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. 2019 Oct 1;8:e41239. doi: 10.7554/eLife.41239

Figure 3. SPNS2-derived S1P controls interactions of PNAd+ HEVs with lymph-derived dendritic cells in pLNs.

(A) FACS analysis of CCR7-expression on endogenous conventional mDCs (CD3-/CD19-/CD11cint/MHC-IIhi) and rDCs (CD3-/CD19-/CD11chi/MHC-IIint) isolated from pLNs of Spns2f/f and Lyve1;Spns2Δ/Δ mice. (B) Confocal microscopy of pLNs of Spns2f/f (left) and Lyve1;Spns2Δ/Δ (right) mice for CD11c+ (green) DCs, PNAd+ (red) HEVs and Lyve-1+ (blue) LECs. (C) TEM images of HEVs in pLNs of Spns2f/f and Lyve1;Spns2Δ/Δ mice. (D) Flow-cytometric TUNEL assay on CD45-/CD31+/PNAd+ high-endothelial cells isolated from pLNs of Spns2f/f and Lyve1;Spns2Δ/Δ mice. (E) Experimental flow-chart of BMDC-differentiation in vitro, and lymphatic homing assays of footpad injected BMDCs to quantify DC-immigration from afferent lymphatics into pLNs of Spns2f/f and Lyve1;Spns2Δ/Δ mice. (F) Confocal microscopy of pLNs of Spns2f/f (left) and Lyve1;Spns2Δ/Δ (right) mice for CMTMR+ BMDCs (red), PNAd+ (green) HEVs and ERTR7+ (blue) fibroblastic tissue networks. (G) Visualisation of the automated detection of individual CMTMR+ BMDCs (white spheres) from PNAd+ HEVs (green surface) in pLNs of Spns2f/f (left) and Lyve1;Spns2Δ/Δ (right) mice. Grey gradients visualise the distance transformation from HEVs (green surface) defined by PNAd-staining. (H) Total numbers of BMDCs (white spheres in (F)) in distances from 0 μm - 100 μm from HEVs counted in 10 μm radial areas around HEVs in pLNs of Spns2f/f and Lyve1;Spns2Δ/Δ mice. Each circle represents an individual mouse (A, D) or total numbers of BMDCs around HEVs in the visual field of a micrograph (H); bars indicate the mean. Scale bars, 5 μm (C), 50 μm (B, F, G). *p<0.05; **p<0.005; ***p<0.0005 (two-tailed unpaired Student’s t-test (A, D, H)). Data are representative for six mice per group pooled from two (A, B) or three (D) independent experiments with n = 3 (A) or n = 4 per (D) mice group, for 2x pLNs and 2x iLNs of three mice per group (C), for 36x representative individual sections of 2x analyzed popliteal LNs per mouse pooled from two independent experiments (H) with n = 6 mice per group (H).

Figure 3.

Figure 3—figure supplement 1. Endogenous DCs do not co-localize with HEVs in pLNs of Lyve1;Spns2Δ/Δ mice.

Figure 3—figure supplement 1.

(A) FACS analysis of CCR7- and CCR7+ conventional mDCs (CD19-/CD3-/CD11c+/MHC-II+) in blood, spleen, and BM of Spns2f/f and Lyve1;Spns2Δ/Δ mice. (B) Confocal microscopy of pLNs of Spns2f/f (left) and Lyve1;Spns2Δ/Δ (right) mice for CD11c+ (green) DCs, PNAd+ (red) HEVs and Lyve-1+ (blue) LECs. White squares show the regions of interest magnified and shown in Figure 3 (B). (C) qRT-PCR analysis of the expression levels of PNAd scaffolds (GlyCAM-1, CD34, MadCAM-1), of Glycan and LPA synthetic enzymes (GlcNAc6ST-2, FucT-VII, ENPP2), and vascular associated genes (CD31, VCAM-1, ICAM-1, VE-cadherin, LTbR) in total mRNA isolated from CD45-/CD31+/PNAd+ high-endothelial cells sorted from pLNs of Spns2f/f and Lyve1;Spns2Δ/Δ mice. Each circle (A) represents an individual mouse (A) or the relative chemokine expression levels in mRNA extracted from the total CD45-/CD31+/PNAd+ high-endothelial cells (C); bars indicate the mean. Data are pooled from two independent experiments (A) with n = 3 or n = 4 mice per group (A), are representative for 18x individual sections of 2x analyzed pLNs, iLNs and bLNs per mouse pooled from three mice per group in two individual experiments (B), three independent mRNA preparations of 2x pLNs, iLNs and bLNs per mouse pooled from five mice per group (C), or seven (D) individual mice. Scale bars, 200 μm (B). *p<0.05; **p<0.005; ***p<0.0005 (two-tailed unpaired Student’s t-test (A, C)).