Garrett Dunlap1, Aaron Wagner2, Nida Meednu3, Anna Jonsson4, Fan Zhang5, Kevin Wei6, Paul Utz7, William Robinson7, Holden Maecker7, Judith James8, Joel Guthridge8, S. Louis Bridges, Jr.9, Laura Donlin9, Susan Goodman9, Edward DiCarlo9, Vivian Bykerk9, Christopher Ritchlin10, Darren Tabechian11, James Lederer4, Ellen Gravallese12, Mandy McGeachy13, Gary S. Firestein14, Peter Gregersen15, Diane Horowitz16, David Boyle17, Laura Geraldino-Pardilla18, Harris Perlman19, Arthur Mandelin20, Joan Bathon18, Laura Hughes21, V. Michael Holers22, Kevin D Deane23, Larry Moreland22, Andrew Filer24, Costantino Pitzalis25, Lindsy Forbess26, Ami Ben-artzi27, Karen Salomon-Escoto28, Soumya Raychaudhuri4, Michael Brenner12, Accelerating Medicines Partership (AMP) RA/SLE Network29, Andrew McDavid2, Jennifer Anolik3 and Deepak Rao4, 1Harvard University, Boston, MA, 2University of Rochester, Rochester, NY, 3University of Rochester Medical center, Rochester, NY, 4Brigham and Women's Hospital, Boston, MA, 5University of Colorado, Aurora, CO, 6Brigham and Women's Hospital and Harvard Medical School, Boston, MA, 7Stanford University School of Medicine, Palo Alto, CA, 8Oklahoma Medical Research Foundation, Oklahoma City, OK, 9Hospital for Special Surgery, New York, NY, 10Allergy, Immunology and Rheumatology Division, University of Rochester Medical School, Canandaigua, NY, 11URMC, Rochester, NY, 12Brigham and Women's Hospital, Harvard Medical School, Boston, MA, 13University of Pittsburgh, Pittsburgh, PA, 14University of California, San Diego, San Diego, CA, 15The Feinstein Inst for Med Research, Larchmont, NY, 16Northwell Health, Jericho, NY, 17UCSD, La Jolla, CA, 18Columbia University, New York, NY, 19Northwestern University, Chicago, IL, 20Northwestern University Feinberg School of Medicine, Chicago, IL, 21University of Alabama at Birmingham, Birmingham, AL, 22University of Colorado, Denver, CO, 23University of Colorado Denver Anschutz Medical Campus, Denver, CO, 24University of Birmingham, Birmingham, United Kingdom, 25Queen Mary University of London, London, United Kingdom, 26Cedars-Sinai Medical Center, Los Angeles, CA, 27Ami Ben-Artzi, MD Inc., Beverly Hills, CA, 28University of Massachusetts Medical School, Worcester, MA, 29National Institutes of Health, Washington, DC
Background/Purpose: In rheumatoid arthritis (RA), T cells represent a large proportion of the immune population present in inflamed joint synovium, and subsets of both CD4+ and CD8+ T cells are suspected to contribute to the pathology of the disease. Here, we use single cell RNA-sequencing (scRNA-seq) to better understand T cell receptor (TCR) repertoire differences and relationships both across cellular subsets and between tissue compartments in RA.
Methods: We performed 5' droplet-based scRNA-seq on lymphocytes sorted from synovial tissue biopsies (n = 12) and matched peripheral blood samples (n = 10) to obtain paired transcriptomic and repertoire information for captured cells. After filtering low-quality cells and performing an initial round of clustering, T cells were identified, populations of CD4+, CD8+, and innate T cells were separately re-clustered, and a combination of differentially-expressed gene analysis, gene-set enrichment, and reference mapping was utilized to clearly define the cell subpopulations present. Paired TCR information for each cell was then incorporated, allowing for metrics of clonal overlap, diversity, and expansion to be calculated among the subpopulations.
Results: Among 14 CD4+ clusters, we identified populations of naïve, memory, regulatory, T peripheral helper (Tph), T follicular helper (Tfh), cytotoxic, and proliferating T cells (Fig 1A). Naïve CD4+ cells had increased representation in the blood compared to the synovial tissue, while Tph, Tfh, and CD25high Treg cells were among the clusters significantly increased in synovial tissue. When analyzing the repertoire of these cells, we found the largest clonal expansion among the cytotoxic, Tph, and granzyme A (GZMA+) memory populations. An examination of shared clones between clusters found the Tph cluster to be clonally related to the Tfh cluster and to comprise a significant proportion of proliferating CD4+ cells in the synovial tissue (Fig 1B). A detailed comparison of clonal and non-clonal Tph cells revealed that expanded clones had elevated markers of effector function, including TNFRSF18, IFNG, and PRF1. Analysis of CD8+ T cells revealed 9 clusters, including populations of naïve, memory, cytotoxic, and proliferating cells (Fig 1C). We identified a population of cells expressing GZMK and GZMB, along with a TEMRA population, to be highly clonally expanded (Fig 1D). We then further characterized a potentially viral-reactive set of these CD8 clones by referencing a public database of viral TCRs. Finally, a focus on innate T cells confirmed the presence of a MAIT population by TCR, of which we could identify expanded clones shared between tissue and blood in multiple patients.
Conclusion: T cells are crucial for the development and progression of RA but features of the TCR repertoire in relation to the disease have not been adequately studied. Here, we provide a comprehensive look at the TCR repertoire of RA patients, both within synovial tissue and in the blood. We identify unique clonal attributes related to each of these compartments and identify clonal relationships among transcriptomically different populations of cells, which together deepen our understanding of the pathogenic role of certain T cell subsets in RA.
Disclosures: G. Dunlap, None; A. Wagner, None; N. Meednu, None; A. Jonsson, Amgen; F. Zhang, None; K. Wei, Gilead sciences, Mestag, nanoString, 10X Genomics; P. Utz, None; W. Robinson, None; H. Maecker, None; J. James, Bristol-Myers Squibb(BMS), AstraZeneca, Novartis, Progentec Biosciences; J. Guthridge, None; S. Bridges, Jr., Bristol Myers Squibb; L. Donlin, None; S. Goodman, Novartis, UCB; E. DiCarlo, None; V. Bykerk, Janssen, Bristol Myers Squibb, Crossbridge, Pfizer, Sanofi Aventis, Brainstorm Therapeutics, Amgen, UCB, Gilead, Genzyme Corporation, Regeneron; C. Ritchlin, UCB, AbbVie, Eli Lilly, Pfizer Inc, Novartis, Janssen, Bristol-Myers Squibb; D. Tabechian, None; J. Lederer, VeloceBio, LLC, Alloplex Biotherapeutics, Inc; E. Gravallese, New England Journal of Medicine, Textbook Rheumatology, AMN healthcare, CVS, American Well Corporation, Cerner Corp; M. McGeachy, None; G. Firestein, Eli Lilly; P. Gregersen, None; D. Horowitz, None; D. Boyle, None; L. Geraldino-Pardilla, None; H. Perlman, Janssen, kininska, exagen, LEK consultating, Guidepoint; A. Mandelin, AbbVie, Pfizer, Bristol-Myers Squibb(BMS), Horizon, CVS Caremark; J. Bathon, None; L. Hughes, None; V. Holers, Janssen; K. Deane, Werfen; L. Moreland, None; A. Filer, None; C. Pitzalis, AbbVie/Abbott, Astellas, Astra-Zeneca/MedImmune, BMS, CelGene, Grunenthal, GSK, Johnson/J&J, Kiniksa, MSD, Pfizer, Sanofi, Roche/Genentech/Chugai, UCB; L. Forbess, None; A. Ben-artzi, None; K. Salomon-Escoto, None; S. Raychaudhuri, Mestag, Inc, Rheos Medicines, Janssen, Pfizer, Biogen; M. Brenner, GSK, 4FO Ventures, Mestag Therapeutics; A. RA/SLE Network, None; A. McDavid, None; J. Anolik, None; D. Rao, Janssen, Merck, Bristol-Myers Squibb, Scipher Medicine, HiFiBio, Inc., AstraZeneca, Pfizer.