Fig 2: TIL immunophenotypes in CT26 tumors from mice treated with mIL-21 +/− mPD-1 or mCTLA-4 mAbs. TILs from mice implanted SC with CT26 tumor cells and treated with control mIgG, mIL-21, mCTLA-4 mAb (9D9-mIgG2b), mPD-1 mAb (4H2-mIgG1), or mIL-21 + mPD-1 or mCTLA-4 mAbs were isolated on study day 16, stained with various markers of immune cell subsets, and evaluated by flow cytometry. The (A) %CD8+ of live CD45+, (B) %CD25+FoxP3+ of CD4+, (C) %CD335+ of live CD45+, and (D) %PD-1+CD69- of CD8+ cells, are shown for each treatment group, indicated on the x-axes. The (E) ratios of the % CD8+ T cells of live CD45+ (Teff) to the % CD4+CD25+ of live CD45+ (Tregs) are plotted. Each symbol represents data from one mouse in the group and mean values are indicated with horizontal lines. Asterisks (*, **, ***, ****) indicate p < 0.05, p < 0.01, p < 0.001 or p < 0.0001, respectively, for differences between the groups indicated by 1-way ANOVA. Study was conducted one time.

Fig 3: 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).

Fig 4: PD-L1 impairs CD8+ T-cell priming by LECs in vitro. A and B, Example histograms (A) and quantification (B) of PD-L1 expression in wild-type (WT) and PD-L1ko LECs determined by FACS (N = 3 independent experiments). IFNγ was used as positive control to further induce PD-L1 expression. C–G, WT and PD-L1ko LECs were loaded with SIINFEKL peptide and cocultured with naive CD8+ OT-1 cells O/N. OT-1 expression of CD69 (C), IFNγ (D), Ki67 (E, left), proliferation (CFSE dilution; E, right), PD-L1 (F), and PD-1 (G) was determined by FACS. One representative of three independent experiments is shown (N = 5). *, P < 0.01; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, one-way ANOVA with paired Sidak post-test.

Fig 5: Characterization of m-reMAIT cells.(A) Flow cytometric profiles of m-reMAIT cells. mMR1-tet staining and expression of T-cell receptor ß (TCRß), CD4, CD8, CD25, and CD44 and the transcription factors PLZF and ROR?t in m-reMAIT cells on differentiation day 18. (B) 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) dose-dependent activation of m-reMAIT cells. The percentages of CD69+ cells among m-reMAIT cells challenged with the indicated concentration of 5-OP-RU in the presence of WT3 (?), WT3/mMR1 (?), CH27 (?), and CH27/mMR1(¦). (C) MR1-dependent activation of m-reMAIT cells. The percentage of CD69+ cells among m-reMAIT cells cultured with CH27/mMR1, challenged as in (B) in the presence of the anti-MR1 antibody (¦) or the isotype control antibody (?). (D) 5-OP-RU dose-dependent activation. The percentage of m-reMAIT cells expressing CD69 upon a challenge with various concentrations of 5-OP-RU. Representative data from two independent experiments are shown. (E) mMR1-tet dose-dependent activation. The percentage of m-reMAIT cells expressing CD69 upon a challenge with the indicated amounts of mMR1-tet. Representative data from two independent experiments are shown. (F) 5-OP-RU- and mMR1-tet-induced cytokines and chemokines. m-reMAIT cells were stimulated with various concentrations of 5-OP-RU (?) or mMR1-tet (¦) and the resultant cytokines and chemokines were quantified with LegendPlex. The concentrations at which each reagent induced a similar degree of activation (% CD69) are shown as relative concentrations (0.1–100 nM for 5-OP-RU and 0.01–10 µg/ml for mMR1-tet). The number on the X-axis corresponds to that in (D) and (E). (G) Tyrosine phosphorylation elicited with 5-OP-RU. A Western blot analysis with PY99 (anti-phosphotyrosine). Upon a challenge with different concentrations of 5-OP-RU for 30 min, the cell lysate from m-reMAIT cells was separated on SDS-PAGE (5 × 105/lane), and subjected to Western blotting. Lane 1, 0; lane 2, 0.1; lane 3, 1.0; lane 4, 10; lane 5, 100; lane 6, 1000; and lane 7, 10,000 (nM). Phosphorylated proteins are indicated with arrows. (H) Time course of tyrosine phosphorylation. A Western blot analysis with PY99. The cell lysate from m-reMAIT cells challenged with 100 nM of 5-OP-RU for the indicated time was separated on SDS-PAGE (5 × 105/lane) and subjected to Western blotting. Lane 1, 0; lane 2, 15; lane 3, 30; lane 4, 60; lane 5, 150; lane 6, 300 (min) . Arrows indicate phosphorylated proteins. (I) Linker for the activation of T cells (LAT) as a phosphorylated 37-kD protein. A Western blot analysis with PY99, anti-LAT, and anti-ß-actin. The cell lysate prepared as described in (H) for 60 min was subjected to Western blotting. A blot with PY99 (upper panel), anti-LAT (middle panel), and anti-ß-actin (lower panel). Phosphorylated LAT and LAT as well as ß-actin are indicated (arrow). Lane 1, 0; lane 2, 0.1; lane 3, 1.0; lane 4, 10; lane 5, 100; lane 6, 1000; lane 7, 10,000 (nM). (J) Tyrosine phosphorylation induced by mMR1-tet. A Western blot analysis with PY99. The cell lysate from m-reMAIT cells challenged with the indicated amounts of unlabeled mMR1-tet for 60 min was subjected to Western blotting (5 × 105/lane). Lane 1, 0; lane 2, 0.43; lane 3, 1.3; lane 4, 4.3; lane 5, 13 (µg/ml). Arrows indicate phosphorylated proteins. MAIT: mucosal-associated invariant T cell. Figure 1—source data 1.Characterization of m-reMAIT cells.The type of antigen-presenting cell (APC), reMAIT cells, and the concentration of 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) (nM) used for activation assays. Activation is shown as the percentage of CD69+ cells among reMAIT cells (B). The percentage of CD69+ MAIT cells challenged with 5-OP-RU in the presence of isotype control or anti-MR1 antibody (in the presence of CH27m as APC) (C). The percentage of CD69+ MAIT cells challenged with 5-OP-RU or mMR1-tet (D, E). Production of the cytokines and chemokines from m-reMAIT cells challenged with 5-OP-RU or mMR1-tet (F). MAIT: mucosal-associated invariant T cell. Figure 1—source data 2.Original gel electrophoresis panels for Figure 1G–J.