Comprehensive T cell Immunophenotyping

Immunophenotype of T cells was performed using standard staining and flow cytometry techniques as described before^{41 (link),42 (link)}. Briefly, combinations of fluorophore-conjugated anti-human monoclonal antibodies specific for CD3 (BioLegend, OKT3, Cat#317344, 1:500), CD4 (BioLegend, OKT4, Cat#317438, 1:500), CD8 (BioLegend, SK1, Cat#344724, 1:500), CCR7 (BioLegend, G043H7, Cat#353214, 1:500), CD45RO (BioLegend, UCHL1, Cat#304244, 1:500), PD1 (eBioscience, EBIOJ105, Cat#12-2799-42, 1:500), TIM3 (BioLegend, F38-2E2, Cat#345006, 1:500), LAG3 (eBioscience, 3DS223H, Cat#15-2239-42, 1:500) were used to label T cells in flow cytometry staining buffer for 30 min on ice after Fc blocking, followed by intracellular cytokine staining with the BD Fixation/Permeabilization Solution Kit. Data were acquired on LSRFortessa (BD Biosciences) and analyzed with FlowJo software (Treestar). Cytokine antibodies included IFN-γ (eBioscience, B27, Cat#MHCIFG04, 1:500), IL9 (BioLegend, MH9A4, Cat#507614, 1:500), IL2 (BioLegend, MQ1-17H12, Cat#500342, 1:500), GrzB (BD Biosciences, GB11, Cat#561142, 1:500), or annexin V (BD Bioscience, Cat#550475, 1:50).