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Intracellular cytokine staining (ICS) assays were performed as previously described [19 (link)]. In brief, splenocytes and lung lymphocytes were isolated and seeded into 96-well plates, incubated with peptide pools of Ag85A or Mtb32 (2 μg/ml per peptide) or with 40 ng/ml Phorbol 12-myristate-13-acetate (PMA) and 1000 ng/ml ionomycin (Sigma-Aldrich, St. Louis, MO). One hour later, brefeldin A (10 μg/ml, BD Biosciences, San Diego, CA) was added and the PMA+ionomycin-stimulated cells were incubated for additional 5 h, whereas the peptide pool-stimulated cells were incubated for additional 10 h. The cells were harvested and stained with surface antibodies (CD3-PerCP, CD4-FITC, CD8-APC; BD Biosciences, San Diego, CA) for 1 h and were then washed, permeabilized, and stained with intracellular antibodies (IFN-γ-PE; BD Biosciences, San Diego, CA). Finally, the cells were detected with a BD Accuri™ C6 instrument.
To assess Tregs in the spleen and mediastinal lymph nodes (MLN), lymphocytes were isolated and stained with surface antibodies (CD3-Pacific Blue, CD4-FITC, CD25-APC; BD Biosciences, San Diego, CA) for 1 h and then washed, permeabilized, and stained with intracellular antibody (FoxP3-PE; BD Biosciences, San Diego, CA). The cells were detected with a BD Accuri™ C6 instrument.

Human IgD was purchased from Abcam (Cambridge, MA, USA). Anti-CD3 and CD28 antibodies were provided by T&L Biological Technology (Beijing, China). A770041 was purchased from Axon Medchem (Groningen, Netherlands). rhTNFR:Fc fusion protein was purchased from Guojian Pharmaceutical Company (Shanghai, China). Anti-mouse IgD antibodies were purchased from eBioscience (San Diego, CA, USA). Anti-human CD69-PE-cy5, CD154-PE, CD4-FITC, CD25-APC, IL-4-APC, IFN-γ-PE-cy7, IL-17-PE, and FoxP3-PE, along with anti-mouse CD3e-PerCP-Cy^TM5.5, CD4-FITC, CD25-APC, CD154-PE, IFN-γ-PerCP-Cy^TM5.5, IL4-APC, IL-17-PE, and FoxP3-PE antibodies were provided by BD Pharmingen (San Diego, CA, USA). Anti-Lck, anti-phospho-ZAP70, anti-phospho-Lck, anti-ZAP70, and anti-β-actin were purchased from Affinity Biosciences, Sigma and Abcam.