The CD4⁺CD25⁺FoxP3⁺ regulatory T (Treg) cells are present in small numbers in peripheral blood and lymphoid organs and play an important role in regulating immune response. Due to their suppressive action on T and B cell response, they have a high potential for immunotherapy in clinics. We have demonstrated earlier that naturally occurring Treg cells inhibit pig to baboon xenogeneic response in vitro. Due to their small number (< 1% of all lymphocytes) these Treg cells cannot be used for immunotherapy in vivo and have been expanded in-vitro by different methods using IL-2 and TCR stimulation. Ex-vivo expanded Treg cells exhibit similar characteristics to the natural occurring Tregs and have a high therapeutic potential for immunotherapy. We have also investigated the effect of ex vivo expanded baboon Treg cells on effector T and B cells. Highly purified CD4+CD25^Hi Treg cells and CD4+CD25^neg cells were expanded for 4 weeks in presence of IL-2 and dynal IgG beads coated with anti non-human primate (NHP) CD3 and CD28 antibodies. After 4 weeks of stimulation CD4+CD25^Hi expanded more than 400X and these expanded Treg cells retained the high expression of FoxP3. To demonstrate the suppression of T cells, mixed lymphocyte reaction (MLR) was performed by using CD4+CD25^neg cells and irradiated pig APC along with expanded Treg cells.  Ex vivo expanded Treg cells suppress the effector T cells proliferation (> 90%) in presence of xenogeneic immune response. Further, effect of expanded Treg cells on the B cells was examined by co-culturing them together with PMA and Ionomycin. B cells were first labeled with CFSE and then were co-cultured with either irradiated ex-vivo expanded CD4+CD25Hi Treg cells or CD4+CD25neg cells for 5 days in presence of PMA/Ionomycin.  On FACS analysis, we found that more than 90% CFSE labeled B cells proliferated on stimulation with PMA/Ionomycin. This proliferation was reduced to only 40-50% when expanded Treg cells were added to the culture at a 1:1 ratio whereas; the addition of CD4+CD25neg cells induced vigorous proliferation. We propose that this versatile capacity of ex-vivo expanded CD4+CD25^Hi Treg cells might be important to efficiently limit T and B cell responses that might help to prevent the hyperacute and delayed rejection in xenotransplantation. Further, mechanism of inhibition of both T and B cell proliferation by Treg cells is being currently investigated in our laboratory.