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Natural killer T cells are immunoregulatory cells, which have important roles in tolerance and autoimmunity, as demonstrated primarily in mice and humans. In this study, we define the phenotype and function of Valpha24(+) T cells derived from the spleens of rhesus macaques, a species increasingly used in models of immune tolerance. Valpha24(+) cells were isolated and expanded with monocyte-derived immature dendritic cells in the presence of alpha-galactosylceramide, IL-2, and IL-15. Rhesus NKT cells were stained with mAbs against both Valpha24 and the invariant complementarity-determining region 3 epitope of the human Valpha24/JalphaQ TCR. The cells were CD4, CD8 double negative and expressed CD56. Rhesus NKT cells also exhibited moderate to high expression of CD95, CD45RO, CD11a, and beta(7) integrin, but did not express CD45 RA, CD62L, CCR7, CD28, and other activation, costimulatory molecules (CD69 and CD40L). By intracellular staining, &gt;90% of unstimulated rhesus NKT cells expressed IL-10, but not IFN-gamma. However, the latter was strongly expressed after stimulation. Rhesus NKT secreted large amounts of TGF-beta, IL-13, and IL-6, and modest levels of IFN-gamma, whereas IL-10 secretion was negligible and no detectable IL-4 was observed either intracellularly or in culture supernatants. Functionally, the NKT cells and their supernatants suppressed T cell proliferation in allogeneic MLR. We conclude that long-term cultured rhesus macaque spleen-derived Valpha24(+) T cells are semi-invariant double-negative cells with effector memory phenotype. These cells are semianergic, polarized to a uniquely Th3 &gt; T regulatory-1 regulatory cell phenotype, and have regulatory/suppressive function in vitro.

Phenotypic and Functional Characterization of Long-Term Cultured Rhesus Macaque Spleen-Derived NKT Cells