(A) Ccl20 mRNA abundance in sorted LN lymphatic endothelial cells (LEC), blood endothelial cells (BEC), fibroblastic reticular cells (FRC) and double negative stromal cells (DN) determined by qRT-PCR, shown relative to Hprt. (B) CCL20 staining of LN section (red). The control (no primary) section was stained with the secondary anti-goat-Cy3 antibody alone. B cells were detected in blue (B220). (C) Transwell migration of IL7RαhiCcr6+ cells to CCL20. (D) Distribution of Cxcr6GFP/+ cells in LNs 3 hr after saline or CCL20 s.c. injection. Sections were stained to detect Cxcr6-GFP (green) and Lyve1 (blue). (E) Representative FACS plots show Ccr6 surface level, in vivo Thy1-PE labeling and CD169 macrophage bleb level on IL7RαhiCcr6+ cells from control (con) or CCL20 injected mice. Summary graph shows comparison of Thy1-PE labeling and CD169+ staining frequency of IL7RαhiCcr6+ and Vγ4+Ccr6+ cells from control or CCL20 injected mice. (F) LN sections from Ccr6GFP/+ or Ccr6GFP/GFP mice stained for EGFP (green) and B220 (blue). White arrow indicates subcapsular sinus area; FO: B cell follicle; T: T zone. (G) Comparison of Thy1-PE labeling on IL7RαhiCcr6+ cells from WT and Ccr6GFP/+ or Ccr6GFP/GFP mice. Ccr6 in Het and KO mice was detected based on GFP reporter expression. (H) Comparison of CD169 staining on IL7RαhiCcr6+ cells from WT, Ccr6GFP/+ or Ccr6GFP/GFP mice. (I) Representative FACS plots showing Vγ4+Scart2+ cells amongst γδT cells and the fraction that are Ccr6+ (upper), and in vivo Scart2-BV605 labeling and CD169 staining (lower). Graph shows summary data. (J) LN sections from Ccr6 Het or KO mice stained for Scart2+ (green) and B220 (blue). White arrow indicates subcapsular sinus area; FO: B cell follicle; T: T zone. (K) Representative histogram plot and summary mean fluorescence intensity (MFI) data of IL17 intracellular staining in IL7RαhiCcr6+ LN cells from Ccr6 Het or KO mice 3 hr after S. aureus bioparticle challenge. *p<0.05, **p<0.01, ***p<0.001, by student’s t test. Data are representative of at least two experiments for panel A–D, F, J. Data are representative of two or more experiments with at least two mice per group for panels E, G–I, K. LN, Lymph node; SCS, Subcapsular sinus.