Intracellular Cytokine Staining of T Cells

Intracellular cytokine staining was performed as previously reported [1 (link),5 (link)]. In brief, immune cells isolated from the lung and spleen were stimulated with a 9-mer Ag85B CD8 epitope peptide (YYQSGLSIV) (Pep8) [1 (link)] and a 15-mer Ag85B CD4 epitope peptide (peptide-25: FQDAYNAAGGHNAVF) [6 (link)] for 6 h. The cells were then incubated with LIVE/DEAD Fixable Dead Cell Stains (Thermo Fisher scientific, Waltham, MA, USA) to identify dead cells, followed by surface staining with the antibodies APC-conjugated anti-CD3 (145-2C11, BioLegend, San Diego, CA, USA), PerCP-Cy5.5-conjugated anti-CD8 ((53-6.7, BioLegend), and PE-Cy7-conjugated anti-CD4 (GK1.5, BioLegend). The cells were then fixed and permeabilized using BD Cytofix/Cytoperm (BD Biosciences, Franklin Lakes, NJ, USA) and stained for IFN-γ (PE) (XMG1.2, BD Biosciences), IL-2 (APC-Cy7) (JES6-5H4, BD Biosciences), and TNF (Alexa Fluor 488) (MP6-XT22, BioLegend). The gating strategy used to identify cytokine-producing CD4⁺ and CD8⁺ T cells is shown in Figure 3C. Polyfunctional cells were defined as those producing two or more cytokines using Boolean combinations Figure 3D. FACS analysis was performed using a FACSVerse flow cytometer (BD Biosciences) with FlowJo software (version 10) (BD Biosciences).