1630: Single-cell Profiling of B Cell Repertoire and Gene Expression in the RA Synovium Reveals Tissue Specific Clonal Expansion

Aaron Wagner¹, Nida Meednu², Garrett Dunlap³, Fan Zhang⁴, Anna Jonsson⁵, Kevin Wei⁶, Ami Ben-artzi⁷, Lindsy Forbess⁸, Laura Geraldino-Pardilla⁹, Diane Horowitz¹⁰, Laura Hughes¹¹, Arthur Mandelin¹², Karen Salomon-Escoto¹³, Darren Tabechian¹⁴, Edward DiCarlo¹⁵, Ellen Gravallese¹⁶, Brendan Boyce¹⁷, Christopher Ritchlin¹⁸, James Lederer⁵, Mandy McGeachy¹⁹, Peter Gregersen²⁰, Paul Utz²¹, William Robinson²¹, Holden Maecker²¹, Judith James²², Joel Guthridge²², Harris Perlman²³, Joan Bathon⁹, Susan Goodman¹⁵, Gary S. Firestein²⁴, David Boyle²⁵, S. Louis Bridges, Jr.¹⁵, Kevin D Deane²⁶, V. Michael Holers²⁷, Larry Moreland²⁷, Andrew Filer²⁸, Costantino Pitzalis²⁹, Vivian Bykerk¹⁵, Laura Donlin¹⁵, Soumya Raychaudhuri⁵, Michael Brenner¹⁶, AMP RA/Lupus¹⁷, Deepak Rao⁵, Andrew McDavid¹ and Jennifer Anolik², ¹University of Rochester, Rochester, NY, ²University of Rochester Medical center, Rochester, NY, ³Harvard University, Boston, MA, ⁴University of Colorado, Aurora, CO, ⁵Brigham and Women's Hospital, Boston, MA, ⁶Brigham and Women's Hospital and Harvard Medical School, Boston, MA, ⁷Ami Ben-Artzi, MD Inc., Beverly Hills, CA, ⁸Cedars-Sinai Medical Center, Los Angeles, CA, ⁹Columbia University, New York, NY, ¹⁰Northwell Health, Jericho, NY, ¹¹University of Alabama at Birmingham, Birmingham, AL, ¹²Northwestern University Feinberg School of Medicine, Chicago, IL, ¹³University of Massachusetts Medical School, Worcester, MA, ¹⁴URMC, Rochester, NY, ¹⁵Hospital for Special Surgery, New York, NY, ¹⁶Brigham and Women's Hospital, Harvard Medical School, Boston, MA, ¹⁷University of Rochester, Rochester, ¹⁸Allergy, Immunology and Rheumatology Division, University of Rochester Medical School, Canandaigua, NY, ¹⁹University of Pittsburgh, Pittsburgh, PA, ²⁰The Feinstein Inst for Med Research, Larchmont, NY, ²¹Stanford University School of Medicine, Palo Alto, CA, ²²Oklahoma Medical Research Foundation, Oklahoma City, OK, ²³Northwestern University, Chicago, IL, ²⁴University of California, San Diego, San Diego, CA, ²⁵UCSD, La Jolla, CA, ²⁶University of Colorado Denver Anschutz Medical Campus, Denver, CO, ²⁷University of Colorado, Denver, CO, ²⁸University of Birmingham, Birmingham, United Kingdom, ²⁹Queen Mary University of London, London, United Kingdom

Background/Purpose: Ectopic lymphoid structures can develop in rheumatoid arthritis (RA) synovial tissue, but the precise pathways of B cell activation and selection are not well understood. Here, we use single cell RNA-sequencing (scRNA-seq) to better understand B cell receptor (BCR) repertoire differences and relationships both across cellular subsets and between tissue compartments in RA.

Methods: We sorted B cells from synovial tissue biopsies (n = 12) and matched peripheral blood samples (n = 10) to obtain paired scRNA-seq and repertoire (BCR) information from captured cells. After filtering low-quality cells and performing an initial round of clustering, B cells were identified and characterized. Analysis of cluster-specific markers and reference mapping were utilized to define the cell subpopulations present. Paired BCR information for each cell was incorporated, and the overlap, diversity, and clonal expansion were calculated using the IGH CDR3 to define clonal families. We also analyzed somatic hypermutation (SHM) and immunoglobulin isotype usage.

Results: After applying QC, 27,869 B cells were captured across all samples with > 80% of B cells having BCR information. Clustering of B cells identified 10 populations including multiple naïve-like (varying in IgD expression), memory, activated (Nur77+), age-associated B cells (ABCs), plasmablast and plasma cells. We observed 5-20 fold synovial enrichment of activated B cells and plasma cells whereas naïve-like clusters are predominant in the blood. ABCs and Activated B cells in the synovium had 0.5-1.2 percentage point higher SHM compared to PBL (p< 0.002). In every subject, we observed substantial clonal expansion in both the synovium and peripheral blood, as well as clones shared between these tissues, though most clones tended to segregate by tissue source. Sub-populations had more expanded clones in the synovium than periphery. Naïve B cells exhibited fewer expanded clonotypes than non-naïve populations. There was clonal sharing across multiple sub-populations, including between IgG and IgA plasma cells, and ABCs with Activated, Plasma and Memory B cells.

Conclusion: Our data demonstrate the value of integrating gene expression and repertoire data for the study of B cell responses in RA synovial tissue and provide evidence of in situ selection. Together, our findings suggest in situ differentiation of selected B cell clones and trafficking of these expanded clones between blood, synovial, and mucosal tissues.

Disclosures: A. Wagner, None; N. Meednu, None; G. Dunlap, None; F. Zhang, None; A. Jonsson, Amgen; K. Wei, Gilead sciences, Mestag, nanoString, 10X Genomics; A. Ben-artzi, None; L. Forbess, None; L. Geraldino-Pardilla, None; D. Horowitz, None; L. Hughes, None; A. Mandelin, AbbVie, Pfizer, Bristol-Myers Squibb(BMS), Horizon, CVS Caremark; K. Salomon-Escoto, None; D. Tabechian, None; E. DiCarlo, None; E. Gravallese, New England Journal of Medicine, Textbook Rheumatology, AMN healthcare, CVS, American Well Corporation, Cerner Corp; B. Boyce, None; C. Ritchlin, UCB, AbbVie, Eli Lilly, Pfizer Inc, Novartis, Janssen, Bristol-Myers Squibb; J. Lederer, VeloceBio, LLC, Alloplex Biotherapeutics, Inc; M. McGeachy, None; P. Gregersen, None; P. Utz, None; W. Robinson, None; H. Maecker, None; J. James, Bristol-Myers Squibb(BMS), AstraZeneca, Novartis, Progentec Biosciences; J. Guthridge, None; H. Perlman, Janssen, kininska, exagen, LEK consultating, Guidepoint; J. Bathon, None; S. Goodman, Novartis, UCB; G. Firestein, Eli Lilly; D. Boyle, None; S. Bridges, Jr., Bristol Myers Squibb; K. Deane, Werfen; V. Holers, Janssen; 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; V. Bykerk, Amgen, Bristol-Myers Squibb(BMS), Genzyme, Brainstorm, Gilead, Regeneron, UCB, Pfizer, Sanofi, Aventis; L. Donlin, None; S. Raychaudhuri, Mestag, Inc, Rheos Medicines, Janssen, Pfizer, Biogen; M. Brenner, GSK, 4FO Ventures, Mestag Therapeutics; A. RA/Lupus, None; D. Rao, Janssen, Merck, Bristol-Myers Squibb, Scipher Medicine, HiFiBio, Inc., AstraZeneca, Pfizer; A. McDavid, None; J. Anolik, None.