Fig. 3: Affinity analysis of TCR-HLA-DR4^{Vim-64cit59-71}/^{α-eno-15cit10-22} interactions, antigen reactivity of RA2.7 TCR towards HLA-DR4^{α-eno-15cit10-22/α-eno-15cit10-22V20G} and TCR gene segment usage of ACPA⁺ RA donor and healthy control CD4⁺ T cells binding to HLA-DR4^{α-eno-15cit10-22V20G} tetramer.

a Binding of TCRs A03, A07 and RA2A03 to HLA-DR4^{Vim-64cit59-71} and (b) Binding of RA2.7 TCR to HLA-DR4^{α-eno-15cit10-22/α-eno-15cit10-22V20G}. For K_D determination in (a) and (b) all data were derived from two or more independent experiments, with A03 TCR (n = 7), A07 TCR (n = 6), RA2A3 TCR (n = 2), RA2.7 (n = 2) and curve fits using a single ligand binding model. For each concentration the points represent the mean and error bars correspond to SD. Expression of CD69 on the surface of RA2.7 TCR transduced SKW-3 cell lines stimulated overnight with serial dilution of (c) α-eno-15cit 10-22 peptide-pulsed BLCL 9031 or (e) α-eno-15cit 10-22V20G peptide- pulsed BLCL 9031. Expression of CD3 on the surface of RA2.7 TCR transduced SKW-3 cell lines stimulated overnight with serial dilution of (d) α-eno-15cit 10-22 peptide-pulsed BLCL 9031 or (f) α-eno-15cit 10-22V20G peptide- pulsed BLCL 9031. For (c–f) the black dots are presented as the mean fluorescence intensity (MFI) of average of duplicated values from three independent experiments. Anti-HLA-DR4 antibody used as control. For (c–f) P-values were determined by one-way ANOVA with Dunnett’s multiple comparison testing, *P < 0.05, **P < 0.01, ***P < 0.002, ****P < 0.0001 and error bars represent \(\pm\) s.e.m. g TCR TRAV and TRBV gene segment usage of CD4⁺ T cells isolated from the peripheral blood of ACPA⁺ RA donor 2, ACPA^- RA donor 3, and HLA-DR4⁺ healthy control (HC) donors 10195 and 4737 using HLA-DR4^{α-eno-15cit10-22V20G} tetramer. All TCR clones were unique except for one TRAV26-1⁺ TRBV5-4⁺ clone from HC donor 4727 for which two sequences were isolated (Supplementary Table 1). Source data are provided as a Source Data file.