Journal: Cancer Communications
Article Title: CEBPB Expression in Tumor Cells Drives Immune Evasion in Colorectal Cancer via CTLA4 Up-regulation in T Cells
doi: 10.34133/cancomm.0013
Figure Lengend Snippet: Tumor cell C/EBPβ increases CTLA-4 expression in CD4 + T cells. (A) CT26 cells expressing Cebpb -oe and EV control were injected subcutaneously into immunocompetent BALB/c mice ( n = 4; representative of 3 independent experiments, top) and immunodeficient BALB/c nude mice ( n = 5; single experiment, bottom). (B) Subcutaneous tumors from EV and Cebpb -oe CT26 cells injected into BALB/c mice were collected approximately 21 days post-inoculation. The percentages of CD4 + and CD8 + cells among CD45 + cells, CTLA-4 + cells among CD4 + and CD8 + cells, Foxp3 + CTLA-4 + and Foxp3 − CTLA-4 + cells among CD4 + cells, and GzmB + and IFN-γ + cells among CD8 + cells were compared by flow cytometry. (C) Mouse splenic T cells isolated from BALB/c mice were activated with anti-CD3ε and anti-CD28, then co-cultured with EV or Cebpb -oe CT26 cells for 72 h. The culture ratio of T cells to CT26 cells is indicated. Flow cytometry was used to analyze CTLA-4 + and Foxp3 + levels among CD4 + cells, as well as CTLA-4 + , PD-1 + , GzmB + , and IFN-γ levels among CD8 + cells. Representative histograms of CTLA-4 + expression in CD4 + T are presented. (D) mRNA expression of immune checkpoint genes in Jurkat (left) and EL4 cells (right) after co-culture with indicated cells, measured by real-time PCR. (E) Proliferation analysis of CFSE-labeled, activated mouse splenic T cells co-cultured with indicated cells for 72 h. CFSE dilution in CD4 + T and CD8 + T cells were assessed by flow cytometry and presented as histograms (representative of 2 independent experiments). (F) Transwell migration assay of mouse splenic T cells (left) and EL4 (right) cultured for 24 h in CM from Cebpb -oe or EV CT26 cells or in fresh media, in the presence of CXCL12 (200 ng/ml). (G) Tumor growth curves of BALB/c mice bearing Cebpb -oe CT26 tumors treated with isotype control IgG, anti-PD-1, anti-CTLA-4, or the combination ( n = 5 per group). Tumor volumes are shown as mean ± SD; statistical comparisons were made using 2-tailed Student’s t tests based on the final tumor volume. Bar graphs represent the mean ± SD; each dot represents an independent biological replicate. P values were calculated using 2-tailed Student’s t tests. Abbreviations: CEBPB , CCAAT enhancer binding protein beta; Cebpb -oe, Cebpb -overexpression; CFSE, carboxyfluorescein succinimidyl ester; CM, conditioned media; CTLA-4, cytotoxic T-lymphocyte associated protein 4; CXCL12, C-X-C motif chemokine ligand 12; EV, empty vector; Foxp3, forkhead box protein P3; LAG3 , lymphocyte activating 3; PD-1, programmed cell death protein 1; PDCD1 , programmed cell death 1; SD, standard deviation.
Article Snippet: Before co-culturing with cancer cells, isolated T cells (1 × 10 6 ) were pre-incubated with plate-coated anti-mouse CD3ε (1.25 μg/ml; BE0001-1, Bio X Cell) and soluble anti-mouse CD28 (1 μg/ml; BE0015-1, Bio X Cell) for polyclonal T cell activation, and seeded at a density of (2 × 10 5 ) cells on 96-well plates.
Techniques: Expressing, Control, Injection, Flow Cytometry, Isolation, Cell Culture, Co-Culture Assay, Real-time Polymerase Chain Reaction, Labeling, Transwell Migration Assay, Binding Assay, Over Expression, Plasmid Preparation, Standard Deviation
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