Figure 3

Increase in Th17 cells in T-bet KO mice following S. rectivirgula exposure. C57BL/6 or T-bet-/- 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 overnight with S. rectivirgula or media alone prior to intracellular cytokine staining. Lung cells from WT/saline exposed mice were pooled due to the low number of CD4+ T cells in the lungs of these mice. 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) RNA was isolated from lungs of individual mice, reverse transcribed, and real-time PCR performed using primers specific for IFNγ and IL-17 the results were normalized to the housekeeping gene HPRT and expressed as fold induction over unexposed mice. * p < 0.05; T-bet-/- mice exposed to S. rectivirgula compared to WT mice exposed to S. rectivirgula.