Journal: Cancer Communications

Article Title: High baseline tumor burden‐associated macrophages promote an immunosuppressive microenvironment and reduce the efficacy of immune checkpoint inhibitors through the IGFBP2‐STAT3‐PD‐L1 pathway

doi: 10.1002/cac2.12420

Figure Lengend Snippet: HTB‐associated PD‐L1 + M2‐like macrophages inhibited CD8 + T cells through PD‐1/PD‐L1, (A) Schematic diagram of co‐cultured system of macrophages and T cells. In the first model, we co‐cultured CD8 + T cells with control macrophages (mac‐Ps, from tumor free mice), LTB MC38 subcutaneous tumor‐infiltrating macrophages (mac‐LTB), and HTB MC38 subcutaneous tumor‐infiltrating macrophages (mac‐HTB), respectively. In the second model, the mac‐Ps would first be exposed to the control medium, LTCS, or HTCS for 24 h and then co‐cultured with CD8 + T cells as described for the first model. Anti‐PD‐L1 was added to supernatants of mac‐HTB & CD8 + T cell and HTCS‐exposed mac‐Ps & CD8 + T cell co‐culture systems, (B) Purified peripheral CD8 + T cells were co‐cultured with control macrophages (mac‐Ps) or mac‐LTB or mac‐HTB 2:1 for 5 days with or without anti‐PD‐L1 antibody (5 μg/mL). Ki‐67 + CD8 + T cells were detected by flow cytometry ( n = 3), (C) Purified peripheral CD8 + T cells were co‐cultured with mac‐Ps or mac‐LTB or mac‐HTB from B16 subcutaneous tumor 2:1 for 5 days with or without anti‐PD‐L1 antibody (5 μg/mL). Ki‐67 + CD8 + T cells were detected by flow cytometry ( n = 3), (D) Purified peripheral CD8 + T cells were co‐cultured with mac‐Ps which stimulated with medium (50% complete medium + 50% serum free medium) or MC38 subcutaneous tumor tissue culture supernatants 2:1 for 5 days with or without anti‐PD‐L1 antibody (5 μg/mL). Ki‐67 + CD8 + T cells were detected by flow cytometry ( n = 3), (E) Purified peripheral CD8 + T cells were co‐cultured with mac‐Ps which stimulated with medium or B16 subcutaneous tumor tissue culture supernatants 2:1 for 5 days with or without anti‐PD‐L1 antibody (5 μg/mL). Ki‐67 + CD8 + T cells were detected by flow cytometry ( n = 3). ns, not significant; **, P < 0.01; ***, P < 0.001. Abbreviations: LTB, low tumor burden; HTB, high tumor burden; PD‐1, programmed cell death protein 1; PD‐L1, programmed death‐ligand 1; mac‐P, peritoneal macrophage; mac‐T, tumor‐infiltrating macrophage; mac‐LTB, macrophage from low tumor burden subcutaneous tumor; mac‐HTB, macrophage from high tumor burden subcutaneous tumor; LTCS, low tumor burden subcutaneous tumor tissue culture supernatants (50% complete medium + 50% MC38 or B16 low tumor burden subcutaneous tumor tissue culture supernatants); HTCS, high tumor burden subcutaneous tumor tissue culture supernatants (50% complete medium + 50% MC38 or B16 high tumor burden subcutaneous tumor tissue culture supernatants); rhIL‐2, recombinant human interleukin‐2.

Article Snippet: Purified spleen CD8 + T cells (1 × 10 5 cells/well in 96‐well plates) and supernatant‐stimulated macrophages (0.5 × 10 5 cells/well in 96‐well plates) were co‐cultured in 1640 RPMI supplemented with 10% FBS, recombinant mouse interleukin‐2 (rmIL‐2, 2 ng/mL, P00198, Solarbio), and CD3/CD28 antibody (2 μL per 8 × 10 4 cells, ThermoFisher, Wilmington, Massachusetts, USA) with or without anti‐PD‐L1 antibody (5 μg/mL, BE0101, BioXCell, Beijing, China) for 5 days.

Techniques: Cell Culture, Co-Culture Assay, Purification, Flow Cytometry, Recombinant