Fig 2: Specific epigenomic profiles induced by alarmins and adaptive cytokine. All the differential H3K27ac peaks after 3 h of stimulation with IFNβ, IL-15 or IL-21 (FDR ≤ 0.01) are depicted in (A) barplots and (B) used for PCA to depict epigenetic responses of IE-CTLs upon stimulation. (C) Scatter plot displaying the association between changes in H3K27ac (Y-axis) and expression (X-axis) upon stimulation. Gene-peak pairs were grouped based on the direction of the fold change in expression (Exp+ and Exp-) and the direction (or lack of changes) in H3K27ac after stimulation (Epi+, Epi - and Epi NA). Red dots (Epi + Exp+) indicate a fold change >1 in both expression and H3K27ac occupancy. Green dots (Epi- Exp-) indicate a fold change < 1 in both expression and H3K27ac occupancy. Dark blue (Exp + EpiNA) and light blue (Exp- EpiNA) are gene-peak pairs with fold change >1 or <1 in expression, respectively, and no change in H3K27ac. (D) Violin plots showing the distribution of gene expression levels (left, log2(VST+1)) and H3K27ac occupation (right, log2(Counts+1)) in up-regulated genes responding to IFNβ (upper panel) and IL-15 (lower panel) stimulation. Upregulated genes with H3K27ac |fold change| >1 (Epi+/Exp+) are shown in red and those without H3K27ac changes (EpiNA/Exp+) are shown in blue. Significant differences were assessed by Wilcoxon test (*p < 0.05; ***p < 0.01; ****p < 0.001, NS non-significant). (E) Representative H3K27ac tracks illustrating the concordance between epigenomics and gene expression after IFNβ or IL-15 treatment. n = 8 samples per time point and condition. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig 3: Alarmin-induced genes and biological pathways potentially regulated by epigenetic modifications. Heatmap showing the differential expression at all time points of cytokine stimulation of (A) IFNβ and (B) IL-15 upregulated genes (log2 fold change >1, FDR ≤ 0.01) with concordant epigenomic changes (Epi+/Exp+). The number of Epi+/Exp + genes per cluster are indicated. (n = 8 samples per time point and condition). Network-based representation of enriched biological pathways identified by GSEA on Epi+/Exp + genes responding to (C) IFNβ or (D) IL-15 stimulation.

Fig 4: Challenge of SKBR3-based 3D models of the immune microenvironment of HER2-OE BC with anti-HER2 antibodies induced cancer cell apoptosis and downmodulation of CD16 in NK cells. (A) Viability assessment with the fluorescent probes Mitoview (live cells, red) and Nucview (apoptotic cells, green) revealed an increase in apoptosis in SKBR3 aggregates upon 4 days of single (trastuzumab, Tmab) or dual (trastuzumab plus pertuzumab, Tmab + Pmab) anti- epidermal growth factor receptor 2 (HER2) antibody challenge. Co-cultures were supplemented with IL-15. Representative pictures depict one of 11 independent experiments, performed with different immune donors. Scale bars: 150 µm. Full panel for 2 timepoints and all culture conditions in Supplementary Figure 3 . (B) Percentage of NK cells (CD3−CD56+) and CD16+ NK cells (CD3−CD56+CD16+) within the viable immune cell population (CD45+ DAPI−), detected by flow cytometry. NK cells representativeness was not impacted by the 4-day single and dual anti-HER2 antibody challenges but the CD16+ NK subset was partially depleted. Co-cultures were supplemented with IL-15. (C) Percentage of the two major NK cell subsets (CD3−CD56dimCD16+ and CD3−CD56+CD16–) in the viable immune cell population (CD45+DAPI−), detected by flow cytometry. Both NK cell subsets were modulated upon the 4-day single and dual anti-HER2 antibody challenges. Co-cultures were supplemented with IL-15. (B, C) Cell populations were identified and quantified according to gating strategy supplied in Supplementary Figure 2A (Panel 2). Bars represent mean ± S.D. of N = 11 independent experiments, performed with different immune donors. Pairwise statistical comparisons, relative to co-cultures with IL-15 and without antibody challenge, evaluated by a paired t-test: #, p<0.05, ##, p<0.01, ###, p<0.001.

Fig 5: 3D models of the immune microenvironment of HER2-OE BC retained the immune cell compartment, including NK cells, for at least 7 days. (A) Viability assessment with the fluorescent probes Mitoview (live cells, red) and Nucview (apoptotic cells, green) showed an increase in apoptotic cells from day 4 to day 7, scattered throughout the aggregates of HCC1954 (left) and SKBR3 (right) co-cultured with PBMCs, regardless of IL-15 supplementation. Representative pictures depict one of 6 independent experiments, performed with different immune donors. Scale bars: 150 µm. Full panel for 3 timepoints and all culture conditions in Supplementary Figure 1 . (B) Fold change in the percentage of immune cells (CD45+) within all viable cells (DAPI−), from day 4 to day 7 of culture, detected by flow cytometry; there was a sharp decrease in the proportion of the immune compartment, in both HCC1954 (blue) and SKBR3 (yellow), which was partially rescued in SKBR3 co-cultures supplemented with IL-15. (C) Percentage of CD56+ lymphoid cells, including NK cells within the viable immune cell population (CD45+ DAPI−), detected by flow cytometry. IL-15 supplementation had a positive effect in the NK cell population, in co-cultures with HCC1954 and SKBR3 cell lines. (D) Percentage of CD16+ CD56+ lymphoid cells within the viable CD56+ lymphoid cell population (CD56+ CD45+ DAPI−), by flow cytometry. Despite the small reduction of CD16+ cells induced by 4-day cytokine stimulation in both co-cultures with HCC1954 and SKBR3, by day 7 the percentages were similar to non-supplemented conditions. (B–D) Cell populations were identified and quantified according to gating strategy depicted in Supplementary Figure 2A (Panel 1). Day 0 corresponds to the PBMC population immediately before encapsulation. Bars represent mean ± S.D. from N = 6 independent experiments, performed with different immune donors. Pairwise statistical comparisons between indicated groups (*) or relative to day 0 (#), performed with paired t-test; *,# p<0.05; **,##, p<0.01, ###, p<0.001. (E) Immunodetection of epidermal growth factor receptor 2 (HER2, green) and CD45 (red), in co-cultures of HCC1954 or SKBR3 with PBMCs (left and right panels, respectively), without or with IL-15 supplementation (upper and lower row, respectively), by 2-photon emission fluorescence microscopy. At day 4, of CD45+ immune cells had an elongated morphology (arrowheads) and established direct contact with HCC1954 and SKBR3 HER2-OE breast cancer cells (arrows). Each image displays a maximum intensity projection of two consecutive optical slices of 1 µm within an entire capsule. Representative pictures depict one of 3 independent experiments, performed with different immune donors. Scale bars: 150 µm (main), 75 µm (inset).