IL23R as well as TRAF3IP2 (Act1), a molecule downstream of the IL-17 receptor (IL-17R), have linked the IL-23/IL-17 axis to disease pathology. Although both cytokines are implicated in PsA, a full picture of their cellular targets and pathogenic mechanisms has not yet emerged. In this review, we focus on the IL-23/IL-17 axis-elicited responses mediated by osteoclasts, keratinocytes and neutrophils. Expanding our understanding of the cellular and molecular mechanisms that dictate pathogenicity in PsA will contribute to developing novel treatment strategies to combat disease.

View details for DOI 10.1016/j.autrev.2014.01.050

View details for PubMedID 24424175
Transgene expression in various organs post BM-HSC transplantation STEM CELL RESEARCH Wang, N., Rajasekaran, N., Hou, T., Mellins, E. D. 2014; 12 (1): 209-221
Abstract

Gene therapy mediated by bone marrow-derived hematopoietic stem cells (BM-HSC) has been widely used in treating genetic deficiencies in both pre-clinical and clinical settings. Using mitotically inactive cell-targeting lentivirus with separate promoters for our gene of interest (the murine MHC class II (MHCII) chaperone, invariant chain (Ii)) and a GFP reporter, we monitored the expression and function of introduced Ii in various types of professional antigen presenting cells (B cells, macrophages and DC) from different organs (spleen, pancreatic lymph nodes (PLN), BM and blood). Ii and GFP were detected. Ii levels correlated with GFP levels only in macrophages and monocytes from spleen, monocytes from PLN and macrophage precursors from blood. By cell type, Ii levels in PLN cells were more similar to those in spleen cells than to those in blood or BM cells. Functionally, Ii expressed in PLN or spleen had more effect on MHCII abundance than Ii expressed in BM or blood. The results have implications for analysis of the outcomes of gene therapy when both therapeutic and reporter genes are introduced. The findings also have implications for understanding the development of immune molecule function.

View details for DOI 10.1016/j.scr.2013.10.010

View details for Web of Science ID 000342286200017
Comparison of transduction efficiency among various lentiviruses containing GFP reporter in bone marrow hematopoietic stem cell transplantation EXPERIMENTAL HEMATOLOGY Wang, N., Rajasekaran, N., Hou, T., Lisowski, L., Mellins, E. D. 2013; 41 (11): 934-943
Abstract

HIV-derived lentiviral vectors have been used widely to transduce non-dividing cells, such as hematopoietic stem cells (HSCs), in the setting of gene therapy. In this study, we screened lentiviral vectors for their ability to drive expression of the murine MHC class II chaperone, invariant chain (Ii) and a GFP reporter. The vectors included T2A vector with T2A-separated Ii and GFP under the same MSCV promoter, dual-promoter vectors with separate promoters for Ii and GFP (called MSCV or EF1a according to the promoter driving Ii expression), and a vector with EF1a driving a fusion of Ii/GFP (called Fusion vector). T2A and MSCV induced the highest levels of Ii and GFP expression, respectively, after direct transfection of 293T cells. All vectors except the Fusion vector drove expression of functional Ii, based on the enhancement of MHC class II level, which is a known consequence of Ii expression. Comparing the vectors after they were packaged into lentiviruses and used to transduce 293T, we found that MSCV and EF1a vectors mediated higher Ii and GFP expression. In ckit(+) bone marrow (BM) cells, MSCV still induced the highest Ii and GFP expression, whereas EF1a induced only robust Ii expression. Regardless of the vector, both Ii and GFP levels were significantly reduced in BM cells compared to 293T cells. When in vivo expression was assessed in cells derived from MSCV-transduced BM-HSCs, up to 80% of myeloid cells were GFP(+), but no Ii expression was observed. In contrast, transplantation of EF1a-transduced BM-HSCs led to much higher in vivo Ii expression. Thus, among those compared, dual-promoter vector-based lentivirus with the EF1a promoter driving the gene of interest is optimal for murine BM-HSC transduction.

View details for DOI 10.1016/j.exphem.2013.07.002

View details for Web of Science ID 000327416700003

View details for PubMedID 23954710
B6.g7 mice reconstituted with BDC2 center dot 5 non-obese diabetic (BDC2 center dot 5NOD) stem cells do not develop autoimmune diabetes CLINICAL AND EXPERIMENTAL IMMUNOLOGY Rajasekaran, N., Wang, N., Hang, Y., Macaubas, C., Rinderknecht, C., Beilhack, G. F., Shizuru, J. A., Mellins, E. D. 2013; 174 (1): 27-37
Abstract

In BDC2.5NOD mice, a spontaneous model of Type 1 diabetes, CD4(+) T cells express a transgene-encoded T-cell receptor (TCR) with reactivity against a pancreatic antigen, chromogranin. This leads to massive infiltration and destruction of the pancreatic islets and subsequent diabetes. When we reconstituted lethally irradiated, lymphocyte-deficient B6.g7 (I-A(g7+) ) Rag(-/-) mice with BDC2.5NOD (ckit(+) Lin(-) Sca-1(hi) ) hematopoietic stem and progenitor cells (HSPC), the recipients exhibited hyperglycemia and succumbed to diabetes. Surprisingly, lymphocyte-sufficient B6.g7 mice reconstituted with BDC2.5NOD HSPCs were protected from diabetes. In this study, we investigated the factors responsible for attenuation of diabetes in the B6.g7 recipients. Analysis of chimerism in the B6.g7 recipients showed that, although B cells and myeloid cells were 98% donor-derived, the CD4(+) T cell compartment contained ?50% host-derived cells. These host-derived CD4(+) T cells were enriched for conventional Tregs (CD25(+) Foxp3(+) ) and also for host-derived CD25(-) Foxp3(-) CD4(+) T cells that express markers of suppressive function, CD73, FR4 and CD39. Though negative selection did not eliminate donor-derived CD4(+) T cells in the B6.g7 recipients, these cells were functionally suppressed. Thus, host-derived CD4(+) T cells that emerge in mice following myeloablation exhibit a regulatory phenoytpe and likely attenuate autoimmune diabetes. These cells may provide new therapeutic strategies to suppress autoimmunity.

View details for DOI 10.1111/cei.12163

View details for Web of Science ID 000324045900004
Susceptibility to Childhood-Onset Rheumatoid Arthritis: Investigation of a Weighted Genetic Risk Score That Integrates Cumulative Effects of Variants at Five Genetic Loci ARTHRITIS AND RHEUMATISM Prahalad, S., Conneely, K. N., Jiang, Y., Sudman, M., Wallace, C. A., Brown, M. R., Ponder, L. A., Rohani-Pichavant, M., Zwick, M. E., Cutler, D. J., Angeles-Han, S. T., Vogler, L. B., Kennedy, C., Rouster-Stevens, K., Wise, C. A., Punaro, M., Reed, A. M., Mellins, E. D., Bohnsack, J. F., Glass, D. N., Thompson, S. D. 2013; 65 (6): 1663-1667
Abstract

Children with childhood-onset rheumatoid arthritis (RA) include those with rheumatoid factor or anti-citrullinated protein antibody-positive juvenile idiopathic arthritis. To test the hypothesis that adult-onset RA-associated variants are also associated with childhood-onset RA, we investigated RA-associated variants at 5 loci in a cohort of patients with childhood-onset RA. We also assessed the cumulative association of these variants in susceptibility to childhood-onset RA using a weighted genetic risk score (wGRS).A total of 155 children with childhood-onset RA and 684 healthy controls were genotyped for 5 variants in the PTPN22, TRAF1/C5, STAT4, and TNFAIP3 loci. High-resolution HLA-DRB1 genotypes were available for 149 cases and 373 controls. We tested each locus for association with childhood-onset RA via logistic regression. We also computed a wGRS for each subject, with weights based on the natural log of the published odds ratios (ORs) for the alleles investigated, and used logistic regression to test the wGRS for association with childhood-onset RA.Childhood-onset RA was associated with TNFAIP3 rs10499194 (OR 0.60 [95% confidence interval 0.44-0.83]), PTPN22 rs2476601 (OR 1.61 [95% confidence interval 1.11-2.31]), and STAT4 rs7574865 (OR 1.41 [95% confidence interval 1.06-1.87]) variants. The wGRS was significantly different between cases and controls (P < 2 � 10(-16) ). Individuals in the third to fifth quintiles of wGRS had a significantly increased disease risk compared to baseline (individuals in the first quintile). Higher wGRS was associated with increased risk of childhood-onset RA, especially among males.The magnitude and direction of the association between TNFAIP3, STAT4, and PTPN22 variants and childhood-onset RA are similar to those observed in RA, suggesting that adult-onset RA and childhood-onset RA share common genetic risk factors. Using a wGRS, we have demonstrated the cumulative association of RA-associated variants with susceptibility to childhood-onset RA.

View details for DOI 10.1002/art.37913

View details for Web of Science ID 000319740600029
Host-derived CD4+T cells attenuate stem cellmediated transfer of autoimmune arthritis in lethally irradiated C57BL/6.g7 mice ARTHRITIS AND RHEUMATISM Rajasekaran, N., Wang, N., Phi Truong, P., Rinderknecht, C., Macaubas, C., Beilhack, G. F., Shizuru, J. A., Mellins, E. D. 2013; 65 (3): 681-692
Abstract

In the K/BxN mouse model of inflammatory arthritis, T cells carrying a transgenic T cell receptor initiate disease by helping B cells to produce arthritogenic anti-glucose-6-phosphate isomerase (anti-GPI) autoantibodies. We found that lethally- irradiated lymphocyte-deficient C57BL/6 (B6).g7 (I-A(g7) +) recombinase-activating gene-deficient (Rag(-/-)) mice reconstituted with K/BxN hematopoietic stem and progenitor cells exhibit arthritis by week 4. In contrast, healthy B6.g7 recipients of K/BxN hematopoietic stem and progenitor cells show only mild arthritis, with limited extent and duration. The objective of this study was to investigate the factors responsible for the attenuation of arthritis in B6.g7 recipients.Antibody responses were measured by enzyme-linked immunosorbent assay. Fluorescence-activated cell sorting analyses were performed for testing chimerism, expression of markers of activation and suppression, tetramer binding, and intracellular cytokines in CD4+ T cells. Suppressive activity of CD4+ T cells was studied by adoptive transfer.Titers of anti-GPI antibodies in reconstituted B6.g7 mice were ?60-fold lower than in reconstituted B6.g7 Rag(-/-) mice. Examination of chimerism in the reconstituted B6.g7 mice showed that B cells and myeloid cells in these mice were donor derived, but CD4+ T cells were primarily host derived and enriched for cells expressing the conventional regulatory markers CD25 and FoxP3. Notably, CD4+CD25-FoxP3- T cells expressed markers of suppressive function (CD73 and folate receptor 4), and delayed disease after adoptive transfer. Activation of donor-derived CD4+ T cells was reduced, and thymic deletion of these cells appeared increased.Despite myeloablation, host CD4+ T cells having a regulatory phenotype emerge in these mice and attenuate autoimmunity.

View details for DOI 10.1002/art.37800

View details for Web of Science ID 000315452400017
HLA-DO acts as a substrate mimic to inhibit HLA-DM by a competitive mechanism NATURE STRUCTURAL & MOLECULAR BIOLOGY Guce, A. I., Mortimer, S. E., Yoon, T., Painter, C. A., Jiang, W., Mellins, E. D., Stern, L. J. 2013; 20 (1): 90-U118
Abstract

Mammalian class II major histocompatibility (MHCII) proteins bind peptide antigens in endosomal compartments of antigen-presenting cells. The nonclassical MHCII protein HLA-DM chaperones peptide-free MHCII, protecting it against inactivation, and catalyzes peptide exchange on loaded MHCII. Another nonclassical MHCII protein, HLA-DO, binds HLA-DM and influences the repertoire of peptides presented by MHCII proteins. However, the mechanism by which HLA-DO functions is unclear. Here we have used X-ray crystallography, enzyme kinetics and mutagenesis approaches to investigate human HLA-DO structure and function. In complex with HLA-DM, HLA-DO adopts a classical MHCII structure, with alterations near the ? subunit's 3?? helix. HLA-DO binds to HLA-DM at the same sites implicated in MHCII interaction, and kinetic analysis showed that HLA-DO acts as a competitive inhibitor. These results show that HLA-DO inhibits HLA-DM function by acting as a substrate mimic, and the findings also limit the possible functional roles for HLA-DO in antigen presentation.

View details for DOI 10.1038/nsmb.2460

View details for Web of Science ID 000313072400015

View details for PubMedID 23222639
Pulse-chase analysis for studies of MHC class II biosynthesis, maturation, and peptide loading. Methods in molecular biology (Clifton, N.J.) Hou, T., Rinderknecht, C. H., Hadjinicolaou, A. V., Busch, R., Mellins, E. 2013; 960: 411-432
Abstract

Pulse-chase analysis is a commonly used technique for studying the synthesis, processing and transport of proteins. Cultured cells expressing proteins of interest are allowed to take up radioactively labeled amino acids for a brief interval ("pulse"), during which all newly synthesized proteins incorporate the label. The cells are then returned to nonradioactive culture medium for various times ("chase"), during which proteins may undergo conformational changes, trafficking, or degradation. Proteins of interest are isolated (usually by immunoprecipitation) and resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the fate of radiolabeled molecules is examined by autoradiography. This chapter describes a pulse-chase protocol suitable for studies of major histocompatibility complex (MHC) class II biosynthesis and maturation. We discuss how results are affected by the recognition by certain anti-class II antibodies of distinct class II conformations associated with particular biosynthetic states. Our protocol can be adapted to follow the fate of many other endogenously synthesized proteins, including viral or transfected gene products, in cultured cells.

View details for DOI 10.1007/978-1-62703-218-6_31

View details for PubMedID 23329504
A New Era in the Treatment of Systemic Juvenile Idiopathic Arthritis NEW ENGLAND JOURNAL OF MEDICINE Sandborg, C., Mellins, E. D. 2012; 367 (25): 2439-2440
View details for DOI 10.1056/NEJMe1212640

View details for Web of Science ID 000312531600015

View details for PubMedID 23252530
Self-antigen recognition by follicular lymphoma B-cell receptors BLOOD Sachen, K. L., Strohman, M. J., Singletary, J., Alizadeh, A. A., Kattah, N. H., Lossos, C., Mellins, E. D., Levy, S., Levy, R. 2012; 120 (20): 4182-4190
Abstract

Follicular lymphoma is a monoclonal B-cell malignancy with each patient's tumor expressing a unique cell surface immunoglobulin (Ig), or B-cell receptor (BCR), that can potentially recognize antigens and/or transduce signals into the tumor cell. Here we evaluated the reactivity of tumor derived Igs for human tissue antigens. Self-reactivity was observed in 26% of tumor Igs (25 of 98). For one follicular lymphoma patient, the recognized self-antigen was identified as myoferlin. This patient's tumor cells bound recombinant myoferlin in proportion to their level of BCR expression, and the binding to myoferlin was preserved despite ongoing somatic hypermutation of Ig variable regions. Furthermore, BCR-mediated signaling was induced after culture of tumor cells with myoferlin. These results suggest that antigen stimulation may provide survival signals to tumor cells and that there is a selective pressure to preserve antigen recognition as the tumor evolves.

View details for DOI 10.1182/blood-2012-05-427534

View details for Web of Science ID 000311637400013

View details for PubMedID 23024238
Genome-Wide Association Meta-Analysis of Eight Independent Systemic Juvenile Idiopathic Arthritis Collections Reveals Regional Association Spanning the Major Histocompatibility Complex Class II and III Gene Cluster Ombrello, M. J., Remmers, E., Grom, A. A., Thomson, W., Martini, A., Gattorno, M., Ozen, S., Prahalad, S., Bohnsack, J. F., Zeft, A., Ilowite, N. T., Mellins, E. D., Russo, R. A., Len, C., Oliveira, S. K., Yeung, R. S., Wedderburn, L. R., Lopez, J. A., Sato-Rius, C., Tachmazidou, I., Langefeld, C. D., Zeggini, E., Thompson, S. D., Woo, P., Kastner, D. L. WILEY-BLACKWELL. 2012: S1126-S1126
View details for Web of Science ID 000309748306167
Mapping the HLA-DO/HLA-DM complex by FRET and mutagenesis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Yoon, T., Macmillan, H., Mortimer, S. E., Jiang, W., Rinderknecht, C. H., Stern, L. J., Mellins, E. D. 2012; 109 (28): 11276-11281
Abstract

HLA-DO (DO) is a nonclassic class II heterodimer that inhibits the action of the class II peptide exchange catalyst, HLA-DM (DM), and influences DM localization within late endosomes and exosomes. In addition, DM acts as a chaperone for DO and is required for its egress from the endoplasmic reticulum (ER). These reciprocal functions are based on direct DO/DM binding, but the topology of DO/DM complexes is not known, in part, because of technical limitations stemming from DO instability. We generated two variants of recombinant soluble DO with increased stability [zippered DO?P11A (szDOv) and chimeric sDO-Fc] and confirmed their conformational integrity and ability to inhibit DM. Notably, we found that our constructs, as well as wild-type sDO, are inhibitory in the full pH range where DM is active (4.7 to ?6.0). To probe the nature of DO/DM complexes, we used intermolecular fluorescence resonance energy transfer (FRET) and mutagenesis and identified a lateral surface spanning the ?1 and ?2 domains of szDO as the apparent binding site for sDM. We also analyzed several sDM mutants for binding to szDOv and susceptibility to DO inhibition. Results of these assays identified a region of DM important for interaction with DO. Collectively, our data define a putative binding surface and an overall orientation of the szDOv/sDM complex and have implications for the mechanism of DO inhibition of DM.

View details for DOI 10.1073/pnas.1113966109

View details for Web of Science ID 000306642100053

View details for PubMedID 22733780
On the perils of poor editing: regulation of peptide loading by HLA-DQ and H2-A molecules associated with celiac disease and type 1 diabetes EXPERT REVIEWS IN MOLECULAR MEDICINE Busch, R., De Riva, A., Hadjinicolaou, A. V., Jiang, W., Hou, T., Mellins, E. D. 2012; 14
Abstract

This review discusses mechanisms that link allelic variants of major histocompatibility complex (MHC) class II molecules (MHCII) to immune pathology. We focus on HLA (human leukocyte antigen)-DQ (DQ) alleles associated with celiac disease (CD) and type 1 diabetes (T1D) and the role of the murine DQ-like allele, H2-Ag7 (I-Ag7 or Ag7), in murine T1D. MHCII molecules bind peptides, and alleles vary in their peptide-binding specificity. Disease-associated alleles permit binding of disease-inducing peptides, such as gluten-derived, Glu-/Pro-rich gliadin peptides in CD and peptides from islet autoantigens, including insulin, in T1D. In addition, the CD-associated DQ2.5 and DQ8 alleles are unusual in their interactions with factors that regulate their peptide loading, invariant chain (Ii) and HLA-DM (DM). The same alleles, as well as other T1D DQ risk alleles (and Ag7), share nonpolar residues in place of Asp at ?57 and prefer peptides that place acidic side chains in a pocket in the MHCII groove (P9). Antigen-presenting cells from T1D-susceptible mice and humans retain CLIP because of poor DM editing, although underlying mechanisms differ between species. We propose that these effects on peptide presentation make key contributions to CD and T1D pathogenesis.

View details for DOI 10.1017/erm.2012.9

View details for Web of Science ID 000307169300001

View details for PubMedID 22805744
Crystal structure of the peptide-exchange inhibitor HLA-DO bound to HLA-DM and insight into the mechanism of DM-facilitated peptide exchange Stern, L., Guce, A., Mortimer, S., Yoon, T., Painter, C., Jiang, W., Mellins, E. PERGAMON-ELSEVIER SCIENCE LTD. 2012: 32-32
View details for DOI 10.1016/j.molimm.2012.02.089

View details for Web of Science ID 000303429800083
Cryptic Fate of Gliadin Epitopes: Implication for Celiac Disease Hou, T., Qiao, S., Jin, X., Sidney, J., Sollid, L., Mellins, E. AMER ASSOC IMMUNOLOGISTS. 2012
View details for Web of Science ID 000304659700626
Crystal structure of the HLA-DM/DO complex and insight into the mechanism of DM-catalyzed peptide exchange Stern, L., Guce, A., Mortimer, S., Yoon, T., Painter, C., Jiang, W., Mellins, E. AMER ASSOC IMMUNOLOGISTS. 2012
View details for Web of Science ID 000304659700628
Interaction of HLA-DM and HLA-DQ2 in antigen presentation: Implications for celiac disease association Hou, T., Qiao, S., Jin, X., Sidney, J., Sollid, L., Macmillan, H., Strohman, M., Yoon, T., Mellins, E. D. PERGAMON-ELSEVIER SCIENCE LTD. 2012: 40-40
View details for DOI 10.1016/j.molimm.2012.03.005

View details for Web of Science ID 000303429800109
Comparison of protein expression by different lentiviral vectors Wang, N., Rajasekaran, N., Hou, T., Mellins, E. AMER ASSOC IMMUNOLOGISTS. 2012
View details for Web of Science ID 000304659700277
Alternative activation in systemic juvenile idiopathic arthritis monocytes CLINICAL IMMUNOLOGY Macaubas, C., Nguyen, K. D., Peck, A., Buckingham, J., Deshpande, C., Wong, E., Alexander, H. C., Chang, S., Begovich, A., Sun, Y., Park, J. L., Pan, K., Lin, R., Lih, C., Augustine, E. M., Phillips, C., Hadjinicolaou, A. V., Lee, T., Mellins, E. D. 2012; 142 (3): 362-372
Abstract

Systemic juvenile idiopathic arthritis (SJIA) is a chronic autoinflammatory condition. The association with macrophage activation syndrome, and the therapeutic efficacy of inhibiting monocyte-derived cytokines, has implicated these cells in SJIA pathogenesis. To characterize the activation state (classical/M1 vs. alternative/M2) of SJIA monocytes, we immunophenotyped monocytes using several approaches. Monocyte transcripts were analyzed by microarray and quantitative PCR. Surface proteins were measured at the single cell level using flow cytometry. Cytokine production was evaluated by intracellular staining and ELISA. CD14(++)CD16(-) and CD14(+)CD16(+) monocyte subsets are activated in SJIA. A mixed M1/M2 activation phenotype is apparent at the single cell level, especially during flare. Consistent with an M2 phenotype, SJIA monocytes produce IL-1? after LPS exposure, but do not secrete it. Despite the inflammatory nature of active SJIA, circulating monocytes demonstrate significant anti-inflammatory features. The persistence of some of these phenotypes during clinically inactive disease argues that this state reflects compensated inflammation.

View details for DOI 10.1016/j.clim.2011.12.008

View details for Web of Science ID 000301036700016

View details for PubMedID 22281427
Hierarchy of risk of childhood-onset rheumatoid arthritis conferred by HLA-DRB1 alleles encoding the shared epitope ARTHRITIS AND RHEUMATISM Prahalad, S., Thompson, S. D., Conneely, K. N., Jiang, Y., Leong, T., Prozonic, J., Brown, M. R., Ponder, L. A., Angeles-Han, S. T., Vogler, L. B., Kennedy, C., Wallace, C. A., Wise, C. A., Punaro, M., Reed, A., Park, J. L., Mellins, E. D., Zeft, A. S., Bohnsack, J. F., Glass, D. N. 2012; 64 (3): 925-930
Abstract

Associations between shared epitope (SE)-encoding HLA-DRB1 alleles and rheumatoid arthritis (RA) are well established. However, only a limited number of studies have investigated these alleles in patients with childhood-onset RA, which is defined as rheumatoid factor- and/or anti-citrullinated protein antibody-positive juvenile idiopathic arthritis. The aims of this study were to investigate the largest cohort of patients with childhood-onset RA for association with SE alleles and to determine whether there is a hierarchy of risk based on the amino acid sequence of the SE.High-resolution HLA-DRB1 genotypes were obtained for 204 patients with childhood-onset RA and 373 healthy control subjects. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated for different SE-encoding HLA-DRB1 alleles. In addition, genotype ORs were calculated for combinations of SE alleles classified into S(2) , S(3P) , or L alleles, based on amino acid sequences in position 70-74 of the DR?1 chain, as proposed by Tezenas du Montcel et al.We confirmed associations between HLA-DRB1 SE alleles and childhood-onset RA (76% of patients carried 1 or 2 SE alleles compared with 46% of control subjects; OR 3.81, 95% CI 2.4-6.0, P < 1 � 10(-7) ). We also observed associations between individual SE alleles (HLA-DRB1*0101, *0401, *0404, *0405, *0408, and *1001) and childhood-onset RA. Genotype-specific risk estimates suggested a hierarchy of risk, with the highest risk among individuals heterozygous for S(2) /S(3P) (OR 22.3, 95% CI 9.9-50.5, P < 0.0001).We confirm the association between SE-encoding HLA-DRB1 alleles and susceptibility to childhood-onset RA. The excess risk conferred by carriage of the combination of S(2) and S(3P) risk alleles suggests that children with DR?1 chains containing the KRAA and QRRAA or RRRAA sequences are especially susceptible to RA.

View details for DOI 10.1002/art.33376

View details for Web of Science ID 000300835900037

View details for PubMedID 21953520
Major Histocompatibility Complex Class II Gene Cluster Harbors Systemic Juvenile Idiopathic Arthritis Susceptibility Locus Ombrello, M. J., Remmers, E., Grom, A. A., Thomson, W., Martini, A., Gattorno, M., Ozen, S., Gul, A., Bohnsack, J. F., Prahalad, S., Zeft, A. S., Mellins, E. D., Satorius, C., Park, J. L., Langefeld, C. D., Zeggini, E., Glass, D. N., Thompson, S. D., Kastner, D. L., Woo, P. WILEY-BLACKWELL. 2011: S659-S660
View details for Web of Science ID 000297621502072
PD-L1 in Systemic Juvenile Idiopathic Arthritis Shenoi, S., Ou, J., Macaubas, C., Mellins, E. D., Wallace, C., Stevens, A. M. WILEY-BLACKWELL. 2011: S98-S98
View details for Web of Science ID 000297621500263
An Insertion Mutant in DQA1*0501 Restores Susceptibility to HLA-DM: Implications for Disease Associations JOURNAL OF IMMUNOLOGY Hou, T., Macmillan, H., Chen, Z., Keech, C. L., Jin, X., Sidney, J., Strohman, M., Yoon, T., Mellins, E. D. 2011; 187 (5): 2442-2452
Abstract

HLA-DM (DM) catalyzes CLIP release, stabilizes MHC class II molecules, and edits the peptide repertoire presented by class II. Impaired DM function may have profound effects on Ag presentation events in the thymus and periphery that are critical for maintenance of self-tolerance. The associations of the HLA-DQ2 (DQ2) allele with celiac disease and type 1 diabetes mellitus have been appreciated for a long time. The explanation for these associations, however, remains unknown. We previously found that DQ2 is a poor substrate for DM. In this study, to further characterize DQ2-DM interaction, we introduced point mutations into DQ2 on the proposed DQ2-DM interface to restore the sensitivity of DQ2 to DM. The effects of mutations were investigated by measuring the peptide dissociation and exchange rate in vitro, CLIP and DQ2 expression on the cell surface, and the presentation of ?-II-gliadin epitope (residues 62-70) to murine, DQ2-restricted T cell hybridomas. We found that the three ?-chain mutations (?+53G, ?+53R, or ?Y22F) decreased the intrinsic stability of peptide-class II complex. More interestingly, the ?+53G mutant restored DQ2 sensitivity to DM, likely due to improved interaction with DM. Our data also suggest that ?-II-gliadin 62-70 is a DM-suppressed epitope. The DQ2 resistance to DM changes the fate of this peptide from a cryptic to an immunodominant epitope. Our findings elucidate the structural basis for reduced DQ2-DM interaction and have implications for mechanisms underlying disease associations of DQ2.

View details for DOI 10.4049/jimmunol.1100255

View details for Web of Science ID 000294059500048

View details for PubMedID 21775680
Pathogenesis of systemic juvenile idiopathic arthritis: some answers, more questions NATURE REVIEWS RHEUMATOLOGY Mellins, E. D., Macaubas, C., Grom, A. A. 2011; 7 (7): 416-426
Abstract

Systemic juvenile idiopathic arthritis (sJIA) has long been recognized as unique among childhood arthritides, because of its distinctive clinical and epidemiological features, including an association with macrophage activation syndrome. Here, we summarize research into sJIA pathogenesis. The triggers of disease are unknown, although infections are suspects. Once initiated, sJIA seems to be driven by innate proinflammatory cytokines. Endogenous Toll-like receptor ligands, including S100 proteins, probably synergize with cytokines to perpetuate inflammation. These and other findings support the hypothesis that sJIA is an autoinflammatory condition. Indeed, IL-1 is implicated as a pivotal cytokine, but the source of excess IL-1 activity remains obscure and the role of IL-1 in chronic arthritis is less clear. Another hypothesis is that a form of hemophagocytic lymphohistiocytosis underlies sJIA, with varying degrees of its expression across the spectrum of disease. Alternatively, sJIA with MAS might be a genetically distinct subtype. Yet another hypothesis proposes that inadequate downregulation of immune activation is central to sJIA, supporting evidence for which includes 'alternative activation' of monocyte and macrophages and possible deficiencies in IL-10 and T regulatory cells. Some altered immune phenotypes persist during clinically inactive disease, which suggests that this stage might represent compensated inflammation. Despite much progress being made, many questions remain, providing fertile ground for future research.

View details for DOI 10.1038/nrrheum.2011.68

View details for Web of Science ID 000292303900007

View details for PubMedID 21647204
Human cytomegalovirus decreases constitutive transcription of MHC class II genes in mature Langerhans cells by reducing CIITA transcript levels MOLECULAR IMMUNOLOGY Lee, A. W., Wang, N., Hornell, T. M., Harding, J. J., Deshpande, C., Hertel, L., Lacaille, V., Pashine, A., Macaubas, C., Mocarski, E. S., Mellins, E. D. 2011; 48 (9-10): 1160-1167
Abstract

Human cytomegalovirus (HCMV) productively infects CD34(+) progenitor-derived, mature Langerhans-type dendritic cells (matLC) and reduces surface expression of MHC class II complexes (MHC II) by increasing intracellular retention of these molecules. To determine whether HCMV also inhibits MHC II expression by other mechanisms, we assessed mRNA levels of the class II transcriptional regulator, CIITA, and several of its target genes in infected matLC. Levels of CIITA, HLA-DRA (DRA) and DRB transcripts, and new DR protein synthesis were compared in mock-infected and HCMV-infected cells by quantitative PCR and pulse-chase immunoprecipitation analyses, respectively. CIITA mRNA levels were significantly lower in HCMV-infected matLC as compared to mock-infected cells. When assessed in the presence of Actinomycin D, the stability of CIITA transcripts was not diminished by HCMV. Analysis of promoter-specific CIITA isoforms revealed that types I, III and IV all were decreased by HCMV, a result that differs from changes after incubation of these cells with lipopolysaccharide (LPS). Exposure to UV-inactivated virus failed to reduce CIITA mRNA levels, implicating de novo viral gene expression in this effect. HCMV-infected matLC also expressed lower levels of DR transcripts and reduced DR protein synthesis rates compared to mock-infected matLC. In summary, we demonstrate that HCMV infection of a human dendritic cell subset inhibits constitutive CIITA expression, most likely at the transcriptional level, resulting in reduced MHC II biosynthesis. We suggest this represents a new mechanism of modulation of mature LC by HCMV.

View details for DOI 10.1016/j.molimm.2011.02.010

View details for Web of Science ID 000290929400010

View details for PubMedID 21458073
Serum amyloid A overrides T-reg anergy via monocyte-dependent and T-reg-intrinsic, SOCS3-associated pathways BLOOD Nguyen, K. D., Macaubas, C., Nadeau, K. C., Phi Truong, T., Yoon, T., Lee, T., Park, J. L., Mellins, E. D. 2011; 117 (14): 3793-3798
Abstract

The acute phase protein serum amyloid A (SAA) has been well characterized as an indicator of inflammation. Nevertheless, its functions in pro versus anti-inflammatory processes remain obscure. Here we provide unexpected evidences that SAA induces the proliferation of the tolerogenic subset of regulatory T cells (T(reg)). Intriguingly, SAA reverses T(reg) anergy via its interaction with monocytes to activate distinct mitogenic pathways in T(reg) but not effector T cells. This selective responsiveness of T(reg) correlates with their diminished expression of SOCS3 and is antagonized by T(reg)-specific induction of this regulator of cytokine signaling. Collectively, these evidences suggest a novel anti-inflammatory role of SAA in the induction of a micro-environment that supports T(reg) expansion at sites of infection or tissue injury, likely to curb (auto)-inflammatory responses.

View details for DOI 10.1182/blood-2010-11-318832

View details for Web of Science ID 000289265500014

View details for PubMedID 21325601
Chaperone Activity of alpha B-Crystallin Is Responsible for Its Incorrect Assignment as an Autoantigen in Multiple Sclerosis JOURNAL OF IMMUNOLOGY Rothbard, J. B., Zhao, X., Sharpe, O., Strohman, M. J., Kurnellas, M., Mellins, E. D., Robinson, W. H., Steinman, L. 2011; 186 (7): 4263-4268
Abstract

For 15 y, ? B-crystallin (heat shock protein [Hsp] B5) has been labeled an autoantigen in multiple sclerosis (MS) based on humoral and cellular responses found in humans and animal models. However, there have been several scientific inconsistencies with this assignment, ranging from studies demonstrating small differences in anticrystallin responses between patients and healthy individuals to the inability of crystallin-specific T cells to induce symptoms of experimental allergic encephalomyelitis in animal models. Experiments in this article demonstrate that the putative anti-HspB5 Abs from 23 MS patients cross-react with 7 other members of the human small Hsp family and were equally present in normal plasma. Biolayer interferometry demonstrates that the binding was temperature dependent, and that the calculated K(a) increased as the concentration of the sHsp decreased. These two patterns are characteristic of multiple binding sites with varying affinities, the composition of which changes with temperature, supporting the hypothesis that HspB5 bound the Ab and not the reverse. HspB5 also precipitated Ig heavy and L chains from sera from patients with MS. These results establish that small Hsps bind Igs with high affinity and refute much of the serological data used to assign ? B-crystallin as an autoantigen.

View details for DOI 10.4049/jimmunol.1003934

View details for Web of Science ID 000288751200051

View details for PubMedID 21357544
Plasma profiles in active systemic juvenile idiopathic arthritis: Biomarkers and biological implications PROTEOMICS Ling, X. B., Park, J. L., Carroll, T., Nguyen, K. D., Lau, K., Macaubas, C., Chen, E., Lee, T., Sandborg, C., Milojevic, D., Kanegaye, J. T., Gao, S., Burns, J., Schilling, J., Mellins, E. D. 2010; 10 (24): 4415-4430
Abstract

Systemic juvenile idiopathic arthritis (SJIA) is a chronic arthritis of children characterized by a combination of arthritis and systemic inflammation. There is usually non-specific laboratory evidence of inflammation at diagnosis but no diagnostic test. Normalized volumes from 89/889 2-D protein spots representing 26 proteins revealed a plasma pattern that distinguishes SJIA flare from quiescence. Highly discriminating spots derived from 15 proteins constitute a robust SJIA flare signature and show specificity for SJIA flare in comparison to active polyarticular juvenile idiopathic arthritis or acute febrile illness. We used 7 available ELISA assays, including one to the complex of S100A8/S100A9, to measure levels of 8 of the15 proteins. Validating our DIGE results, this ELISA panel correctly classified independent SJIA flare samples, and distinguished them from acute febrile illness. Notably, data using the panel suggest its ability to improve on erythrocyte sedimentation rate or C-reactive protein or S100A8/S100A9, either alone or in combination in SJIA F/Q discriminations. Our results also support the panel's potential clinical utility as a predictor of incipient flare (within 9?wk) in SJIA subjects with clinically inactive disease. Pathway analyses of the 15 proteins in the SJIA flare versus quiescence signature corroborate growing evidence for a key role for IL-1 at disease flare.

View details for DOI 10.1002/pmic.201000298

View details for Web of Science ID 000285882200008

View details for PubMedID 21136595
DM influences the abundance of major histocompatibility complex class II alleles with low affinity for class II-associated invariant chain peptides via multiple mechanisms IMMUNOLOGY Rinderknecht, C. H., Roh, S., Pashine, A., Belmares, M. P., Patil, N. S., Lu, N., Truong, P., Hou, T., Macaubas, C., Yoon, T., Wang, N., Busch, R., Mellins, E. D. 2010; 131 (1): 18-32
Abstract

DM catalyses class II-associated invariant chain peptide (CLIP) release, edits the repertoire of peptides bound to major histocompatibility complex (MHC) class II molecules, affects class II structure, and thereby modulates binding of conformation-sensitive anti-class II antibodies. Here, we investigate the ability of DM to enhance the cell surface binding of monomorphic antibodies. We show that this enhancement reflects increases in cell surface class II expression and total cellular abundance, but notably these effects are selective for particular alleles. Evidence from analysis of cellular class II levels after cycloheximide treatment and from pulse-chase experiments indicates that DM increases the half-life of affected alleles. Unexpectedly, the pulse-chase experiments also revealed an early effect of DM on assembly of these alleles. The allelically variant feature that correlates with susceptibility to these DM effects is low affinity for CLIP; DM-dependent changes in abundance are reduced by invariant chain (CLIP) mutants that enhance CLIP binding to class II. We found evidence that DM mediates rescue of peptide-receptive DR0404 molecules from inactive forms in vitro and evidence suggesting that a similar process occurs in cells. Thus, multiple mechanisms, operating along the biosynthetic pathway of class II molecules, contribute to DM-mediated increases in the abundance of low-CLIP-affinity alleles.

View details for DOI 10.1111/j.1365-2567.2010.03282.x

View details for Web of Science ID 000280660800003

View details for PubMedID 20408893
Macrophage activation syndrome: advances towards understanding pathogenesis CURRENT OPINION IN RHEUMATOLOGY Grom, A. A., Mellins, E. D. 2010; 22 (5): 561-566
Abstract

Macrophage activation syndrome (MAS), a major cause of morbidity and mortality in pediatric rheumatology, is most strongly associated with systemic juvenile idiopathic arthritis (SJIA). There are no validated diagnostic criteria and early diagnosis is difficult. This review summarizes the progress in understanding of MAS pathophysiology that may help define specific diagnostic biomarkers.MAS is similar to the autosomal recessive disorders collectively known as familial hemophagocytic lymphohistiocytosis (FHLH), all associated with various genetic defects affecting the cytolytic pathway. Cytolytic function is profoundly depressed in SJIA with MAS as well. This immunologic abnormality distinguishes SJIA from other rheumatic diseases and is caused by both genetic and acquired factors. Phenotypic characterization of hemophagocytic macrophages has been another focus of research. These macrophages express CD163, a scavenger receptor that binds hemoglobin-haptoglobin complexes, and initiate pathways important for adaptation to oxidative stress induced by free iron. Expansion of these macrophages is seen in more than 30% of SJIA patients perhaps representing early stages of MAS. Recent gene expression studies linked expansion of these macrophages to distinct signatures.Recent advances in understanding of pathophysiologic conditions that favor expansion of hemophagocytic macrophages provide a source of new MAS biomarkers with applicability to clinical practice.

View details for DOI 10.1097/01.bor.0000381996.69261.71

View details for Web of Science ID 000280457800015

View details for PubMedID 20517154
Monocytes are resistant to apoptosis in systemic juvenile idiopathic arthritis CLINICAL IMMUNOLOGY Srivastava, S., Macaubas, C., Deshpande, C., Alexander, H. C., Chang, S., Sun, Y., Park, J. L., Lee, T., Begovich, A., Mellins, E. D. 2010; 136 (2): 257-268
Abstract

We investigated whether circulating monocytes from patients with systemic juvenile idiopathic arthritis (SJIA) are resistant to apoptosis and which apoptotic pathway(s) may mediate this resistance. A microarray analysis of peripheral blood mononuclear cells (PBMC) of SJIA samples and RT-PCR analysis of isolated monocytes showed that monocytes from active SJIA patients express transcripts that imply resistance to apoptosis. SJIA monocytes incubated in low serum show reduced annexin binding and diminished FasL up-regulation compared to controls. SJIA monocytes are less susceptible to anti-Fas-induced apoptosis and, upon activation of the mitochondrial pathway with staurosporine, show diminished Bid cleavage and Bcl-w down-regulation compared to controls. Exposure to SJIA plasma reduces responses to apoptotic triggers in normal monocytes. Thus, SJIA monocytes are resistant to apoptosis due to alterations in both the extrinsic and intrinsic apoptosis pathways, and circulating factors associated with active SJIA may confer this phenotype.

View details for DOI 10.1016/j.clim.2010.04.003

View details for Web of Science ID 000280222200012

View details for PubMedID 20462799
I-A(g7) is subject to post-translational chaperoning by CLIP INTERNATIONAL IMMUNOLOGY Rinderknecht, C. H., Lu, N., Crespo, O., Truong, P., Hou, T., Wang, N., Rajasekaran, N., Mellins, E. D. 2010; 22 (8): 705-716
Abstract

Several MHC class II alleles linked with autoimmune diseases form unusually low-stability complexes with class II-associated invariant chain peptides (CLIP), leading us to hypothesize that this is an important feature contributing to autoimmune pathogenesis. We recently demonstrated a novel post-endoplasmic reticulum (ER) chaperoning role of the CLIP peptides for the murine class II allele I-E(d). In the current study, we tested the generality of this CLIP chaperone function using a series of invariant chain (Ii) mutants designed to have varying CLIP affinity for I-A(g7). In cells expressing these Ii CLIP mutants, I-A(g7) abundance, turnover and antigen presentation are all subject to regulation by CLIP affinity, similar to I-E(d). However, I-A(g7) undergoes much greater quantitative changes than observed for I-E(d). In addition, we find that Ii with a CLIP region optimized for I-A(g7) binding may be preferentially assembled with I-A(g7) even in the presence of higher levels of wild-type Ii. This finding indicates that, although other regions of Ii interact with class II, CLIP binding to the groove is likely to be a dominant event in assembly of nascent class II molecules with Ii in the ER.

View details for DOI 10.1093/intimm/dxq056

View details for Web of Science ID 000280281000009

View details for PubMedID 20547545
Masking of a cathepsin G cleavage site in vivo contributes to the proteolytic resistance of major histocompatibility complex class II molecules IMMUNOLOGY Burster, T., Macmillan, H., Hou, T., Schilling, J., Truong, P., Boehm, B. O., Zou, F., Lau, K., Strohman, M., Schaffert, S., Busch, R., Mellins, E. D. 2010; 130 (3): 436-446
Abstract

The expression of major histocompatibility complex class II (MHC II) molecules is post-translationally regulated by endocytic protein turnover. Here, we identified the serine protease cathepsin G (CatG) as an MHC II-degrading protease by in vitro screening and examined its role in MHC II turnover in vivo. CatG, uniquely among endocytic proteases tested, initiated cleavage of detergent-solubilized native and recombinant soluble MHC II molecules. CatG cleaved human leukocyte antigen (HLA)-DR isolated from both HLA-DM-expressing and DM-null cells. Even following CatG cleavage, peptide binding was retained by pre-loaded, soluble recombinant HLA-DR. MHC II cleavage occurred on the loop between fx1 and fx2 of the membrane-proximal beta2 domain. All allelic variants of HLA-DR tested and murine I-A(g7) class II molecules were susceptible, whereas murine I-E(k) and HLA-DM were not, consistent with their altered sequence at the P1' position of the CatG cleavage site. CatG effects were reduced on HLA-DR molecules with DRB mutations in the region implicated in interaction with HLA-DM. In contrast, addition of CatG to intact B-lymphoblastoid cell lines (B-LCLs) did not cause degradation of membrane-bound MHC II. Moreover, inhibition or genetic ablation of CatG in primary antigen-presenting cells did not cause accumulation of MHC II molecules. Thus, in vivo, the CatG cleavage site is sterically inaccessible or masked by associated molecules. A combination of intrinsic and context-dependent proteolytic resistance may allow peptide capture by MHC II molecules in harshly proteolytic endocytic compartments, as well as persistent antigen presentation in acute inflammatory settings with extracellular proteolysis.

View details for DOI 10.1111/j.1365-2567.2010.03247.x

View details for Web of Science ID 000278619800014

View details for PubMedID 20331476
Distribution of circulating cells in systemic juvenile idiopathic arthritis across disease activity states CLINICAL IMMUNOLOGY Macaubas, C., Nguyen, K., Deshpande, C., Phillips, C., Peck, A., Lee, T., Park, J. L., Sandborg, C., Mellins, E. D. 2010; 134 (2): 206-216
Abstract

Juvenile idiopathic arthritis (JIA) encompasses a group of chronic childhood arthritides of unknown etiology. One subtype, systemic JIA (SJIA), is characterized by a combination of arthritis and systemic inflammation. Its systemic nature suggests that clues to SJIA pathogenesis may be found in examination of peripheral blood cells. To determine the immunophenotypic profiles of circulating mononuclear cells in SJIA patients with different degrees of disease activity, we studied PBMC from 31 SJIA patients, 20 polyarticular JIA patients (similar to adult rheumatoid arthritis), and 31 age-matched controls. During SJIA disease flare, blood monocyte numbers were increased, whereas levels of myeloid dendritic cells (DC) and gammadelta T cells were reduced. At both flare and quiescence, increased levels of CD14 and CD16 were found on SJIA monocytes. Levels of CD16-DC were elevated at SJIA quiescence compared both to healthy controls and to SJIA subjects with active disease. Overall, our findings suggest dysregulation of innate immunity in SJIA and raise the possibility that quiescence represents a state of compensated inflammation.

View details for DOI 10.1016/j.clim.2009.09.010

View details for Web of Science ID 000273701800013

View details for PubMedID 19879195
Plasticity of T-cell phenotype and function: the T helper type 17 example IMMUNOLOGY Peck, A., Mellins, E. D. 2010; 129 (2): 147-153
Abstract

Mature T helper type 1 (Th1) and Th2 cells antagonize the development of the opposing subset to sustain lineage-specific responses. However, the recent identification of a third distinct subset of helper T cells - the Th17 lineage - collapses the established Th1/Th2 dichotomy and raises intriguing questions about T-cell fate. In this review, we discuss the Th17 subset in the context of the effector and regulatory T-cell lineages. Initial studies suggested reciprocal developmental pathways between Th17/Th1 subsets and between Th17/regulatory T-cell subsets, and identified multiple mechanisms by which Th1 and Th2 cells antagonize the generation of Th17 cells. However, recent observations reveal the susceptibility of differentiated Th17 cells to Th1 polarization and the enhancement of Th17 memory cells by the Th1 factors interferon-gamma and T-bet. In addition, new data indicate late-stage plasticity of a subpopulation of regulatory T cells, which can be selectively induced to adopt a Th17 phenotype. Elucidating the mechanisms that undermine cross-lineage suppression and facilitate these phenotype shifts will not only clarify the flexibility of T-cell differentiation, but may also shed insight into the pathogenesis of autoimmunity and cancer. Furthermore, understanding these phenomena will be critical for the design of immunotherapy that seeks to disrupt lineage-specific T-cell responses and may suggest ways to manipulate the balance between pathogenic and regulatory lymphocytes for the restoration of homeostasis.

View details for DOI 10.1111/j.1365-2567.2009.03189.x

View details for Web of Science ID 000273458000001

View details for PubMedID 19922424
Precarious Balance: Th17 Cells in Host Defense INFECTION AND IMMUNITY Peck, A., Mellins, E. D. 2010; 78 (1): 32-38
Abstract

Lineage-specific responses from the effector T-cell repertoire form a critical component of adaptive immunity. The recent identification of Th17 cells-a third, distinct lineage of helper T cells-collapses the long-accepted paradigm in which Th1 and Th2 cells distinctly mediate cellular and humoral immunity, respectively. In this minireview, we discuss the involvement of the Th17 lineage during infection by extracellular bacteria, intracellular bacteria, and fungi. Emerging trends suggest that the Th17 population bridges innate and adaptive immunity to produce a robust antimicrobial inflammatory response. However, because Th17 cells mediate both host defense and pathological inflammation, elucidation of mechanisms that attenuate but do not completely abolish the Th17 response may have powerful implications for therapy.

View details for DOI 10.1128/IAI.00929-09

View details for Web of Science ID 000272984300002

View details for PubMedID 19901061
Cathepsin G: Roles in antigen presentation and beyond MOLECULAR IMMUNOLOGY Burster, T., Macmillan, H., Hou, T., Boehm, B. O., Mellins, E. D. 2010; 47 (4): 658-665
Abstract

Contributions from multiple cathepsins within endosomal antigen processing compartments are necessary to process antigenic proteins into antigenic peptides. Cysteine and aspartyl cathepsins have been known to digest antigenic proteins. A role for the serine protease, cathepsin G (CatG), in this process has been described only recently, although CatG has long been known to be a granule-associated proteolytic enzyme of neutrophils. In line with a role for this enzyme in antigen presentation, CatG is found in endocytic compartments of a variety of antigen presenting cells. CatG is found in primary human monocytes, B cells, myeloid dendritic cells 1 (mDC1), mDC2, plasmacytoid DC (pDC), and murine microglia, but is not expressed in B cell lines or monocyte-derived DC. Purified CatG can be internalized into endocytic compartments in CatG non-expressing cells, widening the range of cells where this enzyme may play a role in antigen processing. Functional assays have implicated CatG as a critical enzyme in processing of several antigens and autoantigens. In this review, historical and recent data on CatG expression, distribution, function and involvement in disease will be summarized and discussed, with a focus on its role in antigen presentation and immune-related events.

View details for DOI 10.1016/j.molimm.2009.10.003

View details for Web of Science ID 000274507400004

View details for PubMedID 19910052
URINE PEPTIDOMICS FOR CLINICAL BIOMARKER DISCOVERY ADVANCES IN CLINICAL CHEMISTRY, VOL 51 Ling, X. B., Mellins, E. D., Sylvester, K. G., Cohen, H. J. 2010; 51: 181-213
Abstract

Urine-based proteomic profiling is a novel approach that may result in the discovery of noninvasive biomarkers for diagnosing patients with different diseases, with the aim to ultimately improve clinical outcomes. Given new and emerging analytical technologies and data mining algorithms, the urine peptidome has become a rich resource to uncover naturally occurring peptide biomarkers for both systemic and renal diseases. However, significant analytical hurdles remain in sample collection and storage, experimental design, data analysis, and statistical inference. This study summarizes, focusing on our experiences and perspectives, the progress in addressing these challenges to enable high-throughput urine peptidomics-based biomarker discovery.

View details for DOI 10.1016/S0065-2423(10)51007-2

View details for Web of Science ID 000281865700007

View details for PubMedID 20857622
Urine Peptidomic and Targeted Plasma Protein Analyses in the Diagnosis and Monitoring of Systemic Juvenile Idiopathic Arthritis. Clinical proteomics Ling, X. B., Lau, K., Deshpande, C., Park, J. L., Milojevic, D., Macaubas, C., Xiao, C., Lopez-Avila, V., Kanegaye, J., Burns, J. C., Cohen, H., Schilling, J., Mellins, E. D. 2010; 6 (4): 175-193
Abstract

PURPOSE: Systemic juvenile idiopathic arthritis is a chronic pediatric disease. The initial clinical presentation can mimic other pediatric inflammatory conditions, which often leads to significant delays in diagnosis and appropriate therapy. SJIA biomarker development is an unmet diagnostic/prognostic need to prevent disease complications. EXPERIMENTAL DESIGN: We profiled the urine peptidome to analyze a set of 102 urine samples, from patients with SJIA, Kawasaki disease (KD), febrile illnesses (FI), and healthy controls. A set of 91 plasma samples, from SJIA flare and quiescent patients, were profiled using a customized antibody array against 43 proteins known to be involved in inflammatory and protein catabolic processes. RESULTS: We identified a 17-urine-peptide biomarker panel that could effectively discriminate SJIA patients at active, quiescent, and remission disease states, and patients with active SJIA from confounding conditions including KD and FI. Targeted sequencing of these peptides revealed that they fall into several tight clusters from seven different proteins, suggesting disease-specific proteolytic activities. The antibody array plasma profiling identified an SJIA plasma flare signature consisting of tissue inhibitor of metalloproteinase-1 (TIMP1), interleukin (IL)-18, regulated upon activation, normal T cell expressed and secreted (RANTES), P-Selectin, MMP9, and L-Selectin. CONCLUSIONS AND CLINICAL RELEVANCE: The urine peptidomic and plasma protein analyses have the potential to improve SJIA care and suggest that SJIA urine peptide biomarkers may be an outcome of inflammation-driven effects on catabolic pathways operating at multiple sites. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12014-010-9058-8) contains supplementary material, which is available to authorized users.

View details for PubMedID 21124648
Oligoarticular and polyarticular JIA: epidemiology and pathogenesis NATURE REVIEWS RHEUMATOLOGY Macaubas, C., Nguyen, K., Milojevic, D., Park, J. L., Mellins, E. D. 2009; 5 (11): 616-626
Abstract

Juvenile idiopathic arthritis (JIA) refers to a group of chronic childhood arthropathies of unknown etiology, currently classified into subtypes primarily on the basis of clinical features. Research has focused on the hypothesis that these subtypes arise through distinct etiologic pathways. In this Review, we discuss four subtypes of JIA: persistent oligoarticular, extended oligoarticular, rheumatoid-factor-positive polyarticular and rheumatoid-factor-negative polyarticular. These subtypes differ in prevalence between ethnic groups and are associated with different HLA alleles. Non-HLA genetic risk factors have also been identified, some of which reveal further molecular differences between these subtypes, while others suggest mechanistic overlap. Investigations of immunophenotypes also provide insights into subtype differences: adaptive immunity seems to have a prominent role in both polyarticular and oligoarticular JIA, and the more-limited arthritis observed in persistent oligoarticular JIA as compared with extended oligoarticular JIA may reflect more-potent immunoregulatory T-cell activity in the former. Tumor necrosis factor seems to be a key mediator of both polyarticular and oligoarticular JIA, especially in the extended oligoarticular subtype, although elevated levels of other cytokines are also observed. Limited data on monocytes, dendritic cells, B cells, natural killer T cells and neutrophils suggest that the contributions of these cells differ across subtypes of JIA. Within each subtype, however, common pathways seem to drive joint damage.

View details for DOI 10.1038/nrrheum.2009.209

View details for Web of Science ID 000271246900007

View details for PubMedID 19806151
Breaking old paradigms: Th17 cells in autoimmune arthritis CLINICAL IMMUNOLOGY Peck, A., Mellins, E. D. 2009; 132 (3): 295-304
Abstract

Aberrant helper T cell activation has been implicated in the pathogenesis of an array of autoimmune diseases. In this review, we summarize evidence that suggests the involvement of a novel T cell subset, the Th17 lineage, in rheumatoid arthritis. In particular, we focus on the role of Th17 cells in inducing and perpetuating the chronic inflammation, cartilage damage, and bone erosion that are hallmark phases of joint destruction and consider current and emerging therapies that seek to disrupt the inflammatory Th17 network and shift the immune system back towards homeostasis.

View details for DOI 10.1016/j.clim.2009.03.522

View details for Web of Science ID 000268783900001

View details for PubMedID 19403336
Complexes of two cohorts of CLIP peptides and HLA-DQ2 of the autoimmune DR3-DQ2 haplotype are poor substrates for HLA-DM JOURNAL OF IMMUNOLOGY Fallang, L., Roh, S., Holm, A., Bergseng, E., Yoon, T., Fleckenstein, B., Bandyopadhyay, A., Mellins, E. D., Sollid, L. M. 2008; 181 (8): 5451-5461
Abstract

Atypical invariant chain (Ii) CLIP fragments (CLIP2) have been found in association with HLA-DQ2 (DQ2) purified from cell lysates. We mapped the binding register of CLIP2 (Ii 96-104) to DQ2 and found proline at the P1 position, in contrast to the canonical CLIP1 (Ii 83-101) register with methionine at P1. CLIP1/2 peptides are the predominant peptide species, even for DQ2 from HLA-DM (DM)-expressing cells. We hypothesized that DQ2-CLIP1/2 might be poor substrates for DM. We measured DM-mediated exchange of CLIP and other peptides for high-affinity indicator peptides and found it is inefficient for DQ2. DM-DQ-binding and DM chaperone effects on conformation and levels of DQ are also reduced for DQ2, compared with DQ1. We suggest that the unusual interaction of DQ2 with Ii and DM may provide a basis for the known disease associations of DQ2.

View details for Web of Science ID 000260025300037

View details for PubMedID 18832702
Modulation of Peripheral B Cell Tolerance by CD72 in a Murine Model ARTHRITIS AND RHEUMATISM Li, D. H., Winslow, M. M., Cao, T. M., Chen, A. H., Davis, C. R., Mellins, E. D., Utz, P. J., Crabtree, G. R., Parnes, J. R. 2008; 58 (10): 3192-3204
Abstract

B cells play a dominant role in the pathogenesis of several autoimmune diseases, including systemic lupus erythematosus. It is not well understood how B cell signaling contributes to autoantibody production. The goal of this study was to elucidate the role of CD72 in modulating B cell receptor (BCR)-mediated tolerogenic signaling and peripheral B cell tolerance.A mouse model utilizing hen egg lysozyme (HEL) "anergic" B cells was studied. CD72-deficient mice carrying the BCR-specific IgHEL and/or soluble HEL (sHEL) transgenes were generated by breeding IgHEL-transgenic MD4 mice and/or sHEL-transgenic ML5 mice with congenic, CD72-deficient C57BL/6J mice. Normal and anergic B cells were isolated for analyses of B cell signaling. Aged wild-type and CD72-deficient mice were also examined for autoimmune phenomena.In the absence of CD72, anergic B cells inappropriately proliferated and survived in response to stimulation with self antigen. Biochemical analyses indicated that in anergic B cells, CD72 dominantly down-regulated BCR signaling to limit the antigen-induced elevation in [Ca2+]i and the activation of NFATc1, NF-kappaB, MAPK, and Akt. Mechanistically, CD72 was associated with, and regulated, the molecular adaptor Cbl-b in anergic B cells, suggesting that Cbl-b may play a role in mediating the negative effects of CD72 on BCR signaling. Moreover, in aged CD72-deficient mice, spontaneous production of antinuclear and anti-double-stranded DNA autoantibodies and features of lupus-like autoimmune disease were observed.CD72 is required to maintain B cell anergy and functions as a regulator of peripheral B cell tolerance. Thus, altered CD72 expression may play a role during the development of systemic lupus erythematosus.

View details for DOI 10.1002/art.23812

View details for Web of Science ID 000260024400029

View details for PubMedID 18821699
Reduced levels of FasL and apoptosis resistance in SJIA monocytes Srivastava, S., Macaubas, C., Lee, T., Sandborg, C., Mellins, E. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2008: S93-S93
View details for DOI 10.1016/j.clim.2008.03.260

View details for Web of Science ID 000255533200265
Posttranslational regulation of I-E-d by affinity for CLIP JOURNAL OF IMMUNOLOGY Rinderknecht, C. H., Belmares, M. P., Catanzarite, T. L., Bankovich, A. J., Holmes, T. H., Garcia, K. C., Nanda, N. K., Busch, R., Kovats, S., Mellins, E. D. 2007; 179 (9): 5907-5915
Abstract

Several MHC class II alleles linked with autoimmune diseases form unusually low stability complexes with CLIP, leading us to hypothesize that this is an important feature contributing to autoimmune pathogenesis. To investigate cellular consequences of altering class II/CLIP affinity, we evaluated invariant chain (Ii) mutants with varying CLIP affinity for a mouse class II allele, I-E(d), which has low affinity for wild-type CLIP and is associated with a mouse model of spontaneous, autoimmune joint inflammation. Increasing CLIP affinity for I-E(d) resulted in increased cell surface and total cellular abundance and half-life of I-E(d). This reveals a post-endoplasmic reticulum chaperoning capacity of Ii via its CLIP peptides. Quantitative effects on I-E(d) were less pronounced in DM-expressing cells, suggesting complementary chaperoning effects mediated by Ii and DM, and implying that the impact of allelic variation in CLIP affinity on immune responses will be highest in cells with limited DM activity. Differences in the ability of cell lines expressing wild-type or high-CLIP-affinity mutant Ii to present Ag to T cells suggest a model in which increased CLIP affinity for class II serves to restrict peptide loading to DM-containing compartments, ensuring proper editing of antigenic peptides.

View details for Web of Science ID 000250388000035

View details for PubMedID 17947664
Influenza A virus elevates active cathepsin B in primary murine DC INTERNATIONAL IMMUNOLOGY Burster, T., Giffon, T., Dahl, M. E., Bjorck, P., Bogy, M., Weber, E., Mahmood, K., Lewis, D. B., Mellins, E. D. 2007; 19 (5): 645-655
Abstract

Dendritic cells (DCs) act as a first-line recognition system for invading pathogens, such as influenza A. The interaction of DC with influenza A virus results in DC activation via endosomal Toll-like receptors and also leads to presentation of viral peptides on MHC class II molecules. Prior work demonstrated that influenza A virus (A/HKx31; H3N2) infection of BALB/c mice activates lung DCs for antigen presentation, and that the enhanced function of these cells persists long after viral clearance and resolution of the virus-induced inflammatory response. Whether influenza A virus has acute or longer-lasting effects on the endo/lysosomal antigen-processing machinery of DCs has not been studied. Here, we show that antigen presentation from intact protein antigen, but not peptide presentation, results in increased T cell stimulation by influenza-exposed lung DCs, suggesting increased antigen processing/loading in these DCs. We find that cathepsin (Cat) B levels and activity are substantially up-regulated in murine lung DCs, harvested 30 days after A/HKx31 infection. CatB levels and activity are also increased in murine splenic and bone marrow-derived DCs, following short-term in vitro exposure to UV-inactivated influenza A virus. Modest effects on CatX are also seen during in vivo and in vitro exposure to influenza A virus. Using a cell permeable Cat inhibitor, we show Cats in influenza-exposed DCs to be functional and required for generation of a T cell epitope from intact ovalbumin. Our findings indicate that influenza A virus affects the MHC class II antigen-processing pathway, an essential pathway for CD4(+) T cell activation.

View details for DOI 10.1093/intimm/dxm030

View details for Web of Science ID 000246964500007

View details for PubMedID 17446210
Clinical research networks: a step towards evidence-based practice in pediatric rheumatology NATURE CLINICAL PRACTICE RHEUMATOLOGY Mellins, E. D., Rider, L. G. 2007; 3 (2): 59-59
View details for DOI 10.1038/ncprheum0405

View details for Web of Science ID 000243966500001

View details for PubMedID 17299440
Distinct molecular and cellular aspects of systemic juvenile idiopathic arthritis (SJIA) and polyarticular (PolyJIA) Macaubas, C., Nguyen, K., Pan, K., Lee, T., Deshpande, C., Sandborg, C., Cohen, S., Mellins, E. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2007: S94-S95
View details for DOI 10.1016/j.clim.2007.03.450

View details for Web of Science ID 000247137200246
Candidate early predictors for progression to joint damage in systemic juvenile idiopathic arthritis JOURNAL OF RHEUMATOLOGY Sandborg, C., Holmes, T. H., Lee, T., Biederman, K., Bloch, D. A., Emery, H., McCurdy, D., Mellins, E. D. 2006; 33 (11): 2322-2329
Abstract

To assess if joint damage at 2 years after diagnosis in patients with systemic juvenile idiopathic arthritis (SJIA) can be predicted by clinical or laboratory features assessed up to 3 or 6 months after diagnosis.Medical records from 70 children were retrospectively reviewed. The primary outcome measure was presence of joint damage at 2 years after diagnosis (JD2) as defined by presence of erosions or fusion in one or more joints. Potential predictor variables for JD2 in the first 3 and 6 months after diagnosis consisted of the highest observed white blood cell count, platelet count, erythrocyte sedimentation rate, active joint count, and presence of symptomatic pulmonary or cardiac disease or macrophage activation syndrome, and treatment data.The outcome of interest, JD2, was identified in 15/70 patients. Classification-tree analysis identified a pair of variables (highest observed platelet count and number of active joints) measured within the first 3 months after diagnosis that together predicted progression to JD2 with an estimated sensitivity of 87%, specificity of 82%, and positive predictive value of 57%. Multivariate logistic regression analyses at 3 months found that higher quantities of joints with active arthritis and early use of methotrexate (MTX) were factors significantly associated with increased odds of progression to JD2 (active joints odds ratio = 1.08, 95% CI 1.00-1.16, p = 0.04; MTX OR = 11.85, 95% CI 1.89-74.26, p = 0.01). Unsupervised cluster analysis identified 2 major phenotypes of patients at 3 months characterized by different ages at onset, acute phase markers, active joint counts, and presence of serositis. These phenotypes differed 3-fold in proportion of subjects progressing to JD2 (p < 0.05).By 3 months after diagnosis, a clinical phenotype based on active joint count and platelet count may be prognostic of an increased risk of progression to JD2. Use of corticosteroids did not appear to change the risk of joint damage. In contrast, the presence of serositis appeared to be associated with decreased risk of joint damage.

View details for Web of Science ID 000242010700035

View details for PubMedID 16960920
Human cytomegalovirus alters localization of MHC class II and dendrite morphology in mature Langerhans cells JOURNAL OF IMMUNOLOGY Lee, A. W., Hertel, L., Louie, R. K., Burster, T., Lacaille, V., Pashine, A., Abate, D. A., Mocarski, E. S., Mellins, E. D. 2006; 177 (6): 3960-3971
Abstract

Hemopoietic stem cell-derived mature Langerhans-type dendritic cells (LC) are susceptible to productive infection by human CMV (HCMV). To investigate the impact of infection on this cell type, we examined HLA-DR biosynthesis and trafficking in mature LC cultures exposed to HCMV. We found decreased surface HLA-DR levels in viral Ag-positive as well as in Ag-negative mature LC. Inhibition of HLA-DR was independent of expression of unique short US2-US11 region gene products by HCMV. Indeed, exposure to UV-inactivated virus, but not to conditioned medium from infected cells, was sufficient to reduce HLA-DR on mature LC, implicating particle binding/penetration in this effect. Reduced surface levels reflected an altered distribution of HLA-DR because total cellular HLA-DR was not diminished. Accumulation of HLA-DR was not explained by altered cathepsin S activity. Mature, peptide-loaded HLA-DR molecules were retained within cells, as assessed by the proportion of SDS-stable HLA-DR dimers. A block in egress was implicated, as endocytosis of surface HLA-DR was not increased. Immunofluorescence microscopy corroborated the intracellular retention of HLA-DR and revealed markedly fewer HLA-DR-positive dendritic projections in infected mature LC. Unexpectedly, light microscopic analyses showed a dramatic loss of the dendrites themselves and immunofluorescence revealed that cytoskeletal elements crucial for the formation and maintenance of dendrites are disrupted in viral Ag-positive cells. Consistent with these dendrite effects, HCMV-infected mature LC exhibit markedly reduced chemotaxis in response to lymphoid chemokines. Thus, HCMV impedes MHC class II molecule trafficking, dendritic projections, and migration of mature LC. These changes likely contribute to the reduced activation of CD4+ T cells by HCMV-infected mature LC.

View details for Web of Science ID 000240475300052

View details for PubMedID 16951359
Medicine on a need-to-know basis NATURE IMMUNOLOGY Busch, R., Byrne, B., Gandrud, L., Sears, D., Meyer, E., Kattah, M., Kurihara, C., Haertel, E., Parnes, J. R., Mellins, E. D. 2006; 7 (6): 543-547
Abstract

Disease-oriented, introductory medical curricula can help overcome educational and institutional barriers that separate aspiring translational scientists in PhD programs from the world of medicine.

View details for Web of Science ID 000237751200004

View details for PubMedID 16715061
Human dendritic cell expression of HLA-DO is subset-specific and regulated by maturation Hornell, T. M., Burster, T., Jahnsen, F. L., Pashine, A., Ochoa, M. T., Harding, J. J., Macaubas, C., Lee, A., Modlin, R. L., Mellins, E. AMER ASSOC IMMUNOLOGISTS. 2006: S49-S49
View details for Web of Science ID 000238837100226
Human dendritic cell expression of HLA-DO is subset specific and regulated by maturation JOURNAL OF IMMUNOLOGY Hornell, T. M., Burster, T., Jahnsen, F. L., Pashine, A., Ochoa, M. T., Harding, J. J., Macaubas, C., Lee, A. W., Modlin, R. L., Mellins, E. D. 2006; 176 (6): 3536-3547
Abstract

Expression of HLA-DO (DO) in cells that express HLA-DM (DM) results in an altered repertoire of MHC class II/peptide complexes, indicating that DO modulates DM function. Human and murine B cells and thymic epithelial cells express DO, while monocytes/macrophages do not. Monocyte-derived dendritic cells (DC) also have been found to be DO-negative, leading to the assumption that DC do not express DO. In this study, we report that, in fact, certain types of human primary DC express DO. These include Langerhans cells (LC) and some subtypes of circulating blood DC. Specifically, the majority of BDCA-3(+) DC, a small subset of uncertain function, are DO(+), while smaller proportions of CD11c(+), BDCA-1(+) (myeloid) DC, at most a minority of CD123(+)/BDCA-2(+) (plasmacytoid) DC, and no detectable CD16(+) (myeloid) DC, express DO. Immunohistochemistry of human tonsil sections demonstrates that tonsillar interdigitating DC are also DO(+). In a subset of immature LC with higher DO expression, an increased fraction of surface DR molecules carry CLIP peptides, indicating that DO functions as a DM inhibitor in these cells. LC expression of DO is down-regulated by maturation stimuli. DM levels also decrease under these conditions, but the DM:DO ratio generally increases. In the myeloid cell types tested, DO expression correlates with levels of DObeta, but not DOalpha, implying that modulation of DObeta regulates DO dimer abundance in these cells. The range of APC types shown to express DO suggests a broader role for DO in immune function than previously appreciated.

View details for Web of Science ID 000238768400029

View details for PubMedID 16517722
Human cytomegalovirus alters localization of MHC class II and dendrite morphology in mature Langerhans cells. Lee, A., Hertel, L., Louie, R., Burster, T., Lacaille, V., Pashine, A., Abate, D., Mocarski, E., Mellins, E. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2006: S22-S23
View details for DOI 10.1016/j.clim.2006.04.206

View details for Web of Science ID 000237924300051
Gene expression profiling of peripheral blood mononuclear cells (PBMC) from SJIA patients. Deshpande, C., Sun, Y., Mocaubas, C., Alexander, H., Pan, K., Lee, T., Chang, S., Lih, C., Lin, R., Sandborg, C., Tibshirani, R., Begovich, A. B., Cohen, S., Mellins, E. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2006: S68-S68
View details for DOI 10.1016/j.clim.2006.04.090

View details for Web of Science ID 000237924300172
Diagnostic and prognostic biomarkers in systemic juvenile idiopathic arthritis. Park, J., Carroll, T., Lau, K., Lee, T., Sandborg, C., Schilling, J., Mellins, E. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2006: S71-S71
View details for DOI 10.1016/j.clim.2006.04.099

View details for Web of Science ID 000237924300181
Differential expression of immune parameters in systemic juvenile idiopathic arthritis (SJIA) flare and quiescence. Nguyen, K., Macaubas, C., Deshpande, C., Lee, T., Sandborg, C., Mellins, E. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2006: S72-S72
View details for DOI 10.1016/j.clim.2006.04.100

View details for Web of Science ID 000237924300182
Achieving stability through editing and chaperoning: regulation of MHC class II peptide binding and expression IMMUNOLOGICAL REVIEWS Busch, R., Rinderknecht, C. H., Roh, S., Lee, A. W., Harding, J. J., Burster, T., Hornell, T. M., Mellins, E. D. 2005; 207: 242-260
Abstract

In antigen-presenting cells (APCs), loading of major histocompatibility complex class II (MHC II) molecules with peptides is regulated by invariant chain (Ii), which blocks MHC II antigen-binding sites in pre-endosomal compartments. Several molecules then act upon MHC II molecules in endosomes to facilitate peptide loading: Ii-degrading proteases, the peptide exchange factor, human leukocyte antigen-DM (HLA-DM), and its modulator, HLA-DO (DO). Here, we review our findings arguing that DM stabilizes a globally altered conformation of the antigen-binding groove by binding to a lateral surface of the MHC II molecule. Our data imply changes in the interactions between specificity pockets and peptide side chains, complementing data from others that suggest DM affects hydrogen bonds. Selective weakening of peptide/MHC interactions allows DM to alter the peptide repertoire. We also review our studies in cells that highlight the ability of several factors to modulate surface expression of MHC II molecules via post-Golgi mechanisms; these factors include MHC class II-associated Ii peptides (CLIP), DM, and microbial products that modulate MHC II traffic from endosomes to the plasma membrane. In this context, we discuss possible mechanisms by which the association of some MHC II alleles with autoimmune diseases may be linked to their low CLIP affinity.

View details for Web of Science ID 000232048300020

View details for PubMedID 16181341
Selective developmental defects of cord blood antigen-presenting cell subsets HUMAN IMMUNOLOGY Drohan, L., Harding, J. J., Holm, B., Cordoba-Tongson, E., Dekker, C. L., Holmes, T., Maecker, H., Mellins, E. D. 2004; 65 (11): 1356-1369
Abstract

Defective antigen-presenting cell (APC) function has been hypothesized to contribute to increased infection susceptibility in newborns. We used multiparameter flow cytometry to characterize APC subsets in adult peripheral blood (APB) and cord blood (CB). APB had a higher proportion of CD11c+ dendritic cells (DC), whereas CB mainly contained CD123+ DC. APB was enriched in CD16+CD11c+ DC subset, whereas CD34+CD11c-CD123lo cells were prominent in CB. Lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-alpha production was dampened in myeloid DC and monocytes from CB, whereas IL-1alpha production was not different. The reduction in TNF-alpha response did not appear to result from reduced surface detection of LPS, because CD14, toll-like receptor (TLR)-4 and TLR-2 levels were not reduced in CB APC compared with APB cells. Also, there was no correlation between TLR-2 or TLR-4 levels and TNF-alpha production in myeloid DC and monocytes. CB monocytes had lower surface HLA-DR immediately ex vivo. Both APB and CB monocytes upregulated HLA-DR after incubation, but an additional LPS-induced increase in HLA-DR was suggested only in APB monocytes. APB monocytes also showed a greater LPS-induced increase in CD40 expression. Together, our data show significant, selective differences in circulating APC between neonates and adults.

View details for DOI 10.1016/j.humimm.2004.09.011

View details for Web of Science ID 000225728800010

View details for PubMedID 15556686
Cord blood antigen presenting cells show selective deficits in inflammatory responses Drohan, L., Holm, B., Holmes, T., Tongson, E. C., Dekker, C., Maecker, H., Mellins, E. NATURE PUBLISHING GROUP. 2004: 390A-390A
View details for Web of Science ID 000220591102285
Bordetella pertussis infection of primary human monocytes alters HLA-DR expression INFECTION AND IMMUNITY Shumilla, J. A., Lacaille, V., Hornell, T. M., Huang, J., Narasimhan, S., Relman, D. A., Mellins, E. D. 2004; 72 (3): 1450-1462
Abstract

Bordetella pertussis is the causative agent of whooping cough, a potentially lethal respiratory disease in children. In immunocompetent individuals, B. pertussis infection elicits an effective adaptive immune response driven by activated CD4(+) T cells. However, live B. pertussis persists in the host for 3 to 4 weeks prior to clearance. Thus, B. pertussis appears to have evolved short-term mechanisms for immune system evasion. We investigated the effects of B. pertussis wild-type strain BP338 on antigen presentation in primary human monocytes. BP338 infection reduced cell surface expression of HLA-DR and CD86 but not that of major histocompatibility complex class I proteins. This change in cell surface HLA-DR expression reflected intracellular redistribution of HLA-DR. The proportion of peptide-loaded molecules was unchanged in infected cells, suggesting that intracellular retention occurred after peptide loading. Although B. pertussis infection of monocytes induced rapid and robust expression of interleukin-10 (IL-10), HLA-DR redistribution did not appear to be explained by increased IL-10 levels. BP338-infected monocytes exhibited reduced synthesis of HLA-DR dimers. Interestingly, those HLA-DR proteins that were generated appeared to be longer-lived than HLA-DR in uninfected monocytes. BP338 infection also prevented gamma interferon (IFN-gamma) induction of HLA-DR protein synthesis. Using mutant strains of B. pertussis, we found that reduction in HLA-DR surface expression was due in part to the presence of pertussis toxin whereas the inhibition of IFN-gamma induction of HLA-DR could not be linked to any of the virulence factors tested. These data demonstrate that B. pertussis utilizes several mechanisms to modulate HLA-DR expression.

View details for DOI 10.1128/IAI.72.3.145-1462.2004

View details for Web of Science ID 000189270800029

View details for PubMedID 14977950
Regulation of the class II MHC pathway in primary human monocytes by granulocyte-macrophage colony-stimulating factor JOURNAL OF IMMUNOLOGY Hornell, T. M., Beresford, G. W., Bushey, A., Boss, J. M., Mellins, E. D. 2003; 171 (5): 2374-2383
Abstract

GM-CSF stimulates the growth and differentiation of hematopoietic progenitors and also affects mature cell function. These effects have led to the use of GM-CSF as a vaccine adjuvant with promising results; however, the mechanisms underlying GM-CSF-mediated immune potentiation are incompletely understood. In this study, we investigated the hypothesis that the immune stimulatory role of GM-CSF is in part due to effects on class II MHC Ag presentation. We find that, in primary human monocytes treated for 24-48 h, GM-CSF increases surface class II MHC expression and decreases the relative level of the invariant chain-derived peptide, CLIP, bound to surface class II molecules. GM-CSF also increases expression of the costimulatory molecules CD86 and CD40, but not the differentiation marker CD1a or CD16. Furthermore, GM-CSF-treated monocytes are better stimulators in a mixed leukocyte reaction. Additional analyses of the class II pathway revealed that GM-CSF increases total protein and RNA levels of HLA-DR, DM, and DOalpha. Expression of class II transactivator (CIITA) types I and III, but not IV, transcripts increases in response to GM-CSF. Furthermore, GM-CSF increases the amount of CIITA associated with the DR promoter. Thus, our data argue that the proinflammatory role of GM-CSF is mediated in part through increased expression of key molecules involved in the class II MHC pathway via induction of CIITA.

View details for Web of Science ID 000184970900025

View details for PubMedID 12928384
Interaction of HLA-DR with an acidic face of HLA-DM disrupts sequence-dependent interactions with peptides IMMUNITY Pashine, A., Busch, R., Belmares, M. P., Munning, J. N., Doebele, R. C., Buckingham, M., Nolan, G. P., Mellins, E. D. 2003; 19 (2): 183-192
Abstract

HLA-DM (DM) edits major histocompatibility complex class II (MHCII)-bound peptides in endocytic compartments and stabilizes empty MHCII molecules. Crystal structures of DM have revealed similarity to MHCII but not how DM and MHCII interact. We used mutagenesis to map a MHCII-interacting surface on DM. Mutations on this surface impair DM action on HLA-DR and -DP in cells and DM-dependent peptide loading in vitro. The orientation of DM and MHCII predicted by these studies guided design of soluble DM and DR molecules fused to leucine zippers via their beta chains, resulting in stable DM/DR complexes. Peptide release from the complexes was fast and only weakly sequence dependent, arguing that DM diminishes the selectivity of the MHCII groove. Analysis of soluble DM action on soluble DR/peptide complexes corroborates this conclusion.

View details for Web of Science ID 000184929000006

View details for PubMedID 12932352
Cytokines elicited by synovial T cell epitopes from a autoantigen - Altered peptide ligands can reduce interferon-gamma and interleukin-10 production ARTHRITIS AND RHEUMATISM Hall, F. C., Visconti, K. C., Ahmad, R. C., Parry, S. L., Miltenburg, A. M., McConnell, H. M., Mellins, E. D., Sonderstrup, G. 2003; 48 (8): 2375-2385
Abstract

To explore the cytokine responses associated with T cell epitopes from human cartilage glycoprotein 39 (HC gp-39) and the potential for modifying cytokine secretion using altered peptide ligands (APLs).Draining lymph node cells were harvested from HLA-DR*0401 transgenic mice that had been immunized with HC gp-39. Cytokine responses to 5 previously identified HLA-DR*0401-restricted HC gp-39 T cell epitopes were studied in vitro. The anchor and T cell receptor (TCR) contact residues of peptide 322-337 were identified, and this information was used to design alanine-substituted APLs. T cells were primed in vivo with wild-type peptide 322-337, restimulated with wild-type peptide or APLs, and the cytokine profiles were compared.Restimulation with individual peptides elicited distinct cytokine profiles. HC gp-39 (peptide 322-337) elicited a dominant interferon-gamma (IFNgamma) response. Residues within the core (positions P1-P9) 322-337 peptide sequence were critical for T cell recognition. Surprisingly, the N-terminal flanking region was also important for recognition by 6 of 10 specific T cell hybridomas. Substitutions of charged TCR contact residues in the 322-337 core epitope (E332A and K335A) were associated with a significant reduction in the IFNgamma and interleukin-10 (IL-10) stimulation indices. Restimulation with peptides W325A and V326A was also associated with a trend toward reduced IFNgamma and IL-10 secretion. In contrast, restimulation with peptide D330N elicited cytokine profiles more comparable with those resulting from restimulation with wild-type peptide.This study indicates that APLs of a proinflammatory HC gp-39 T cell epitope may be used to alter the cytokine response from a memory T cell population.

View details for DOI 10.1002/art.11132

View details for Web of Science ID 000184585000035
Susceptibility of immature and mature Langerhans cell-type dendritic cells to infection and immunomodulation by human cytomegalovirus JOURNAL OF VIROLOGY Hertel, L., Lacaille, V. G., Strobl, H., Mellins, E. D., Mocarski, E. S. 2003; 77 (13): 7563-7574
Abstract

Human cytomegalovirus (CMV) infection initiates in mucosal epithelia and disseminates via leukocytes throughout the body. Langerhans cells (LCs), the immature dendritic cells (DCs) that reside in epithelial tissues, are among the first cells to encounter virus and may play important roles in the immune response, as well as in pathogenesis as hosts for viral replication and as vehicles for dissemination. Here, we demonstrate that CD34(+) progenitor cell-derived LC-type DCs exhibit a differentiation state-dependent susceptibility to CMV infection. In contrast to the small percentage (3 to 4%) of the immature LCs that supported infection, a high percentage (48 to 74%) of mature, LC-derived DCs were susceptible to infection with endotheliotropic strains (TB40/E or VHL/E) of CMV. These cells were much less susceptible to viral strains AD169varATCC, TownevarRIT(3), and Toledo. When exposed to endotheliotropic strains, viral gene expression (IE1/IE2 and other viral gene products) and viral replication proceeded efficiently in LC-derived mature DCs (mDCs). Productive infection was associated with downmodulation of cell surface CD83, CD1a, CD80, CD86, ICAM-1, major histocompatibility complex (MHC) class I, and MHC class II on these cells. In addition, the T-cell proliferative response to allogeneic LC-derived mDCs was attenuated when CMV-infected cultures were used as stimulators. This investigation revealed important characteristics of the interaction between CMV and the LC lineage of DCs, suggesting that LC-derived mDCs are important to viral pathogenesis and immunity through their increased susceptibility to virus replication and virus-mediated immune escape.

View details for DOI 10.1128/JVI.77.13.7563-7574.2003

View details for Web of Science ID 000183598600043

View details for PubMedID 12805456
Point mutations in or near the antigen-binding groove of HLA-DR3 implicate class II-associated invariant chain peptide affinity as a constraint on MHC class II polymorphism JOURNAL OF IMMUNOLOGY Doebele, R. C., Pashine, A., Liu, W., Zaller, D. M., Belmares, M., Busch, R., Mellins, E. D. 2003; 170 (9): 4683-4692
Abstract

During maturation of MHC II molecules, newly synthesized and assembled complexes of MHC II alphabeta dimers with invariant chain (Ii) are targeted to endosomes, where Ii is proteolyzed, leaving remnant class II-associated Ii peptides (CLIP) in the MHC II peptide binding groove. CLIP must be released, usually with assistance from the endosomal MHC II peptide exchange factor, HLA-DM, before MHC II molecules can bind endosomal peptides. Structural factors that control rates of CLIP release remain poorly understood, although peptide side chain-MHC II specificity pocket interactions and MHC II polymorphism are important. Here we report that mutations betaS11F, betaS13Y, betaQ70R, betaK71E, betaK71N, and betaR74Q, which map to the P4 and P6 pockets of the groove of HLA-DR3 molecules, as well as alphaG20E adjacent to the groove, are associated with elevated CLIP in cells. Most of these mutations increase the resistance of CLIP-DR3 complexes to dissociation by SDS. In vitro, the groove mutations increase the stability of CLIP-DR3 complexes to dissociation. Dissociation rates in the presence of DM, as well as coimmunoprecipitation of some mutant DR3 molecules with DM, are also diminished. The profound phenotypes associated with some of these point mutations suggest that the need to maintain efficient CLIP release represents a constraint on naturally occurring MHC II polymorphism.

View details for Web of Science ID 000182528100034

View details for PubMedID 12707347
Formation of two peptide/MHC II isomers is catalyzed differentially by HLA-DM BIOCHEMISTRY Belmares, M. P., Busch, R., Mellins, E. D., McConnell, H. M. 2003; 42 (3): 838-847
Abstract

Major histocompatability class II proteins are transmembrane alphabeta-heterodimers that present peptides to T-cells. MHC II may bind exogenous peptides directly at the cell surface. Alternatively, peptides derived from processing of endosomal protein may bind to MHC II in endosomal compartments. There, HLA-DM catalyzes the formation of peptide/MHC complexes, which are then transported to the cell surface. Here we report evidence that the peptide Ii CLIP 81-104 binds to DR*0404 in two alternate registries, whose dissociation rates, while kinetically indistinguishable at pH 5.3 and 37 degrees C, are kinetically resolved in the presence of HLA-DM. In one registry isomer, CLIP Met 91 is placed in the N-terminal P1 pocket of DR*0404, and peptide dissociation is readily catalyzed by HLA-DM. In a second proposed registry, likely with CLIP Leu 97 in the P1 pocket, the complex is substantially less sensitive to HLA-DM catalysis. Without HLA-DM, or at pH 7, the fraction of each isomer formed in solution is relatively insensitive to the duration of incubation with peptide. However, with HLA-DM, the fraction of the DM-insensitive isomer is dramatically influenced by peptide incubation time. The mechanism of isomer formation appears to be determined by the HLA-DM-modified relative association to the two registries, followed by HLA-DM-catalyzed dissociation of each isomer and rebinding, leading to a final isomer composition determined by these kinetic constants. Intramolecular isomer interconversion does not appear to be involved. The behavior of these complexes may provide a model for peptide editing by DM in endosomes.

View details for DOI 10.1021/bi020466p

View details for Web of Science ID 000180568900027

View details for PubMedID 12534297
Growth delay in systemic JRA: Interplay between steroid usage and disease severity Lee, T., Sandborg, C., Biederman, K., Mellins, E. WILEY-LISS. 2002: 3405-3405
View details for Web of Science ID 000179754000053
Structural factors contributing to DM susceptibility of MHC class II/Peptide complexes JOURNAL OF IMMUNOLOGY Belmares, M. P., Busch, R., Wucherpfennig, K. W., McConnell, H. M., Mellins, E. D. 2002; 169 (9): 5109-5117
Abstract

Peptide loading of MHC class II (MHCII) molecules is assisted by HLA-DM, which releases invariant chain peptides from newly synthesized MHCII and edits the peptide repertoire. Determinants of susceptibility of peptide/MHCII complexes to DM remain controversial, however. Here we have measured peptide dissociation in the presence and the absence of DM for 36 different complexes of varying intrinsic stability. We found large variations in DM susceptibility for different complexes using either soluble or full-length HLA-DM. The DM effect was significantly less for unstable complexes than for stable ones, although this correlation was modest. Peptide sequence- and allele-dependent interactions along the entire length of the Ag binding groove influenced DM susceptibility. We also observed differences in DM susceptibility during peptide association. Thus, the peptide repertoire displayed to CD4(+) T cells is the result of a mechanistically complicated editing process and cannot be simply predicted from the intrinsic stability of the complexes in the absence of DM.

View details for Web of Science ID 000178777400053

View details for PubMedID 12391227
Determinants of joint damage in systemic onset JRA (SOJRA). Sandberg, C., Lee, T. L., Biederman, K., Chira, P., Saper, V., Emery, H., Kennedy, J., Zehran, S., McCurdy, D., Mellins, E. WILEY-BLACKWELL. 2002: S478-S478
View details for Web of Science ID 000178421801297
Stabilization of soluble, low-affinity HLA-DM/HLA-DR1 complexes by leucine zippers JOURNAL OF IMMUNOLOGICAL METHODS Busch, R., Paschine, A., Garcia, K. C., Mellins, E. D. 2002; 263 (1-2): 111-121
Abstract

The ectodomains of interacting membrane-bound proteins, when expressed as recombinant soluble molecules, often have low affinities for each other, hampering studies of their interaction. We reasoned that stabilization of unstable protein-protein complexes should aid our understanding of the structural and functional consequences of complex formation. Here, we have used fusion with leucine zipper (LZ) domains to stabilize a complex formed between the class II major histocompatibility complex (MHC-II) protein, HLA-DR1 (which binds peptides for presentation to CD4+ T cells) and HLA-DM (which catalyzes peptide exchange of MHC-II molecules). To this end, the DM beta chain ectodomains were fused to acidic LZ domains (AcidP1 or Fos); similarly, the DR1 beta chain ectodomains were fused to basic LZ domains (BaseP1 or Jun). We expressed LZ-modified soluble DM or DR1 alphabeta dimers, or both, in insect cells and purified the secreted sDM-AcidP1 and sDR1-BaseP1 molecules as well as the complex. LZ modification greatly enhanced DM-catalyzed peptide binding to DR1 compared to unmodified soluble DM and DR1. We readily detected LZ-modified DM/DR complexes on native PAGE gels and by coimmunoprecipitation. Thus, fusion with artificial LZ domains can stabilize unstable protein-protein complexes for biochemical and structural studies of interactions within the complex.

View details for Web of Science ID 000176076300010

View details for PubMedID 12009208
Relationship between kinetic stability and immunogenicity of HLA-DR4/peptide complexes EUROPEAN JOURNAL OF IMMUNOLOGY Hall, F. C., Rabinowitz, J. D., Busch, R., Visconti, K. C., Balmares, M., Patil, N. S., Cope, A. O., Patel, S., McConnell, H. M., Mellins, E. D., Sonderstrup, G. 2002; 32 (3): 662-670
Abstract

Immunodominant T cell epitopes from the autoantigen human cartilage glycoprotein 39 have previously been mapped in the context of HLA-DR*0401 and *0402, using mice expressing HLA-DR4 transgenes. We measured the dissociation rates of these epitopes from soluble recombinant DR*0401 and DR*0402 to assess the relationship between peptide/HLA-DR4 kinetic stability and immunogenicity. Experiments were performed at endosomal pH (5.5) and at cell surface pH (7), in the absence and presence of soluble recombinant HLA-DM (sDM). All (4/4) immunodominant peptide/HLA-DR complexes exhibit dissociation half-times of 1h to several days. In contrast, most (3/4) non-immunodominant complexes dissociate with half-times <30 min under at least one of these conditions. Interestingly, a complex which is stable except in the presence of HLA-DM at pH 5.5 is immunogenic only following peptide immunization, while a complex which is stable at acidic but not at neutral pH, is non-immunogenic following either whole protein or peptide immunization. These data indicate that kinetic stability of peptide/MHC complexes in vivo is a key determinant of immunogenicity.

View details for Web of Science ID 000174439000008

View details for PubMedID 11857340
Rheumatoid arthritis (RA)-associated HLA-DR alleles form less stable complexes with class II-associated invariant chain peptide than non-RA-associated HLA-DR alleles JOURNAL OF IMMUNOLOGY Patil, N. S., Pashine, A., Belmares, M. P., Liu, W., Kaneshiro, B., Rabinowitz, J., McConnell, H., Mellins, E. D. 2001; 167 (12): 7157-7168
Abstract

Certain HLA-DR alleles confer strong susceptibility to the autoimmune disease rheumatoid arthritis (RA). We compared RA-associated alleles, HLA-DR*0401, HLA-DR*0404, and HLA-DR*0405, with closely related, non-RA-associated alleles, HLA-DR*0402 and HLA-DR*0403, to determine whether they differ in their interactions with the class II chaperone, invariant chain (Ii). Ii binds to class II molecules in the endoplasmic reticulum, inhibits binding of other ligands, and directs class II-Ii complexes to endosomes, where Ii is degraded to class II-associated Ii peptide (CLIP). To evaluate the interaction of Ii and CLIP with these DR4 alleles, we introduced HLA-DR*0401, *0402, and *0404 alleles into a human B cell line that lacked endogenous HLA-DR or HLA-DM molecules. In a similar experiment, we introduced HLA-DR*0403 and *0405 into an HLA-DM-expressing B cell line, 8.1.6, and its DM-negative derivative, 9.5.3. Surface abundance of DR4-CLIP peptide complexes and their susceptibility to SDS-induced denaturation suggested that the different DR4-CLIP complexes had different stabilities. Pulse-chase experiments showed CLIP dissociated more rapidly from RA-associated DR molecules in B cell lines. In vitro assays using soluble rDR4 molecules showed that DR-CLIP complexes of DR*0401 and DR*0404 were less stable than complexes of DR*0402. Using CLIP peptide variants, we mapped the reduced CLIP interaction of RA-associated alleles to the shared epitope region. The reduced interaction of RA-associated HLA-DR4 molecules with CLIP may contribute to the pathophysiology of autoimmunity in RA.

View details for Web of Science ID 000172613400058

View details for PubMedID 11739539
Autoantigenic HCgp39 epitopes are presented by the HLA-DM-dependent presentation pathway in human B cells JOURNAL OF IMMUNOLOGY Patil, N. S., Hall, F. C., Drover, S., Spurrell, D. R., BOS, E., Cope, A. P., Sonderstrup, G., Mellins, E. D. 2001; 166 (1): 33-41
Abstract

It is hypothesized that autoimmune diseases manifest when tolerance to self-Ags fails. One possible mechanism to break tolerance is presentation of self-Ag in an altered form. Most Ags are presented by APCs via the traditional presentation pathway that includes "epitope editing" by intracellular HLA-DM, a molecule that selects for stable MHC-peptide complexes. We were interested in testing the hypothesis that autoreactive MHC-peptide complexes may reach the cell surface by an alternate pathway without being edited by HLA-DM. We selected a cartilage autoantigen human cartilage glycoprotein 39 to which T cell responses are observed in rheumatoid arthritis (RA) patients and some DR(*)04 healthy subjects. RA is genetically associated with certain DRB1 alleles, including DRB1(*)0401 but closely related allele DRB1(*)0402 is either neutral or mildly protective with respect to RA. We generated human B lymphoblastoid cell line cells expressing DR(*)0401 or DR(*)0402 in the presence or absence of intracellular HLA-DM and assessed their ability to present a candidate autoantigen, human cartilage glycoprotein 39. Our results show that the presence of intracellular HLA-DM is critical for presentation of this autoantigen to CD4(+) T cell hybridomas generated from DR(*)04-transgenic mice. Presentation of an autoantigen by the traditional HLA-DM-dependent pathway has implications for Ag presentation events in RA.

View details for Web of Science ID 000166012400007

View details for PubMedID 11123274
pH stability of HLA-DR4 complexes with antigenic peptides BIOCHEMISTRY Belmares, M. P., Rabinowitz, J. D., Liu, W., Mellins, E. D., McConnell, H. M. 2000; 39 (47): 14558-14566
Abstract

Complexes between antigenic peptides and class II proteins of the major histocompatibility complex (MHC) trigger cellular immune responses. These complexes usually dissociate more rapidly at mildly acidic pH, where they are formed intracellularly, as compared to neutral pH, where they function at the cell surface. This paper describes the pH dependence of the dissociation kinetics of complexes between MHC proteins and antigenic peptides containing aspartic and glutamic acid residues. Some of these complexes show an unusual pH dependence, dissociating much more rapidly at pH 7 than at pH 5.3. This occurs when the carboxylate group of the aspartic or glutamic acid residue is located in a neutral pocket of the protein. In contrast, solvent-exposed carboxylate groups or carboxylate groups buried in pockets where they form salt bridges with the protein do not show this unusual pH dependence. The kinetic data having the unusual pH dependence conform closely to a model in which there is a rapid reversible equilibration between a less stable deprotonated complex and a more stable protonated complex. In this model, the pK(a) of the protonation reaction for the partially buried peptide carboxylate group ranges from 7.7 to 8.3, reflecting the strongly basic conditions required for deprotonation. One of the few peptide/MHC complexes demonstrated to play a role in autoimmunity in humans contains a buried peptide carboxylate and shows this unusual pH dependence. The relevance of this finding to understanding the chemical basis of autoimmunity is briefly discussed.

View details for Web of Science ID 000165602400024

View details for PubMedID 11087411
Determination of the HLA-DM interaction site on HLA-DR molecules IMMUNITY Doebele, R. C., Busch, R., Scott, H. M., Pashine, A., Mellins, E. D. 2000; 13 (4): 517-527
Abstract

HLA-DM removes CLIP and other loosely bound peptides from MHC class II molecules. The crystal structures of class II molecules and of HLA-DM have not permitted identification of their interaction sites. Here, we describe mutations in class II that impair interactions with DM. Libraries of randomly mutagenized DR3 alpha and beta chains were screened for their ability to cause cell surface accumulation of CLIP/DR3 complexes in EBV-B cells. Seven mutations were associated with impaired peptide loading in vivo, as detected by SDS stability assays. In vitro, these mutant DR3 molecules were resistant to DM-catalyzed CLIP release and showed reduced binding to DM. All mutations localize to a single lateral face of HLA-DR, which we propose interacts with DM during peptide exchange.

View details for Web of Science ID 000090079100011

View details for PubMedID 11070170
Accessory molecules for MHC class II peptide loading CURRENT OPINION IN IMMUNOLOGY Busch, R., Doebele, R. C., Patil, N. S., Pashine, A., Mellins, E. D. 2000; 12 (1): 99-106
Abstract

Accessory molecules, such as HLA-DM and invariant chain, modulate the ligands bound to MHC class II molecules in antigen-presenting cells. Recent investigations, including gene targeting experiments, have shed light on the functions of these molecules, their mechanisms of action, interactions with class II molecules, and the relationships with associated molecules such as tetraspanins and HLA-DO.

View details for Web of Science ID 000085306900014

View details for PubMedID 10679402
Secondary structure composition and pH-dependent conformational changes of soluble recombinant HLA-DM JOURNAL OF BIOLOGICAL CHEMISTRY Busch, R., Reich, Z., Zaller, D. M., Sloan, V., Mellins, E. D. 1998; 273 (42): 27557-27564
Abstract

HLA-DM catalyzes the release of invariant chain fragments from newly synthesized major histocompatibility complex (MHC) class II molecules, stabilizes empty class II molecules, and edits class II-associated peptides by preferentially releasing those that are loosely bound. The ability of HLA-DM to carry out these functions in vitro is pH dependent, with an optimum at pH 4.5-5.5 and poor activity at pH 7. The structural basis for these properties of HLA-DM is unknown. Sequence homology suggests that HLA-DM resembles classical, peptide-binding MHC class II molecules. In this study, we examined whether HLA-DM has a secondary structure composition consistent with an MHC fold and whether HLA-DM changes conformation between pH 5 and pH 7. Far-UV circular dichroism (CD) spectra of recombinant soluble HLA-DM (sDM) indicate that HLA-DM belongs to the alpha/beta class of proteins and structurally resembles both MHC class I and class II molecules. The CD peak around 198 nm increases upon going from neutral to endosomal pH and drops sharply upon denaturation below pH 3.5, distinguishing at least three states of sDM: the denatured state and two highly similar folded states. Fluorescence emission spectra show a slight blue-shift and a approximately 20% drop in intensity at pH 5 compared with pH 7. Unfolding experiments using guanidinium chloride show that the stability of sDM is somewhat reduced but not lost at pH 5. These results indicate that sDM undergoes a pH-dependent conformational change between neutral and endosomal pH. The change seems to involve both hydrogen bonding patterns and the hydrophobic core of sDM and may contribute to the pH dependence of DM activity.

View details for Web of Science ID 000076448000075

View details for PubMedID 9765288
Aberrant intermolecular disulfide bonding in a mutant HLA-DM molecule: Implications for assembly, maturation, and function JOURNAL OF IMMUNOLOGY Busch, R., Doebele, R. C., von Scheven, E., Fahrni, J., Mellins, E. D. 1998; 160 (2): 734-743
Abstract

HLA-DM (abbreviated DM) is an MHC-encoded glycoprotein that catalyzes the selective release of peptides, including class II-associated invariant chain peptides, from MHC class II molecules. To perform its function, DM must assemble in the endoplasmic reticulum (ER), travel to endosomes, and interact productively with class II molecules. We have described previously an EBV-transformed B cell line, 7.12.6, which displays a partial Ag presentation defect and expresses a mutated DM beta-chain with Cys79 replaced by Tyr. In this study, we show that HLA-DR molecules in 7.12.6 have a defect in peptide loading and accumulate class II-associated invariant chain peptides (CLIP). Peptide loading is restored by transfection of wild-type DMB. The mutant DM molecules exit the ER slowly and are degraded rapidly, resulting in greatly reduced levels of mutant DM in post-Golgi compartments. Whereas wild-type DM forms noncovalent alphabeta dimers, such dimers form inefficiently in 7.12.6; many mutant DM beta-chains instead form a disulfide-bonded dimer with DM alpha. Homodimers of DM beta are also detected in 7.12.6 and in the alpha-chain defective mutant, 2.2.93. We conclude that during folding of wild-type DM, the native conformation is stabilized by a conserved disulfide bond involving Cys79beta and by noncovalent contacts with DM alpha. Without these interactions, DM beta can form malfolded structures containing interchain disulfide bonds; malfolding is correlated with ER retention and accelerated degradation.

View details for Web of Science ID 000071915200027

View details for PubMedID 9551909
MEDIATION BY HLA-DM OF DISSOCIATION OF PEPTIDES FROM HLA-DR NATURE Sloan, V. S., Cameron, P., Porter, G., Gammon, M., Amaya, M., Mellins, E., Zaller, D. M. 1995; 375 (6534): 802-806
Abstract

Human leukocyte antigen (HLA)-DM is an unconventional major histocompatibility complex (MHC) class II heterodimer that is important for B-cell-mediated antigen processing and presentation to MHC class II-restricted T cells. HLA-DM is encoded by two genes, DMA and DMB, which map to the MHC class II region, and shares some homology with MHC class I and class II proteins. Here we define the biochemical role of HLA-DM. Recombinant soluble HLA-DM heterodimers have been purified from culture supernatants of insect cell transformants. At pH 5.0, they induce the dissociation of a subset of peptides bound to HLA-DR, including a nested set of class-II-associated invariant chain peptides (CLIP). This process liberates HLA-DR and leads to the enhanced binding of exogenous peptides.

View details for Web of Science ID A1995RF98900082

View details for PubMedID 7596415
AN ESSENTIAL ROLE FOR HLA-DM IN ANTIGEN PRESENTATION BY CLASS-II MAJOR HISTOCOMPATIBILITY MOLECULES NATURE Morris, P., Shaman, J., ATTAYA, M., Amaya, M., Goodman, S., Bergman, C., Monaco, J. J., Mellins, E. 1994; 368 (6471): 551-554
Abstract

In antigen-presenting cells, class II molecules of the major histocompatibility complex (MHC) bind peptides derived from endocytosed proteins. In certain B-lymphoblastoid cell mutants, MHC class II molecule-peptide complex formation is impaired, resulting in deficient antigen-presenting function. MHC deletion mutants with this defect map the responsible gene(s) to the class II region of the MHC. Here we report that multiple independent mutants with the class II presentation defect harbour lesions in HLA-DMB, an MHC-linked gene encoding a class II-like beta-chain. Expression of DMB complementary DNA in mutants lacking DMB messenger RNA restores the wild-type phenotype. These results establish HLA-DM as a critical regulatory molecule in class II-restricted antigen presentation and suggest that it functions at an intracellular site to promote class II molecule-peptide association.

View details for Web of Science ID A1994NE33500056

View details for PubMedID 8139689

Elizabeth Mellins

Professor of Pediatrics (Human Gene Therapy) at the Lucile Salter Packard Children's Hospital

Pediatrics - Human Gene Therapy

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