National Institutes of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), National Institutes of Health (NIH) Chevy Chase, MD, United States
Urvashi Kaundal1, Chloe Borden1, Cihan Oguz2, Jinghua Lu2, Emilee Stenson1, Ami Shah3, Maureen Mayes4, Ayo Doumatey5, Amy Bentley5, Daniel Shriner5, Robyn Domsic6, Thomas Medsger7, Paula Ramos8, Richard Silver8, Virginia Steen9, John Varga10, Vivien Hsu11, Lesley Ann Saketkoo12, Elena Schiopu13, Dinesh Khanna14, Jessica Gordon15, Lindsey Criswell16, Heather Gladue17, Chris Derk18, Elana Bernstein19, S. Louis Bridges, Jr.15, Victoria Shanmugam20, Lorinda Chung21, Suzanne Kafaja22, Reem Jan23, Marcin Trojanowski24, Avram Goldberg25, Benjamin Korman26, Settara Chandrasekharappa5, Faiza Naz27, Stefania Dell'Orso1, Adebowale Adeyemo5, Charles Rotimi5, Elaine Remmers5, Francesco Boin28, Fredrick Wigley29, Peter Sun2, Daniel Kastner5 and Pravitt Gourh30, 1National Institutes of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), National Institutes of Health (NIH), Bethesda, MD, 2National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, 3Johns Hopkins Rheumatology, Baltimore, MD, 4Division of Rheumatology and Clinical Immunogenetics, University of Texas McGovern Medical School, Houston, TX, 5National Human Genome Research Institute, Bethesda, MD, 6University of Pittsburgh, Pittsburgh, PA, 7University of Pittsburgh School of Medicine, Pittsburgh, PA, 8Medical University of South Carolina, Charleston, SC, 9Georgetown University School of Medicine, Washington, DC, 10University of Michigan, Ann Arbor, MI, 11Rutgers-RWJ Medical School, South Plainfield, NJ, 12University Medical Center - Comprehensive Pulmonary Hypertension Center and ILD Clinic Programs // New Orleans Scleroderma and Sarcoidosis Patient Care & Research Centeris, New Orleans, LA, 13Michigan Medicine, Ann Arbor, MI, 14Division of Rheumatology, Department of Internal Medicine, Scleroderma Program, University of Michigan, Ann Arbor, MI, 15Hospital for Special Surgery, New York, NY, 16National Human Genome Research Institute, NIH, Bethesda, MD, 17Arthritis & Osteoporosis Consultants of the Carolinas, Charlotte, NC, 18University of Pennsylvania, Philadelphia, PA, 19Columbia University, New York, NY, 20George Washington University, Great Falls, VA, 21Stanford University, Stanford, CA, 22UCLA Department of Medicine, Division of Rheumatology, Los Angeles, CA, 23University of Chicago, Chicago, IL, 24Boston University School of Medicine, Boston, MA, 25NYU Langone Medical Center - NYU Hospital for Joint Diseases, Lake Success, NY, 26University of Rochester, Rochester, NY, 27National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, MD, 28Cedars-Sinai Medical Center, Los Angeles, CA, 29Johns Hopkins University, Baltimore, MD, 30National Institutes of Health, Bethesda, MD
Background/Purpose: Systemic sclerosis (SSc) is an autoimmune, fibrotic disorder that disproportionately affects African Americans (AA). Previous work from our lab and others has suggested a pivotal role of HLA-II genes in SSc pathogenesis. HLA-II alleles encode for variations in antigen binding grooves of HLA proteins that present antigens to T-cell receptors (TCRs) and can contribute to antigen-specific immune responses. The TCR repertoire is HLA-restricted, antigen-specific, and recognizes antigens presented by HLA proteins. In this study, we explored the diversity of the hypervariable complementarity determining region 3 (CDR3) and TCR repertoire in SSc patients. We also examined the role of HLA-II alleles, especially HLA-DPB1*13:01, in the selection of the specific sequence and amino acid composition of the TCR in SSc patients.
Methods: Genomic DNA from 132 AA SSc patients from the GRASP consortium along with 50 AA healthy control samples were extracted and the TCRβ chain was deep sequenced using the immunoSEQ assay (Adaptive Biotechnologies, USA). Data was analyzed using Immunarch to identify differences in unique clones, CDR3 length, V gene usage, J gene usage, and amino acid usage in the FG-Loop of the TCRβ chain between the two SSc groups. Benjamini Hochberg FDR was used to correct for multiple testing.
Results: The CDR3 length was similar between SSc patients and controls (Figure 1A, B). The TCRβ repertoire was clonally expanded in HLA-DPB1*13:01+ SSc patients as compared to patients who did not carry this allele (Figure 1C). The proportion of small and hyperexpanded TCRβ clonotypes was increased in HLA-DPB1*13:01+ SSc patients (Figure 1D). Analysis of V gene and J gene usage identified statistically significantly increased usage of TRBV5-1, TRBV7-3, TRBJ2-1, and TRBJ2-2 genes and a decreased usage of TRBV6-1, TRBV6-4, TRBV6-5, TRBV25-1, and TRBJ1-5 genes in SSc patients compared to controls and the differences were statistically significant (Figure 2A, B). Overall amino acid usage of the FG loop of the TCRβ chain (antigen binding region) identified increased usage of hydrophobic amino acids, leucine and valine, and reduced usage of negatively charged glutamine in the FG loop of SSc patients compared to controls. Positional amino acid usage analysis of the FG loop of the TCRβ chain revealed increased usage of leucine at position 108 with decreased usage of tyrosine, glutamine, and aspartate.
Conclusion: This is the first study to report an association between HLA-II alleles and specific CDR3 composition in SSc patients. We examined the TCRβ repertoire of a large number of AA SSc patients and identified differential V and J gene and FG-loop amino acid usage that correlated with the presence of a specific HLA-II allele. Hydrophobic amino acid usage in the FG loop of the TCRβ chain has been reported to increase the avidity of TCR-MHC interaction and increased autoreactivity. Based on our findings, we can hypothesize that the TCRβ sequences favored by the SSc-associated HLA-II alleles are involved in autoantigen recognition and autoimmunity. These results highlight the important role of HLA-II alleles and T cells in SSc pathogenesis.
Figure 1. A. CDR3 length distribution in SSc patients (SSc) and controls; B. CDR3 length distribution in control, HLA-DPB1*1301- , HLA-DPB1*1301+ group; C. Number of unique clones in control, HLA-DPB1*1301- , HLA-DPB1*1301+ group; D. Proportion of TCR clonotype groups within frequency ranges: rare (0 to 10-5); small- (10-5 to 10-4); medium (10-4 to 10-3); large (10-3 to 10-2); and hyperexpanded (10-2 to 1).
Figure 2. TCR A. V-gene usage; B. J-gene usage Disclosures: U. Kaundal, None; C. Borden, None; C. Oguz, None; J. Lu, None; E. Stenson, None; A. Shah, Arena Pharmaceuticals, Medpace/Eicos, Kadmon Corporation; M. Mayes, Actelion Pharma, Mitsubishi-Tanabe, Boehringer Ingelheim, EICOS, Horizon Pharma, Prometheus, Corbus, Medtelligence; A. Doumatey, None; A. Bentley, None; D. Shriner, None; R. Domsic, None; T. Medsger, None; P. Ramos, None; R. Silver, None; V. Steen, None; J. Varga, Boehringer-Ingelheim; V. Hsu, None; L. Saketkoo, None; E. Schiopu, None; D. Khanna, Boehringer Ingelheim, Genentech, Prometheus, Horizon, Chemomab, Talaris, Gesynta, Amgen, Acceleron, Actelion, Bayer, CSL Behring, Paracrine Cell Therapy, Mitsubishi Tanabe, Theraly, Eicos Sciences; J. Gordon, None; L. Criswell, None; H. Gladue, GlaxoSmithKlein(GSK), AstraZeneca; C. Derk, None; E. Bernstein, Boehringer-Ingelheim, Kadmon, Pfizer; S. Bridges, Jr., Bristol Myers Squibb; V. Shanmugam, None; L. Chung, Kyverna, Mitsubishi Tanabe, Eicos, Boehringer-Ingelheim, Jasper, Genentech; S. Kafaja, None; R. Jan, None; M. Trojanowski, None; A. Goldberg, None; B. Korman, None; S. Chandrasekharappa, None; F. Naz, None; S. Dell'Orso, None; A. Adeyemo, None; C. Rotimi, None; E. Remmers, None; F. Boin, None; F. Wigley, None; P. Sun, None; D. Kastner, None; P. Gourh, None.