In vitro T cell proliferation and activation assay

Mouse T cells were isolated from spleens using the Mouse Pan T Kit (Invitrogen), labeled with carboxyfluorescein succinimidyl ester (CFSE) (2 μM; Invitrogen), then were plated in 48-well anti-CD3 (5 μg/ml; BD) coated plates plus soluble anti-CD28 (1 μg/ml; BD) added to the medium. According to protocol described previously [35], BMDMs pretreated with TRAPs were added 3 h after T cell activation at indicated ratios. Cells were cocultured with or without neutralizing monoclonal antibodies against PD-L1, IL-10 or rat IgG isotype control (eBioscience). For transwell assay, TRAP-pretreated macrophages and T cells were seeded in the top chamber and bottom chamber of the transwell insert, respectively. For antigen-specific T cell proliferation, TRAP-pretreated macrophages were cocultured with CFSE labeled OT-I splenocytes in the presence of OVA_257–264 peptide (1 μg/ml; GL Biochem). 72 h later, CD4⁺ and CD8⁺ T cell division was determined by flow cytometry. For human T cell proliferation assay, peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by Ficoll-Paque PLUS (GE Healthcare), and CD14⁺ monocytes and autologous CD3⁺ T cells were purified from PBMCs using the Human monocyte isolation kit (Stemcell) and Human CD3 Kit (Invitrogen), respectively. Subsequently, monocytes were exposed to TRAPs derived from cancer patients for 72 h, and then cocultured with CFSE labeled CD3⁺ T cells at a ratio of 1:3 in 96-well anti-CD3 (5 μg/ml; BD) coated plates plus soluble anti-CD28 (1 μg/ml; BD) for 5 d. For T cell activation assay, TRAP-pretreated monocytes were incubated with autologous T cells at a ratio of 1:3 in anti-CD3 (0.5 μg/ml; BD) coated plates for 20 h, CD25 expression of CD4⁺ and CD8⁺ T cells was examined by flow cytometry. Breferdin A plus monensin was added during the last 8 h of culture, the frequency of IFN-γ⁺ T cells was determined by intracellular staining.