Figure 5. Co-localization of PNAd⁺ HEVs with lymph-derived BMDCs in pLNs is dependent on S1PR1- but not S1PR3-signalling.

(A) Experimental flow-chart for the administration of the non-specific S1PR-antagonist FTY720 i.p. and lymphatic homing assays of footpad injected BMDCs to quantify HEV-DC interactions in pLNs in situ. (B) Confocal microscopy of pLNs of vehicle (left) or FTY720 (right) treated mice for CMTMR⁺ BMDCs (red), PNAd⁺ (green) HEVs and ERTR7⁺ (blue) fibroblastic tissue networks. (C) Visualisation of the distance of individual CMTMR⁺ BMDCs (white spheres) from PNAd⁺ HEVs (green surface) in pLNs of vehicle (left) or FTY720 (right) treated mice. Grey gradients visualise the distance transformation from HEVs (green surface) defined by PNAd-staining. (D) Total numbers of BMDCs (white spheres in (B)) in distances from 0 μm - 100 μm from HEVs (green surface in (B)) counted in 10 μm radial areas around HEVs in pLNs of vehicle or FTY720 treated mice. (E) Experimental flow-chart for the administration of the specific S1PR1-antagonist W146 and the S1PR3-antagonist TY52156, and lymphatic homing assays of BMDCs to quantify HEV-DC interactions in pLNs in situ. (F, G) Total numbers of BMDCs (white spheres as shown in (C)) in distances from 0 μm - 100 μm from HEVs counted in 10 μm radial areas around HEVs in pLNs of treated mice. Each circle represents the total numbers of BMDCs around HEVs in the visual field of a micrograph (D, F, G); bars indicate the mean. Scale bars, 50 μm (B, C). **p<0.005; ***p<0.0005 (two-tailed unpaired Student’s t-test (F, G)). Data are representative for 37x representative individual sections of 2x analyzed popliteal LNs per mouse pooled from two independent experiments (B, C, D) with n = 6 mice per group (B, C, D) and for 34x (F) or 26x (G) representative individual sections of 2x analyzed popliteal LNs per mouse pooled from 5x mice per group (F, G).

Figure 5—figure supplement 1. Co-localization of lymph-derived BMDCs with PNAd⁺ HEVs in pLNs is dependent on S1PR1- but not S1PR3-signaling.

(A) Confocal microscopy of pLNs of vehicle (left) or W146 (right) treated mice for CMTMR⁺ BMDCs (red), PNAd⁺ (green) HEVs and ERTR7⁺ (blue) fibroblastic tissue networks. (B) Visualisation of the automated quantification of the distance of individual CMTMR⁺ BMDCs (white spheres) from PNAd⁺ HEVs (green surface) in pLNs of vehicle (left) or W146 (right) treated mice. Grey gradients visualise the distance transformation from HEVs (green surface) defined by PNAd-staining. (C) Confocal microscopy of pLNs of vehicle (left) or TY52156 (right) treated mice for CMTMR⁺ BMDCs (red), PNAd⁺ (green) HEVs and ERTR7⁺ (blue) fibroblastic tissue networks. (D) Visualisation of the distance of individual CMTMR⁺ BMDCs (white spheres) from PNAd⁺ HEVs (green surface) in pLNs of vehicle (left) or TY52156 (right) treated mice. Grey gradients visualise the distance transformation from HEVs (green surface) defined by PNAd-staining. (E) Fluorescence microscopy of pLNs of Spns2^f/f (left) and Lyve1;Spns2^Δ/Δ (right) mice for isotype (top, green)/pAkt (bottom, green) on PNAd+ (red) HEVs and CD45+ (white) hematopoietic cells. Scale bars, 50 μm (A–D) and 20 μm (E). Data are representative for 2x analyzed popliteal LNs per mouse pooled from 5x mice per group (A–D), or are representative for five mice per group pooled from two independent experiments (E) with n = 2 or n = 3 mice per group (E).