T Regulatory cells (Treg) are CD4⁺CD25⁺CD127l^oFoxp3⁺T cells in humans that have potential as therapy in autoimmunity and transplant rejection. Current clinical studies of Treg use in vitro expanded tTreg that only suppress effector CD4⁺T cells at a >1:1 ratio and are not antigen-specific.

Our rodent studies demonstrate in vitro antigen-induced activation of tTreg induces potent antigen-specific Treg. Antigen-activated Treg exposed to IL-4 are induced to express receptor for the Th2 cytokine IL-5. Those exposed to IL-2 and antigen express receptors for Th1 cytokine IFN-g and IL-12.

We aim to extend our studies on antigen-specific Treg to humans by culture of human tTreg with antigen and IL-2 or IL-4, to characterize their phenotype.

CD4⁺CD25⁺CD127^loTreg isolated from peripheral blood from healthy donors using a commercial Treg isolation kit were cultured with antigen and IL-2 or IL-4 for 3-4 days. Pre- and post-culture phenotype was analysed by multicolour flow cytometry, specifically with anti-CD45RA to assess naïve and activated Treg as well as chemokine receptors. RT-PCR assayed expression of cytokine receptors.

There was an increase in CD45RA^-, CD45RO⁺ and CD45RB⁺ population, which were FOXP3^hi and CD25^hi. CD62L expression remained similar. Culture with IL-4 appears to induce polyclonal activation of tTreg as well. Similar to our rat studies, RT-PCR of cultured Treg showed induction of IL-5Rα, the specific chain of IL-5 receptor, only in tTreg cultured with both IL-4 and antigen.

Increased CD45RA^-/CD45RO⁺ population is consistent with activation of antigen-specific Treg. This suggests culture with IL-4 as a potential pathway for activation of tTreg in producing potent antigen-specific Treg, circumventing the need for high numbers of tTreg for therapy. Protocols for generation of human antigen specific Treg that may be useful in preventing allograft rejection, GVH and autoimmune diseases such as MS.