Fig 2: BRG1 is critical to induce EOMES-dependent program. a Immunoblot (IB) of lysates from 293 cells transfected with an Eomes vector and immunoprecipitated (IP) with an anti-EOMES (up) or anti-BRG1 (down) antibody. b ChIP experiment for EOMES and BRG1 from WT and EomesTg CD3+CD8SP thymocytes followed by qPCR at the indicated loci. Each horizontal bar represents an individual data point from a single experiment. c Representative flow cytometry plots gated on CD3+CD8SP thymocytes from mice lacking Smarca4 expression in T cells (Smarca4fl/flxCD4Cre: Smarca4?T, down) and their littermate controls (Smarca4fl/fl, up) after 4 days of daily intra-peritoneal injection of anti-IL-4/rIL-4 (IL-4c) or an isotype control (left panel). Percentage of EOMEShi CD3+CD8SP thymocytes after isotype control or IL-4c injections (right panel). d Mixed bone marrow chimera experimental scheme (upper panel). cGy centigrays. Percentages of EOMEShi cells among WT vs. Smarca4?T CD3+CD8SP thymocytes (lower left panel), and proportions of WT vs. Smarca4?T cells among CD3+CD8SP thymocytes (lower right panel) after isotype control or IL-4c injections. e Expression (MFI) of cell markers in EOMESlo and EOMEShi CD3+CD8SP thymocytes from Smarca4fl/fl vs. Smarca4?T mice after IL-4c injections. f Representative correlation plots of EOMES and CXCR3 single-cell (sc) fluorescences in CD3+CD8SP thymocytes from Smarca4fl/fl and Smarca4?T mice after IL-4c injections. Kendall’s Tau correlation coefficients and Loess fitting curves (red lines) and their corresponding confidence interval (gray area) are shown. g Representative flow cytometry plots gated on CD8+ splenocytes from Smarca4fl/fl and Smarca4?T mice at steady-state, showing the proportions of CD44hiCD49dlo (virtual memory cells, VM) and CD44hiCD49dhi (true memory cells, TM) (left panel). VM proportions in CD8+ splenocytes from Smarca4?T and Smarca4fl/fl mice after isotype control or IL-4c injections are shown (right panel). h Expression (MFI) of cell markers in naïve (N), TM, and VM subpopulations of CD8+ splenocytes from Smarca4fl/fl and Smarca4?Tmice. In c, d, e, g, and h, horizontal bars indicate median ± interquartile range (each point represents a single mouse, n = 5 mice per group). Statistics were calculated using Mann–Whitney test. ns not significant, *P < 0.05, **P < 0.01. Source data are provided as a Source Data file. Source Data file

Fig 3: EOMES expression is sufficient to drive TIM transcriptional program. a Representative flow cytometry plots of total thymocytes from WT and EomesTg mice showing DN, DP, CD4SP, and CD8SP proportions. Histograms indicate the frequency of each population and their EOMES expression (MFI). b Expression of the indicated markers in WT and EomesTg CD3+CD8SP thymocytes and in EOMESlo and EOMEShi CD3+CD8SP thymocytes from Balb/c mice, shown as a reference. SPADE trees indicate cell frequency of CD3+CD8SP thymocytes from WT and EomesTg mice using the tree constructed from WT Balb/c CD3+CD8SP thymocytes as shown in Fig. 1. c IFN? expression measured by flow cytometry in CD3+CD8SP thymocytes from WT and EomesTg mice cultured with IL-12+IL-18 for 16 h or with phorbol-myristate-acetate and ionomycin (P/I) for 4 h. For P/I, EOMESlo and EOMEShi CD3+CD8SP thymocytes from Balb/c mice are shown. d Volcano plot of RNA-seq data of CD3+CD8SP thymocytes from WT versus EomesTg mice show the adjusted P-value versus fold-change (up in EomesTg, red; up in WT, green). The numbers of differentially expressed genes are indicated. e GSEA plots of innate memory (TIM)-specific (left) or naïve (N)-specific (right) gene sets in CD3+CD8SP thymocytes from EomesTg mice. NES normalized enrichment score. f Expression heatmaps of RNA-seq data comparing mean fold-change (FC) of TIM and EomesTg CD3+CD8SP thymocytes in comparison to their respective naïve and WT controls, respectively. CPM counts per million. In a–c horizontal bars indicate median ± interquartile range and are representative of more than three experiments. Each point represents an individual mouse. Statistics were calculated using Mann–Whitney test. ns not significant, *P < 0.05; **P < 0.01; and ***P < 0.001

Fig 4: EOMES and RUNX3 interact within chromatin-associated complexes. a Selected EOMES-interacting proteins identified by RIME in activated CD8+ T cells. Total spectral counts for each replicate (anti-EOMES or control IgG) are shown. b Genomic distribution of EOMES (4306) and RUNX3 (6741)-binding sites in innate memory (TIM) cells. c Venn diagram illustrating the intersection between EOMES (in TIM) and RUNX3 (in naïve [N] and TIM cells) peaks at active promoters and enhancers. Density plots centered on common EOMES/RUNX3 or EOMES-specific peaks (±250 bp) represent the distribution of the best predicted sites of the JASPAR RUNX3 (MA0684.1) and EOMES (MA0800.1) motifs. d Representative EOMES, RUNX3, H3K4me1, H3K27Ac ChIP-seq, and ATAC-seq tracks at the Ly6c2 locus showing the co-localisation of EOMES and RUNX3 highlighted in grey (top). Sequence of the highlighted region and the location of EOMES and RUNX motifs with their position P-value are indicated (down). e Cumulative distribution plot generated by BETA algorithm showing the predicted activating/repressive functions of RUNX3 and EOMES with the indicated P-values determined by the Kolmogorov–Smirnov test. f Heatmap showing expression of genes from e that are predicted to be targets of EOMES. Selected genes are shown in the right margin. g Normalized coverage plot of histones modifications (H3K4me1, H3K4me3, and H3K27ac) and chromatin accessibility (ATAC-seq) centered on EOMES-binding sites at promoters and enhancers annotated to predicted genes from e. ChIP-seq was performed on three independent IPs (n = 7 mice per sample)

Fig 5: Ifnlr1-/- mice show reduced numbers of cNK cells in the kidney after pristane treatment. (A) Flow cytometry gating strategy. Total cell numbers per kidney of CD4+ cells (B) NKp46+Lin- NK cells (C), NKp46+Lin-Eomes+ conventional NK cells (D,F) and NKp46+Lin-Eomes- ILC1 cells (E,G) were determined by flow cytometry at 6 months after pristane treatment. Lin = Lineage (CD3, CD5, CD19, Ly6G). Each symbol represents an individual sample, bars represent median with interquartile range. For some mice, not enough tissue was available for flow cytometry. Statistical analyses to compare WT + pristane vs. Ifnlr1-/- + pristane were performed using Mann–Whitney U test. * = p < 0.05; ** = p < 0.005; ns = not significant. Values of WT without pristane are shown as reference. Findings were confirmed in at least one other independent experiment.