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* Department of Surgery, Division of Transplantation Immunobiology, University of Alabama, Birmingham, AL 35294;
Diabetes Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02215; and
Cancer Biology Program, Hematology/Oncology Division, Beth Israel-Deaconess Medical Center, Harvard Medical School, Boston, MA 02215
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 V
24+ T cells derived from the spleens of rhesus macaques, a species increasingly used in models of immune tolerance. V24+ cells were isolated and expanded with monocyte-derived immature dendritic cells in the presence of -galactosylceramide, IL-2, and IL-15. Rhesus NKT cells were stained with mAbs against both V24 and the invariant complementarity-determining region 3 epitope of the human V24/JQ 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 
7 integrin, but did not express CD45 RA, CD62L, CCR7, CD28, and other activation, costimulatory molecules (CD69 and CD40L). By intracellular staining, >90% of unstimulated rhesus NKT cells expressed IL-10, but not IFN-
. However, the latter was strongly expressed after stimulation. Rhesus NKT secreted large amounts of TGF-, IL-13, and IL-6, and modest levels of IFN-, 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 V24+ T cells are semi-invariant double-negative cells with effector memory phenotype. These cells are semianergic, polarized to a uniquely Th3 > T regulatory-1 regulatory cell phenotype, and have regulatory/suppressive function in vitro.
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