Activation of human alloantigen specific CD4⁺CD25⁺CD127^loFoxp3⁺ Treg that induce tolerance is a key goal. In rats, CD4⁺CD25⁺Treg activated with alloantigen and rIL-2 express more Foxp3 and CD25 and express receptors for IFN-γ (IFNGR) and IL-12 (IL-12Rβ2). These cells are more potent suppressors of specific alloactivation. Here we examined activation of human Treg.
Human Treg are heterogenous with three populations; Pop I (Foxp3⁺CD25⁺CD45RA⁺) as resting Treg; Pop II (Foxp3^hiCD25^hiCD45RA^-) as activated Treg and Pop III (Foxp3⁺CD25⁺CD45RA^-) include both Treg and activated effector T cells. Pop II and III express chemokine receptors of activated T cells, including CXCR3 (Th1), CCR6 (Th17), CCR4 (Th2) which promote migration to inflammation.
CD4⁺CD127^loCD25⁺ Treg isolated by FACS from blood of healthy volunteers were cultured for 4 days with rIL-2 and irradiated allostimulators (AlloS).  Cells were examined using multicolour flow cytometry for shifts in Pop I, II, III and chemokine receptors. Cells were stained for foxp3 and ifngr using RNAscope.
First, enriched Treg were cultured with AlloS and rIL-2 for 4 days. tTreg cultured alone had reduced Pop II compared to the fresh starting population (1.3% vs 8.6%).   Culture with AlloS preserved the Pop II (12% vs 8.6%). IL-2 alone increased Pop II in 5/8 experiments, similar to the culture with AlloS and rIL-2 (6/8 experiments).  CXCR3 expression in Pop II was similar in unfractionated Treg to those cultured with IL-2 or AlloS alone or IL-2 with AlloS.
RNAscope showed freshly isolated Treg expressed foxp3⁺, but not ifngr. Treg cultured with AlloS and rIL-2 had foxp3⁺ifngr⁺ double positive cells but also single foxp3⁺ and ifngr⁺ cells.    Treg cultured with rIL-2 alone had no ifngr+ cells whereas Treg cultured with AlloS only had single positive foxp3⁺ and ifngr⁺ cells, none were double positive.   Treg cultured alone had fewer foxp3⁺ cells and no ifngr⁺ cells. 
Enriched Pop I cells lost Foxp3 in absence of AlloS or rIL-2. Culture with both AlloS and rIL-2 produced Foxp3^hi and CD25⁺ but remained CD45RA⁺. CXCR3^- and CCR6^- were not induced.  A new subpopulation of CD45RA⁺Foxp3^hiCD25^hi cells appeared.
Cells in Pop II died when cultured alone. Activation with rIL-2 alone or with AlloS increased expression of Foxp3, CD25, CCR6 and maintained CCR4 and CXCR3 expression.
Pop III showed two shifts, some expressed less Foxp3 while others increased Foxp3 and CD25 expression, shifting to Pop II.
Treg activation can be monitored by changes in expression of CD25, Foxp3, CD45RA and chemokine and cytokine receptors. Human tTreg stimulated with AlloS and rIL-2 induced IFNGR, like our rat studies. IFNγ may induce potent antigen specific Treg for therapy.