Resistance to IL-10 inhibition of IFN-γ production in RA CD4+ T cells
The CD28 costimulatory pathway is crucial for effective antigen-specific T-cell cytokine production, and IL-10 can directly suppress this response by inhibiting CD28 tyrosine phosphorylation and binding of phosphatidylinositol 3-kinase [24]. To evaluate the responsiveness of RA CD4+ T cells to IL-10, purified PB CD4+ T cells from three patients with active RA and from three healthy controls were stimulated by immobilized anti-CD3 antibody and anti-CD28 antibody with or without diluted concentrations of IL-10 for 36 hours, and IFN-γ production was measured by ELISA. As shown in Fig. 1, IFN-γ production by activated normal CD4+ T cells was mostly inhibited at concentrations as low as 1 ng/ml IL-10. However, RA CD4+ T cells were able to produce significant amounts of IFN-γ in the presence of 1 ng/ml IL-10, and the maximal but not complete inhibition by IL-10 was obtained at 10–100 ng/ml.
We thus compared the levels of IFN-γ production by CD4+ T cells after CD3 and CD28 costimulation in the presence of 1 ng/ml IL-10 in RA patients with active disease (multiple inflammatory joints, CRP level ≥ 10 mg/l) and inactive disease (in remission, CRP level < 10 mg/l) [26] and in healthy controls. There were no statistically significant differences in IFN-γ production without IL-10 among these three groups (Fig. 2a), but the inhibitory effect of IL-10 on IFN-γ production was significantly limited in the active RA group as compared with the inactive RA group and healthy controls (percentage decrease: active RA, 2.9 ± 14.4%; inactive RA, 45.6 ± 14.4%; controls, 65.8 ± 7.9%) (Fig. 2b). As a consequence, CD4+ T cells from active RA patients produced higher levels of IFN-γ in the presence of 1 ng/ml IL-10 than did normal CD4+ T cells (Fig. 2a).
In addition, we compared IL-2 production by CD4+ T cells after CD3 and CD28 costimulation in the presence of IL-10 in active RA patients and in healthy controls. Similarly, IL-2 production was not affected by 1 ng/ml IL-10 in RA patients (percentage decrease, -2.1 ± 13.8%), while it was significantly reduced in healthy controls (61.1 ± 13.7%; P < 0.05). Taken together, these results indicate that RA CD4+ T cells become less susceptible to the immunoregulatory effect of IL-10 during the active phase.