Journal: Journal of experimental & clinical cancer research : CR

Article Title: Regnase-1 downregulation promotes pancreatic cancer through myeloid-derived suppressor cell-mediated evasion of anticancer immunity.

doi: 10.1186/s13046-023-02831-w

Figure Lengend Snippet: Fig. 6 Suppression of CTLs by CD11b+ MDSCs is responsible for the acceleration of tumor progression by Regnase-1 downregulation. A-D Evaluation of phenotypes of orthotopic syngeneic tumors of WT or Regnase-1 KO murine pancreatic cancer cells. Representative macro images of pancreatic tumors (A). Relative mRNA levels of Cd8a, Ifng, Fasl, and Gzmb (B) (N = 6 per group). Representative images of HE (C, left panel) and CD8a immunostaining (C, right panel) and the number of CD8-positive cells (C, right) (N = 6 per group). Dot plots of CD3+CD8+ cells evaluated by flow cytometry (D, left) and the proportion of CD8 + cells among CD45+ cells (D, right) (N = 3 per group). E–H Evaluation of phenotypes of orthotopic syngeneic tumors of WT or Regnase-1-KO murine pancreatic cancer cells with or without depletion of CD8+ cells upon anti-CD8a antibody or IgG treatment. Experimental schematic (E). Dot plots of CD3+ and CD8.+ cells in WT or Regnase-1-KO syngeneic tumors upon anti-CD8a antibody or IgG treatment evaluated by flow cytometry (F). Tumor weights (G) (N = 6 per group). The relative mRNA levels of Cd8a, Ifng, Fasl, and Gzmb (H) (N = 6 per group). Student’s t test was used to evaluate differences between 2 groups. One-way ANOVA with Tukey’s post hoc test was used to compare differences among 4 groups. *P < 0.05, scale bars: 100 μm (insets)

Article Snippet: BE0061, a fully neutralizing mAb recognizing CD8a, and control IgG were obtained from Bioxcell.

Techniques: Immunostaining, Flow Cytometry

Journal: International Journal of Biological Sciences

Article Title: BRD7 Inhibited Immune Escape in Nasopharyngeal Carcinoma via Inhibiting PD-L1 Expression

doi: 10.7150/ijbs.103703

Figure Lengend Snippet: BRD7 downregulated PD-L1 expression and enhanced the cytotoxicity of T lymphocytes against NPC cells. (A) CIBERSORT determined the proportion of immune cell populations in NPC. (B) CD8 + T cell infiltration plays a vital role in NPC. (C) Correlation analysis between the expression of BRD7 and immune cells abundance. (D) Correlation analysis between BRD7 expression level and immune cell subtypes in GSE102349 of NPC. (E) Clonogenic assays of 5-8F and CNE2 cells stably transfected with BRD7 overexpression and knockdown or empty vector plasmids with or without T cells co-culture and PD-L1 antibody incubation. Atezolizumab: the PD-L1 antibody. (F) CCK-8 assay of 5-8F and CNE2 cells stably transfected with BRD7 overexpression and knockdown or empty vector plasmids with or without T cells co-culture and PD-L1 antibody incubation. Absorbance values were detected at 450 nm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. (G) Flow cytometry detecting the apoptosis ratio of CD8 + T cells in T cells co-cultured with 5-8F or CNE2 cells stably transfected with BRD7 overexpression or empty vector plasmids with or without PD-L1 antibody incubation. (H) Flow cytometry detecting the apoptosis ratio of CD8 + T cells in T cells co-cultured with 5-8F or CNE2 cells stably transfected with BRD7 knockdown or empty vector plasmids. (I) Flow cytometry detecting the ratio of PD-1 + CD8 + T cells in T cells co-cultured with 5-8F or CNE2 cells stably transfected with BRD7 overexpression or empty vector plasmids with or without PD-L1 antibody incubation. (J) Flow cytometry detecting the ratio of PD-1 + CD8 + T cells in T cells co-cultured with 5-8F or CNE2 cells stably transfected with BRD7 knockdown or empty vector plasmids. (K) ELISA detecting the IFN-γ content in the culture medium of T cells co-cultured with 5-8F cells stably transfected with BRD7 overexpression or empty vector plasmids and CNE2 cells stably transfected with BRD7 knockdown or empty vector plasmids with or without PD-L1 antibody incubation for 24 hours. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

Article Snippet: T lymphocytes and tumor cells were cocultured at a ratio of 10:1 for 24 h. T cells were stained with an Annexin V-FITC/PI Apoptosis Detection Kit (Biosharp, China), APC Anti-Human CD3 Antibody (Elabscience, China), PerCP Anti-Human CD8a Antibody (Elabscience, China), and FITC Anti-Human CD279/PD-1 Antibody (Elabscience, China) according to the instructions.

Techniques: Expressing, Stable Transfection, Transfection, Over Expression, Knockdown, Plasmid Preparation, Co-Culture Assay, Incubation, CCK-8 Assay, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Biological Sciences

Article Title: BRD7 Inhibited Immune Escape in Nasopharyngeal Carcinoma via Inhibiting PD-L1 Expression

doi: 10.7150/ijbs.103703

Figure Lengend Snippet: BRD7 could inhibit the immune escape of NPC. (A) Design of the experiment in vivo . (B) Images of the appearance of subcutaneous tumors in mice. n = 5 per group. (C) Statistical analysis of subcutaneous tumor weight. n = 5 per group. (D) Tumor growth curve. n = 5 per group. (E) Relative BRD7 and PD-L1 mRNA levels measured by q-PCR in tumor tissues. n = 5 per group. (F) Western blot analysis of BRD7, PD-L1, and PI3K/AKT pathway molecules in tumor tissues. (G) Representative images of immunohistochemical staining for BRD7, PD-L1, PI3K/AKT pathway molecules, and CD8 expression in tumor tissues. The scale bar is 20 μm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

Article Snippet: T lymphocytes and tumor cells were cocultured at a ratio of 10:1 for 24 h. T cells were stained with an Annexin V-FITC/PI Apoptosis Detection Kit (Biosharp, China), APC Anti-Human CD3 Antibody (Elabscience, China), PerCP Anti-Human CD8a Antibody (Elabscience, China), and FITC Anti-Human CD279/PD-1 Antibody (Elabscience, China) according to the instructions.

Techniques: In Vivo, Western Blot, Immunohistochemical staining, Staining, Expressing

Journal: iScience

Article Title: TRAIL-dependent apoptosis of peritoneal mesothelial cells by NK cells promotes ovarian cancer invasion

doi: 10.1016/j.isci.2023.108401

Figure Lengend Snippet: Ascites blocks NK cell degranulation but not TRAIL-dependent HPMC apoptosis (A and B) TAL were pre-cultured in RPMI/5% AB media or 100% ascites pool (+/− α-CD3 Ab stimulation) prior to co-culture with HPMC. (A) Apoptosis of HPMC is shown as percentage of annexin V+ cells after gating on CD45 − cells (n = 7 patients). (B) Degranulating NK cells in response to HPMC were measured via flow cytometry and depicted as percentage of CD335+/CD107a+ cells (n = 6 patients). (C and D) TAL were pretreated with α-TRAIL blocking Ab prior to HPMC co-culture. An irrelevant mouse IgG was included as control. Experiments were conducted with TAL cultured in RPMI/5% AB media (n = 11 patients) (C) and 100% ascites pool (n = 5 patients) (D). The amount of Annexin V+ HPMC was determined as described previously. (E) Schematic representation of co-culture experiments applying T cell CM for NK cell activation. CM were collected from CD3 + , CD3+/CD4+, and CD3+/CD8+ T cell subsets cultured in media +/− α-CD3 Ab stimulation. Purified NK cells were then stimulated with T cell CM prior to HPMC co-cultures. (F) The amount of apoptotic HPMC induced by NK cells activated with CM of CD3 + T cells was compared with CM of CD3+/CD4+ and CD3+/CD8+ T cells (n = 4 matched pairs of different patients). (G) Analysis of TRAIL signaling was performed by applying an α-TRAIL blocking Ab to NK cells stimulated with CD3 + T cell CM (α-CD3 Ab activated) prior to the co-culture with HPMC (n = 5 patients). An irrelevant mouse IgG was included as control. The mean is shown by horizontal bars or boxes; vertical error bars represent the standard deviation in A–D, F, and G. ∗ FDR < 0.05; ∗∗ FDR < 0.01; ∗∗∗ FDR < 0.001; determined by paired t test and Benjamini-Hochberg adjustment (ns, not significant).

Article Snippet: To further divide the CD3 + T cell fraction into CD3+/CD4+ T helper cells and CD3+/CD8+ cytotoxic T-cells, the preselected CD14 − lymphocytes were stained with APC-labelled anti-human CD8 antibody (Miltenyi Biotec), incubated with anti-APC microbeads and isolated via MACS.

Techniques: Cell Culture, Co-Culture Assay, Flow Cytometry, Blocking Assay, Control, Activation Assay, Purification, Standard Deviation

Journal: iScience

Article Title: TRAIL-dependent apoptosis of peritoneal mesothelial cells by NK cells promotes ovarian cancer invasion

doi: 10.1016/j.isci.2023.108401

Figure Lengend Snippet:

Article Snippet: To further divide the CD3 + T cell fraction into CD3+/CD4+ T helper cells and CD3+/CD8+ cytotoxic T-cells, the preselected CD14 − lymphocytes were stained with APC-labelled anti-human CD8 antibody (Miltenyi Biotec), incubated with anti-APC microbeads and isolated via MACS.

Techniques: Control, Recombinant, Polymer, Software