Fig. 1: RA patients display a circulating TLR4⁺ T-cell population that is expanded in the synovial fluid.

a Gating strategy and cumulative frequency of CD3⁺CD4⁺TLR4⁺ cells in freshly obtained synovial fluid (n = 12 RA patients). b Representative histogram and cumulative plot of relative cell size (FSC-A) in TLR4⁻ (gray) and TLR4⁺ (red) synovial fluid T cells (n = 12 RA patients). c Representative histogram and cumulative plot of relative cell complexity (SSC-A) of TLR4⁻ (gray) and TLR4⁺ (red) synovial fluid T cells (n = 12 RA patients). d Gating strategy and cumulative frequency of CD3⁺CD4⁺TLR4⁺ cells in freshly obtained peripheral blood (n = 100 RA patients). e Representative histogram and cumulative plot of relative cell size (FSC-A) in TLR4⁻ (gray) and TLR4⁺ (red) peripheral blood T cells (n = 100 RA patients). f Representative histogram and cumulative plot of relative cell complexity (SSC-A) of TLR4⁻ (gray) and TLR4⁺ (red) peripheral blood T cells (n = 100 RA patients). g Donor-matched analysis of the frequency of TLR4 expression by CD3⁺CD4⁺ T cells in peripheral blood (closed circles; PB) and in synovial fluid (open circles; SF) (n = 12 RA patients). h Donor-matched analysis of the MFI of TLR4 expression by CD3⁺CD4⁺ T cells in peripheral blood (closed circles; PB) and in synovial fluid (open circles; SF) (n = 11 RA patients). i Correlation between the frequency of CD3⁺CD4⁺ TLR4⁺ T cells in the blood (PB) and in synovial fluid (SF) (n = 12 RA patients). j t-SNE plots of peripheral blood total CD4⁺ T cells. The color indicates cell expression levels of labeled markers (TLR4, HLA-DR, and PD-1). Circle demarks TLR4⁺ cells (n = 26 RA patients). k–n Confocal microscopy of FACS-purified HLA-DR⁻ and HLA-DR⁺ CD4⁺ T cells. k Cells were surface labeled for CD3 and TLR4, stained for DAPI, and analyzed by 3D confocal microscopy. Bar, 5 μm. l Cumulative graphs of 3D volume (n = 76 HLA-DR⁻CD4⁺ T cells; n = 47 HLA-DR⁺CD4⁺ T cells); m larger diameter (n = 76 HLA-DR⁻CD4⁺ T cells; n = 47 HLA-DR⁺CD4⁺ T cells), and n roundness index (n = 75 HLA-DR⁻CD4⁺ T cells; n = 44 HLA-DR⁺CD4⁺ T cells). Data are presented as mean ± SD, for parametric statistical tests, or median ± IQR, for non-parametric statistical tests. Sample normality distribution was tested by using D’Agostino & Pearson normality test. P values ****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05 were determined by (b, c, g) Paired t test; (e, f, h) Wilcoxon matched-pairs rank test; (i) Pearson Correlation and (l–n) Mann–Whitney test. Effect size measures ⁺⁺⁺high, ⁺⁺medium, ⁺small were determined by (b, c, g) d – Cohen’s d; (e, f, h, l–n) r – correlation coefficient r, and (i) r_p – Pearson’s correlation coefficient.