pd1 j116  (Thermo Fisher)

Journal: Nature Communications

Article Title: Breast cancer cell-derived extracellular vesicles promote CD8 + T cell exhaustion via TGF-β type II receptor signaling

doi: 10.1038/s41467-022-31250-2

Figure Lengend Snippet: a – j Experimental analysis in vivo: BALB/c mice were nipple injectied with 4T1 cells (2 × 10 5 cells per mouse) expressing control shRNA or doxycycline-inducible shRNA targeting TβRII (shTβRII) 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 TβRII + crEVs in plasma from mice b . Immunoblot analysis of TβRII 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 IFNγ + 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 × 10 5 cells per mouse), followed by tail vein-injection of EVs derived from control 4T1 cells (TβRII + ) or TβRII knock-out 4T1 cell (TβRII − ) (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), IFNγ + 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 TβRII + 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-γ and TβRII + 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.

Article Snippet: Anti-human PD1 antibody (clone J116, #BE0188, BioXcell) or anti-human PD-L1 antibody (clone 29E.2A3, #BE0285, BioXcell) were added at a final concentration of 100 ng/ml.

Techniques: In Vivo, Expressing, Control, shRNA, Clinical Proteomics, Western Blot, Isolation, Injection, Derivative Assay, Knock-Out, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Breast cancer cell-derived extracellular vesicles promote CD8 + T cell exhaustion via TGF-β type II receptor signaling

doi: 10.1038/s41467-022-31250-2

Figure Lengend Snippet: a Experimental analysis in vivo: BALB/c mice were nipple injectied with 4T1 cells (2 × 10 5 cells per mouse) expressing control shRNA or doxycycline-inducible shRNA targeting TβRII (shTβRII) and tumors were grown for 3 weeks ( n = 6 mice per group), followed by the administration of doxycycline (Dox) as in Fig. (left panel). Frequency of CD8 + T cells expressing TNFα, IFNγ, 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 TβRII-GFP for 48 h. Scale bar, 5 μm. d – f Experimental analysis in vivo: BALB/c mice were tail vein-injected of control EVs, TβRII-GFP + EVs, or TβRII − 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 + , IFNγ + , IFNγ + &TNFα + , 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 (TβRII + ) or TβRII knock-out (TβRII − ) 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 + , IFNγ + , TNFα + , 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.

Article Snippet: Anti-human PD1 antibody (clone J116, #BE0188, BioXcell) or anti-human PD-L1 antibody (clone 29E.2A3, #BE0285, BioXcell) were added at a final concentration of 100 ng/ml.

Techniques: In Vivo, Expressing, Control, shRNA, Quantitative RT-PCR, Confocal Microscopy, Purification, Derivative Assay, Injection, Transgenic Assay, Knock-Out, Two Tailed Test