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. 2022 Jan 13;81(4):480–489. doi: 10.1136/annrheumdis-2021-220500

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© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.

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Detection of human antigen-specific T cells in the peripheral blood of HLA-DRB1*04:01 carriers by flow cytometry using ^#DR4 (HLADRB1*04:01)-tetramers with specificity for the gal₂₆₄Col2_259-273 or nCol2_259-273 peptide. (A) Representative dot blots: the CD4⁺ enriched T cells from PBMCs were stained with a dead/live marker and DRB1*04:01/Col2 peptide tetramers conjugated with two different fluorophores (PE and APC). Subsequent flowcytometric analysis reveals the Col2-specific cells in the live double positive stained subpopulation. Representative dot blots of specific DR4/gal₂₆₄Col2 and DR4/nCol2 tetramer binding to T helper cells in samples from a patient with RA and a HD are shown. Biotin-streptavidin complexes without a specific peptide conjugate served as a negative control. (B) Specific tetramer binding of T helper cells from patients with RA (n=55) and HD (n=20) using ^#DR4 (DRB1*04:01)-tetramers with different peptide specificity (gal₂₆₄Col2 vs nCol2). In PBMCs of patients with RA, the frequencies of DR4/nCol2 and DR4/gal₂₆₄Col2 staining CD4+T cells do not differ significantly (n.s.). Depicted values represent processed data in which for each tetramer staining datapoint the respective biotin background has already been subtracted from the raw value. (C) Detection of antigen-specific T cells in PBMCs of patients with RA (HLA-DRB1*04:01) on in vitro stimulation by synthetic Col2 peptides using flow cytometry. PBMCs from patients with RA were stimulated with a Col2 peptide (gal₂₆₄Col2, nCol2) and anti-CD40 for 7 hours. Subsequently, peptide-induced upregulation of the activation marker CD154 on the surface of the live CD4+T cell population was detected by flow cytometry. The frequency of CD154-positive CD4-positive T helper cells in PBMCs was significantly elevated in response to vitro challenge by the galCol2 peptide (n=41) compared with unmodified nCol2 (n=35, p=0.0262). Statistical significance was determined using the Mann-Whitney test. APC, allophycocyanin; HD, healthy donors; PBMC, peripheral blood mononuclear cells; PE, phycoerythrin; RA, rheumatoid arthritis; TCR, T-cell receptor.