Figure 7.

STAT6 phosphorylation and myeloid effector cell recruitment in the tracheal mucosa are associated with CD4⁺ T-cell clusters. “HDM-primed” polyclonal CD4⁺ T cells, isolated from the bronchial lymph node of CAG-DsRed mice, were adoptively transferred into TCRα^−/− mice on d-7. At d0, recipients received HDM/CT o.ph. and at different time points, perfusion-fixed tracheas were collected for whole-mount confocal analysis. (A-C): Analysis of p-STAT6 distribution at d3. (A) Co-localization of a p-STAT6⁺ cell cluster with a cluster of DsRed⁺ polyclonal effector T cells (block arrows indicate the clusters). (B) Lack of bright p-STAT6⁺ cells in mice without transferred T cells (arrows indicate a few dim p-STAT6⁺ cells). Images shown in A and B were acquired using the same laser intensity and detector settings (bars=50 μm). (C): XY, XZ and YZ representations of a subepithelial effector cluster (slicer positions are indicated by tick marks). The tracheal epithelium is shown between dashed lines. Arrows show bright p-STAT6⁺ cells. DsRed⁺ p-STAT6⁺ cells were excluded from the final images.

(D-F): Mice were challenged on d7 and on d9, perfusion-fixed tracheas were collected for whole-mount confocal analysis of eosinophil and neutrophil accumulation. (D): Localization of Siglec-F⁺ eosinophils in a DsRed⁺ CD4⁺ effector cluster. Boxed region in the low-magnification image (left panels, bar=100 μm) has been enlarged (right panel) and shown as XY, XZ and YZ representations (a range of slices is shown, as indicated by tick marks). (E): Upper panels: low-magnification image of two DsRed⁺ CD4⁺ effector clusters (dashed lines) showing the partial or complete co-localization of CD11b⁺ neutrophils and Siglec-F⁺ eosinophils, respectively (bar=100μm). Lower panels: high-magnification image of a DsRed⁺ CD4⁺ effector cluster showing the positioning of both eosinophils and neutrophils within the clusters (bar=20 μm). (F): Quantitation of eosinophil vs. neutrophil localization within T-cell clusters. Six clusters (n=6) were analyzed in 3 tracheas (**:p < 0.01 as determined using Mann-Whitney test). All images are representative of at least two independent experiments.