Fig 1: Antitumor activity of m-reMAIT cells bolstered by NK cells.(A) Activation of m-reMAIT cells by NK cells. CD69 expression in m-reMAIT cells (reMAIT ?) and NK cells (NK ?) upon incubation at the indicated ratio. The percentage of cells expressing CD69 (left panel) and the intensity of CD69 in each cell population (right panel) are shown. (B) Cytokines and chemokines upon a coculture. Cytokines and chemokines released upon a coculture of m-reMAIT cells and NK cells at the indicated ratio are shown (reMAIT:NK). Amounts were quantified with LegendPlex. Representative data from two independent experiments are shown. (C) Transcripts relevant to cytolytic activity in m-reMAIT cells. Ifng, Gzma, Gzmb, Tbf, Gzmk, Pfr1, Fasl, Tnfsf10, Il6, Il17a, Il22, Ccl3, Ccl4, Ccl5, and Ccl22 in m-reMAIT cells cultured with NK cells were quantified with qRT-PCR. m-reMAIT cells and NK cells were sort-purified after the coculture (purity >98%) or cultured individually. The expression of each transcript was normalized with Gapdh, and fold changes in the relative expression of the transcript in m-reMAIT cells cultured with NK cells relative to that in m-reMAIT cells cultured alone are shown. Data are representative of three independent experiments. (D) Transcripts relevant to cytolytic activity in NK cells. Fold changes in the relative expression of the indicated transcript as described in (C) in NK cells cultured with m-reMAIT cells relative to that in NK cells alone are shown. Representative data from three independent experiments are shown. (E) Activation and degranulation of NK cells and m-reMAIT cells. The expression of CD69, an activation marker, and CD107a, a marker for the exocytosis of cytolytic granules, was assessed under various culture conditions. The percentages of CD69+ cells and CD69+CD107a+ cells among NK cells alone (control), NK cells cocultured with m-reMAIT cells (+reMAIT), NK cells cultured with Yac-1 (+Yac-1), and NK cells cocultured with m-reMAIT cells and Yac-1 (+reMAIT/Yac-1) (upper panels). The percentages of CD69+ cells and CD69+CD107a+ cells among m-reMAIT cells alone (control), m-reMAIT cells cocultured with NK cells (+NK), m-reMAIT cells cocultured with Yac-1 (+Yac-1), and m-reMAIT cells cocultured with NK cells and Yac-1 (+NK/Yac-1) (lower panels). Data are representative of three independent experiments. (F) Cytolytic activity against Yac-1. Cytolytic activity of m-reMAIT cells (reMAIT ?), NK cells (NK ?), and NK cells plus m-reMAIT cells (NK+reMAIT ?). Cytolytic activities (% lysis) at different effector (NK cells, m-reMAIT cells, and NK cell+m-reMAIT cells)/Target (Yac-1) (E/T) ratios are shown. Representative data from three experiments are shown. The significance of differences between the groups at the indicated E/T ratio assessed with a two-way ANOVA is shown (*p<0.05, **p<0.01, ***p<0.005). From the top, NK+reMAIT vs. reMAIT, NK+reMAIT vs. NK, and reMAIT vs. NK. Data are representative of three independent experiments. (G) Cytolytic activity against Lewis lung carcinoma (LLC). The cytolytic activities of m-reMAIT cells (?), NK cells (?), and m-reMAIT cells plus NK cells (?) against LLC at the indicated E/T ratio are shown as % lysis. The significance of differences between the groups is calculated as in (E). Data are representative of three independent experiments. (H) NK cell-dependent extension of survival. C57BL/6 mice were divided into two groups, one that received 1 × 106 m-reMAIT cells (reMAIT) and another that was left untreated (n). Each group was further divided into two subgroups, one that received consecutive injections of anti-Asialo GM1 (AsG) (–1 and 16 days, 50 µg/mouse) after the LLC inoculation (3 × 105 i.v.) and another that was left untreated (n). Survival was monitored thereafter. Representative data from two independent experiments are shown (n = 10–14/group). p-Values between the indicated groups are shown (the log-rank test). Figure 4—source data 1.Antitumor activity of m-reMAIT cells bolstered by NK cells.Activation of m-reMAIT cells by NK cells (A). Cytokines and chemokines produced upon a coculture (B). Transcripts relevant to cytolytic activity in m-reMAIT cells (C). Transcripts relevant to cytolytic activity in NK cells (D). Activation and degranulation of NK cells and m-reMAIT cells (E). Cytolytic activity against Yac-1 (F). Cytolytic activity against LLC (G). NK cell-dependent extension of survival (H).