Fig 1: EV-TßRII antagonizes the anti-tumor immunity and thus elevates tumor growth.a–j Experimental analysis in vivo: BALB/c mice were nipple injectied with 4T1 cells (2 × 105 cells per mouse) expressing control shRNA or doxycycline-inducible shRNA targeting TßRII (shTßRII) and tumors were grown for 3 weeks, followed by the administration of doxycycline (Dox) (a left panel). Primary tumor formation in each group at day 21 after implantation is shown (n = 6 mice per group); Normalized BLI signals (a right panel). Percentage of TßRII+ crEVs in plasma from mice b. Immunoblot analysis of TßRII in primary tumor, metastatic tumor and circulating EVs from plasma in mice c. Representative BLI signals of primary tumor d. Normalized BLI signals (left panel) and representative bioluminescent view of isolated lung (right panel) e; Scale bar, 2 mm. FACS analysis and quantification of the percentage of CD8+ or CD4+ cells from lymph nodes f and spleen g; FACS analysis and quantification of IFN?+ h or GZMB+ i of CD8+ cells in tumor-infiltrating lymphocytes (TIL) populations from mice. FACS analysis and quantification of the percentage of tregs j. k Experimental analysis in vivo: BALB/c mice were nipple injected with 4T1 cells (5 × 105 cells per mouse), followed by tail vein-injection of EVs derived from control 4T1 cells (TßRII+) or TßRII knock-out 4T1 cell (TßRII-) (50 µg per mouse every other day) for 3 weeks (left) (n = 5 mice per group). Tumor volume measured in time (right). l Quantification of the percentage of GZMB+ of CD8+ cells (left panel), IFN?+ of CD8+ cells (middle panel) from draining lymph node (DLN) and tumor-infiltrating lymphocyte (TIL) and PD1+, TIM3+ of CD8+ cells from TIL (right panel). m BLI signals of lung metastasis (left panel), number of lung metastasis nodules (middle panel) and percentage of TßRII+ crEVs in plasma (right panel) of all mice in each experimental group at week 5 were shown. n Pearson correlation analysis of the ELISA-detected levels of IFN-? and TßRII+ crEVs in patients with breast cancer (n = 46 samples). ns, not significant (p > 0.05) and *p < 0.05 (unpaired two-tailed Student’s t test b, e, f–j, l, m or two-way ANOVA a, k, n). Data are analyzed from three independent experiments and shown as mean + SD f–j, l or as means ± SD a, b, e, k, m. Source data are provided as a Source Data file.
Fig 2: EV-TßRII mediates the exhaustion of CD8+ T cells by TEVs.a Experimental analysis in vivo: BALB/c mice were nipple injectied with 4T1 cells (2 × 105 cells per mouse) expressing control shRNA or doxycycline-inducible shRNA targeting TßRII (shTßRII) and tumors were grown for 3 weeks (n = 6 mice per group), followed by the administration of doxycycline (Dox) as in Fig. 6a (left panel). Frequency of CD8+ T cells expressing TNFa, IFN?, PD1, TIM3, LAG-3, and Ki-67 in tumor-infiltrating lymphocytes (TIL) populations from mice (right panel). b qRT-PCR analysis of CD8 T cells in TILs from mice and the results shown as a heatmap. c Confocal microscopy (left panel) and FACS analysis (right panel) of purified CD8+ T cells pre-treated with control EVs or EVs (40 µg) derived from MDA-MB-231 cells expressing TßRII-GFP for 48 h. Scale bar, 5 µm. d–f Experimental analysis in vivo: BALB/c mice were tail vein-injected of control EVs, TßRII-GFP+ EVs, or TßRII- EVs for 3 weeks (n = 6 mice per group); then the percentage of GFP positive CD8+ T cells of mice blood were quantified d. The percentage of CD8+ T cells in lym-node or spllen and TIM3+, PD1+, IFN?+, IFN?+&TNFa+, LAG3+ of CD8+ T cells in TIL were analyzed by FACS (n = 6 mice per group) e. The cellular proliferation of CD8+ T cells were analyzed by CFSE dilution f. g Experimental analysis in vivo: OT-I TCR transgenic mice were subcutaneous injected with B16 melanoma cells expressing MHC class I specific epitope of OVA (B16-OVA), followed by tail vein-injection of EVs derived from control (TßRII+) or TßRII knock-out (TßRII-) 4T1 cell (50 µg per mouse every other day) for 3 weeks (n = 5 mice per group) (left); Quantification of the percentage of PD1+, LAG3+, IFN?+, TNFa+, Ki67+, T-bet+, Eomes+ of CD8+ T cells and qPCR analysis of TOX mRNA from tumor-infiltrating lymphocyte (TIL). *p < 0.05 (two-tailed Student’s t-test a, d, e, g). Data are analyzed of three independent experiments and shown as mean + SD a, d, e, g. Source data are provided as a Source Data file.
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