Figure 3.

Visualization of effector cytokine production by CD4⁺ T cells in the trachea. (A) C57Bl/6 mice were treated with HDM/CT o.ph. and 8 days later, effector cytokine production by CD4⁺ T cells from digested tracheas was analyzed after re-stimulation with PMA/Ionomycin or treatment with Brefeldin A (BFA) only. Numbers indicate percentages. (B) C57Bl/6 mice were treated with HDM/CT o.ph. on d0 and received HDM o.ph. challenge on d7. Tracheas were analyzed on d9. Confocal microscopic analysis of cytokine-producing cells in tracheal frozen sections. Boxed regions were enlarged to show individual cytokine-positive cells (left panels, bars=50μm, right enlarged panels, bars=20μm, asterisk shows the tracheal lumen). Data are representative of at least two independent experiments. (C, D) Analysis of T-cell cytokine production directly after tissue digest (C) or after in vitro re-stimulation (D). 3 tracheas were pooled for each measurement; numbers indicate percentages (isotype control vs. specific anti-cytokine staining was done using the same digest). Plots were gated on extravascular, CD4⁺CD44^hi cells. (E, F) Quantitative representation of T-cell cytokine production measured directly ex vivo 12h after HDM challenge (E) or after in vitro re-stimulation (F). Data were pooled from two independent experiments; each dot indicates a single measurement. n=5 measurements for E and n=4 measurements for F; three tracheas were pooled and used for each measurement (*:p < 0.05; **:p < 0.01 as determined using Mann-Whitney test).