Mapping Oligoclonally Expanded T Cells Within the Peripheral and Synovial Immune Landscape of Untreated ACPA+ Rheumatoid Arthritis Patients at the Single Cell Level

Background/Purpose: Clonal T cell expansions – some large – have been identified in the peripheral blood (PB) and in synovial tissue (ST) of multiple joints from recently-diagnosed rheumatoid arthritis (RA) patients. As the degree of ST clonal dominance decreased with longer disease duration, new-onset RA presents the ideal opportunity to study the transcriptome and TCRα/β of individual clonotypes. This can be achieved using single-cell RNA-sequencing, which already identified novel T cell subsets in longstanding RA. To elucidate the TCR usage and transcriptome of oligoclonally expanded T cells in the PB and ST compartments in newly-diagnosed RA, we combined TCR repertoire, clonality and transcriptomic analysis of individual ST and paired PB mononuclear cells (PBMC) in untreated HLA-DRB1*0401+ ACPA+ RA patients.

Methods: PBMC and whole cryopreserved ST samples were collected from 4 treatment-naïve RA patients undergoing arthroscopic synovial biopsy. CD45+ leukocytes from cryopreserved PBMC, and CD45+ leukocytes and CD45– fibroblasts isolated from paired disaggregated ST, by fluorescence-activated cell sorting, were sequenced using 5’ RNA/TCR single-cell 10x Genomics kits. Single-cell transcriptomic analysis, using the Seurat package in R, reconstructed the compartment-specific composition of PB and ST.

Results: Myeloid cells were expanded in the ST relative to PB. Myeloid and NK cell expansion was related to arthroscopic synovitis severity. NK and B cell clusters were proportionately smaller among ST than PB leukocytes. Transcriptomics identified 10 unique CD3+TCR+ populations in PB and ST, including cytotoxic, helper and regulatory T cells. In PB, the most expanded clonotypes were CD8+ and CD4+ cytotoxic T cells. ST expanded clonotypes included CD8+ and CD4+ T cells expressing cytotoxic and multiple cytokine genes, CD4+ICOS+ T cells with evidence of IL-6 signalling, and CD4+GATA3+ tissue-resident T cells. Shared ST and PB T cell clonotypes were predominantly cytotoxic polyfunctional CD8+ T cells. A proportion of PB cytotoxic clonotypes matched known EBV and CMV-reactive clones, and a proportion of shared PB/ST cytotoxic clonotypes matched CMV-reactive clones.

Conclusion: Similar T cell clusters were identified in ST and PB. Transcriptomes of non-cytotoxic expanded CD4+ T cell clonotypes in the ST of untreated patients suggest B cell interaction and help. The most expanded clonotypes in circulation include T cells with cytotoxic and proinflammatory capability, including known viral-specific clones, some of which reach and expand in RA ST. Antigen derived and presented from persistent viruses may expand responsive clones to be bystander drivers of synovial inflammation.