Il 10

For in vitro stimulation, 2 × 10⁵ human or murine cancer cell lines were plated in a six well plate to adhere overnight. On the subsequent day, cells were stimulated with either human or mouse IL-10, TNFα, or IFN-γ (Tonbo Biosciences) at 50 ng/mL for 48 h and evaluated for expression of MHC-I, MHC-II, PD-L1, and B7x by FACS.

Mouse macrophage cell line J774A.1 was purchased from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; GIBCO, Thermo Fisher Scientific, Waltham, MA, USA). Cells were induced to differentiate into M2Ms by adding 50 ng/mL IL-4, IL-10, and TGF-β (Tonbo Biosciences, San Diego, CA, USA) to DMEM and incubating for 4 days. Cell surface antigens were identified by flow cytometry and protein expression in culture supernatants was measured by ELISA (Supplementary information).