The ability of dendritic cells to stimulate CD1-restricted T cells is determined by their CD1 expression profile. To determine the ability of DCs to present CD1-restricted antigens to T cells, human monocytes were cultured in IL-4, GM-CSF, and FCS (•) or AS (□) for 6 days and plated in 96-well plates and pulsed with decreasing concentrations of antigen presented by CD1a (dideoxymycobactin), CD1b (glucose monomycolate), CD1c (mannosyl-b-1-phosphomycoketide), or CD1d (α-galactosylceramide). CD1-restricted T cells were then added at a 1:1 ratio to T-cell lines J.RT-3/CD8–2 (CD1a), LDN5 (CD1b), CD8–1 (CD1c), or sorted NKT cells (CD1d). Activation of CD1a-, CD1b-, and CD1c-restricted T cells was determined by their production of IL-2 using the HT-2 cells bioassay (A-C). Activation of CD1d-restricted NKT cells was measured after 6 hours of coculture by determining the frequency of IFNγ-producing cells by intracellular cytokine staining and flow cytometry (D). The graphs A-C show the average IL-2 release (± standard deviation) of triplicates in 1 representative donor of 3. The average frequency of IFNγ⁺ NKT cells (± standard deviation) in 2 individual donors (1 representative experiment of 2; 4 donors tested in total) is presented (D).