Figure 3.

Polyclonal stimulation in vitro suggests local Th17 cell bias in MEA and indicates a systemic IL−17 increase in PBMC from MEA and SEA. BALC and PBMC from HE (BALC, n=9; PBMC n=7) or horses with MEA (n=3) or SEA (BALC n=5; PBMC n=8) were incubated in vitro for 24 h, with PMA and ionomycin added for the last 6 h (P/i) in comparison with medium alone, and analysed by flow cytometry. Representative examples of BALC (MEA) are shown. (A) Singlet-live-lymphocytes were gated and cytokine expression evaluated in quadrant gates, as illustrated for IL-17. P/i-stimulated expression was medium subtracted (net percentage) and analysed (e.g., CD4^-IL-17⁺ + CD4⁺IL-17⁺) in BALC or PBMC lymphocytes (D, G). (B) CD4⁺cytokine⁺ cell subsets were quantified in relation to all CD4⁺ lymphocytes [e.g., CD4⁺IL-17⁺/(CD4⁺IL17⁺ + CD4⁺IL-17^-)] and medium subtracted [(E) BALC; (H) PBMC]. (C) CD8⁺cytokine⁺ lymphocyte subsets were analysed in the same manner as CD4⁺ and compared between the groups [(F) BALC; (I) PBMC]. (D–I) Bars represent group medians. Statistical comparisons in Mann-Whitney-tests are indicated by lines with p-values if p≦0.1. Group comparisons indicated increased CD4⁺IL-17⁺ net percentages in BALC from MEA compared with HE (E). Net percentages of IL-17⁺ lymphocytes were increased in MEA or SEA PBMC compared with HE (G).