Background/Purpose: Citrulline (cit) autoimmunity is central in rheumatoid arthritis (RA), including both anti-citrulline protein antibodies (ACPA) and autoreactive CD4+ T cells. While the ACPA are abundant, studies of cit-reactive T cells have been hampered by their scarcity. Still, cit-reactive T cells have been reported in the RA joint and in peripheral blood by peptide-HLA tetramer technology. We have set up an in-vitro antigen(ag)-specific expansion method [1] for generating TCR sequences to dissect the diversity of the autoreactive T cell repertoire. Hereby we can look for dominant T cell clones amongst different patients sharing the same HLA genes.

Methods: We subjected synovial fluid-derived cells (SFMC) from HLA-DRB1*04:04 RA patients (n=4) and peripheral blood (PBMC) from HLA-DRB1*04:04 healthy controls (n=4) to our ag-specific expansion method [1]. A cit-peptide from cartilage intermediate layer protein (cit-CILP2) was used to query autoimmunity whereas an influenza matrix protein derived peptide (MP54) was used as positive control. In-vitro expanded tetramer positive cells were single sorted and processed for PCR based TCR amplification, followed by barcoding and TCR sequencing using miSeq. The data was demultiplexed by our in-house script and clones identified by comparison of both CDR3α and β amino acid sequences. TCRs were further validated for specificity by transient re-expression into HEK293 cells, followed by staining with the same tetramers initially used for sorting.

Results: Cit-CILP2+ T cells could be expanded in vitro in all samples and clonal TCR expansions were prominent. Collectively, we could get 27 cit-CILP2 specific (PB n=6 and SF n=21) and 56 MP54 specific (PB n=37 and SF n=19) expanded T-cell clones. We also captured another 101 cit-CILP2 specific and 190 MP54-specific unique TCRs. The proportion of unique and expanded clones for cit-CILP2 and MP54 was similar in the joint-derived samples. The CDR3 length distribution for cit-CILP2 specific clones was different from MP54 specific clones for both the TCR α and β chain. Longer CDR3 length were more common in cit-CILP2 specific clones and there were differences in distribution of amino acids at specific positions. Clones derived from same patient showed shared usage of V and J genes for both α and β chains. HEK cell Re-expression of TCRs validated their target specificity.

Conclusion: This method provides a rapid tool for generation and validation of ag-specific TCRs. So far, the gene usage and CDR3 profiles appear subject-driven with limited sharing among subjects. Today, many studies are generating transcriptomic data (including TCR) from sites of inflammation and generating orphan TCRs, i.e. without known ag-specificity. We are accumulating cit-peptide ag-specific TCR sequences, which may in the future be used to identify ag-specific TCR signatures among “orphan” TCRs in such public big data sets. Until then, cit-reactive TCRs will be used in functional in vitro studies as well as in establishing relevant mice models for RA. Finally, we will also evaluate longitudinal changes in the autoreactive TCR repertoire during disease progression and after therapeutic intervention.

Reference:

Kumar R et. al. DOI: 1136/annrheumdis-2020-eular.1093

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/identification-and-validation-of-citrulline-specific-tcrs-in-cd4t-cells-in-rheumatoid-arthritis/