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Batched stored samples were thawed quickly at 37°C and washed twice with 1X BD PermWash buffer. Cells were then incubated in 1X BD PermWash for 10 min before incubation with the antibody cocktail mix in 2% FCS at 4°C for 45 min. After incubation, cells were washed twice with PBS (2% FCS) and the resuspended in 0.3 mL PBS for cell acquisition using a Beckton Dickinson LSRII flow cytometer (SORF model). The following monoclonal antibody-fluorochrome conjugates were used: IL-2-R-phycoerythrin (PE), CD8-V500, IFN-γ-Alexa Fluor-700, TNF-α-PE-Cy7, Ki67-Fluorescein isothiocyanate (FITC), all from BD, CD27 PE-Cy5, HLA-DR- Allophycocyanin-Cy7 (APC-Cy7), CD3-BV650 (Biolegend), CD4 PE-Cy5.5 (Invitrogen), CD45RA PE-Texas Red-X (Beckman Coulter). A minimum of 50,000 ViViD negative (viable) CD3 events were collected using FACS DIVA v6 software. Post-acquisition compensation and analysis was performed in FlowJo version 9 (FlowJo, LLC). Supplementary Figure 1 shows the gating strategy employed.

Staining and flow cytometry were conducted as previously described (34 (link)). In brief, batched stored samples were thawed quickly and washed, incubated in BD PermWash, and then stained with the antibody cocktail mix. After incubation, cells were washed and then resuspended in PBS for cell acquisition using a BD LSRII flow cytometer (SORF model). The following monoclonal antibody–fluorochrome conjugates were used: IL-2-R–phycoerythrin (PE), CD8-V500, IFN-γ–Alexa Fluor 700, TNF-α–PE-Cy7, Ki67–fluorescein isothiocyanate (FITC), all from BD; CD27–PE-Cy5, HLA-DR–allophycocyanin-Cy7 (APC-Cy7), CD3-BV650 (BioLegend); CD4–PE-Cy5.5 (Invitrogen); and CD45RA–PE-Texas Red-X (Beckman Coulter). A minimum of 50,000 ViViD-negative (viable) CD3⁺ events were collected using BD FACSDiva v6 software. Postacquisition compensation and analysis was performed in FlowJo version 9 (FlowJo, LLC). Supplemental Figure 2 shows the gating strategy employed. Measurable response to BCG was characterized by the polyfunctionality of the CD3⁺CD4⁺ T cell response assessed by analyzing permutations of TNF-α, IL-2, and IFN-γ expression after stimulation using combinatorial polyfunctionality analysis of antigen-specific T cell subsets (COMPASS) (35 (link)). PFS and FS, which summarize the functional profile of each subject, were calculated from posterior probabilities as described previously (35 (link)).