Anti cd3 cd28 mabs

CD4⁺ T cells was isolated from mesenteric lymph node (mLN) from wild type mice using mouse CD4 T lymphocyte enrichment Set (BD Biosciences, CA, United States). CD4⁺ T cells were labeled with CellTrace^TM Violet (CTV) Cell Proliferation Kit (Thermo Fisher Scientific, Rockford, IL, United States). CD4⁺ T cells (2 × 10⁵ cells/well) were co-cultured with Duolac-treated BMDCs (2 × 10⁴ cells/well) incubated on anti-CD3/CD28 mAbs (BD Biosciences)-coated 96-well plate. After the incubation for 72 h, the cells were examined for proliferation of Foxp3⁺CD4⁺ T cells and the supernatant was collected and examined for IL-10 and IFN-gamma concentration.

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).