Mhcii

For TLR and MHC-II blockades, PBMC was pre-treated with 10 μg of either anti-TLR-2 (eBioscience), TLR-4 (eBioscience) or MHC-II (eBioscience) blocking antibodies for 30 min. PBMC were then stimulated with FBCs from 10 HIV + ART-MSM subjects for 4 days and T cell activation was enumerated. For cytokine blockades, PBMC were treated with 10 μg of anti-TNF-α (eBioscience), IFN-γ (eBioscience) or IL-6 (eBioscience) monoclonal antibodies, then stimulated with FBCs and T cell activation was enumerated as above.

The tumor tissues were diced and digested in PBS containing 1 mg/mL collagenase-IV (Worthington Bio, Lakewood, USA), 0.1 mg/mL DNase-I (Roche, Mannheim, Germany) and 2% fetal bovine serum at 37℃ for 1 to 2 hours. Single cell suspension was obtained through 80 mesh-sized cell sieve. To block Fc receptor on immune cells, the cells were incubated with CD16/32 (10 μg/mL, clone 2.4G2, BioXcell) at 4℃ for 10 min before staining. To analyze PD-L1 expression on tumor cells (CD45⁻ for MC-38 and 4T1 tumor cells, and CD19⁺ for A20 tumor cells) and DCs (CD45⁺CD11c⁺MHCII⁺), the cells were incubated with CD45 (1 µg/mL, Cat. 56-0451-82, eBioscience), CD19 (1 µg/mL, Cat. 115512, Biolegend), CD11c (1 µg/mL, Cat. 15-0114-82), MHC-II (1 µg/mL, Cat. 25-5321-82, eBioscience) and PD-L1 (1 µg/mL, Cat. 12-5982-82, eBioscience) antibodies at 4℃ for 30 min. To analyze tumor infiltration numbers of CD8⁺ T cells (CD45⁺CD3e⁺CD8⁺) and CD4⁺T cells (CD45⁺CD3e⁺CD4⁺), the cells were incubated with CD45 (1 µg/mL), CD3e (1 µg/mL, Cat. 25-0031-82, eBioscience), CD4 (1 µg/mL, Cat. 45-0042-82, eBioscience) and CD8a (2.5 µg/mL, Cat. 11-0081-82, eBioscience) antibodies at 4℃ for 30 min. Samples were analyzed via a FACS Calibur (BD Biosciences, USA) or Gallios (Beckman Coulter, USA) flow cytometer.