Fig 2: Characterization of CD4+ TIL, dLN, and Arm Transcriptomes by scRNA-Seq(A–D) TILs and dLN cells from wild-type (WT) mice at day 14 after MC38-GP injection analyzed by scRNA-seq and flow cytometry.(A) Heatmap shows row-standardized expression of selected genes across TIL and dLN clusters. Bar plot indicates the percentage of cells in each cluster relative to the total TIL or dLN cell number.(B) Flow cytometry contour plots of Foxp3 versus T-bet in CD44hi GP66+ dLN cells (left) and in CD44hiCD4+ splenocytes from tumor-free control mice (right).(C) Flow cytometry contour plots of Foxp3 versus T-bet in PD-1+ and GP66+ TILs (left) and in CD44hi CD4+ splenocytes from tumor-free control mice (right).(B and C) Data representative from 18 tumor-bearing mice analyzed in four separate experiments.(D) t-SNE display of TILs and dLN cells, shaded gray by tissue origin (left) or color coded by main group (right, as defined in A).(E) t-SNE (TIL and dLN cell positioning as shown in B) display of normalized expression levels of selected genes.(F) Heatmap shows Pearson correlation between cluster fold change vectors (as defined in the text) across the two replicate experiments for TILs (left) and dLN cells (right).See also Figures S1 and S2 and Tables S1 and S6.

Fig 3: MPT70-specific CD4 T cells maintain a low differentiation state compared to ESAT-6.(a) The functional differentiation score (FDS) of MPT70 and ESAT-6-specific CD4 T cells over the course of Mtb Erdman infection (n=4). The FDS is defined as the ratio of all IFN-? producing CD4 T cell subsets divided by subsets producing other cytokines (IL-2, TNF-a), but not IFN-? (high FDS = high IFN-? production). Multiple t-tests with correction for multiple testing using the Holm-Sidak method. Shown as average mean ± SEM. Flow Cytometry gating as depicted in Figure S1, using antibody panel 2. (b) Frequencies of KLRG1 expressing MPT70 and ESAT-6-specific CD4 T cells throughout infection (n=4). Shown as average mean ± SEM. Multiple t-tests with correction for multiple testing using the Holm-Sidak method. (c) Frequency of CD45-labelled MPT70 and ESAT-6 specific CD4 T cells in the lung-associated vasculature (CD45+) 20 weeks post-infection (p.i.) with Mtb (n=4). Paired t-test, two-tailed. (d, upper) Schematic representation of custom-made I-Ab:MPT7038–52 MHC-II tetramer. (d, lower) Representative concatenated FACS plots showing frequencies of I-Ab:MPT7038–52 and I-Ab:ESAT-64–17 tetramer+ CD4 T cells or corresponding hClip tetramer+ CD4 T cells in lungs of mice 12 weeks post Mtb infection (n=4). (e) Frequency of I-Ab:MPT7038–52 and I-Ab:ESAT-64-17 CD4 T cells 12–16 weeks post Mtb infection expressing CXCR3, KLRG1, and T-bet. Parametric, paired t-test, two-tailed (n=12). Flow Cytometry gating as depicted in Figure S3 using antibody panel 1. (f) Concatenated FACS plot of CX3CR1+KLRG1+ co-expressing ESAT-64-17 CD4 T cells (n=4).

Fig 4: Optimal lung MAIT1 subset response during systemic Francisella tularensis infection is independent of inducible T-cell costimulator (ICOS) and CD28. (a) Representative flow cytometric plots showing transcription factor profiles of MAIT cells isolated from lungs of C57BL/6 [wild-type (WT)] mice uninfected, or at day 7 after intranasal infection with 106 Salmonella Typhimurium or 104 Legionella longbeachae, or at day 7 after intravenous infection with 104 CFU F. tularensis. Frequency of MAIT1 (T-bet+ ROR?t-) and MAIT17 (ROR?t+) subsets is indicated. (b–e) WT, Cd80/86 -/- and Icos -/- mice were infected with 104 F. tularensis via the intravenous route. Lungs and livers were harvested for analysis at day 7 after infection. (b) Absolute numbers of non-MAIT aß T cells, MAIT cells and (c) MAIT1 cells in the lungs. Data show means ± s.e.m. and individual mice (n = 9 or 13) from three independent experiments. ****P < 0.0001, one-way ANOVA with Dunnett's multiple comparisons. (d) Representative plots and (e) stacked plots showing the frequency of MAIT1 and MAIT17 subsets in the lungs. Data show means ± s.e.m. (n = 9) from two independent experiments. *P < 0.05, ***P < 0.001, one-way ANOVA with Dunnett's multiple comparisons performed on MAIT1 cell% between WT and each genetic knockout strain. See also Supplementary figures 1 and 2. MAIT, mucosal-associated invariant T; ns, not significant.

Fig 5: IL‐12 and IL‐23 promote MAIT1 cell responses during Francisella tularensis infection. Wild‐type (WT), Il‐23p19 −/− , Il‐12p35 −/− and Il‐12p40 −/− mice were infected with 103 colony‐forming units of F. tularensis via the intravenous route. Lungs and livers were harvested for analysis at day 7 after infection. (a) Absolute numbers of non‐MAIT αβ T cells and MAIT cells in lungs. Data show means ± s.e.m. and individual mice (n = 8) from two independent experiments. **P < 0.01, ****P < 0.0001, one‐way ANOVA with Dunnett's multiple comparisons. (b) Absolute numbers of MAIT1 and MAIT17 cells in lungs. Data show means ± s.e.m. and individual mice (n = 8). *P < 0.05, ***P < 0.001, ****P < 0.0001, one‐way ANOVA with Tukey's multiple comparisons performed between all groups. (c) Representative flow cytometry plots and (d) stacked plots showing frequency of MAIT1 and MAIT17 subsets in lungs. Data show means ± s.e.m. (n = 8). ****P < 0.0001, one‐way ANOVA with Dunnett's multiple comparisons performed on MAIT1 cell percentage between WT and each genetic knockout strain. (e) Expression of T‐bet in lung MAIT1 cells. Data show means ± s.e.m. and individual mice (n = 8). ****P < 0.0001, one‐way ANOVA with Dunnett's multiple comparisons. (f) Representative flow cytometric plots and (g) scatter plots showing IFNγ production in MAIT1 cells isolated from lungs and livers of WT and Il‐12p35 −/− mice. Cells were cultured with Golgi plug for 4 h without any further stimulation. Data show means ± s.e.m. and individual mice (n = 7) from two independent experiments. **P < 0.01, ****P < 0.0001, unpaired t‐test. See also Supplementary figure 3. 5‐OP‐RU, 5‐(2‐oxopropylideneamino)‐6‐d‐ribitylaminouracil; IL, interleukin; MAIT, mucosal‐associated invariant T; MR1, MHC‐related protein 1; ns, not significant.