Anti cd3 af488

All antibody staining was preceded by 15 min of 1:50 FcγR block in FC buffer, on ice. Extracellular antibodies were then added to FC buffer containing FcγR block, and incubated for 45 min on ice. Intracellular staining was accomplished after surface staining using the Foxp3 staining kit (eBioscience). Myeloid subsets were stained with anti-CD1lb-FITC, anti-Ly6C-AF700, anti-MR-PE-Cy7, anti-CD11c-PE/Dazzle594, anti-Ly6G-BV605 (BioLegend), anti-MHCII-eFluor450 (eBioscience), and anti-F4/80-APC (BioRad). To evaluate Tregs, cells were stained with anti-CD3-AF488, anti-CD4-APC, anti-CD25-PerCP-Cy5.5 (BioLegend), and anti-Foxp3-PE (BD Pharmingen). To assess T cell activation, total tumor cells were incubated for 4 hr at 37°C in a 5% CO2 incubator with Protein Transport Inhibitor Cocktail containing brefeldin A and monensin, or with Cell Stimulation Cocktail containing protein transport inhibitors and PMA/ionomycin (eBioscience). Cells were then stained with anti-CD3-AF488, anti-CD4-PE, and anti-CD8-PerCP-Cy5.5 followed by intracellular stain with anti-IFNγ-APC (BioLegend). In addition, 1E5 CD3⁺ cells were stimulated with 4E4 CD3/CD28 Dynabeads (ThermoFisher) for 3 days, followed by staining using anti-CD3-PerCP-Cy5.5, anti-CD8-BV650, anti-CD4-BV605, anti-IFNγ-APC, anti-TNFα-BV421 (BioLegend), and anti-GranzymeB-PE (eBioscience).

T-cell subsets were enumerated in freshly thawed cryopreserved PBMC. After washing and counting viable cells, PBMCs were surface-stained with the following conjugated mAbs: anti-CD3-AF488 (Biolegend; clone HIT3a), anti CD4-APC/Cy7 (Biolegend; RPA-T4), anti-CD25-PE/Cy7 (Biolegend; BC96), anti-HLA-DR-PerCP/Cy5.5 (Biolegend; L243), anti-CD39-APC (Biolegend; A1), and anti-CD38-PECy7 (Biolegend; HIT2). Cells were fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences), and stained with anti-IL10-APC (R & D Systems; 127107), anti-FOXP3-PE (Biolegend; 206D), anti-TGFβ-PerCP/Cy5.5 (Biolegend; TW4-2F8) and anti-IL35-PE (eBioscience; ebic6) and analyzed with Guava easyCyte 8HT and FlowJo (Treestar).
Subsets were expressed as percentages of the parent CD4+ and CD8+ T-cell populations. The gating strategy is presented in Fig S1.