IL-6 is required for development of Th17 cells during HP. C57BL/6 or IL-6-/- mice (5/group) were intranasally exposed to S. rectivirgula (150 μg) or saline for 3 weeks. Lungs were removed and cells were isolated from individual mice as described in materials and methods. A). Lung cells were stimulated with media alone or PMA and ionomycin for 4 hrs prior to intracellular cytokine staining. Cells were surface stained with antibodies to CD45, βTcR chain and CD4 followed by permeabilization and incubation with anti-IL-17 and IFNγ antibodies and run on a BD LSRII flow cytometer. The data was analyzed using DIVA software and the % of cells expressing IL-17 or IFNγ was obtained by gating on CD45+/βTcR+/CD4+ T cells. B) RT-PCR was performed on RNA isolated from one lung lobe from individual WT or IL-6-/- mice (n = 4/group) exposed to saline or S. rectivirgula using primers specific for IL-17A and IL-17F. The housekeeping gene β-actin was used as an internal control.