Regulatory Cells Generated by Testicular Tolerization to Retinal S-Antigen: Possible Involvement of IL-4, IL-10, and TGF-β in the Suppression of Experimental Autoimmune Uveoretinitis

Intratesticular injection of a retinal protein (S-antigen) into Lewis rats induces systemic immunotolerance (designated orchidic tolerance) and renders animals refractory to experimental autoimmune uveoretinitis (EAU) produced by S-antigen immunization. We demonstrated in this work that the immunotolerance could be transferred to syngeneic naive rats by both CD4+and CD8+regulatory cells. Attempts were then made to characterize the cytokines involved in the immunosuppressive activity of these regulatory cells. Using thein vitrolymphoproliferation assay, the inhibitory effect of CD4+cells on effector cells was found to be reversed by antibodies against IL-4 and IL-10 but not by anti-TGF-β antibody. IL-4 (IC50= 1.6 ng/106cells) and IL-10 (IC50= 0.6 ng/106cells) added to the assay medium were potent inhibitors of effector cell proliferation. Increased immunoreactivity and mRNA expression for IL-4 and IL-10 was observed for CD4+regulatory cells. The inhibitory effect of CD8+regulatory cells was reversed by anti-TGF-β antibody but not by antibodies against IL-4 and IL-10. Compared with control CD8+cells, CD8+cells from tolerized rats demonstrated higher immunoreactivity for TGF-β but did not show an enhanced expression of mRNA for TGF-β. TGF-β1 and TGF-β2 added to effector cells showed dichotomous effects; both isoforms stimulated cell proliferation at 2.5 ng/106cells and inhibited at lower or higher concentrations. These results led us to conclude that IL-4 and IL-10 are important cytokines for the immunosuppressive effect of CD4+regulatory cells generated in orchidic tolerance. TGF-β is an important immunosuppressive cytokine for CD8+regulatory cells but further studies will determine whether other cytokines are also involved.