Journal: Respiratory Research

Article Title: TMPRSS2 is a tumor suppressor and its downregulation promotes antitumor immunity and immunotherapy response in lung adenocarcinoma

doi: 10.1186/s12931-024-02870-7

Figure Lengend Snippet: In vivo and in vitro experimental validation of the bioinformatics findings. TMPRSS2-knockdown tumors display increased tumor-infiltrating lymphocytes, expression of immune checkpoint molecules, and sensitization to immune checkpoint inhibitors. a TMPRSS2 knockdown markedly promoted proliferative and invasive abilities of A549 cells. b TMPRSS2 knockdown increased tumor volume and progression in Lewis tumor mouse models. Lewis tumor cells transfected with ShCon or ShTMPRSS2 lentivirus were subcutaneously injected into mice. The tumor volumes were measured every three days from the fifth day to the fifteenth. Data represent mean ± SEM. SEM, standard error of mean. ShTMPRSS2 versus ShCon group, n = 6 for each group, two-tailed Student’s t test, * p < 0.05, ** p < 0.01, *** p < 0.001. c TMPRSS2 knockdown increased MSH6 expression in A549 cells, as evidenced by Western blotting. d TMPRSS2 knockdown enhanced the expression of MHC class I genes ( HLA-A , HLA-B , and HLA-C ) in A549 cells, as evidenced by real-time qPCR. e NK cells co-cultured with TMPRSS2 -knockdown A549 cells showing higher proliferation capacity than NK cells co-cultured with TMPRSS2 -wildtype A549 cells, as evidenced by the EDU proliferation assay. f CD8, CD49b, and PD-L1 immunofluorescence staining in Lewis orthotopic tumors and H-score analysis. ShTMPRSS2 versus shCon group, n = 6 for each group, two-tailed Student’s t test, *** p < 0.001. g - j Comparisons of TNF-α, IFN-γ, PD-1, and LAG3 expression on CD8 + T cells from tumor-infiltrating lymphocytes (TILs) in tumor-bearing mice between TMPRSS2 -knockdown and TMPRSS2 -wildtype group (ShTMPRSS2 versus ShCon group, n = 6 for each group, two-tailed Student’s t test, * p < 0.05, ** p < 0.01, *** p < 0.001). TILs were stained with CD3, CD8, TNF-α, and IFN-γ and were then analyzed by flow cytometry. Lymphocytes were gated according to forward scatter and side scatter. CD3 and CD8 staining was used to identify CD8 + T cells. k - m TMPRSS2 -knockdown tumors formed by subcutaneous injection of Lewis cells, as mentioned in ( b ). shCon and shTMPRSS2 tumor-bearing mice were divided into vehicle and BMS-1 groups. The vehicle and BMS-1 groups of mice were treated with solvent and BMS-1, respectively. k Representative images of tumor-bearing mice shown on the left. The right graph showing the change of tumor size in the tumor-bearing mice over time. Data represent mean ± SEM ( n = 6 for each group, two-tailed Student’s t test, * p < 0.05, ** p < 0.01, *** p < 0.001); Comparison of the volume ratios of mice tumors after and before treatment with BMS-1 between TMPRSS2 -knockdown and TMPRSS2 -wildtype groups (two-tailed Student’s t test, *** p < 0.001). Comparisons of TNF-α ( l ) and IFN-γ ( m ) expression on CD8 + T cells from TILs in tumor-bearing mice ( n = 6 for each group, two-tailed Student’s t test, * p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: Anti-PD-L1 (66248-1-Ig) and anti-MSH6 (66172-1-Ig) in supplementary materials were purchased from Proteintech Group, Inc.PE anti-mouse TNF-α antibody (12-7321-81), APC anti-mouse IFN-γ antibody (17-7311-81), APC anti-mouse CD279 (PD-1) antibody (12-9985-81), and APC anti-mouse CD223 (LAG-3) antibody (12-2231-81) were purchased from eBioscience (San Diego, CA).

Techniques: In Vivo, In Vitro, Biomarker Discovery, Knockdown, Expressing, Transfection, Injection, Two Tailed Test, Western Blot, Cell Culture, Proliferation Assay, Immunofluorescence, Staining, Flow Cytometry, Solvent, Comparison

Journal: Science Advances

Article Title: Immune evasion mediated by PD-L1 on glioblastoma-derived extracellular vesicles

doi: 10.1126/sciadv.aar2766

Figure Lengend Snippet: ( A and B ) PD1 blockade prevents the inhibition of PD-L1 high GSC EVs on PBMCs. Percent change in CD69 expression for CD4 + (A) and CD8 + (B) T cells. PD1 blocking antibody (10 μg/ml) or isotype control (10 μg/ml) was added at day 0 ( n = 7 PBMC donors, means ± SD). ( C and D ) PD1 blockade furthermore prevents the inhibition of PD-L1 high GSC EVs on CD3 + isolated cells. CD3 + CD4 + (C) and CD3 + CD8 + (D) cells ( n = 3) after treatment. ( E ) PD-L1–carrying, palmtdT-labeled PD-L1 high GSC EVs can bind to wells coated with recombinant PD1, whereas PD1 antibody blockade inhibits EV binding. Representative confocal images are shown on the left, whereas quantification is provided on the right. Spots per field of view (FOV) on the y axis represent palmtdT-positive dots. Scale bar, 50 μm; ×500 magnification inserts; quadruplicates as means ± SD. One-way ANOVA, with post hoc Bonferroni’s correction, was used to differentiate multiple groups (**** P < 0.0001, *** P < 0.001, ** P < 0.01, and * P < 0.05).

Article Snippet: Human PD1 Fc fusion protein (2 μg/ml; BPS Bioscience) was coated on high protein-binding 96-well plates (#3590, Costar).

Techniques: Inhibition, Expressing, Blocking Assay, Isolation, Labeling, Recombinant, Binding Assay

Journal: Science Advances

Article Title: Immune evasion mediated by PD-L1 on glioblastoma-derived extracellular vesicles

doi: 10.1126/sciadv.aar2766

Figure Lengend Snippet: ( A ) Glioma GSCs up-regulate PD-L1 in vitro in response to activated PBMC supernatants. PBMCs were stimulated with anti-CD3, and supernatants were collected and co-incubated with GSCs (G44, a PD-L1 low GSC) in the presence or absence of anti–IFN-γ. PD-L1 expression was measured by flow cytometry. DMEM, Dulbecco’s modified Eagle’s medium. ( B ) IFN-γ–mediated increase of PD-L1 expression levels in PD-L1 High and PD-L1 low GSCs as shown by Western blots of four different GSCs. ( C and D ) EVs derived from IFN-γ–treated PD-L1 low GSCs inhibit anti-CD3–stimulated T cell activation, and this can be partially reversed by PD1 blockade. Inhibition potential was measured by the percentage change of CD69 + levels on anti-CD3–stimulated CD3 + CD4 + (C) or CD3 + CD8 + (D) cells, isolated from five human volunteers (means ± SD). Representative dot plots for (C) and (D) can be found in fig. S4C. ( E ) PD-L1 low EVs up-regulated indoleamine 2,3-dioxygenase (IDO) mRNA in PBMCs treated with PD-L1 low EVs. Quantitative polymerase chain reaction (qPCR) expression levels are shown ( n = 3). ( F ) PD-L1 low EVs cause interleukin-10 (IL-10) up-regulation in PBMCs. IL-10 cytokine (left) and qPCR expression levels (right) are shown ( n = 3). ( G and H ) Immunosuppressive molecules IDO and IL-10 primarily derive from the CD3-negative population. IDO (G) and IL-10 (H) mRNA levels are shown after CD3 + magnetic-activated cell sorting ( n = 3). Data sets consist of EVs from four different glioblastoma cell lines with means ± SD. One-way ANOVA, with post hoc Bonferroni’s correction, was used to differentiate multiple groups (**** P < 0.0001, *** P < 0.001, ** P < 0.01, and * P < 0.05). Student’s t test was used to differentiate between two groups, and one-way ANOVA with post hoc Bonferroni’s correction was used for multiple groups (**** P < 0.0001, *** P < 0.001, ** P < 0.01, and * P < 0.05).

Article Snippet: Human PD1 Fc fusion protein (2 μg/ml; BPS Bioscience) was coated on high protein-binding 96-well plates (#3590, Costar).

Techniques: In Vitro, Incubation, Expressing, Flow Cytometry, Modification, Western Blot, Derivative Assay, Activation Assay, Inhibition, Isolation, Real-time Polymerase Chain Reaction, FACS

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Sex Differences in Genomic Features of Hepatitis B–Associated Hepatocellular Carcinoma With Distinct Antitumor Immunity

doi: 10.1016/j.jcmgh.2022.10.009

Figure Lengend Snippet: List of Antibodies and Reagents

Article Snippet: Human PD-1 , IHC , Proteintech (Rosemont, IL) , 18106-1-AP , 1:200.

Techniques: Cell Culture