Biased TCR gene usage in citrullinated Tenascin C specific T-cells in rheumatoid arthritis

Genadiy Kozhukh,William W Kwok,Sara Turcinov,Vivianne Malmström,Niyaz Yoosuf,Anatoly Dubnovitsky,Lars Klareskog,Ravi K Sharma,Fredrik Wermeling,Sanjay V Boddul

doi:10.1038/s41598-021-04291-8

Abstract

We aimed to search for common features in the autoreactive T cell receptor (TCR) repertoire in patients with rheumatoid arthritis (RA), focusing on the newly identified candidate antigen citrullinated Tenascin C (cit-TNC). Mononuclear cells from peripheral blood or synovial fluid of eight RA-patients positive for the RA-associated HLA-DRB1*04:01 allele were in-vitro cultured with recently identified citrullinated peptides from Tenascin C. Antigen-specific T cells were isolated using peptide-HLA tetramer staining and subsequently single-cell sequenced for paired alpha/beta TCR analyses by bioinformatic tools. TCRs were re-expressed for further studies of antigen-specificity and T cell responses. Autoreactive T cell lines could be grown out from both peripheral blood and synovial fluid. We demonstrate the feasibility of retrieving true autoreactive TCR sequences by validating antigen-specificity in T cell lines with re-expressed TCRs. One of the Tenascin C peptides, cit-TNC22, gave the most robust T cell responses including biased TCR gene usage patterns. The shared TCR-beta chain signature among the cit-TNC22-specific TCRs was evident in blood and synovial fluid of different patients. The identification of common elements in the autoreactive TCR repertoire gives promise to the possibility of both immune monitoring of the autoimmune components in RA and of future antigen- or TCR-targeted specific intervention in subsets of patients.

Highlights

MHC/HLA class II-restricted and antigen-specific CD4 + T cells are considered essential in the pathogenesis of ACPA-positive rheumatoid arthritis (RA) and increased knowledge of the specificity and composition of T cell receptors (TCRs) of such T cells will be valuable for the development of novel T-cell directed therapies[1]
We could identify a biased TCR beta gene usage in response to citrullinated Tenascin C that is shared among patients, indicating structural similarities in the TCR repertoire in RA
We have focused on recently identified T-cell epitopes from citrullinated Tenascin C, to which T cells have been found to be prevalent, in some cases in close parity to that of virus-specific T c ells[5]

Summary

Introduction

MHC/HLA class II-restricted and antigen-specific CD4 + T cells are considered essential in the pathogenesis of ACPA-positive rheumatoid arthritis (RA) and increased knowledge of the specificity and composition of T cell receptors (TCRs) of such T cells will be valuable for the development of novel T-cell directed therapies[1]. We have established a pipeline (Fig. 1a) for the isolation and re-expression of paired αβ TCR sequences from RA patient-derived single T c ells[9,10] In this proof-of-principle study utilizing the RA candidate autoantigen citrullinated Tenascin C, we could demonstrate the feasibility of isolation of in-vitro expanded autoreactive CD4 + T cells after stimulation with a cocktail of citrullinated peptides, and their subsequent paired alpha–beta TCR sequencing and validation. Specific immune responses in clinical manifestations and joint damage in this RA subset This knowledge motivates future efforts towards design of tolerogenic approaches for restoring immune homeostasis to this extracellular matrix protein

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Conclusion