Fig. 1.

A heterogeneous population of Clec4a4⁺ and Clec4a4⁻ eosinophils resides in the small intestine. (A) Targeting strategy to generate Clec4a4^mCherry/+ mice. CDS, coding sequence; H2B, human histone H2B; T2A, T2A self-cleaving peptide; UTR, untranslated region. (B) Expression of Clec4a4 and mCherry in splenic CD11b⁺ cDCs. (C) Frequency of mCherry⁺ cells among CD45⁺ cells (gated on live cells) in the indicated tissues (n = 3 to 7). DLN, skin-draining lymph node; LP, lamina propria; MLN, mesenteric lymph node. (D) mCherry expression on CD11c^high and CD11c^low CD45⁺ cells derived from siLP (gated on live cells). (E) Expression of CD11b, CD103, Siglec-F, and MHC class II on mCherry⁺ CD11c^high CD45⁺ cells and mCherry⁺ CD11c^low CD45⁺ cells derived from siLP (gated on live cells). (F) mCherry expression in Siglec-F⁺ eosinophils derived from siLP (gated on SSC^high MHC class II⁻ CD11b⁺ cells). (G) Representative pictures of FACS-sorted mCherry⁺ and mCherry⁻ eosinophils from siLP. (H) CCR3 expression in mCherry⁺ and mCherry⁻ eosinophils in siLP. (I) Representative image showing localization of Siglec-F⁺mCherry⁺ eosinophils in siLP villi of Clec4a4^mCherry/+ mice. Inset is an enlargement of I. DAPI, 4′,6-diamidino-2-phenylindole. (J) mCherry expression in eosinophils in the indicated tissues. (K) Parabiosis of CD45.1 WT and CD45.2 Clec4a4^mCherry/+ mice indicates that eosinophils recirculate in siLP, blood, and lung but much less so in BM; Clec4A4 mCherry is only expressed by eosinophils in the siLP. (L) Frequency of parabiont-derived eosinophils in the indicated tissues. (M) Frequency of parabiont-derived mCherry⁺ eosinophils in the indicated tissues. Results are shown as mean ± SEM. P values were calculated using one-way ANOVA and the Tukey's multiple comparisons test or the two-tailed Student’s t test. ns, not significant. ***P < 0.001; ****P < 0.0001.