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

Abstract

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 Vα24+ T cells derived from the spleens of rhesus macaques, a species increasingly used in models of immune tolerance. Vα24+ cells were isolated and expanded with monocyte-derived immature dendritic cells in the presence of α-galactosylceramide, IL-2, and IL-15. Rhesus NKT cells were stained with mAbs against both Vα24 and the invariant complementarity-determining region 3 epitope of the human Vα24/JαQ 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 β7 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-γ. However, the latter was strongly expressed after stimulation. Rhesus NKT secreted large amounts of TGF-β, IL-13, and IL-6, and modest levels of IFN-γ, 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 Vα24+ 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.

Circulating natural killer and γδ T cells decrease soon after infection of rhesus macaques with lymphocytic choriomeningitis virus

“…NKT cells have been identified in the mouse as either double negative for CD4 and CD8 or as positive for CD4 with a very limited diversity of α/β TCR, most commonly Vα14 Vβ281 (Emoto & Kaufmann 2003). In human beings, the NKT TCR is Vα24, which cross-reacts with the rhesus monkey cell determinant for NKT (Gansuvd et al 2003). In rhesus macaques, 80% of Vα24TCR + cells from the spleen also express CD56 (Gansuvd et al 2003).…”

“…In human beings, the NKT TCR is Vα24, which cross-reacts with the rhesus monkey cell determinant for NKT (Gansuvd et al 2003). In rhesus macaques, 80% of Vα24TCR + cells from the spleen also express CD56 (Gansuvd et al 2003). Although it has been accepted that rhesus macaque NKTs are CD56 + , there is some controversy about the presence of CD16 on their surface (Gansuvd et al 2003), so we used both the CD16 and CD56 as markers.…”

“…In rhesus macaques, 80% of Vα24TCR + cells from the spleen also express CD56 (Gansuvd et al 2003). Although it has been accepted that rhesus macaque NKTs are CD56 + , there is some controversy about the presence of CD16 on their surface (Gansuvd et al 2003), so we used both the CD16 and CD56 as markers. Unlike the CD3 -NK cells, the NKTs in our monkey studies seemed unaffected or even a little increased by day 7 of the study.…”

Peripheral NKT Cells in Simian Immunodeficiency Virus-Infected Macaques

“…This is in contrast with some previous studies of NKT cells from rhesus macaques, where there seems to be little agreement in the cell surface phenotype of NKT cells with regard to CD4 and CD8 expression. In one study (15), NKT cells defined as being V␣24 ϩ 6B11 ϩ (an antibody specific for the invariant NKT cell TCR-␣ chain) were nearly all CD4…”

“…SIV and SHIV viruses, like HIV-1 strains, vary in their uses of either CXCR4 or CCR5 chemokines to enter CD4-bearing cells. There have been only a limited number of studies of NKT cells in macaques (14,15,20,27), often studying NKT cells within lymphoid organs such as the spleen. We undertook a comprehensive kinetic study of NKT cells in pigtail macaques using an ␣-galactosylceramide (␣-GalCer)-loaded CD1d tetramer prior to, and throughout, both CCR5-using SIV mac251 infection and CXCR4-using SHIV mn229 infection.…”