Figure 1.
iNKT cells are critically necessary for antimyeloma immunity elicited by the CD3/BCMA BsAb. (A) The expression level of BCMA on Vk14451 myeloma cells. FMO indicates the fluorescence minus one controls. (B) CellTrace Violet (CTV)–labeled T cells were cocultured with Vk14451 cells for 3 days in the presence of indicated concentrations of the CD3/BCMA BsAb. CD8 T-cell proliferation. (C) Expression levels of BCMA on parental 5TGM1 cells and 5TGM1-BCMA cells . (D) Cell viability of 5TGM1 and 5TGM1-BCMA cells 2 days after coculture with T cells in the presence of indicated concentrations of the CD3/BCMA BsAb. (E) The experimental design. (F) Expression levels of CD69 in T cell subsets in the BM from naïve and tumor-bearing mice. (G-H) Flow cytometry showed the frequencies of CD8 T cells and NK cells expressing CD69 and CD107a (G) and IFN-γ (H) in the myeloma BM (n = 4). (I) WT mice and Jα18−/− mice were challenged with Vk14451 cells, and treated with a single dose of CD3/BCMA BsAb. WT mice were pretreated with anti-CD8β or anti-CD4–depleting mAb 3 days before BsAb treatment. Violin plots showing the number of BM tumor cells 2 weeks after treatment with the BsAb. The middle lines indicate the median values. Results are pooled from 2 experiments (n = 6-9). (J) Frequencies of live iNKT cells and CD8 T cells in the BM. (K) Frequencies of annexin V+ apoptotic cells in mice treated with the CD3/BCMA BsAb (red) or control (blue). (L) The number of live BM iNKT cells and CD8 T cells at the indicated time points after treatment (n = 8). (M-N) Tumor-bearing WT and Jα18−/− mice were treated with or without the CD3/BCMA BsAb, and BM CD8 T and NK cells were analyzed 16 hours after treatment. (M) Expression levels of CD69. Green histograms indicate the untreated control. (N) Frequencies of IFN-γ–producing cells. Results are pooled from 2 or 3 experiments (n = 6-10). Data are expressed as the mean ± SEM (D,G-H,L-N), and differences were tested for statistical significance using an unpaired Student t test (D,G-H), a Kruskal-Wallis test with Dunn's post hoc test (I), a Mann-Whitney U test (L), and 2-way analysis of variance with Sidak’s multiple comparison (M-N). *P < .05, **P < .01, ***P < .001, ****P < .0001.

iNKT cells are critically necessary for antimyeloma immunity elicited by the CD3/BCMA BsAb. (A) The expression level of BCMA on Vk14451 myeloma cells. FMO indicates the fluorescence minus one controls. (B) CellTrace Violet (CTV)–labeled T cells were cocultured with Vk14451 cells for 3 days in the presence of indicated concentrations of the CD3/BCMA BsAb. CD8 T-cell proliferation. (C) Expression levels of BCMA on parental 5TGM1 cells and 5TGM1-BCMA cells . (D) Cell viability of 5TGM1 and 5TGM1-BCMA cells 2 days after coculture with T cells in the presence of indicated concentrations of the CD3/BCMA BsAb. (E) The experimental design. (F) Expression levels of CD69 in T cell subsets in the BM from naïve and tumor-bearing mice. (G-H) Flow cytometry showed the frequencies of CD8 T cells and NK cells expressing CD69 and CD107a (G) and IFN-γ (H) in the myeloma BM (n = 4). (I) WT mice and Jα18−/− mice were challenged with Vk14451 cells, and treated with a single dose of CD3/BCMA BsAb. WT mice were pretreated with anti-CD8β or anti-CD4–depleting mAb 3 days before BsAb treatment. Violin plots showing the number of BM tumor cells 2 weeks after treatment with the BsAb. The middle lines indicate the median values. Results are pooled from 2 experiments (n = 6-9). (J) Frequencies of live iNKT cells and CD8 T cells in the BM. (K) Frequencies of annexin V+ apoptotic cells in mice treated with the CD3/BCMA BsAb (red) or control (blue). (L) The number of live BM iNKT cells and CD8 T cells at the indicated time points after treatment (n = 8). (M-N) Tumor-bearing WT and Jα18−/− mice were treated with or without the CD3/BCMA BsAb, and BM CD8 T and NK cells were analyzed 16 hours after treatment. (M) Expression levels of CD69. Green histograms indicate the untreated control. (N) Frequencies of IFN-γ–producing cells. Results are pooled from 2 or 3 experiments (n = 6-10). Data are expressed as the mean ± SEM (D,G-H,L-N), and differences were tested for statistical significance using an unpaired Student t test (D,G-H), a Kruskal-Wallis test with Dunn's post hoc test (I), a Mann-Whitney U test (L), and 2-way analysis of variance with Sidak’s multiple comparison (M-N). *P < .05, **P < .01, ***P < .001, ****P < .0001.

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