1739: Single-Cell Characterization of the TCR Repertoire Across Tissue and Blood in Rheumatoid Arthritis

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 CD25^high 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 T_EMRA 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.