TCRß Sequencing Reveals Spatial and Temporal Evolution of Clonal CD4 T cell Responses in a Breach of Tolerance Model of Inflammatory Arthritis

Objectives: Development of effective tolerogenic therapies for Rheumatoid Arthritis (RA) relies on the understanding the development and evolution of articular antigen specific CD4 T cell responses. We aimed to assess the clonality of the initial endogenous CD4 T cell infiltrate in early inflammatory arthritis and monitor the evolution and dynamics of CD4 antigen specific responses in both the inflamed joint and its associated draining popliteal lymph node (pLN).Methods: To recapitulate the early and late phases of inflammatory arthritis reported in patients, we used mouse models of antigen-induced breach of self-tolerance and chronic polyarthritis and analysed the endogenous CD4 T cell articular infiltrate in inflamed joints and pLNs. Flow cytometry and TCRß sequencing were used to phenotypically characterise and assess the clonality of infiltrating, antigen experienced, endogenous CD4 T cells.Results: The TCR repertoires from pLNs displayed increased clonality and diversity with disease progression, while inflamed joints maintain similar CD4 T cell repertoire clonality and diversity with time. The increase in clonality and diversity in pLNs at the late phase results from the accumulation of clones with a diverse range of Variable chain genes of the T cell receptor ß chain (TRBV), while inflamed joints at both phases contain clones with similar TRBV genes, reflecting differences in antigen specificities between these sites with time. Repertoires from pLNs and joints at the late phase have a reduced number of overlapping CDR3ß sequences compared with pLNs and joints in early disease phases, however correlation analysis revealed that the most abundant clones present at both sites accumulate in the joint at the later phase.Conclusions: Antigen specific responses broaden and evolve with the progression of inflammatory arthritis and are reflected in pLNs before potentially being mirrored in the joint over time. These observations imply that antigen-specific tolerogenic therapies will be more easily developed and more effective at earlier phases of the disease, when CD4 T cell clonality is least diverse.