αGC-loaded fusion promotes hu-iNKT cell cytotoxicity in vitro. (A-B) Freshly isolated hu-iNKT cells were mixed with autologous PBMCs and then added to wells pre-coated with αGC-loaded or unloaded CD1d-CD19 fusion as in Figure 3. After 24 hours, cells were harvested and analyzed for different immune cell populations by flow cytometry. (A) Representative density plots showing percent CD19⁺ B, percent CD4⁺, and percent CD8⁺ cells in PBMCs + iNKT cell mix after culture on plate-bound unloaded or loaded fusion protein (1 μg/mL) for 24 hours. (B) Frequencies of B (CD19⁺), CD4⁺, and CD8⁺ cells in PBMCs and iNKT cell cocultures plated on either unloaded or αGC-loaded CD1d-CD19 fusion protein. Data are pooled from 4 independent experiments. Each symbol in the graphs corresponds to 1 donor. (C) Freshly isolated human were plated with ⁵¹Cr-labeled EBV-LCLs at varying effector:target (E:T) ratios with no stimulus (no Ag), PBS44, or unloaded (ULFP) or αGC-loaded fusion (LFP, 1 μg/mL). After 16 to 18 hours, culture supernatants were collected, radioactivity quantified, and percent specific lysis calculated. Depicted are results obtained using 3 representative EBV-LCLs. Data are from 1 of 6 independent experiments. (D) Mean percent increase ± SEM in cytolysis of the EBV-LCLs in the presence of unloaded or αGC-loaded fusion compared with cytolysis in the presence of PBS44. Data are averaged from 6 EBV-LCL lines. (E) hu-iNKTs were plated with nonradiolabeled EBV-LCLs at varying E:T = 20:1 (40 × 10³ NKT cells and 2 × 10³ EBV-LCLs) ratio with no stimulus, or the unloaded or αGC-loaded CD1d-CD19 fusion (1 μg/mL). After 16 to 18 hours, supernatants were collected and IFN-γ levels were measured by enzyme-linked immunosorbent assay. Representative data (mean ± standard deviation [SD]) from 1 of 3 experiments is shown. (F) Data are as in panel D, except that in these experiments Jurkat (human, left) or EL4 (murine, right) T cells were used as targets. (G-H) Freshly isolated hu-iNKT cells were added to wells coated with plate-bound αGC-loaded or unloaded CD1d-CD19 fusion (1 μg/mL) or left untreated. (G) After 24 hours, cells were harvested and analyzed for intracellular levels of lytic molecule (granzyme B) and degranulation (CD107a). Data are representative of 3 independent experiments. Numbers in the histograms indicate MFI. (H) Compiled data (mean ± SD) from 3 independent experiments showing fold increase in granzyme B (GrB) and CD107a expression on iNKT cells plated on αGC-loaded CD1d-CD19 fusion compared with those plated on unloaded fusion. Significance in panels B, D, and H was determined by unpaired 2-tailed Student t test; and in panels C and F by 2-way analysis of variance (ANOVA) test. *P < .05, **P < .01.