CD4⁺CD25^hi Tregs from RA patients recover their suppressive function after infliximab therapy. (A) FOXP3 mRNA expression is reduced in CD4⁺CD25^hi Tregs of patients with active RA and recovers following treatment with infliximab. CD4⁺CD25^- and CD4⁺CD25^hi T cells were sorted as described from RA patients before (RA pre) and after (RA post) treatment with infliximab. Real-time PCR was carried out in triplicate for FOXP3 mRNA and relative fold changes were normalized to GAPDH. Data represent the mean ± SEM of 5 different experiments. The same RA patients were examined following 3 months of therapy with infliximab. Following infliximab therapy, FOXP3 mRNA levels in CD4⁺CD25^hi Tregs of RA patients were significantly increased (P = .05) and not significantly different from that found in healthy controls. (B) FoxP3 protein expression in CD4⁺CD25^hi Tregs recovers after anti-TNF treatment of RA patients. CD4⁺CD25^- and CD4⁺CD25^hi T cells were sorted as described. Intracellular FoxP3 staining was carried out in CD4⁺CD25^- and CD4⁺CD25^hi T cells sorted from an RA patient before and after infliximab therapy. (C) CD4⁺CD25^- responders (5 × 10⁴/well) and CD4⁺CD25^hi Tregs (5 × 10⁴/well) were isolated from active RA patients before infliximab therapy and after 3 months of infliximab treatment. Cells were cultured with plate-bound anti-CD3 (1 μg/well) either alone or at a 1:1 ratio. After 72 hours, ³H-thymidine incorporation was determined. Data represent the mean ± SE of 15 RA patients before and after infliximab treatment.