Activation and expansion of NKT cells in vivo after A20/αGC vaccination. A20 cells were cocultured overnight with 1 μg/mL αGC. After washing, these cells (A20/αGC) were irradiated (50 Gy) and intravenously injected into BALB/c mice. Lymphoid cells of the spleen from A20/αGC-vaccinated mice were isolated on indicated time points. The cells were stained with anti-TCRβ Ab plus CD1d tetramer and Ab for intracellular IFN-γ before being analyzed by flow cytometry. TCRβ⁺CD1d tetramer⁺ cells were gated and analyzed (bottom panels). Splenocytes from an untreated mouse (Nil) were used as control. Numbers indicate the percentage of each gate.

Therapeutic antitumor activity generated by A20/αGC vaccination. (A,B) BALB/c mice were inoculated with 1 × 10⁶ live A20 cells intravenously at day 0. On day 5 (day +5) or day 10 (day +10), mice were vaccinated with A20/veh, A20/αGC, or left untreated (Nil) (n=7-10 per group), and the survival was monitored. (C) Survivors from panel A were rechallenged with live A20 cells 80 days after primary tumor inoculation, and the survival was monitored. Age-matched naive mice were used as control (Nil; n=5). P values were calculated in comparison with the nonvaccinated group.

Analysis of immune cell composition in the spleens and blood of A20/αGC-vaccinated mice. BALB/c mice were vaccinated with A20/αGC or left untreated (Nil) (n=3 per group). At 8 weeks after vaccination, splenocytes (A) and blood leukocytes (B) were isolated from vaccinated or untreated mice (Nil). Cells were analyzed by flow cytometer after staining with anti-CD19, anti-CD4, anti-CD8, and anti-CD11b Abs (A). Neutrophils were defined by forward scatter (FSC) and side scatter (SSC) profiles in combination with anti-Gr1 and anti-CD11b Ab staining by flow cytometer (B). Values are means (± SEM).

Preventive antitumor activity generated by A20/αGC vaccination. (A) A20 cells were cocultured overnight with 1 μg/mL αGC (A20/αGC) or vehicle (0.5% polysorbate; A20/veh) followed by irradiation (50 Gy). BALB/c mice (n=6-7 per group) were vaccinated with A20/veh, A20/αGC, or left untreated (Nil). After 7 days, all mice received 1 × 10⁶ live A20 cells intravenously, and the survival was checked (day −7). (B) Survivors from panel A were rechallenged with live A20 cells 80 days after primary inoculation (Tumor rechallenge). Age-matched naive mice (n=5) were used as control (Nil). P values were calculated in comparison with the nonvaccinated group. Data shown represent at least 2 independent experiments.

Comparison of antitumor efficacy by free or tumor-loaded αGC. (A) BALB/c mice received 1 × 10⁶ live A20 cells intravenously (day 0) before they were vaccinated with A20/αGC, intravenous injection of αGC (2 μg/mouse) plus A20/veh (A20/veh + αGC), or αGC alone (2 μg/mouse) at day 5 (n=7 per group). (B) Mice were vaccinated with the indicated regimen with nonirradiated A20 cells (live A20/veh, live A20/αGC) on day −7 (n=5 per group). On day 0, all mice received 10⁶ live A20 cells intravenously. Nonvaccinated mice were used as a control (Nil). The survival was monitored daily. P values were calculated in comparison with the nonvaccinated group.

Exclusive role of CD4⁺ T cells in mediating antitumor activity. (A) A group of mice were vaccinated with A20/veh, A20/αGC, or left untreated (Nil) on day −7. A20/αGC-vaccinated mice were injected intraperitoneally with depleting Abs of CD4, CD8, or NK cells, respectively, on days −3 and −1 (n=5-7 mice per group) before all mice were inoculated intravenously with live A20 on day 0. Survival was monitored daily. (B) BALB/c mice were vaccinated with A20/αGC. At 7 days later, CD4⁺ or CD8⁺ cells from lymphoid cells of A20/αGC-vaccinated mice were sorted and transferred intravenously into syngeneic naive mice (2-3 × 10⁷/transfer) on day 0 (n=4-5 mice per group). A20/αGC-vaccinated mice (day −7) were used as a positive control. All mice were inoculated intravenously with live A20 on day 0, and the survival was monitored daily. P values are calculated in comparison with the nonvaccinated (Nil) group. (C) BALB/c mice were vaccinated with A20/αGC. At 7 days later, CD1d tetramer⁻CD4⁺ cells from lymphoid cells of A20/αGC-vaccinated mice were sorted and transferred intravenously into syngeneic naive mice (2 × 10⁷/transfer) on day 0. Nontreated naive mice were used as a control (Nil). All mice were inoculated intravenously with live A20 cells on day 0, and the survival was monitored. P values are calculated in comparison with nontreated group. (D,E) CD1d tetramer⁻CD4⁺ T cells were sorted from vaccinated mice by using FACSAria (n=3 per group). Sorted CD4⁺ T cells (4 × 10⁵/well) were cultured in the presence or absence of irradiated A20 cells (1 × 10⁵/well) for 24 hours, and ELISPOT (D) or intracellular cytokine staining (E) was performed for analysis of IFN-γ and TNF-α. Values in panel D are means (±SEM). Values in panel E are percentages of the gated cells.