Version 9

Lymphocytes were obtained from the pneumonic lung by bronchoalveolar lavage (BAL) and adherent cells were removed by incubating on plastic for 1 h at 37 °C. Single-cell preparations of spleen were enriched for CD8 T cells by panning on tissue culture plates coated previously with AffiniPure goat anti-mouse IgG + IgM (H + L) (Jackson ImmunoResearch Labs). CD8⁺ T-cell responses to each candidate epitope were tested via intracellular cytokine staining. Enriched lymphocytes from spleens or bronchoalveolar lavage of mice infected 10 days previously with IAV were incubated with 10 U/ml IL-2 (Roche) and 1 μg/ml Golgi-plug (BD Biosciences) in the presence or absence of 1 μM synthetic peptide in 96-well plates and cultured for 5 h at 37 °C and 5% CO₂. Cells were then stained for surface expression of CD8α and intracellular IFN-γ and TNF. Data was acquired by flow cytometry (FACSCanto, BD Biosciences) and analysis was performed using Flowjo version 9.6 (FlowJo LLC, Ashland). Single viable live CD8⁺ lymphocytes were gated for analysis (Supplementary Fig. 7). A response was deemed positive if the test response (with background subtracted) was equal to or greater than 2× the average background values.

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)).

Blood was collected from the retro-orbital sinus, facial vein, or tail vein of anesthetized mice to assess engraftment of human cells. Blood was lysed with the BD FACS Lyse Wash Assistant (BD Biosciences, San Jose, CA). Cells were stained with antibodies to human CD45 (BD Biosciences, # 561864, APC), mouse CD45 (BD Biosciences, # 559864, PE). Samples were assayed on the BD LSR II or LSR Fortessa (BD Biosciences) and data were analyzed with FlowJo version 9 (FlowJo, LLC, Ashland, OR). Samples were sorted for hCD45 and YFP on the BD Aria (BD Biosciences).

Expression levels of ABCG2 were assessed by flow cytometry as previously described^{91 (link)}, using PE-conjugated anti-ABCG2 antibody (R&D Systems, Minneapolis, MN). Results were analyzed relative to negative isotype control, PE-conjugated IgG2b (DakoCytomation, Denmark A/S) with an LSRFortessa™ X-20 and FlowJo version 9 (FlowJo LLC, Ashland, OR).