Anti mouse ifn γ xmg1

Mice were vaccinated SC with Bl-Eng2 in CFA twice two weeks apart. On the day of challenge, before yeast was given, mice received 100 μg intravenously of cytokine neutralizing antibody or rat IgG. Antibodies were given every other day until the day of harvest. Lungs were harvested at day 7 post infection, homogenized, and plated for CFU analysis on BHI agar plates. Neutralizing antibodies were purchased from BioXcell: anti-mouse IFN-γ (XMG1.2 cat#BP0055) and anti-mouse IL-17A (17F3 cat#BP0173).

The isolation of intestinal lamina proprial cells and flow cytometry were done as previously described(6 (link)). Splenocytes were made into single cell suspension and CD4⁺ T cells were purified with CD4⁺ T cell isolation kit (Miltenyi) with 90–95% purity. 24-well-plates were coated with anti-hamster antibody (MP Biomedical). CD4⁺ T cells were cultured in IMDM medium (Sigma Aldrich) supplied with soluble hamster-anti-mouse CD3 (0.25 µg/ml unless otherwise indicated in the text), hamster-anti-mouse CD28 (1 µg/ml), Gentamicin (50 µg/ml), anti-mouse IL-4 (11B11, 2 µg/ml, BioXCell), and anti-mouse IFN-γ XMG1.2, 2 µg/ml, BioXCell). For iTreg cell differentiation, 5 ng/ml TGF-β was added to the culture. For Th17 cell differentiation, TGF-β was added at 5 ng/ml and IL-6 was added at 20 ng/ml. In some experiments, FICZ was added at a concentration of 200 nM. For blocking RORγt or Stat3 activity, cells were cultured with 10 µM Digoxin (Sigma) or 15 µM STA21 (Santa Cruz), respectively, with controls of DMSO. For IL-21 neutralization, the cells with cultured with 12.5 µg/ml anti-IL21 (R&D Systems) or control IgG.