Rhesus monkey immature monocyte-derived dendritic cells generate alloantigen-specific regulatory T cells from circulating CD4 ⁺CD127^-/lo T cells

Background. Generation of non-human primate regulatory T cells (Treg) with alloantigen (alloAg) specificity would allow their testing in preclinical transplant models. Low recovery of Treg from peripheral blood limits their potential utility. In small animals and humans, conventional myeloid dendritic cells (DC) have been shown to select or induce alloAg-specific Treg. Methods. We combined enrichment of rhesus macaque blood CD4⁺ Treg based on IL-7Rα(CD127) expression with their stimulation in mixed leukocyte cultures with immature, allogeneic control or vitamin (Vit) D3/IL-10-conditioned monocyte-derived DC. After co-culture in IL-2 and IL-15 for up to 14 days, the ability of the resulting T cells to suppress alloreactive effector T-cell proliferation was assessed. Results. CD4⁺CD127^-/lo T cells represented approximately 7% of normal rhesus circulating CD4⁺ T cells and were enriched for forkhead box P3 (Foxp3)⁺ cells. When stimulated with control allogeneic DC, they exhibited much inferior proliferative responses compared with bulk CD4⁺ or CD4 ⁺CD127⁺ cells. This anergic state was reversed by exogenous IL-2 and IL-15. After 10 to 14 days culture of CD4 ⁺CD127^-/lo T cells with mmature allogeneic DC, particularly maturation-resistant VitD3/IL-10 DC, the frequency of oxp3 ⁺ T cells was increased. The cultured cells markedly inhibited CD4⁺ effector T-cell proliferation in a dose-related and donor alloAg-specific manner. Conclusion. Stimulation of rhesus CD4 ⁺CD127^-/lo T cells with immature and especially maturation-resistant allogeneic DC, generated highly-suppressive, alloAg-specific Treg. Without resorting to a more purified starting population, this approach may have therapeutic utility in clinically relevant transplant models.