Pd 1 pe dazzle

Isolated CD4 T cells or colon-derived mononuclear cells were stained with LIVE/DEAD Fixable Blue Dead Cell Stain Kit (Thermofisher) to exclude nonviable cells and surface phenotyped by staining with fluorochrome-conjugated antibodies at 4°C for 30 min. For intracellular staining, cells were subsequently fixed and permeabilized using Cytofix/Cytoperm solution (BD Biosciences). Flow cytometric data was obtained on a LSRFortessa (Becton Dickinson) and analyzed with FlowJo software (TreeStar) and when applicable, fluorescence minus one (FMO) gating strategies were used to set the manual gates. The following antibodies were used: CD3-BV510 (BD Biosciences, #563109), CD4-PE-Cy7 (Biolegend, #344612), CD45-BV421 (BD Bioscience, #563879), CD69-FITC (BD Bioscience, #555530), CD38-AF700 (BD Bioscience, #560676), TIGIT-PE (eBioscience, #12–9500-41), PD-1-PE/Dazzle (Biolegend, #329939), PD-L1-APC (eBioscience, #17–5983-41), and Ki-67-PE (Biolegend, #350503).

Splenocytes were processed and T cells were stained for CD8-BV786 (Biolegend, clone 53–6.7), CXCR3-BV421 (Biolegend, clone173), CD27-BV605 (BD Biosciences, clone LG.3A10), PD-1-PE-Dazzle (Biolegend, clone 29F.1A12), TIM-3-PerCP-Cy5.5 (Biolegend, clone RMT3–23), TIGIT-PE (BD Biosciences, clone 1G9), or –BV421 (Biolegend, clone 1G9), 2B4-APC (eBioscience, clone 244F4), as well as APC conjugated H-2K^b/p79 (TSINFVKI) tetramers (NIH Tetramer Core, Atlanta, GA). The FITC channel was left open in order to measure YFP fluorescence of virally infected cells as previously reported [23 (link)]. B cells were stained for CD19-PE-Cy7 (Biolegend, clone 6D5), GL7-Pacific Blue (Biolegend, clone GL7), and CD95-APC (Biolegend, clone SA367H8). Absolute numbers were calculated using TruCount bead analysis according to the manufacturer’s instructions (BD Biosciences). Samples were analyzed using a multicolor LSRII (BD). Data were analyzed using FlowJo software (Treestar, San Carlos, CA) and by ViSNE and CITRUS analysis (Cytobank, Inc.) as described in Supplemental Information.