Figure 1: TNF-α in RA-SF impairs T_reg cell function.

(a) Representative FACS data of the frequency of CD4⁺CD25^hi T cells (top) and FOXP3⁺CD127^lo/− cells within gated CD4⁺CD25^hi T cells (bottom) from RA-PBMC, RA-SFMC and PBMC of healthy controls (HC-PBMC) (n = 20 subjects per group). (b) Frequency of CD4⁺CD25^hiFOXP3⁺CD127^lo/− T_reg cells from the indicated groups (left, n = 20 per group) and paired samples (right, n = 10 per group). (c) Inhibition of CD4⁺CD25⁻ T (T_eff) cells by CD4⁺CD25^hiCD127^lo/− T (T_reg) cells isolated from RA-PBMC, RA-SFMC or HC-PBMC (n = 5 per group) in coculture assays (see the Online Methods for a more detailed description of the measures of inhibition). (d) Mean percentage inhibition of T_eff cells from healthy controls (n = 5) by T_reg cells in the absence (dashed line) or presence of RA-SF at the indicated dilutions. (e) Percentages of divided CFSE-labeled T_eff cells pretreated with RA-SF (dilution 1:8) or culture medium (CM) in coculture with T_reg cells. Shown are representative data from five independent experiments. (f) Mean percentage inhibition of T_eff cell proliferation by T_reg cells in the presence of RA-SF (dilution 1:8) pretreated with the indicated antibodies or without pretreatment (dashed line). Data are from five independent experiments. (g) Mean percentage inhibition of T_eff cell proliferation by T_reg cells in the presence of increasing concentrations of recombinant TNF-α. (h) Inhibition assay as performed in e except T_eff cells and T_reg cells were pretreated with TNF-α or culture medium as indicated. Numbers in the FACS plots indicate the frequencies of gated cells. Error bars, s.e.m. *P < 0.05, **P < 0.01 determined by two-tailed Student's t test.