Fig 2: MAIT cells and macrophages crosstalk induces an inflammatory response in vitro.a, b Wild-type (clear background) or MR1−/− (gray background) M0 macrophages were co-cultured for 72 h with spleen MAIT cells, either naive (nMAIT) or pre-activated (acMAIT). a Data showing the frequency of M1 macrophages (CD206-CD11c+) and M2 macrophages (CD206+CD11c−) among CD11b+F4/80+ total macrophages and M1/M2 ratio (n = 12 samples over two independent experiments). b Wild-type or MR1−/− M0 macrophages were co-cultured for 72 h with spleen pre-activated MAIT cells (acMAIT) in presence of cytokine-neutralizing antibodies (anti-IL-17, anti-IFNγ, and/or anti-TNF). Data show the frequency of M1 macrophages (CD206−CD11c+) and M2 macrophages (CD206+CD11c−) among CD11b+F4/80+ total macrophages and M1/M2 ratio (n = 8 samples over two independent experiments). c Relative expression of Mr1 in M0, M2, or M1 macrophages and surface staining for MR1 in in vitro-differentiated macrophages. Culture media contained 100 μM of Ac-6-FP. d–e Spleen nMAIT cells were co-cultured with M0-, M2-, or M1-differentiated macrophages for 48 h in presence of 5-OP-RU agonist molecule. c Data showing the frequency of M1 macrophages (CD206−CD11c+) and M2 macrophages (CD206+CD11c−) among CD11b+F4/80+ total macrophages co-cultured with acMAIT cells in medium with or without anti-IL-17A, anti-IFNγ and/or anti-TNFα neutralizing antibodies (n = 4 samples). d CD44 and CD69 surface marker expression on nMAIT cells and intracellular staining of nMAIT cells for TNFα, IFNγ, and IL-17A (n = 20 samples from four independent experiments). e Intracellular staining of MAIT cells co-cultured either with WT M0, M2, or M1 macrophages (round symbol, clear background) or MR1−/− M0, M2, or M1 macrophages (diamond symbol, gray background) for TNF-α, IFN-γ, and IL-17A (n = 7 samples). Data are representative of at least three independent experiments. For a and a–e, each symbol represents an individual mouse (small horizontal lines indicate the mean ± S.E.M.). In b, in M1/M2 ratio graph, $ symbol represents statistics between WT macrophages vs. MR1−/− macrophages cultured in the same conditions. All statistical analyses were performed by two-tailed Mann–Whitney test. *P < 0.05, **P < 0.01, ***P < 0.001 (see also Supplementary Fig. 11).

Fig 3: Dysfunction Transcriptomes of Th1, Isc, and Treg TILs(A) Heatmap shows row-standardized expression of selected exhaustion genes across TIL, dLN, and Arm clusters from replicate experiments I and II.(B) Overlaid protein expression of PD-1 in GP66+ clone 13 (red trace) and GP66+ TILs (left) or dLN cells (right) (cyan trace). Gray-shaded histograms show PD-1 expression on CD44+CD4+ splenocytes from tumor-free control mice.(C) Flow cytometry contour plots of NKG2A versus CD94 (top) or IRF7 (bottom) in TILs and clone 13 Foxp3−GP66+ T cells. Graphs on the right summarize data from two experiments; each symbol represents one mouse. Two-tailed unpaired t test; ***p < 0.001 and ****p < 0.0001.(B and C) Data are from 10 mice of each condition, analyzed on two separate experiments.(D) Analysis of interleukin-27 (IL-27) signature genes overlapping with TIL subpopulation-characteristic genes. Heatmaps show Pearson correlation (left) and row-standardized expression of overlapping genes across TIL Th1, Treg, Isc, and nRes cells (clusters t1–2, t6–7, t3–4, and t5, respectively, as shown in Figure 1A) (right).See also Figure S5 and Tables S3 and S4.

Fig 4: miR-15/16 Restrict Memory CD8+ T Cell Differentiation(A) Flow cytometry of GP33+ CD8+ T cells from spleens harvested 8 days p.i. with LCMV cultured overnight in vitro (n = 7 biological replicates from two Independent experiments).(B) Quantification of frequencies of Live/Dead− activated caspase-3− GP33+ CD8+ T cells from spleens harvested 8 days p.i. with LCMV cultured overnight in vitro (n = 7 biological replicates from two independent experiments, two-tailed t test).(C) Quantification of frequencies of EdU+ CD44+ CD8+ T cells from spleens harvested 8 days p.i. with LCMV (n = 7 biological replicates from two independent experiments, two-tailed t test).(D) Quantification of frequencies of MP and TE populations among tetramer+ CD8+ T cells from spleens harvested 5 days p.i. with LCMV (n = 8 biological replicates from two independent experiments).*p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S6.

Fig 5: Rac1 inhibition enhances thymus regeneration and peripheral CD4+ naive T cell recovery after acute damage6- to 8-week C57BL/6 WT or Nod2-/- mice were treated with the Rac1 inhibitor EHT1864 (40 mg/kg i.p. injection) at days 3, 5, and 7 following a sublethal dose of TBI (550 cGy), and the thymus was analyzed on day 14.(A) Total thymic cellularity at day 14 after SL-TBI (WT, n = 20/treatment across 4 independent experiments; KO, n = 9–12 across three independent experiments).(B) Flow plots showing CD4 and CD8 expression at day 14 after SL-TBI (gated on CD45+ cells; plots were concatenated of all samples in each treatment group from one experiment).(C) Total number of DP, SP4, or SP8 thymocytes (n = 20/treatment across 4 independent experiments).(D) Expression of miR29c analyzed by qPCR on whole thymic tissue (n = 5/group).(E) Total thymic amounts of BMP4 and IL-23 assessed by ELISA at day 7 (n = 4–5/group, representative of two independent experiments).(F) Total number of cTECs and mTECs (n = 15/group across three independent experiments).(G) Flow plots showing CD4 and CD8 expression in the spleen at day 56 after SL-TBI (gated on CD45+CD3+ cells).(H) Total number of CD4+ and CD8+ T cells in the spleen at d56 (n = 5/group).(I) Plots of CD62L and CD44 on CD4+ and CD8+ T cells (gated on either CD45+CD3+CD4+CD8- or CD45+CD3+CD4-CD8+) cells; plots concatenated of all samples in a given experiment).(J) Ratio of number of naive (CD62Lhi CD44lo) CD4+ or CD8+ to CD4+ or CD8+ EM (CD62Lhi CD44hi) T cells (n = 5/group).Experiments in (C)–(J) were performed only in WT mice. Graphs represent mean ± SEM; each dot represents a biologically independent observation. See also Figure S5.