Surface antibodies

Surface staining and intracellular cytokine staining (ICS) were described previously [42 (link)]. Briefly, single-cell suspensions from peripheral blood, spleen or liver were stained with LIVE/DEAD Fixable Aqua Dye (Invitrogen) to exclude dead cells, then incubated with surface antibodies (eBiosciences) in combination with tetramers (NIH Tetramer Core Facility, Atlanta, GA). For ICS, cells were first cultured at 37°C for 5 hours in the complete medium (RPMI 1640 containing 10% FBS, 1% HEPES, 1% non-essential amino acid, 2 mM L-glutamine, 50 μM β-mercaptoethanol, 100 U/ mL penicillin and 100 μg/mL streptomycin; Gibco), in the presence of LLO_190-201, GP_33-41 or OVA_257-264 peptides (1 μg/mL), or PMA (50 ng/mL) + Ionomycin (0.5 μg/mL) (Sigma Aldrich). Brefeldin A and monensin were added to the culture for the last 4 hours. Cells were stained for viability and surface antigens as described above, followed by permeabilization (BD Cytofix/Perm) for intracellular staining. Samples were acquired on LSRII flow cytometer (BD Biosciences) and data were analyzed with FlowJo software (Treestar). Details about the reagents and softwares we used are shown in S1 Table.

For murine peripheral blood flow cytometry analysis, peripheral blood was collected by retro-orbital bleeding and incubated with surface antibodies (eBioscience) in staining buffer (phosphate-buffered saline (PBS) supplemented with 0.5% FBS). Peripheral blood cell suspensions were treated with Pharm Lyse Lysing Buffer (BD Biosciences) and washed twice with PBS. For apoptosis assays, cells were incubated with allophycocyanin-conjugated Annexin V (BD Bioscience) for 15 min at RT in 1X Annexin V Binding Buffer (BD Bioscience) following staining with 7-aminoactinomycin (eBioscience). Data were acquired on a FACSCanto I and results were analyzed using FlowJo Version 10 (FlowJo). The list of antibodies used for flow cytometry analysis of mouse and human cells is indexed in Supplementary Data 4.