AT delays IL-17 induction in innate γδ T cells and impaired Th17 and Th1 T lymphocyte development.

<p>BALB/c mice (n = 5 on days 1 and3, n = 10 on day 7) were passively immunized (IP) with 2A3 or isotype control mAb R347 (10 mg/kg), followed by ID challenge with WT SF8300 (5×10⁷ cfu) 24 h later. As a positive control mice received <i>Δhla</i> (5×10⁷ cfu). Skin lesions were harvested 1, 3 and 7 days post challenge, digested with liberase and DNAse. Single cell suspensions were stimulated for 4 h at 37°C with leukocyte activation cocktail containing Golgi Plug (BD Biosciences). Following washing and fixation, the cells were labeled for T cell subsets with anti- CD4, CD8, and anti-β and -γ chain specific Abs. After permeabilization, cells were then incubated with anti-IFN-γ and IL-17 Abs. Cells were gated for TCRβ, or TCRβ^−/TCRγ⁺ and then for IL-17 and IFN-γ. Intracellular IL-17 and IFN-γ expression was analyzed by flow cytometry. (A) Representative dot plot for IL-17 and IFN-γ intracellular expression in αβCD4+ and γδT cells from one animal in each group on days 3 and 7. The percentage of cells in each quadrant is indicated. (B) Number of IL-17⁺ γδ T cells, Th1 and Th17 cells as determined by flow cytometry 3 or 7 days post-infection. Cell numbers from each mouse were quantified by flow cytometry after specific staining for TCRδ or CD4 in TCRβ⁺ and intracellular staining for IL-17 and IFN-γ. Data are represented as mean values ± standard deviation. Values were analyzed with a Student’s t-test and considered statistically different if p≤0.05.