Fig 2: Tumor necrosis factor receptor signaling regulates Chst2 expression in PNAd+ TEC. C57BL/6 WT or TNFR1/2-/- mice were I.P. injected with B16-OVA cells and tumors were harvested 14 days after implantation. LN were harvested from non-tumor-bearing mice. (A, B) Endothelial cells were sorted from CD45+ depleted LN and I.P. tumor suspensions, and the expression levels of indicated RNA transcripts were determined by qPCR, as described in Methods. Data from one experiment presented as 2-ΔCT relative to Hprt, n=5 LN or I.P. tumors per group. (A, B) Results are mean ± SD analyzed by unpaired Welch’s t-test. **p<0.01, all other paired bar comparisons are not significant (p>0.05) and have been omitted for clarity.

Fig 3: Balb-ADAM10B-/- LN exhibit WT architecture unlike C57-ADAM10B-/- nodes.Naïve LN sections from C57Bl/6 and Balb/c WT and ADAM10B-/-; (A) B cell (blue, B220), T cell (red, Thy1.2), and HEV (green, pNAD); (B) FDC reticula (red,CR1/2), collagen (green), and cortico-medullary junction (dotted line in inset box); (C) TNF staining (green), B cell (blue, B220) follicle. (D) Average TNF staining intensity representing 12 follicle sections per group. Scale bar, 50μm. **p<0.005.

Fig 4: Biosynthesis of peripheral node addressin in lymph node blood endothelial cells. PNAd refers to the 6-sulfo sialyl Lewis X carbohydrate structure that is displayed on O-linked glycans that decorate multiple mucin-domain containing scaffolding proteins. Generation of 6-sulfo sialyl Lewis X involves a series of post-translational modifications mediated by glycosyl transferases. Polypeptide N-acetylgalactosaminyltransferase 1 (GALNT1) initiates formation of the O-linked glycan by attaching a N-acetyl-galactosamine (GalNAc) residue to serine and/or threonine (35). O-glycans are biantennary structures consisting of Core 1 and Core 2 branches. Glycoprotein-N-acetylgalactosamine 3-beta-galactosyltransferase 1 (C1GALT1, Core 1 synthase) creates the core 1 branch by attaching a galactose (Gal) residue to the O-linked GalNAc residue (36, 37), and this branch is extended by the addition of N-acetylglucosamine (GlcNAc) by beta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3) (37). The Core 2 branch is created by Beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-N-Acetylglucosaminyltransferase 1 (GCNT1, Core 2 synthase), which attaches a GlcNAc residue to the O-linked GalNAc (38). The GlcNAc residues of the Core 1 and Core 2 branches are each modified in 3 additional ways. Galactose/N-acetylglucosamine/N-acetylglucosamine 6-O-sulfotransferase 1 (GLCNAC6ST-1) and 2 (GLCNAC6ST-2) attach sulfate onto the Core 1 and Core 2 GlcNAc residues, although GLCNAC6ST-1 acts only inefficiently to modify Core 1 (39–42). Alpha- (1, 3)-fucosyltransferase 4 (FUT4) (43) and 7 (FUT7) (44) attach fucose to both Core 1 and Core 2 GlcNAc residues. Finally, beta-1,4-galactosyltransferase (B4GALT) attaches Gal residues to GlcNAc in both branches (45), which are then further modified by CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase 1 (ST3GAL1) (46), 4 (ST3GAL4) (47), and 6 (ST3GAL6) (48), which attach N-acetylneuraminic acid (NeuAc) to Gal. This completes formation of the 6-sulfo sialyl Lewis X structure that is recognized by L-selectin expressed on naïve and central memory T- and B-cells. Image created with BioRender.com.

Fig 5: Glycosyl transferases are expressed at significantly lower levels in PNAd+ TEC than in LN PNAd+ BEC. C57BL/6 mice were I.P. injected with B16-OVA cells and tumors were harvested 14 days after implantation. LN were harvested from non-tumor-bearing mice. Endothelial cells were sorted from CD45+ depleted LN and I.P. tumor suspensions, and the expression levels of indicated RNA transcripts were determined by quantitative PCR (qPCR), as described in Methods. Data from one experiment presented as 2-ΔCT relative to Hprt, n=5 LN or I.P. tumors per group. Results are mean ± SD analyzed by unpaired Welch’s t-test. **p<0.01.