Function of CD94⁺ iNKT cells. (A) Representative plot of CD4 and CD94 expression in primary iNKT cells (n = 23). (B) Representative plots from experiment showing mutually exclusive expression of CD4 and CD94. Primary iNKT cells were purity sorted by flow cytometry based on the highlighted populations CD4⁺CD94^– and CD4^–CD94^–. The cells were then cultured for 7 days with or without stimulation with CD3/CD28 Dynabeads and IL-2. Plots on the right show the expression of CD94 gated on the CD4⁺ and the CD4^– population on day 7. (C) Cytotoxicity of iNKT cells. CD1d⁺ Jurkat cells were incubated with α-galactosylceramide (αGC) overnight (far left). Bulk iNKT cells (left) or CD94-depleted iNKT cells (right) were then added in a 1:1 ratio. CD94-depleted iNKT cells induced significantly less apoptosis in target cells compared with bulk iNKT cells (far right, n = 8). (D) Allogeneic cytotoxicity toward APCs. Allo-myeloid dendritic cells (mDCs) were pulsed with αGC and mixed with iNKT cells sorted by FACS in a 1:1 ratio (n = 6). Cell death was measured using 7-aminoactinomycin D (7AAD) and Annexin V (AnV). The plot (far right) shows frequency of 7AAD⁺AnnexinV⁺ mDCs (blue) and 7AAD^–AnnexinV⁺ mDCs (red). CD94⁺CD4^– iNKT cells induced significantly more mDC death than CD94^–CD4^– and CD94^–CD4⁺ iNKT cells. (E) Immune regulatory function of CD94⁺ iNKT cells. Purified CD3⁺ T cells were stimulated with CD3/CD28 Dynabeads for 72 hours. Addition of CD94-depleted iNKT cells in a dose-dependent manner decreased the proliferation of the T cells (n = 6). Means ± standard error of the mean (A,C-E).*P = .05-.01; ****P < .0001.