Fig. 3: TCR and CD28 stimulation lead to CD14 and TLR4 upregulation in RA patients and in healthy donors.

a Representative dot plot and cumulative graph of the frequency of CD14 expression in CD3⁺CD4⁺ T cells of peripheral blood of RA patients (n = 6 RA patients). b Representative dot plot and cumulative graph of CD14 expression by FACS-purified CD14⁻CD3⁺CD4⁺SSC^lowFSC^low T cells from RA patients after 1 and 5 days with and without αCD3 and αCD28 stimulation (n = 4 RA patients). c–d Confocal microscopy of FACS-purified CD3⁺CD4⁺SSC^lowFSC^low T cells from healthy donors (HD) that were either left unstimulated (unst) or were stimulated with αTCR and αCD28 for 5 days in the presence or absence of LPS. c Cells were surface labeled for CD45 and TLR4 and analyzed by 3D confocal microscopy. Bar, 5 μm. d Cumulative graphs of 3D TLR4 fluorescence (n = 143 cells from four different HD, n = 43 cells unstimulated, n = 40 cells TCR CD28 and n = 50 cells TCR CD28 LPS conditions). e Representative histogram and cumulative plot of FSC MFI of CD4⁺ T cells from HD stimulated for 5 days with αTCR and αCD28 in the presence or absence of LPS (n = 30 HD, 53 independent experiments). f Representative dot plots and cumulative graph of the frequency of viable cells CD4⁺ T from HD stimulated for 5 days with αTCR and αCD28 in the presence or absence of LPS and labeled with cell viability dye (n = 30 HD, 39 independent experiments). g Representative histogram and cumulative plot of cell trace MFI of CD4⁺ T cells from HD stimulated for 5 days with αTCR and αCD28 in the presence or absence of LPS (n = 5 HD). h Representative dot plots and cumulative graph of proliferative CD4⁺ T cells from HD stimulated for 5 days with αTCR and αCD28 in the presence or presence or absence of LPS (n = 5 HD). 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 (n > 6) or Shapiro–Wilk normality test (n ≤ 6). P values ****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05 were determined by (b) Ratio-paired t test; (d) Kruskal–Wallis test with posttest Dunn’s multiple comparisons; the p values are adjusted for multiple comparisons; (e, g, h) Wilcoxon matched-pairs rank test and (f) Paired t test. Effect size measures ⁺⁺⁺high, ⁺⁺medium, ⁺small were determined by (b, f) d – Cohen’s d; (d) η² – eta-squared and (e, g, h) r – correlation coefficient r.