Antigen-Expanded T-Cells Infiltrating New-Onset Rheumatoid Arthritis Synovial Tissue Are IL-6-Reponsive, T-Helper 2 and Pro-Inflammatory Viral-Specific Clones

Aims: Seropositive autoimmune rheumatoid arthritis (RA) is characterized by T cells interacting with antigen-presenting cells, autoantibody production and chronic inflammation. While clonal T cell expansions in peripheral blood (PB) and synovial tissue (ST) of recently-diagnosed patients suggest a role for antigen-specific T cells, their contribution to RA inflammation is unclear. Using simultaneous single-cell RNA and T cell receptor (TCR)α/β sequencing, we aimed to identify antigen-expanded T cell clonotypes and matched transcriptomes in PB and ST of newly-diagnosed, seropositive, DMARD-naïve HLA-DRB1*04:01+ RA patients.

Methods: CD45+ leukocytes from cryopreserved PBMC, and CD45+ leukocytes and CD45- fibroblasts isolated from 4 pairs of disaggregated ST were sequenced using 5' RNA/TCR single-cell 10x Genomics kits. Single-cell transcriptomic analysis reconstructed the compartment-specific composition of PB and ST. Imaging mass cytometry (IMC) corroborated transcriptomic and TCR findings and investigated cellular and spatial interactions.

Results: Myeloid cells were enriched in ST relative to PB. Transcriptomics identified four T cell superclusters in PB and ST, with enrichment of Th2-like and polyfunctional cytotoxic effector T cells in ST. In PB, the most expanded clonotypes were CD8+ and CD4+ cytotoxic T cells. Transcriptomic signatures of ST-unique CD4+ clonotypes highlight central memory, Th2 differentiation and IL-6 signaling, while the most expanded circulating CD4+ and CD8+ clonotypes display cytotoxic polyfunctional proinflammatory capacity. Among these, TCR sequencing, IMC and tetramer staining identify EBV- and CMV-specific clonotypes, some of which migrate to ST and lodge adjacent to blood vessels, Thy1+ fibroblasts, and dendritic cells.

Conclusions: Our findings suggest that at disease onset, synovial inflammation is propagated by a two-pronged attack: synovial antigen-specific T cells supporting APC activation, and cytotoxic viral-specific bystander clones with polyfunctional proinflammatory capacity. These findings demonstrate the critical importance of viral immunity and viral-specific bystander T cells to the immunopathogenesis of RA and have important implications for the design of future immunotherapies.