Analyzing T Cell Cytokine Profiles

Lung cells were harvested at day 4 post-infection. Cells (0.5 x 10⁶ cells/ml) were stimulated for 4 to 6 hours with anti-CD3 (clone 145-2C11; 0.1μg/ml) and anti-CD28 (clone 37.51; 1μg/ml) in the presence of Golgi-Stop (BD Biosciences). Stimulation with fungal ligands yielded comparable cytokine production by transgenic T-cells compared to CD3/CD28 stimulation as shown previously [42 (link),60 (link)]. After cells were washed and stained for surface CD4 and CD8 using anti-CD4 PerCP, anti-CD8 PeCy7, and anti-CD44-FITC mAbs (Pharmingen), they were fixed and permeabilized in Cytofix/Cytoperm at 4°C overnight. Permeabilized cells were stained with anti-IL-17A PE and anti-IFN-γ Alexa 700 (clone XMG1.2) conjugated mAbs (Pharmingen) in FACS buffer for 30 min at 4°C, washed, and analyzed by FACS. Cells were gated on CD4 and cytokine expression in each gate analyzed. The number of cytokine positive CD4⁺ T cells per organ was calculated by multiplying the percent of cytokine-producing cells by the number of CD4⁺ cells. Intracellular Caspase 3 Alexa647 (9602S, Cell Signaling), Caspase 8 PE (12602S Cell Signaling), Bcl2 Alexa 647 (633510 Biolegend) and Bcl-xL PE (13835 Cell Signaling) were stained from ex vivo derived lymph node cells and splenocytes.