FIGURE 2.
SLE‐TFR cells are functionally impaired and IL‐2 stimulation restores molecular expression‐mediated suppressor function. (a) Comparison of FoxP3 and CD45RA expression between healthy controls (HCs) and SLE‐TFR cells. Activated TFR cells are gated as CD45RA‐FoxP3high, non‐suppressive TFR cells as CD45RA‐FoxP3low and resting TFR cells as CD45RA+FoxP3low. (b) Comparison of suppressive capacity for TFH cell proliferation by TFR cells. CellTrace Violet‐labeled TFH cells were cultured with or without presence of TFR cells under TCR stimulation for 96 h. Suppressive capacity was calculated using a ratio of TFH cell proliferation under the concentration of TFR cells written in the Figure (SLE, n = 4, HC, n = 4). (c) Comparison of IL‐21 concentrations of TFH‐TFR co‐culture supernatant (SLE, n = 3, HC, n = 3). (d) Comparison of molecular expressions of TFR cells as measured by quantitative polymerase chain reaction (qPCR). The gene expression values were normalized to those of the control gene encoding glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) (SLE, n = 5, HC, n = 5). E) Methylation analysis of CNS2 in FoxP3 locus. Methylation levels of eight cytosine–phosphate–guanine (CpG) islands were quantified by pyrosequencing (SLE, n = 3, HC, n = 3). Methylation levels of TFH cells were analyzed for positive control of methylation. (f) Change of molecular expression after IL‐2 stimulation of SLE‐TFR cells. TFR cells derived from SLE patients were cultured under TCR stimulation and various IL‐2 concentrations for 96 h. Molecular expression in cultured cells was analyzed with flow cytometry. Data are expressed as the mean fluorescent intensity changes from baseline (no IL‐2 stimulation) (SLE, n = 4). Data are mean ± standard deviation (SD). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; NS = not significant; CTV = CellTrace Violet; CTLA‐4 = cytotoxic T lymphocyte antigen 4; GITR = glucocorticoid‐induced tumor necrosis factor (TNF) receptor; SLE = systemic lupus erythematosus; TFR = follicular regulatory T; IL = interleukin; FoxP3 =forkhead box protein 3; TFH = follicular helper T