Introduction: The human T regulatory cells (CD4⁺CD25⁺CD127^loFoxp3⁺) population is heterogeneous.  It can be divided into three subpopulations; Population I (PopI) is CD25⁺CD45RA⁺ and comprise of naïve Treg.  PopII is CD25^hiCD45RA^- and contains highly activated Treg.  Pop III is CD25⁺CD45RA^- and includes both Treg and activated effector T cells. Activated Treg express chemokine receptors of activated T cells, including CXCR3(Th1) and CCR6(Th17) and are mainly expressed in Pop II and Pop III. These promote activated Treg migration to sites of inflammation.  CCR4 is mainly expressed by PopI and promotes migration to peripheral lymphoid organs.  We examined changes in Treg populations following culture of whole Treg or individual Treg populations with rIL-2 and alloantigen to determine the effect of culture on Treg activation.
Methods: To isolate individual Treg populations, PBMC were collected from healthy human blood and were stained with monoclonal antibodies to CD4, CD25, CD127 and CD45RA. Subpopulations within the CD4⁺CD127^loCD25⁺ population were sorted to Pop1 or Pop II &III.   Whole Treg or individual Treg populations were cultured for 4 days with rIL-2 and alloantigen. Multicolour flow cytometry assessed changes in Treg subpopulations.
Results and Discussion: tTreg cultured alone had reduced Pop II compared to the fresh starting population (1.3% vs 8.6%). Culture with alloantigen stimulators alone preserved the PopII (12% vs 8.6%). IL-2 alone increased PopII in 5 of 8 of experiments similar to the culture with alloantigen stimulators and rIL-2 (6/8 experiments).  CXCR3 expression in PopII was similar in fresh Treg to those cultured with IL-2 or alloantigen alone or IL-2 with alloantigen stimulators.
When individual Treg populations were cultured, PopI lost Foxp3 in absence of alloantigen or rIL-2. Culture with both alloantigen stimulators and rIL-2 produced cells with higher Foxp3 and CD25 expression, which expressed CD45RA, and had increased expression of CCR4 but not CXCR3 and CCR6. 
Pop II died when cultured alone or with alloantigen. Activation with rIL-2 alone or with alloantigen increased expression of Foxp3 and CD25, maintained CCR4 and CXCR3 expression and increased CCR6⁺ cells.
Pop III when cultured with IL-2, had mixed changes.  Some cells expressed less Foxp3 while others shifted to Pop II having increased Foxp3 and CD25 expression.
Conclusions: We showed that the naïve Treg in population I were activated to express more CD25 and Foxp3 but did not reduce CD45RA expression, and did not become fully activated Treg in PopII. We also showed that activated Treg in PopII needed allostimulation or IL-2 to survive. PopIII included cells that reverted to effector cells and lost Foxp3 expression.  Others were activated to PopII cells with high CD25 and Foxp3 expression. 
We showed that each Treg subpopulation had a different pathway of activation. Understanding these tTreg activation pathways may produce potent antigen-specific Treg for therapy.