Figure 5

Gating strategy for identification of respiratory lymphocyte subsets and Lin^negleukocytes. Respiratory B and T lymphocytes were identified according to low side scatter characteristics, CD19 and CD3e surface expression. This gating strategy resulted also in exclusion of autofluorescent respiratory macrophages. Out of the CD3e-positive population CD4+ T helper, CD4⁺CD25⁺ T helper and CD8 T cells were gated according to staining with the respective mAbs and γδ T cells were identified by using a γδ TCR mAb. Please note that the CD4⁺CD25⁺ T cells are not CD25^high. A more detailed gating strategy of Treg subsets including isotpye controls is shown in Figure 8 . Natural killer cells were identified according to CD49b⁺Cd3e^neg expression. Respiratory lineage-negative leukocytes were identified by absence of CD3e, CD8a, CD11b, CD11c, CD19, B220, TER-119, FceR1 (Lineage cocktail) and CD4 expression and subsequently separated into CD90⁺ and CD90^neg cells.