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. 2021 Dec 31;11:24512. doi: 10.1038/s41598-021-04291-8

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Identification and validation of antigen-specificity of cit-TNC specific TCRs: (a) The number of sorted cells that gave productive TCRs after sequencing are shown with cit-TNC22/56 specific cells in burgundy and cit-TNC17/45 specific TCRs in blue color (top panel). Clustering of TCRs using CDR3αβ amino acid sequences showed the exact number of TCRs obtained for the two specificity groups in each patient (bottom panel). Collectively, we could retrieve TCR sequences for cit-TNC17/45 specificity from 3 patients, and cit-TNC22/56 from 7 samples. (b) Demonstration of a cit-TNC22 specific CD4 + T cell expansion from peripheral blood. Tetramer staining at day 9 showed an expansion of cit-TNC22 specific CD4 + T cells, comprising one dominant TCR. Transient artificial re-expression of the TCR into HEK293T cells confirmed binding to its cognate tetramer (x-axis), but not to another epitope from cit-TNC17 (y-axis). (c) Stable re-expression of same TCR into 58αβ TCR-negative cell line expressing NFAT-GFP reporter demonstrated responsiveness towards cit-TNC22 loaded HLA-DRB1*04:01 monomers, both in proportion of NFAT + cells and of NFAT-GFP median fluorescence intensity (MFI) after stimulation with cit-TNC22 (red symbols) but not with the irrelevant cit-TNC17 peptide-monomer complexes (green symbols). Anti-CD3/CD28 stimulation is shown in blue color. X axis depicts different amounts of peptide-monomer complexes used for coating the plates. (d) Figure shows the recovery of TCRs belonging to each of the two specificity groups (cit-TNC17/45 or cit-TNC22/56) from both PB and SF from different patients, where each color represents TCRs derived from a different patient. (e) First panel shows TCR repertoire of expanded cit-TNC specific T cells. In total, 19 expanded TCR clones were found in 8 RA patients (4 PB, 4 SF), with varying extent of expansion. Each color in the figure indicates TCRs coming from different patients. Second panel shows all expanded TCRs in context of fine-specificity group (red = cit-TNC22/56 specific, blue = cit-TNC17/45 specific). Among the expanded clones, 9 TCR clones were specific for cit-TNC22/56, while 10 were cit-TNC17/45 specific. Last panel shows the extent of expansion of TCR clones within PB (top) and SF (bottom) compartments, where both sites showed expanded TCR clones.