Fig 2: Tumor inhibitory activity of m-reMAIT cells.(A) Time course of 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU)-dependent MR1 expression. The indicated cancer cell lines were challenged with 5-OP-RU. MR1 expression levels on the cell surface at the indicated time point are shown as relative geometric mean fluorescent intensity (gMFI). Data are representative of three independent experiments. (B) m-reMAIT cell dose-dependent survival extension. C57BL/6 mice received the indicated amounts of m-reMAIT cells 6 days prior to the Lewis lung carcinoma (LLC) inoculation (3 × 105 cells/mouse i.v.), and survival was monitored (n = 10–12/group). Data are representative of three independent experiments. p-Values between the indicated group are shown (the log-rank test). (C) Effects of the multiple transfers of m-reMAIT cells on survival. The survival of C57BL/6 mice that received m-reMAIT cells (1 × 106 /mouse, i.p.) 6 days prior to the LLC inoculation (3 × 105 cells/mouse, i.v.), and of mice that received LLC and two more consecutive transfers of m-reMAIT cells (1 × 106/transfer/mouse) was monitored (n = 10–12/group). Sham-treated mice that only received LLC served as a control. Data are representative of two independent experiments. p-Values between the indicated groups are shown (the log-rank test). (D) Effects of m-reMAIT cells on in situ tumor growth. Growth curve of LLC. LLC (3 × 105 /mouse) was subcutaneously inoculated into the right flank of C57BL/6 mice 6 days after the m-reMAIT cell transfer (i.p.). Tumor size was plotted with time. Sham treated (?), 0.3 × 106 transferred (¦), 1.0 × 106 transferred (?), and 3.0 × 106 m-reMAIT cells transferred (?). Data are shown as SEM (5–6 mice per group). Figure 3—source data 1.Time-dependent MR1 expression in various cancer cell lines upon 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) challenge.(A), m-reMAIT cell dose-dependent mouse survival (B). Effects of the multiple transfers of m-reMAIT cells on mouse survival (C). Effects of m-reMAIT cell dose on in situ tumor growth (D).

Fig 3: 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.

Fig 4: Stable aaMAIT cell subsets differ in localization and protective capacity.a, Il17a-FM YFP mice for FM Il17a expression were analysed at indicated times post BRD509 vaccination. Experimental setup (left). aaMAIT cell YFP expression is shown in representative histograms (middle) and quantified as percentage of YFP+ cells (right). n = 6 (untreated), n = 8 (day 40) mice per group, combined from two independent experiments. Data displayed as mean ± s.d.; statistical significance assessed via ANOVA with Tukey’s multiple comparisons test, ****P < 0.0001. b, Experimental setup and gating (left). The indicated populations of CD45.2 donor lung aaMAIT cells at day 40 post vaccination were sorted and transferred into CD45.1 recipient mice. After gating on MR1:5-OP-RU tetramer+ TCRβ+ MAIT cells and CD45.2 (donor), Klrg1 and CD127 expression was plotted in recipient mice 6 days post vaccination with BRD509 (right). Representative data shown from one of two independent experiments. c, Populations of aaMAIT cells were gated as in b, sorted and cultured for 72 h under indicated stimulation conditions and analysed for expression of Klrg1 and CD127. Representative data shown from one of two independent experiments. d, Vascular localization of indicated aaMAIT cell subsets at indicated times post BRD509 vaccination represented as percentage of cells accessible to staining with intravenously injected CD45 antibody 3 min before tissue collection. Gating (left) and quantification (right). Paired t-test, n = 5 (untreated) n = 6 (day 40) mice per group, ****P < 0.0001. p.i., post infection. e, UMAP presentation of circulatory and tissue residency signatures from scRNA-seq data from pulmonary MAIT cells at day 0 and day 40 post BRD509 vaccination. f–i, Donor mice were vaccinated with BRD509. Total MAIT cells were sorted at day 7 (f and g) or subsets were sorted at day 40 (h and i). MAIT cells were adoptively transferred into recipient mice, and recipients were infected with S. pneumoniae (f–h) or influenza H1N1 (i). f–h show bacterial burden in lung 18 h post infection. In f, n = 8, 6, 8 and 9 mice per group, respectively, combined from two independent experiments, data displayed as mean ± s.d., one-way ANOVA with Tukey’s multiple comparisons test, **P = 0.0094, **P = 0.0068, respectively. In g, n = 7 and 9 mice per group, respectively, unpaired t-test, ***P = 0.0007, combined from two independent experiments. In h, n = 9, 8 and 10 mice per group, respectively, combined from two independent experiments, one-way ANOVA with Tukey’s multiple comparisons test, *P = 0.0274, *P = 0.0397, respectively. In i, n = 11, 8 and 10 mice per group, combined from two independent experiments. Data displayed as mean ± s.e.m. Survival represented as Kaplan–Meier plot (top) and weight loss (bottom) following influenza infection of recipient mice. Log-rank (Mantel–Cox) test *P = 0.0111, **P = 0.0048. Source numerical data are available in source data. n.s., not significant.Source data

Fig 5: Immunostaining of microglia, amyloid-beta (Aβ), and MR1 in the temporal cortex of individuals with Alzheimer’s disease (AD). A Fold change in MR1 gene expression in human AD from the Agora Database. B Representative images of AD and non-AD control tissue showing Dapi (blue), Aβ (green), IBA1-labeled microglia/macrophages (red), MR1 (white), and these images merged. Scale bar = 250 µm. C–E Densitometry analysis presented as the relative mean pixel intensity for Aβ (C), IBA1 (D), and MR1 (E). F Representative images of non-AD and AD tissue at 100 × with co-localization of the boxed regions in the XY, ZX, and ZY planes (arrows). Scale bar = 50 µm. G, H Densitometry analysis presented as the relative mean pixel intensity for IBA1 (G) and MR1 expression within the boundaries of the IBA1 + cells (H). I Densitometry analysis of the MR1 signal within the boundaries of the IBA1 + cells that are determined to be near (visibly touching) or far from Aβ plaques (no visible touching). Statistical analysis was performed using a Student’s t test (C–E, G–I) and a two-way ANOVA with Tukey post hoc multiple comparison test (I). *P < 0.05, **P < 0.01, ***P < 0.001 (n = 5/group). Significance in both males and females was normalized to the age of death. Data are shown as the mean ± standard error of the mean