Intracellular Cytokine Profiling of Activated CD4+ T Cells in Tuberculosis

For the detection of intracellularly expressed IFNγ and IL-17A in activated CD4⁺ T cells in lungs of Mtb-infected mice, an intracellular cytokine staining kit (BD Biosciences Heidelberg, Germany) was used according to the manufacturer’s instructions as described [35 (link)]. Briefly, 2 × 10⁶ cells of single cell suspensions were incubated with medium or with plate-bound anti-CD3/CD28 mAbs at 37 °C for 4 hrs in the presence of brefeldin A-containing GolgiPlug^TM (BD Biosciences). Subsequently, cells were incubated with anti-FcγRIII/II mAb (clone 2.4G2), mouse and rat serum, washed and surface markers were stained with anti-CD44-FITC (clone IM7) and anti-CD4-APC (clone RM4-5) (BD Biosciences). After subsequent fixation and permeablization in Cytofix/Cytoperm™ (BD Biosciences), intracellular IFNγ and IL-17A were detected with PE-labelled mAbs (clone XMG1.2 and clone TC11 18 H10, respectively) (BD Biosciences).
For the intracellular staining of FoxP3, the mouse regulatory T cell staining kit (eBioscience) was employed, and cell surfaces were additionally examined by staining with anti-CD4-FITC and anti-CD25-APC (BD Bioscience).
Fluorescence intensity was acquired on a FACSCanto II (BD Biosciences) (Figure S1).