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CD4+CD25+FOXP3+Treg (tTreg) are main mediators of transplant tolerance. There is a wide interest in use of Treg to promote transplant tolerance. Naïve tTreg are required in large numbers to induce tolerance as they only suppress T effectors at 1: 1 ratio. In tolerant hosts, Treg suppress rejection in an alloantigen specific manner but their numbers are not greatly increased showing their suppressive potential if far greater than tTreg. Methods to identify and expand antigen-specific Treg in vitro are required. In rats, culture of naïve tTreg with rIL-2 or rIL-4 and alloantigen can induce alloantigen-specific Treg.Recent studies show the human Treg population is heterogenous, and comprise of naive Treg expressing CD45RA (Pop I), and CD45RAlo/- activated and memory Treg. Within the CD45RA- population, there are cells with high expression of CD25 and FOXP3, which are considered activated (Pop II) and cells with low expression of FOXP3 and CD25 (Pop III). Additionally, activated Treg express chemokine receptors of activated T cells, including CXCR3 (Th1), CCR8 (Th2) and CCR6 (Th17). Here, we examined changes in phenotype of human Treg after culture with either rIL-2 or rIL-4 alone or with alloantigenic stimulator cellsHealthy human blood CD4+CD127loCD25+Regulatory T cell were isolated by kits that either required PBMC isolation first or direct from whole blood. Multicolour flow cytometry was used to phenotype cells. These enriched tTreg were cultured with human rIL-2 or rIL-4 (200Units/ml) and irradiated allogeneic stimulator cells. Proliferation, changes in surface molecules by FACS and expression of cytokine and cytokine receptors were examined.tTreg had significant proliferation at 3-7d with either rIL-4 or rIL-2 and alloantigen with a peak at 4d. FACS profile of Treg within three populations based on CD45RA and Foxp3 expression showed an increase in Foxp3+CD45RA+ population I, with a marked increase in Foxp3hiCD45RA- population (Pop II, 22% vs 10%). CD45RO+ and CD45RB+ population also increased. Nearly all cells continued to express CD62L. Comparing the two methods of CD4+CD25+CD127loTreg enrichment showed differences related to purity. The first method had enrichment to 90% FOXP3+ and the second methods yielded 70% FOXP3+. The highly enriched population had greater activation of Treg with alloantigen and cytokine, whereas the less enriched populations had marked activation with alloantigen alone and no cytokine, suggesting the FOXP3-T cells may have been activated by alloantigen to produce IL-2 and or IL-4.CD4+CD25+CD127loFOXP3+Treg can be activated by alloantigen and either rIL-2 or rIL-4 to increase the Population II, which is FOXP3hi and CD25hi, and represents the activated Treg population. There increase in population I may represent polyclonal expansion of naïve tTreg These tTreg activation pathways may produce potent antigen-specific Treg for therapy.