Fig 2: TCF1 partners (cooperates) with SMAD3 to impose CD8+ T cell exhaustion.a qRT-PCR analysis of CD8+ T cells from mice in Fig. 7g and the results shown as a heatmap. b Immunoblot (IB) analysis of anti-SMAD3 immunoprecipitates from purified CD8+ T cells pre-incubated with Co.EVs, TßRII+ (RII+) or TßRII- (RII-) (40 µg) for 48 h (left panel); Purified TCF1 and SMAD3 interaction in vitro: Eukaryotic purified TCF1 proteins and prokaryotic purified SMAD3 were incubated and immunoprecipitated with streptavidin beads (middle panel); IB of anti-TCF1 immunoprecipitate derived from purified CD8+ T cells treated with or without TGF-ß (5 ng/ml) as indicated (right panel). c Genome Browser tracks from ENCODE in HepG2 cells representing the binding sites of TCF1, SMAD3, SMAD4 and H3K27ac at the Eomes, Batf or NR4A1 gene locus. d, e ChIP-qPCR assay for TCF1 and SMAD3 at the Eomes or Batf d, Nr4a1 e gene locus in Jurkat cells pre-incubated with Co.EVs, TßRII+ (RII+) or TßRII- (RII-) (40 µg) for 48 h. f qRT-PCR analysis of indicated T cell exhaustion-related genes in Jurkat cells pre-incubated with TßRII+ (RII+) or TßRII- (RII-) (40 µg) for 48 h. g Immunoblot analysis of purified CD8+ T cells pre-treated with TßRII+ (RII+) or TßRII- (RII-) (40 µg). h Schematic that depicts the SMAD3 enhances TCF1-dependent transcription. i T cell-mediated cancer cell killing assay. MDA-MB-231 cells pre-incubated with Co.EVs or TßRII+ (40 µg) were co-cultured for 48 h with or without activated human peripheral blood mononuclear cells (PBMC) in the presence of anti-PD1or anti-PD-L1 antibody at 100 ng/ml. Representative images of crystal violet staining (left) and quantification of cytotoxicity of T cells in different E:T (effector: target) ratio (right). j Experimental analysis in vivo: BALB/c mice were nipple injected with 4T1 cells, followed by tail vein-injection of EVs (TßRII+ or TßRII- EVs, 50 µg per mouse every other day) and/or anti-PD-1 antibody (100 µg per mouse every other day). Treatment protocol is summarized (left). Tumor volume measured in time (right). Data represent mean ± SD. n = 5 mice per group. k Proposed model of the breast cancer EV-TßRII-mediated exhaustion of CD8+ T cells and evasion of anti-tumor immunity. Briefly, aggressive breast cancer tumors secrete TßRII+ EVs to stimulate TGF-ß/SMAD activation in adjacent and remote recipient cells. Up-take of EV-TßRII in the adjacent low-grade tumor cells initiates EMT and increases tumor stemness, drug resistance and metastasis. Meanwhile, EV-TßRII as cargo delivered to CD8+ T cells induces the activation of SMAD3 which cooperates with TCF1 transcription factors to impose exhaustion of CD8+ T cell and the dysregulation of anti-tumor immunity. Therefore, the release of abundant TßRII+ EVs from metastatic breast tumor cells counteract with the anti-tumor immunity systemically. ns, not significant (p > 0.05) and *p < 0.05 (two-tailed Student’s t test d–f, i or two-way ANOVA j). Data are analyzed of three independent experiments and shown as mean + SD i or as means ± SD d–f, j. Source data are provided as a Source Data file.