Figure 8.

Gating strategy for flow cytometric data analysis and calculation of the absolute cell counts of lymphocyte subsets. Lymphocytes were identified based on forward and side scatter (FSC/SSC) properties, and then gated for expression of CD4 or CD8 surface receptors. CD4⁺ T cells were analyzed for expression/co-expression of CD25 and Foxp3. On this basis, Treg (Foxp3⁺CD25⁺CD4⁺) and non-Treg (the remaining CD4⁺ T cells, i.e., non-Foxp3⁺CD25⁺ CD4⁺ T cells) cells were distinguished. Subsequently, IL-4-, IL-10-, IL-17- and TGF-β-producing cells were identified within particular cell subsets. Absolute cell counts of lymphocyte subsets (i.e., number of cells from particular subpopulations per mediastinal lymph node or lung sample) were calculated using the dual platform method, as shown above.