4.2.2. Cell Staining

After stimulations, cells were washed, stained with phycoerythrin (PE)-conjugated chemokine receptor antibody CCR7 (BD Biosciences, San Jose, CA, USA), and subsequently surface stained with the following antibodies: Brilliant violet (BV) 650-conjugated CD3 (BD Biosciences, San Jose, CA, USA); CD4-BV785 (BD); CD45RA-BV570 (BioLegend, San Diego, CA, USA); CD27–BV510 (BD Biosciences, San Jose, CA, USA); Fluorescein Isothiocynate (FITC)-conjugated HLA-DR (BioLegend, San Diego, CA, USA); Phycoerythrin (PECy5)-conjugated CD38 (BD Biosciences, San Jose, CA, USA); Peridinin-chlorophyll-protein complex (PerCP\_eFluor710)-conjugated Killer Lectin-like receptor G (KLRG1, Invitrogen, Carlsbad, CA, USA); and LIVE-DEAD-Far Infrared (IR, Invitrogen, Shenzhen, China). The antibody panel including clone and catalog number in accordance with the MIFlowCyt guidelines [32] is shown in Supplementary Table S1. Cells were then fixed and permeabilized using Cytofix/Cytoperm bu ffer (BD Biosciences, San Jose, CA, USA) and stained with the antibodies Allophycocyanin (APC)-conjugated Interleukin (IL)-2 (BD Biosciences, San Jose, CA, USA); interferon gamma (IFN-γ)-AlexaFluor700 (BD Biosciences, San Jose, CA, USA); tumor necrosis factor (TNF)- α -PeCy7 (BioLegend, San Diego, CA, USA); and Ki67-BV421 (BD Biosciences, San Jose, CA, USA). Cells were fixed in 1% paraformaldehyde and acquired on a BD LSRII flow cytometer within one hour of fixation at the South African Tuberculosis Vaccine Initiative (SATVI). FACS data were analyzed in FlowJo (BD Biosciences, San Jose, CA, USA) and Boolean combination gating was used to calculate frequencies corresponding to eight and sixteen di fferent combinations of cytokines and memory markers, respectively. The gating strategy is shown in Supplementary Figure S1.
