Extended Data Fig. 9: IFN-STAT1 signaling in CTLs and TH1 cells upregulates RGS1 and inhibits T cell trafficking.
a, Representative immunofluorescent staining for IFNG expression in cancer cells (CK+), macrophages (CD68+), DC (CD11c+), T cells (CD3+) and natural killer cells (CD56+) in breast tumor tissues with low or high IFNG production (IFNGlow: n = 7, IFNGhigh: n = 5). Asterisk indicates the location of higher magnification images at right. Scale bar, 50 μm. b, Representative plots of flow cytometry analysis of STAT1 phosphorylation (p-STAT1) in the whole tumors (left: grey: isotype, red: p-STAT1) and gated p-STAT1+ cells for different markers staining. Numbers indicate the percentages of different cell types in p-STAT1+ cells (mean±s.e.m.; IFNGlow: n = 7, IFNGhigh: n = 5). c,d, 4T1 cells transduced with sgStat1 (Stat1KO) or a control sgRNA (WT) were inoculated into the fat pad of BALB/c mice, respectively. c, Tumors were collected for protein extraction and the phosphorylation and total levels of Stat1 were determined by Western blotting. Three independent experiments were performed and yielded similar results. α-Tubulin, loading control. d, Tumors were collected 2 days after T cell transfusion and analyzed by flow cytometry. Representative plots of the infiltration of macrophages (F4/80+CD11b+) and DCs (CD11c+) and the p-STAT1 levels in these cells. Numbers indicate the percentages of macrophages and DCs in the tumors and the percentages of p-Stat1+ cells in gated cells (mean ± s.d.; n = 3 per group). *P = 0.0232, **P = 0.0026, ***P = 0.001, ****P < 0.0001 compared with WT grafts by two-tailed Student’s t-test. e, Representative histograms of Rgs1 expression in CD8+ T cells isolated from the spleens of mice bearing 4T1 tumor grafts and transduced with shRgs1, sgStat1 or control lentivirus (shGFP+ctrl-sgRNA), determined by flow cytometry. Numbers in the plots indicate the MFI of Rgs1 (mean±s.d. ×103, n = 3, ****P < 0.0001 compared with ctrl-CTLs by two-tailed one-way ANOVA with Dunnett’s multiple comparison test). f, The scheme of the mechanism underlying RGS1 expression and contribution to the trafficking of CTLs and TH1 cells.
