Flow cytometry staining

Cryopreserved cells were thawed, washed, and counted. Cells were stained in PBS with Aqua fixable live/dead dye (Invitrogen) for 15 minutes at room temperature and washed.

For surface staining, cells were then stained in PBS with 1% BSA with the following antibodies for 30 minutes at 4°C: anti-CD14-BV510-dump (M5E2), anti-CD25-FITC (M-A251), anti-CD8-BUV395 (RPA-T8), anti-CD4-BV605 (RPA-T4), anti-CD38-PercpCy5.5 (HIT2), anti-CD45RA-BV711 (HI100), anti-PD-1-PE-Cy7 (MIH4), anti-CD3-AF700 (UCHT1), anti-CD127-APC (A019D5) and anti-KLRG1-BV421 (SA231A2) from BioLegend. Cells were washed in cold PBS, passed through a 70μM filter, and data acquired on a BD Fortessa analyzer using FACSDiva software. Data were analyzed using FlowJo 10.4.2. For intracellular staining of transcription factors, cells were processed and stained for viability and indicated surface markers as described above. Cells were washed and incubated with 1x eBioscience Transcription Factor Fixation/Permeabilization Buffer at room temperature for 1 hour. Cells were then washed in 1x eBioscience Permeabilization Buffer twice and incubated with indicated intracellular antibodies including anti-T-bet-BV785 (4B10, BD Biosciences), anti-EOMES-PerCP-ef710 (X4–83, BD Biosciences), anti-granzyme B-AF647 (GB11, Invitrogen), anti-granzyme K-FITC (GM26E7, Invitrogen), anti-perforin-BV421 (dG9, Invitrogen) and anti-Ki-67-BV605 (Ki67, BioLegend) at room temperature for 1 hour. Cells were washed twice in 1x eBioscience Permeabilization Buffer, passed through a 70μM filter, and data acquired on a BD Fortessa analyzer. For intracellular detection of TNF and IFN-γ, sorted cells were stimulated with 1x PMA/ionomycin + Brefeldin A/Monesin (Invitrogen) at 37°C for 2 hours. Cells were stained for viability and indicated surface markers as described above. Cells were washed and incubated with 1x eBioscience Transcription Factor Fixation/Permeabilization Buffer at room temperature for 1 hour. Cells were then washed in 1x eBioscience Permeabilization Buffer twice and incubated with indicated intracellular antibodies including anti-TNF-PE (MAb1) and anti-IFN-γ-APC (4S.B3) from BioLegend at room temperature for 1 hour. Cells were washed twice in 1x eBioscience Permeabilization Buffer, passed through a 70μM filter, and data acquired on a BD Fortessa analyzer.