αGC-loaded CD1d-CD19 fusion promotes hu-iNKT cell activation in vitro. (A) hu-iNKT cells were expanded from PBMCs as detailed in “Materials and methods.” (Top left) Representative density plot showing % human CD1d tetramer (Cd1dtet)⁺CD3⁺ cells in the expanded PBMC cultures after 7 days. (Top right) Representative density plot showing that hu-iNKT cells can be successfully expanded from PBMCs and isolated to >98% purity. (Bottom left) Representative density plot showing percent CD4⁺ and percent CD8⁺ cells gated on iNKT cells from the PBMC cultures described previously. (Bottom right) Frequencies of CD4⁺, CD8⁺, and CD4⁻CD8⁻ (DN) iNKT cells. Data are pooled from 6 independent donors. Each symbol in the graphs corresponds to 1 donor. (B) Freshly isolated hu-iNKT cells were cultured in plates coated with αGC-loaded or unloaded CD1d-CD19 fusion (1.0 μg/mL). After 24 hours, cells were harvested and analyzed for expression of activation markers (CD25 and CD69) by flow cytometry. (C) hu-iNKT cells were labeled with 250 nM of CFSE on day 0 and cultured as described. After 4 days, cells were harvested and analyzed for cell proliferation by flow cytometry. Numbers in the histograms indicate MFI. (D) Culture supernatants from experiments in panel A were analyzed for the levels of cytokines by MILLIPLEX MAP Human High Sensitivity T Cell Magnetic Bead Panel kit. Data are presented as mean ± standard error of the mean (SEM) and compiled from independent experiments using hu-iNKT cells from 10 normal donors. Significance was determined by unpaired 2-tailed Student t test. *P < .05, **P < .01. Ag, αGC; DN, double negative; GM-CSF, granulocyte-macrophage colony-stimulating factor; ns, not significant.