Characterization and expansion of baboon CD4⁺CD25⁺ Treg cells for potential use in a non-human primate xenotransplantation model

Background: It is well established that CD4⁺CD25⁺ regulatory T (Treg) cells can modulate allogeneic immune responses. Xenotransplantation, proposed as a means to address the critical shortage of human organs, may also benefit from similar approaches to avert rejection. Baboons are a preferred preclinical animal model for xenogeneic organ transplantation experiments, and the characterization of baboon Treg cells will be beneficial to future tolerance studies in this animal model. Methods: We analyzed CD4⁺CD25⁺ T cells from baboon lymph nodes, spleens, and blood by flow cytometry, then purified and expanded porcine antigen-specific baboon CD4⁺CD25^high cells in vitro to evaluate their regulatory activity in the baboon anti-pig xenogeneic responses. Results: CD4⁺CD25^high T cells were 1.7%, 3.1%, and 1.9% of baboon splenic, lymph node, and blood T cells, respectively. The CD4 ⁺CD25^high T cells expressed the Treg cell-associated transcription factor, FoxP3. Proliferation/suppression assays using irradiated pig peripheral blood mononuclear cells as stimulators showed that Treg cells suppressed the vigorous baboon CD4⁺CD25^- T-cell anti-pig proliferation response and cytokine secretion. Expanded baboon Treg cells suppressed baboon anti-pig CD4⁺CD25^- T-cell proliferation ∼4- to 10-fold more than freshly isolated Treg cells. Expanded Treg cells suppressed proliferation to primary cells from the same pig used for expansion more effectively than proliferation to stimulators from a different strain of pig, suggesting a level of antigen specificity. Conclusion: We demonstrate that baboon Treg cells suppress immune responses to xenogeneic stimulation. These studies suggest that adoptive transfer of expanded Treg cells into transplant recipients may provide an approach to prevent cell-mediated rejection of grafts and potentially induce tolerance in the pig to baboon xenotransplantation preclinical model.