Expansion of iNKT cells dramatically improves therapeutic efficacy of the CD3/BCMA BsAb against myeloma. (A) Freshly isolated iNKT cells (2 × 10⁵) were cocultured with Vk14451 cells (2 × 10⁴) in the presence or absence of BM-derived DCs (1 × 10⁵). The levels of IL-12p70 in culture supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA) 24 hours after treatment. (B-C) Tumor-bearing IL-12-YFP reporter mice were treated with the CD3/BCMA BsAb. (B) Frequencies of BM CD11c cells expressing MHC-II. (C) Frequencies of BM CD8α⁺ DCs (gated on CD11c⁺MHC2⁺CD11b⁻CD8α⁺) expressing IL-12. Results are pooled from 2 experiments (n = 7-8). (D) Vk14451 cells were cocultured with B-cell–depleted splenocytes in the presence or absence of recombinant IL-12 (rIL-12, 0.5 ng/mL). The levels of IFN-γ and granzyme (Gzm) B in culture supernatants were analyzed by ELISA 48 hours after stimulation with indicated concentrations of the CD3/BCMA BsAb (n = 7). (E-F) Tumor-bearing WT and Il12p35^−/− mice were treated with the CD3/BCMA BsAb. (E) Levels of IFN-γ (left) and Gzm B (right) in blood plasma 6 hours after treatment. (F) Tumor burden and violin plots showing the number of tumor cells in the BM 2 weeks after treatment. The middle lines indicate the median values. Results are pooled from 2 experiments (n = 7-8). (G) DCs were pulsed with α-GalCer overnight and IV injected into tumor-bearing mice (1 × 10⁶). Representative plots (left) and graph (right) show expansion of iNKT cells in the myeloma BM 3 days after adoptive transfer. (H) The experimental design in the VK14451 myeloma model in C57BL/6 WT mice. (I) Serum levels of IgG2b 2 weeks after treatment. The middle lines indicate the median values. Serum IgG2b levels in naïve mice (n = 7) are shown as reference ranges. (J) Kaplan-Meier survival curves of mice after indicated treatment. Results are pooled from 2 experiments (n = 9-10 per group). (K) The experimental design in the 5TGM1-BCMA myeloma model in C57BL/KaLwRij mice. (L) Bioluminescence images showing tumor lesions in mice at the indicated time points after therapy. (M) Kaplan-Meier survival curves of mice after the indicated treatment. Results are pooled from 2 experiments (n = 10 per group). Data are expressed as the mean ± SEM (A-E,G). Differences were tested for statistical significance with a 2-way analysis of variance with Sidak’s multiple-comparisons test (A,E), an unpaired t test (B-C,G), paired t test (D), Kruskal-Wallis test with Dunn’s post hoc test (F,I), and Mantel-Cox test (J,M). *P < .05, **P < .01, ***P < .001, ****P < .0001.