To determine whether the inhibitory function of intraocular CD4
+CD25
+ T cells on infiltrated effector T cells accounted for monophasic or recurrent EAU, we compared the ability of ocular Tregs from animals recovering from m-EAU or r-EAU to suppress the proliferation of R16 effector T cells in vitro. The CD4
+CD25
+ Treg cells were derived from the eyes and spleens of rats with m-EAU (day 16 p.i.) and r-EAU (day 12 after transfer); CD4
+CD25
− T effector cells were derived from the spleens of m-EAU rats (9 days p.i.). The CD4
+CD25
− T effector cells proliferated in response to specific antigen, whereas CD25
+ Tregs did not. Furthermore, the eye-derived CD4
+CD25
+ Treg cells from the m-EAU rats completely abolished the proliferation of responder CD4
+CD25
− T cells at a Treg/T-responder ratio as low as 1:10
(Fig. 3A) , whereas the same eye-derived Treg cells from r-EAU rats
(Fig. 3B)or the spleen of m-EAU rats
(Fig. 3A)suppressed the proliferation of responder CD4
+CD25
− T cells at a Treg/T-responder ratio of only 1:4. Interestingly, there was a hierarchical potency of suppression by CD4
+CD25
+ cells from different sources, with the m-EAU eye > r-EAU eye > m-EAU spleen
(Fig. 3C) . These results are consistent with the idea that CD4
+CD25
+ Tregs in the eye are functionally activated and antigen specific.