University of Rochester Medical center Rochester, NY, United States
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.