Fig 2: ATF3 regulates IL-6-pSTAT3 signaling in intestinal Th17 cells. Flow cytometry analysis of phospho-STAT3 in (A) IL-6 or IL-22 stimulated freshly isolated ileum crypts or IL-6-stimulated peripheral blood mononuclear cell (PBMC) from wild-type mice, or in (B) IL-6-stimulated PBMC from wild-type and ATF3-deficient mice. (C) Flow cytometry analysis of intracellular IL-17A and IL-22 expression in naïve lamina propria T cells from the indicated mice. Cells were treated with PMA, ionomycin and IL-23 in the presence of BFA for 4 h before analysis and gated on live CD45+EpCAM-Lin-CD3+ population as shown. (D) Quantitative real-time PCR analysis of IL-17A and IL-22 mRNA levels in freshly isolated lamina propria (LPL) cells, mesenteric lymph nodes (mLN), or splenocytes. (E) Model of ATF3-mediated mucosal immunity via cross-regulation between IL-22-pSTAT3 signaling in epithelium (associated with AMP production and epithelial fucosylation) and IL-6-pSTAT3 signaling in Th17 cells (associated with signature IL-17A and IL-22 production). “n” refers to the number of mice analyzed. Statistical analysis was done by multiple comparison in Two-way ANOVA test using Prism software. *P < 0.05, **P < 0.005, ***P < 0.0005.

Fig 3: ATF3 promotes IL-22-induced STAT3 phosphorylation by suppressing phosphatases. (A) Freshly isolated ileum crypts, or (B) ileum organoids at day 6 of culture, were stimulated with IL-22, followed by fixation and intracellular staining of phospho-STAT3, and analyzed by flow cytometry. Western blot analysis of (C) IL-22-stimulated CMT93 cells, or (D) IL-22-stimuated colon fragments isolated from the indicated mice, for the expression of the indicated proteins. (E) Quantitative real-time PCR analysis of IL-22R1 and IL-10R2 mRNA levels in freshly isolated ileum crypts from mice. (F) Flow cytometry analysis of IL-22R1 in freshly isolated ileum crypt cells gated on the CD45-EpCAM+ population. (G,H) Western blot analysis of unstimulated or IL-22-stimulated CMT93 cells for the indicated proteins. ATF3-/- CMT93 cells with SHP2 knockdown (ATF3-/-SHP2KD) were indicated. Images were representative of four independent experiments (G–H). Results were from two independent experiments (A–F). “n” refers to the number of mice analyzed (A,B,E,F). Statistical analysis was done by multiple comparison in Two-way ANOVA test using Prism software. *P < 0.05, **P < 0.005, ***P < 0.0005.

Fig 4: Microglia supply co-stimulatory signal 2 for T cell activation in the WNV-NS5-E218A-infected CNS. Mice were fed PLX5622 chow or control chow for 2 weeks, then i.c. infected with WNV-NS5-E218A (104 PFU). At 8 dpi, leukocytes were analyzed from the cortex. a Representative flow cytometry contour plots of P2RY12 expression on CD45+-gated cells to identify resident microglia (P1). b Representative flow cytometry plots of CD11b expression on P2RY12negCD45+ cells to identify infiltrating macrophages (P2). c, d Representative flow cytometry plots of MHCII expression on c P1 microglia and d P2 macrophages. e, f Representative flow cytometry plots of CD80 expression on e P1 microglia and f P2 macrophages. g, h Representative flow cytometry dot plots of CD86 expression on g P1 microglia and h P2 macrophages. i, j Quantification of flow cytometry analysis of i percentages and j total numbers of P1- vs P2-gated cells. k Quantification of percentages and l total numbers of MHCII+CD45+ cells within each P1 and P2 population. m Quantification of percentages and n total number of CD80+CD45+ cells within each P1 and P2 population. o Quantification of percentages and p total numbers of CD86+CD45+ cells within each P1 and P2 population. For quantification panels, each symbol represents an individual control- (black) or PLX5622-treated (red) mouse, and bars indicate mean ± SEM. Data presented are the compilation of two independent experiments with seven to nine mice per group. Statistical significance was calculated using multiple t tests with Welch’s correction. For all data: ns, not significant at P < 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig 5: The onco-fetal signature is conserved across humans and mice(A) Experimental design. LMPPs were sorted as Lin−Sca1+ckit+CD45+B220−CD19−Flt3+.(B) Heatmap of oncogenes overrepresented in FL LMPPs.(C) GO of genes more highly expressed in FL LMPPs.(D) GSEA of genes overrepresented in FL LMPPs.(E) GSEA of genes more highly expressed in BM LMPPs.(F) Heatmap of genes conserved across species and maintained in MLL-AF4+ infant ALL blasts.(G) Hoxa9 and Meis1 expression in murine FL Mll-AF4+ LMPPs and control LMPPs. RNA-seq data are shown as mean ± SD; each dot represents a sample. ∗p < 0.05, ∗∗p < 0.01.(H) Heatmap of the 20 genes selected for further analysis as represented in murine FL LMPPs expressing Mll-AF4 (MA4) and control LMPPs (VEC).See also Figure S2 and Table S1.