Facsuite 5 software

We cultured PBMCs in 48-well plates and added Phorbol 12-myristate 13-acetate (PMA) (50 ng/ml, TOCRIS, USA) plus ionomycin calcium salt (1 μM/ml, Sigma-Aldrich, USA) for 16 h. We added protein transport inhibitor (BD Bioscience, USA) to the co-culture 12 h before we stopped the stimulation. After coculturing, PBMCs were stained for CD4, CD8, interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), and TIM3 expression. The cytokine and immune checkpoint expressions were measured by flow cytometry and were analyzed in BD FACSuite V software (BD, Biosciences, USA).

The PBMCs were stained for CD4, PD-1 and CTLA-4, and then measured using flow cytometry (FACSVerse, BD Biosciences, San Jose, CA, USA). We discriminated the lymphocyte population using forward scatter and side scatter. Within groups, we gated the subgroups of CD4⁺ T lymphocytes and measured the expressions of PD-1, and CTLA-4. The gating cut-off values were based on isotype staining. We also stained the PBMCs for CD4, CD25, and Foxp3, and defined positive staining for all three as showing Treg cells in the lymphocyte population. We measured CD3-/CD14-/HLA-DR- cells and then gated CD11b⁺/CD33⁺ cells to represent MDSCs using a modified protocol [18 (link)].
The staining antibodies were anti-CD4-APC, anti-CD25-FITC, anti-Foxp3-PerCP antibodies (BD Biosciences, San Jose, CA, USA), anti-PD-1-PE, and anti-CTLA-4-PEcy7.0 (eBiosciences, San Diego, CA, USA). Data were analyzed using BD FACSuite V software (BD Biosciences, San Jose, CA, USA).

The components of the PBMCs (1 × 10⁶ cells), including T cells, B cells, NK cells, and monocytes, were measured using flow cytometry (FACSVerse, BD Biosciences, USA) using anti-CD3-PerCP, anti-CD4-APC, anti-CD25-FITC, anti-CD8-FITC, anti-CD14-PerCP, anti-CD19-APC, and anti-CD56-FITC antibodies (BD Biosciences, CA, USA). The PD-1, PD-L1, PD-L2, and apoptosis markers were also stained with anti-PD-1-PE, anti-PD-L1-FITC, anti-PD-L2-APC antibodies (eBiosciences, USA), Annexin V, and SYTOX orange (eBiosciences, USA). Data were analyzed using BD FACSuite V software (BD, Biosciences, USA). We discriminated lymphocyte and monocyte populations using forward scatter (FSC) and side scatter (SSC). We gated the lymphocyte markers CD3, CD19 and CD56 to identify lymphocytes, and CD14 for monocytes. We then gated CD4 and CD8 in CD3-positive lymphocytes and further gated CD25 in CD4-positive lymphocytes. Early apoptosis was defined as Annexin V (+) but SYTOX orange (−), and overall apoptosis was defined as Annexin V (+) regardless of the SYTOX orange results. The PBMCs were stimulated by mock or MAC (MOI 100) for 48 h and stained with PD-1 and T cell and apoptosis markers.