Ex-vivo expanded baboon CD4+ CD25 Hi Treg cells suppress baboon anti-pig T and B cell immune response

Singh AK, Seavey CN, Horvath KA, Mohiuddin MM. Ex‐vivo expanded baboon CD4⁺ CD25^Hi Treg cells suppress baboon anti‐pig T and B cell immune response. Xenotransplantation 2012; 19: 102–111. © 2012 John Wiley & Sons A/S. Abstract: Background: CD4⁺ CD25⁺ FoxP3⁺ regulatory T (Treg) cells play an important role in regulating immune responses. A very small number of Treg cells are present in peripheral blood and lymphoid organs, but due to their ability to suppress the immune response, they have a high potential for immunotherapy in clinics. Successful ex‐vivo expansion of naturally occurring CD4⁺ CD25⁺ T cells has been achieved after TCR stimulation in the presence of T cell growth factors. In this study, we evaluated the role of these Treg cells in suppressing proliferative response of baboon T and B cells to pig xenoantigens. Methods: Naturally occurring baboon CD4⁺ CD25⁺ regulatory T cells (nTreg) were sorted from peripheral blood and expanded in the presence of either anti‐CD3/CD28 beads or irradiated pig peripheral blood mononuclear cells with IL‐2. Treg cells were also enriched directly from CD4⁺ T cells cultured in the presence of rapamycin (0.1–10 nm ). Mixed lymphocyte culture and polyclonal B cell stimulation with ex‐vivo Treg cells were performed to assess the function of ex‐vivo expanded Treg cells. Results: The nTreg cells were expanded to more than 200‐fold in 4 weeks and retained all the nTreg cell phenotypic characteristics, including high levels of FoxP3 expression. 2‐fold increase in enrichment of CD4⁺ CD25⁺ FoxP3⁺ Treg cells from CD4⁺ cells was observed with rapamycin compared to cultures without rapamycin. The ex‐vivo expanded Treg cells obtained from both methods were able to suppress the baboon anti‐porcine xenogeneic T and B cell immune response in‐vitro efficiently (more than 90% suppression at 1 : 1 ratio of T regulatory cells: T effector cells), and their suppression potential was retained even at 1 : 256 ratio. However, freshly isolated nTreg cells had only 70% suppression at 1 : 1 ratio, and their suppressive ability was reduced to ≤50% at 1 : 16 ratio. Furthermore, we have found that ex‐vivo expanded Treg can also suppress the proliferation of B cells after polyclonal stimulation. Forty to 50 percent reduction in B cell proliferation was observed when ex‐vivo expanded Treg cells were added to the culture at a 1 : 1 ratio. The addition of CD4⁺ CD25^Neg cells however induced vigorous proliferation. Conclusion: Ex‐vivo expanded CD4⁺ CD25⁺ FoxP3⁺ Treg cells can be used to efficiently suppress xenogeneic immune responses by inhibiting T and B cell proliferation. These ex‐vivo expanded Treg cells may also be used with other immunosuppressive agents to overcome xenograft rejection in preclinical xenotransplantation models.