T regulatory cells (CD4⁺CD25⁺Foxp3⁺) regulate immune responses. Rodent CD4⁺CD25⁺ cells activated in vitro with alloantigen and rIL-4 express more Foxp3 and CD25, and IL-5Rα is induced. These cells are more potent suppressors of alloactivation in vitro and in vivo.   They are further activated by IL-5.  Activation of human Treg (CD4⁺CD25⁺CD127^loFoxp3⁺) to have higher antigen-specific suppression is a key goal for therapy.  
Human Treg are heterogenous.  Based on expression of CD25/Foxp3 and CD45RA they can be segregated into three populations, resting Treg (Pop I as Foxp3⁺/CD25⁺CD45RA⁺), activated Treg (Pop II as CD25^hiCD45RA^-) and Pop III as Foxp3⁺/CD25⁺CD45RA^- cells that includes both Treg and activated effector T cells. Pop II and III express chemokine receptors of activated T cells, including CXCR3 (Th1), CCR4 but not CXCR3 or CCR6 (Th2), and CCR6(Th17), which promote migration to inflammation. CCR7, which promotes migration to lymphoid tissue is expressed by Pop I. 
We examined activation of human Treg (CD4⁺CD25⁺CD127^lo) by alloantigen (AlloS) and rIL-4 for expression of CCR4 and IL-5Rα.  CD4⁺CD127^loCD25⁺ Treg isolated by FACS from blood of healthy volunteers (HV) were cultured for 4 days with rIL-4 and irradiated PBMC as AlloS.  Cells were examined using multicolour flow cytometry for shifts in Pop I, II, III and expression of chemokine receptors and IL-5Rα.
Enriched tTreg had 85.1±9.5% CD4⁺CD25⁺CD127^lo cells with 14.8±9.9% Pop I, 17.2±10.6% Pop II and 48.4+8.2% Pop III. Culture with different rIL-4 (E.coli, CHO cells, HEK expressed) and concentration with or without irradiated AlloS for 4 days showed 2ng/ml CHO derived rIL-4 with AlloS, was best at promoting Treg survival. When cultured alone CD4⁺CD25⁺CD127^lo cells had <2% Foxp3⁺ cells and no Foxp3^hi. Culture with rIL-4 alone reduced Pop I (7.2±8.7%) with no cells as Pop II (0.27±0.47). With AlloS and rIL-4, Pop I was preserved (25.2 ±15.3) and 1/4th cells in Pop II survived (4.2±1.9%). % of CXCR3⁺, CCR6⁺ were similar pre- and post-culture, however, CCR4⁺CXCR3^-CCR6^- (Th2-like cells) increased with AlloS and rIL-4. IL-5Rα increased in Pop I with AlloS alone or IL-4 and AlloS.  Pop II after culture with AlloS and rIL-4 (26%) had high IL-5Rα expression compared to fresh tTreg (0.24%), AlloS stimulated (11.8%) or rIL-4 alone (0%). Pop III expressed IL-5Rα with rIL-4, which was higher than with AlloS and rIL-4 (15.2 vs 4.41%).
Understanding Treg activation pathways may produce potent antigen-specific Treg for therapy. Treg activation can be monitored by changes in expression of CD25, Foxp3, CD45RA and chemokine/cytokine receptors. Human Treg, cultured with AlloS and rIL-4 had induced expression of IL-5Ra and chemokine receptors consistent with Th2-like Treg. Th2 cytokines IL-4 and IL-5 may promote activation of potent antigen-specific Treg to improve therapy for tolerance induction.