Demonstration that long-term IL-5 treatment suppresses EAN and induces Ag-specific CD4⁺CD25⁺ Tregs that express Il-5rα. (A) Extended treatment with rIL-5 (5000 U/d; (●) from day 9 to day 30 after immunization prevented onset of significant clinical disease (top panel; P < .001 on days 15-27, and P < .02 on days 14 and 29; n = 6/group) compared with controls with EAN (○). Weight loss (bottom panel) was significantly reduced at days 10-22, P < .05. (B) CD4⁺CD25⁺ T cells were prepared from the lymph nodes and spleens of rIL-5–treated animals and from untreated controls in panel A, at the end of the treatment, which was day 30 after immunization. CD4⁺CD25⁺ T cells from rIL-5–treated animals (IL-5Rx) expressed Il-5rα, whereas cells from controls (CTL) had no Il-5rα (**P = .02). (C) The same CD4⁺CD25⁺ T cells from rIL-5–treated animals (■) proliferated to specific immunizing Ag PNM but not autologous stimulator cells with no Ag or RTA (**P = .02). CD4⁺CD25⁺ T cells from untreated EAN controls (□) did not respond to any stimulus. These data show that IL-5 promoted induction of Ag-specific Tregs that expressed Il-5rα mRNA.