1735: Clonally Expanded Cytotoxic CD8⁺ T Cells Recognize Citrullinated Antigens in ACPA⁺ Rheumatoid Arthritis

Jae-Seung Moon¹, Shady Younis², Orr Sharpe², Navin Rao³, Julie Carman³, Eddie James⁴, Jane Buckner⁴, Kevin D Deane⁵, Michael Holers⁶, Laura Donlin⁷, Mark Davis² and William Robinson⁸, ¹Stanford University, Palo Alto, CA, ²Stanford University, Stanford, CA, ³Janssen Research and Development, LLC, Spring House, PA, ⁴Benaroya Research Institute at Virginia Mason, Seattle, WA, ⁵University of Colorado Denver Anschutz Medical Campus, Denver, CO, ⁶Department of Medicine Division of Rheumatology, Aurora, CO, ⁷Hospital for Special Surgery, New York, NY, ⁸Stanford University School of Medicine, Palo Alto, CA

Background/Purpose: Rheumatoid arthritis (RA) is a systemic autoimmune disease associated with MHC polymorphisms. The shared epitope polymorphism in MHC class II genes is by far the strongest genetic association with ACPA⁺ RA, and the shared epitope polymorphisms have been demonstrated to facilitate the binding and presentation of citrullinated peptides. MHC class I gene polymorphisms are also associated with the susceptibility to seropositive RA. To further investigate the potential role of MHC class I polymorphisms in the pathogenesis of RA, we characterized CD8⁺ T cell phenotypes and clonality in ACPA⁺ RA blood using flow cytometric and single cell RNA and TCR sequencing analysis. We also performed in vitro assays to characterize the antigen specificity and functional properties of clonally expanded CD8⁺ T cells in ACPA⁺ RA blood.

Methods: We used flow cytometric and single-cell RNA profiling analyses including VDJ sequencing to analyze the effector phenotypes and clonal expansion of CD8⁺ T cells in ACPA⁺ RA blood. To investigate the antigen targets of the expanded CD8⁺ T cells in ACPA⁺ RA, we performed in vitro protein stimulation in ACPA⁺ RA peripheral blood mononuclear cells (PBMCs) or whole blood with candidate citrullinated RA antigens. To assess the cytotoxic potential of citrullinated antigen-activated cytotoxic of CD8⁺ T cells, we used an in vitro MHC-independent cytotoxicity assay.

Results: We identified GZMB⁺CD8⁺ subpopulations in ACPA⁺ RA blood containing large clonal lineage expansions expressing cytotoxic and tissue homing transcriptional programs, and a GZMK⁺CD8⁺ memory subpopulation comprised of smaller clonal expansions expressing effector T cell transcriptional programs. We demonstrated citrullinated RA autoantigens presented by MHC class I activate ACPA⁺ RA blood-derived GZMB⁺CD8⁺ T cells to expand, express cytotoxic mediators, and mediate killing of target cells. We also demonstrated that these activated GZMB⁺CD8⁺ T cells are present in RA synovium.

Conclusion: Our findings demonstrated that clonally expanded GZMB⁺ CD8⁺ T cells are present in ACPA⁺ RA blood. We further demonstrated that these clonally expanded cytotoxic CD8⁺ T cells are specific for citrullinated antigens and can mediate cytolytic cell killing. Our findings suggest that cytotoxic CD8⁺ T cells targeting citrullinated antigens could contribute to synovitis and joint tissue destruction in ACPA⁺ RA.
Clonally expanded cytotoxic CD8+ T cells reactive to citrullinated antigens are present in ACPA+ RA blood. (A) Percentage of CD69, GPR56 and granzyme B-expressing CD8+ T cells in healthy controls (HC), ACPA- or ACPA+ RA PBMCs. (B) Phenotypic analysis of clonally expanded CD8+ T cells in ACPA+ RA and HC. Heatmap of known gene markers provides the gene expression patterns of clonally expanded cells for: cytotoxicity/Effector, cell trafficking, suppressor, and naïve/memory subsets. (C) Native vimentin or citrullinated vimentin pulsed monocyte-derived dendritic cells were cocultured with Cell Proliferation Dye eF450-labeled CD8+ T cells isolated from ACPA+ RA PBMCs with or without anti-CD8/HLA class I-blocking antibody for 10 days. Proliferation ability of T cells was determined by flow cytometry. (D) Percentage of GzmB+IFNg+ population in CD8+ T cells from ACPA+ RA or HC fresh whole blood stimulation using native or citrullinated vimentin.
Disclosures: J. Moon, None; S. Younis, None; O. Sharpe, None; N. Rao, Janssen; J. Carman, Janssen; E. James, Janssen, Provention Bio, Bristol-Myers Squibb(BMS), Novartis; J. Buckner, Amgen, Bristol Myers Squibb, Gentiobio, Hot Spot Therapeutics, Janssen, Pfizer, Novo Nordisk, Allen Institute for Immunology, Type 1 Diabetes TrialNet Study Group, La Jolla Institute, Oklahoma Medical Research Foundation, Bristol Myers Squibb Immunology, Colton Center for Autoimmunity at Penn, Board of Scientific Counsellors; K. Deane, Werfen; M. Holers, Celgene, Bristol-Myers Squibb(BMS), Janssen; L. Donlin, None; M. Davis, None; W. Robinson, None.