Introduction: Activation of human CD4⁺CD25⁺CD127^loFoxp3⁺ Treg to have higher antigen specific suppression is a key goal for therapy. In rats, CD4⁺CD25⁺Treg activated in vitro with alloantigen and rIL-2 express more Foxp3 and CD25 and are induced to express receptors for IFN-γ (IFNGR) and IL-12 (IL-12Rβ2). These cells are more potent suppressors of alloactivation in vitro and in vivo. Human Treg by FACS can be divided into three populations; Pop I (CD25⁺CD45RA⁺), which are resting Treg; Pop II (CD25^hiCD45RA^-), which are activated Treg and Pop III (CD25⁺CD45RA^-) including both Treg and activated effector T cells. Pop II and III express chemokine receptors of activated T cells, including CXCR3(Th1) and CCR6(Th17), which promote migration to inflammation. CCR7, which promotes migration to lymphoid tissue is expressed by Pop I. 
Methods: CD4⁺CD127^loCD25⁺ Treg isolated by FACS from blood of healthy volunteers were cultured for 4 days with rIL-2 and irradiated allogeneic PBMC (allostimulators).  Cells were examined using multicolour flow cytometry for shifts in Pop 1, II, III and chemokine receptors. Cells were stained using RNAscope for foxp3 and ifngr.
Results: tTreg cultured alone had reduced Pop II compared to the fresh starting population (1.3% vs 8.6%). Culture with allostimulators only preserved the Pop II (12% vs 8.6%). IL-2 alone increased Pop II in 5/8 of experiments, similar to the culture with allostimulators and rIL-2 (6/8 experiments).  CXCR3 expression in Pop II was similar in fresh Treg to those cultured with IL-2 or allostimulators only or IL-2 with allostimulators.
RNAscope showed freshly isolated Treg expressed foxp3, but not ifngr. Treg cultured with allostimulators, and rIL-2 had foxp3⁺, ifngr⁺ and also foxp3⁺ifngr⁺ double positive cells. Treg cultured with rIL-2 alone had no ifngr whereas Treg cultured with allostimulators only had foxp3 and ifngr expressing cells, but none were double positive.   Treg cultured alone had fewer foxp3⁺  and no ifngr⁺ cells. 
Enriched Pop I cells lost Foxp3 in absence of allostimulators or rIL-2. Culture with both allostimulators and rIL-2 produced cells with higher Foxp3 and CD25, retaining CD45RA expression. CXCR3 or CCR6 expression did not increase. They are a new subpopulation of CD45RA⁺ cells higher Foxp3 and CD25 expression than Pop I.
Pop II died when cultured alone or with allostimulators. Activation with rIL-2 alone or with allostimulators increased expression of Foxp3 and CD25, maintained CCR4 and CXCR3 and increased CCR6.
Pop III showed two shifts, some expressed less Foxp3 while others increased Foxp3 and CD25 expression, shifting to Pop II.
Conclusion: Treg activation can be monitored by changes in expression of CD25, Foxp3, CD45RA and chemokine and cytokine receptors. Human tTreg, with allostimulators and rIL-2 induced a Ts1 type cells, consistent with our rat studies. Understanding Treg activation pathways may produce potent antigen specific Treg for therapy.