![TLOs are present in the terminal ileum of S1pr1iΔLECmice, and they are connected to the lymphatic vessels. (A) Mesenteric tissue along the ileum was harvested and analyzed using the lymphatic vessel marker VEGFR3. Stitched images revealed a large number of nodules in S1pr1iΔLEC (TM@P1–7) mice, but not in control littermates. The nodules were observed both in pre-collecting vessels (arrowheads) and in collecting lymphatic vessels (arrows). (B) The number and size of LYVE1+B220+ nodules were measured and quantified. (C) Mesenteries of 8–12-mo-old control mice and S1pr1iΔLEC littermates that were treated with TM from P1–7 were analyzed using markers for the immune cells, stromal cells, and endothelial cells. Some S1pr1iΔLEC mice had R26+/tdTomato reporter, the expression of which was permanently induced in the lymphatic vessels by TM-activated CreERT2. A few other mice had the Prox1-tdTomato reporter. Lymphatic vessels were labelled by VEGFR3, PROX1, LYVE1, and tdTomato. LYVE1 was also expressed in a subset of macrophages. B220, CD3e, CD11b, and S100A9 are markers of B cells, T cells, myeloid lineage cells, and neutrophils, respectively. CCL21 is a marker for lymphatic vessels and the FRCs within TLOs. PECAM1 labels all endothelial cells, but its expression is stronger in blood endothelial cells when compared with LECs. ICAM1 is expressed in HEVs, inflamed blood endothelial cells, and a variety of immune cells. MAdCAM1 is a marker of HEVs. (D) TLOs in the terminal ileum of 1-year-old S1pr1f/f and S1pr1iΔLEC (TM@8W) mice were counted and plotted. (E) Mesenteric lymphatic vessels of S1pr1f/f mice had clear claudin-5+ LVs (arrows) and weak LYVE1 expression. LVs were also observed in S1pr1iΔLEC (TM@8W) mice (arrows), although those that were close to the TLOs appeared defective. The TLOs had GL7+ germinal center B cells, CD11c+ dendritic cells, and F4/80+ macrophages. Statistics: (A) n = 3 S1pr1f/f and n = 3 S1pr1iΔLEC (TM@P1–7) mice; (B) n = 6 S1pr1f/f and n = 8 S1pr1iΔLEC mice. The size of the nodules was quantified, and each dot represents a nodule on the graph; (C and E) representative images from three to five mice/genotype/marker; (D) n = 6 S1pr1f/f and n = 7 S1pr1iΔLEC (TM@8W) mice. Graphs were plotted as mean ± SD. Welch’s t test (B [number of TLOs] and D) and Mann–Whitney test (B [TLO size]) were performed for the statistical analysis. ***P < 0.001; ****P < 0.0001.](https://cdn.rupress.org/rup/content_public/journal/jem/222/9/10.1084_jem.20241799/2/jem_20241799_fig5.png?Expires=1770197322&Signature=GJwZakxuyyAqlcYhlqV-o4ZO8eG~Am7QPRabzD8Gok~l98WQWVdlyli3A6jxJnOYX0~nLjyCSLuN1qs23Hf7vb-fOBZLxvW72-YgbSTt-Tmgw5QcbgaC85O7ISunkuRwRUgt6epgzlvImNnIX91XoaPlgIiBdRk-sCejK1nCvAILdeZSorkVlZzMHOOVI4ZXmB9CiDF---TyunRqw~6vb-MY8UtXuStpSIBnwJUj8LFHOMuUCRgt0sFfuMNaPEGXv2EsarFRiEC3nVLZJdZsFDT00ZdXvfiNxn-tm8GcJViIeJU9OpDRp2wafs-kVh6crVGkko04bsW1oOglWKV6sQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
TLOs are present in the terminal ileum of S1pr1 iΔLEC mice, and they are connected to the lymphatic vessels. (A) Mesenteric tissue along the ileum was harvested and analyzed using the lymphatic vessel marker VEGFR3. Stitched images revealed a large number of nodules in S1pr1iΔLEC (TM@P1–7) mice, but not in control littermates. The nodules were observed both in pre-collecting vessels (arrowheads) and in collecting lymphatic vessels (arrows). (B) The number and size of LYVE1+B220+ nodules were measured and quantified. (C) Mesenteries of 8–12-mo-old control mice and S1pr1iΔLEC littermates that were treated with TM from P1–7 were analyzed using markers for the immune cells, stromal cells, and endothelial cells. Some S1pr1iΔLEC mice had R26+/tdTomato reporter, the expression of which was permanently induced in the lymphatic vessels by TM-activated CreERT2. A few other mice had the Prox1-tdTomato reporter. Lymphatic vessels were labelled by VEGFR3, PROX1, LYVE1, and tdTomato. LYVE1 was also expressed in a subset of macrophages. B220, CD3e, CD11b, and S100A9 are markers of B cells, T cells, myeloid lineage cells, and neutrophils, respectively. CCL21 is a marker for lymphatic vessels and the FRCs within TLOs. PECAM1 labels all endothelial cells, but its expression is stronger in blood endothelial cells when compared with LECs. ICAM1 is expressed in HEVs, inflamed blood endothelial cells, and a variety of immune cells. MAdCAM1 is a marker of HEVs. (D) TLOs in the terminal ileum of 1-year-old S1pr1f/f and S1pr1iΔLEC (TM@8W) mice were counted and plotted. (E) Mesenteric lymphatic vessels of S1pr1f/f mice had clear claudin-5+ LVs (arrows) and weak LYVE1 expression. LVs were also observed in S1pr1iΔLEC (TM@8W) mice (arrows), although those that were close to the TLOs appeared defective. The TLOs had GL7+ germinal center B cells, CD11c+ dendritic cells, and F4/80+ macrophages. Statistics: (A) n = 3 S1pr1f/f and n = 3 S1pr1iΔLEC (TM@P1–7) mice; (B) n = 6 S1pr1f/f and n = 8 S1pr1iΔLEC mice. The size of the nodules was quantified, and each dot represents a nodule on the graph; (C and E) representative images from three to five mice/genotype/marker; (D) n = 6 S1pr1f/f and n = 7 S1pr1iΔLEC (TM@8W) mice. Graphs were plotted as mean ± SD. Welch’s t test (B [number of TLOs] and D) and Mann–Whitney test (B [TLO size]) were performed for the statistical analysis. ***P < 0.001; ****P < 0.0001.
