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Journal Articles

Amyloidosis of bridging veins is a pathologic feature of Alzheimer’s disease

Leon C.D. Smyth, Daan Verhaege, Elio Standen-Bloom, Yue Wu, Yiming Gan, Steffen E. Storck, Pavle Boskovic, Benjamin A. Plog, Tornike Mamuladze, Jose A. Mazzitelli, Zhuoying Wang, Daniel D. Lee, Gwendalyn J. Randolph, Antoine Drieu, Katherine E. Schwetye, Song Hu, Daniel S. Reich, Douglas H. Kelley, Rupal I. Mehta, Jonathan Kipnis
Journal: Journal of Experimental Medicine
J Exp Med (2025) 223 (2): e20251860.
https://doi.org/10.1084/jem.20251860
Published: 02 December 2025
View article titled, Amyloidosis of bridging veins is a pathologic feature of Alzheimer’s disease
Open the PDF for Amyloidosis of bridging veins is a pathologic feature of Alzheimer’s disease in another window
Includes: Supplementary data
Images
Progressive amyloidosis of bridging veins in 5XFAD mice. (A) Representative images of duras from 5-mo-old 5XFAD mice and littermate controls stained for the vascular marker PECAM-1, ACE point marker DPP4, and pan-Aβ antibody D54D2. Scale = 2 mm, inset = 200 μm. (B) Quantification of the area of Aβ within ACE points and the rest of the dura in 5XFAD mice. Two-tailed paired t test, N = 10. ***P < 0.001. (C) Schematic for the quantification of Aβ intensity, relative to ACE points. Line profiles of DPP4 and Aβ intensity, relative to the terminal of the ACE point (0 μm). N = 44 bridging veins from N = 7 5XFAD animals, mean ± SEM. (D) Representative image of a 5-mo-old 5XFAD mouse, stained for the vascular marker PECAM-1, ACE point marker DPP4, and pan-Aβ antibody D54D2, highlighting bridging vein and dural amyloidosis. Scale = 2 mm, insets = 200 μm. (E) Representative stereomicroscopy images of skull bases (with attached dura) stained with the ACE point marker DPP4 and pan-Aβ antibody D54D2. Representative light-sheet microscopy of a cleared skull base with the vascular marker PODXL, ACE point marker GLUT1, and pan-Aβ antibody D54D2. Scale = 2 mm, insets = 200 μm. (F) Representative images of amyloidosis of bridging veins along the superior sagittal sinus of 5XFAD mice up to 10 mo old. Duras are stained for the vascular marker PECAM-1, ACE point marker DPP4, and pan-Aβ antibody D54D2. Scale = 2 mm. (G) Quantification of Aβ staining intensity within the DPP4-positive area of ACE points between 1 and 10 mo in 5XFAD mice. Two-way ANOVA with Tukey’s post hoc test. Mean ± SEM. P values are given for the comparison between 1- and 2-mo-old 5XFAD and other time points. N = 1–22 mice per time point. ns, P > 0.05, nonsignificant; ***P < 0.001. (H) Representative images of dural amyloidosis around lymphatic vessels at the transverse sinus. Blood vasculature is stained with PECAM-1, ACE points with DPP4, lymphatic vessels and macrophages with LYVE-1, as well as the pan-Aβ antibody D54D2. Scale = 200 μm.

Progressive amyloidosis of bridging veins in 5XFAD mice. (A) Representativ...

in Amyloidosis of bridging veins is a pathologic feature of Alzheimer’s disease > Journal of Experimental Medicine
Published: 02 December 2025
Figure 1. Progressive amyloidosis of bridging veins in 5XFAD mice. (A) Representative images of duras from 5-mo-old 5XFAD mice and littermate controls stained for the vascular marker PECAM-1, ACE point marker DPP4, and pan-Aβ antibody D54D2. More about this image found in Progressive amyloidosis of bridging veins in 5XFAD mice. (A) Representativ...
Images
Disrupted CSF flow and vascular function at ACE points in 5XFAD mice. (A) Representative images of ICM OVA in the dura of 10-mo-old 5XFAD mice and littermate controls 2 h following injection. Scale = 2 mm. (B) Quantification of OVA-positive area in 5XFAD and littermate control duras. Two-tailed unpaired Student’s t test. N = 6 5XFAD, N = 5 littermate controls, mean ± SEM. *P < 0.05. (C) Representative images of ICM beads trapped within ACE points of a 10-mo-old 5XFAD mouse. Scale = 2 mm, inset = 200 μm. (D) Workflow and representative images of particle flow along bridging veins in 8–9-mo-old 5XFAD mice and littermate controls. Scale = 200 μm. (E) Quantification of particle flow in 5XFAD and littermate control duras. N = 10 5XFAD, N = 6 littermate controls. Two-tailed unpaired Student’s t test, mean ± SEM, *P < 0.05. (F) Experimental paradigm and representative images of blood flow speed in leptomeningeal vessels by photoacoustic imaging, colored by blood flow speed. (G) Quantification of baseline diameter in 5XFAD and littermate control bridging veins. N = 8 5XFAD, N = 6 littermate controls. Two-tailed unpaired Student’s t test, mean ± SEM. *P < 0.05. (H) Quantification of baseline blood flow in 5XFAD and littermate control bridging veins. N = 8 5XFAD, N = 6 littermate controls. Two-tailed unpaired Student’s t test, mean ± SEM. *P < 0.05. (I) Quantification of baseline oxygen saturation (SO2) in 5XFAD and littermate control bridging veins. N = 8 5XFAD, N = 6 littermate controls. Two-tailed unpaired Student’s t test, mean ± SEM. ns, P > 0.05, nonsignificant. (J) Quantification of the change in diameter in 5XFAD and littermate control bridging veins following hypercapnia. N = 8 5XFAD, N = 6 littermate controls. Two-tailed unpaired Student’s t test, mean ± SEM. ns, P > 0.05, nonsignificant. (K) Quantification of the change in blood flow in 5XFAD and littermate control bridging veins following hypercapnia. N = 8 5XFAD, N = 6 littermate controls. Two-tailed unpaired Student’s t test, mean ± SEM. *P < 0.05. (L) Quantification of the change in oxygen saturation in 5XFAD and littermate control bridging veins following hypercapnia. N = 8 5XFAD, N = 6 littermate controls. Two-tailed unpaired Student’s t test, mean ± SEM. ns, P > 0.05, nonsignificant.

Disrupted CSF flow and vascular function at ACE points in 5XFAD mice. (A) ...

in Amyloidosis of bridging veins is a pathologic feature of Alzheimer’s disease > Journal of Experimental Medicine
Published: 02 December 2025
Figure 2. Disrupted CSF flow and vascular function at ACE points in 5XFAD mice. (A) Representative images of ICM OVA in the dura of 10-mo-old 5XFAD mice and littermate controls 2 h following injection. Scale = 2 mm. (B) Quantification of More about this image found in Disrupted CSF flow and vascular function at ACE points in 5XFAD mice. (A) ...
Images
Amyloidosis of bridging veins in postmortem specimens from cognitively intact adults and cognitively impaired adults with AD pathology and dementia. (A) Top: Representative low-magnification image of a formalin-fixed paraffin-embedded dural sample, sectioned coronally through the superior sagittal sinus (SSS). Bottom: Representative image of a section through a bridging vein from the dura mater as it drains into the SSS. Scale = 2 mm. (B) Representative low-magnification image and schematic of anti–pan-Aβ (4G8) label around a bridging vein in an specimen from an old adult with AD pathology and dementia. Scale = 200 μm. c.s., cross section; l.s., longitudinal section; VL, vein lumen. (C) Representative images of anti-Aβ1-40 and anti-Aβ1-42 labeling around bridging veins from an old adult with AD pathology and dementia. Scale = 200 μm. (D) Representative images of anti–pan-Aβ (4G8) labeling in bridging veins from cognitively normal young and old adults without neurological disease and a cognitively impaired adult with AD pathology and dementia. Scale = 200 μm. (E) Quantification of the proportion (top row) and intensity (bottom row) of bridging veins stained with anti–pan-Aβ antibody (4G8), and antibodies against anti-Aβ1-40 and anti-Aβ1-42. N = 5. One-way ANOVA with Tukey’s post-hoc test, mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ns, P > 0.05, nonsignificant.

Amyloidosis of bridging veins in postmortem specimens from cognitively inta...

in Amyloidosis of bridging veins is a pathologic feature of Alzheimer’s disease > Journal of Experimental Medicine
Published: 02 December 2025
Figure 3. Amyloidosis of bridging veins in postmortem specimens from cognitively intact adults and cognitively impaired adults with AD pathology and dementia. (A) Top: Representative low-magnification image of a formalin-fixed paraffin-embedded More about this image found in Amyloidosis of bridging veins in postmortem specimens from cognitively inta...
Journal Articles

The antigen-presenting molecule MR1 binds host-generated riboflavin catabolites

Mohamed R. Abdelaal, Jieru Deng, Mitchell P. McInerney, Emi Ito, Anthony W. Purcell, Sho Yamasaki, Jose A. Villadangos, Hamish E.G. McWilliam, Nicholas A. Gherardin, Jamie Rossjohn, Wael Awad
Journal: Journal of Experimental Medicine
J Exp Med (2025) 223 (2): e20250711.
https://doi.org/10.1084/jem.20250711
Published: 26 November 2025
View article titled, The antigen-presenting molecule MR1 binds host-generated riboflavin catabolites
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Includes: Supplementary data
Journal Articles

A common progenitor gives rise to fibroblastic reticular cells and vascular smooth muscle cells in murine lymph nodes

Lisa Kurz, Mechthild Lütge, Angelina De Martin, Hung-Wei Cheng, Elina Bugar, Yves Stanossek, Samuel Meili, Joshua D. Brandstadter, Ivan Maillard, Lucas Onder, Burkhard Ludewig
Journal: Journal of Experimental Medicine
J Exp Med (2025) 223 (2): e20242300.
https://doi.org/10.1084/jem.20242300
Published: 26 November 2025
View article titled, A common progenitor gives rise to fibroblastic reticular cells and vascular smooth muscle cells in murine lymph nodes
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Includes: Supplementary data
Journal Articles

Correction: Neonatal microbiota colonization primes maturation of goblet cell–mediated protection in the pre-weaning colon

Åsa Johansson, Mahadevan Venkita Subramani, Bahtiyar Yilmaz, Elisabeth E.L. Nyström, Elena Layunta, Liisa Arike, Felix Sommer, Philip Rosenstiel, Lars Vereecke, Louise Mannerås-Holm, Andy Wullaert, Thaher Pelaseyed, Malin E.V. Johansson, George M.H. Birchenough
Journal: Journal of Experimental Medicine
J Exp Med (2025) 223 (1): e2024159111202025c.
https://doi.org/10.1084/jem.2024159111202025c
Published: 26 November 2025
View article titled, Correction: Neonatal microbiota colonization primes maturation of goblet cell–mediated protection in the pre-weaning colon
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Images
RF and RF catabolites bind MR1. (A) Mechanism of metabolism and photodegradation of RF. (B) Titration curves of the shown ligands binding to MR1 were obtained from the fluorescence polarization–based assay (left). Each data point represents normalized percentage binding from three independent experiments performed in triplicate. Mean values are plotted with SEM represented in error bars. Curve fit for ligands is displayed in the table (right). (C and D) (C) XICs for m/z 243.087 in a lumichrome solvent standard and (D) MS2 fragmentation of the main peak at retention time (RT) 6.82 are depicted. (E) UV-treated MR1 RF XIC for m/z 301.0927. (F) XIC for m/z 243.087 showing lumichrome present at RT 6.82, with an additional peak at RT 7.76. (G) MS2 fragmentation of main peak at RT 6.82, showing aligned fingerprint comparison with lumichrome solvent standard. (H) Thermostability of soluble WT MR1 refolded with the indicated ligands was measured by fluorescence-based thermal shift assay. The graph shows baseline-corrected, normalized emission at 610 nm plotted against temperature (°C). Each point represents the mean of three technical replicates, and error bars represent SD. The Tm50 is indicated by the dotted line at 50%. The table on the right shows the mean Tm50 from three independent experiments, each measured in at least a technical triplicate. XICs, extracted ion chromatograms.

RF and RF catabolites bind MR1. (A) Mechanism of metabolism and photodegra...

in The antigen-presenting molecule MR1 binds host-generated riboflavin catabolites > Journal of Experimental Medicine
Published: 26 November 2025
Figure 1. RF and RF catabolites bind MR1. (A) Mechanism of metabolism and photodegradation of RF. (B) Titration curves of the shown ligands binding to MR1 were obtained from the fluorescence polarization–based assay (left). Each data point More about this image found in RF and RF catabolites bind MR1. (A) Mechanism of metabolism and photodegra...
Images
RF catabolites modulate MR1 expression on the surface of APCs. (A–C) Bar graphs depict the expression of surface MR1*01 on C1R.MR1 cells incubated for 3 h (A) or 16 h (B and C) with titrated quantities of ligands followed by staining with 8F2F9 anti-MR1 antibody. Shown in B are overlay histograms for the expression of surface MR1*01 on C1R.MR1 cells after 16-h treatment with 200 µM of RF catabolites. (D–F) MR1 was then quantified after adding the indicated concentrations of the RF catabolites on THP-1.MR1 cells incubated for 3 h (D) or 16 h (E and F). Shown in E are overlay histograms for the expression of surface MR1*01 on THP-1.MR1 cells after 16-h treatment with 200 µM of RF catabolites. (G) Bars indicate the expression of surface MR1 on C1R.MR1 cells after treatment with lumichrome overnight and then staining with 26.5 vs. 8F2F9 MAb for comparison. (H) MR1 expression on the surface of PBMC-derived B lymphocytes was measured after treatment with RF catabolites for 16 h in the presence or absence of 5-OP-RU. Data in A–G represent the gMFI fold change from three independent experiments performed in duplicates, with standard error (SEM) represented by the error bars. Data in H are the average of three independent experiments done on PBMCs from three different donors performed in duplicate, with standard error (SEM) represented by the error bars. One-way ANOVA statistical analysis was performed for all samples with Dunnett’s multiple comparisons performed using NaOH as a control (ns: not significant, *P < 0.05, **P < 0.01, ****P < 0.0001). Statistical analysis for the 5-OP-RU competition experiment (H) used 5-OP-RU as a control. MR1, MHC-I–related protein. gMFI, geometric mean fluorescence intensity.

RF catabolites modulate MR1 expression on the surface of APCs. (A–C) Bar g...

in The antigen-presenting molecule MR1 binds host-generated riboflavin catabolites > Journal of Experimental Medicine
Published: 26 November 2025
Figure 2. RF catabolites modulate MR1 expression on the surface of APCs. (A–C) Bar graphs depict the expression of surface MR1*01 on C1R.MR1 cells incubated for 3 h (A) or 16 h (B and C) with titrated quantities of ligands followed by staining More about this image found in RF catabolites modulate MR1 expression on the surface of APCs. (A–C) Bar g...
Images
RF catabolites induce retention of MR1*01 in the ER, but not MR1K43A. (A) Analysis of Endo H–treated (+) or untreated (−) MR1 by western blotting with anti-MR1 (8G3) after culturing C1R.MR1 cells with DMSO (vehicle control), Ac-6-FP (10 μM), RF (100 μM), FMF (100 μM), lumiflavin (100 μM), lumichrome (100 μM), or alloxazine (100 μM) for 16 h, at 37°C. S, Endo H–susceptible MR1; R, Endo H–resistant MR1. The bars show the Endo H–susceptible MR1 and Endo H–resistant MR1 fractions quantified in at least two independent experiments. (B) Intracellular total MR1 level was also measured in C1R.MR1 cells by flow cytometry after treating the cells with the indicated ligands (100 µM) for 16 h followed by permeabilization and staining with anti-MR1-PE (8F2.F9). Shown are the overlay histograms (left) and a bar chart depicting the gMFI fold change of intracellular MR1 level (right) from three independent experiments performed in duplicates, with standard error (SEM) represented by the error bars. (C and D) C1R cells expressing (C) MR1R9H mutant or (D) MR1K43A mutant were incubated for the indicated periods with titrated quantities of ligand followed by flow cytometry. (E–G) Competition between RF catabolites and Ac-6-FP in E C1R.MR1 cells and (F) THP-I.MR1 cells, as well as (G) 5-OP-RU in C1R.MR1 cells, was quantified after incubation with the indicated concentrations of RF catabolites for 16 h before the addition of Ac-6-FP/5-OP-RU for further 3 h. Shown in E–G is the average percentage reduction in Ac-6-FP/5-OP-RU–induced MR1 upregulation in three independent experiments performed in duplicates with standard error (SEM) represented by error bars. One-way ANOVA statistical analysis was performed for all samples with Dunnett’s multiple comparisons performed using NaOH, Ac-6-FP, or 5-OP-RU as controls for the comparison (ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). gMFI, geometric mean fluorescence intensity. Source data are available for this figure: SourceData F3.

RF catabolites induce retention of MR1*01 in the ER, but not MR1 K43A...

in The antigen-presenting molecule MR1 binds host-generated riboflavin catabolites > Journal of Experimental Medicine
Published: 26 November 2025
Figure 3. RF catabolites induce retention of MR1*01 in the ER, but not MR1 K43A . (A) Analysis of Endo H–treated (+) or untreated (−) MR1 by western blotting with anti-MR1 (8G3) after culturing C1R.MR1 cells with DMSO (vehicle control), More about this image found in RF catabolites induce retention of MR1*01 in the ER, but not MR1 K43A...
Images
RF catabolites selectively inhibit ex vivo human MAIT cell activity. (A) Bar graph showing the proportion of CTVlow MR1-5-OP-RU tetramer+ MAIT cells as a proportion of total MAIT cells in PBMC after 7-day culture in the presence of 5-OP-RU (10 µM), RF (100 µM), lumiflavin (100 µM), lumichrome (100 µM), or alloxazine (100 µM) on day 7. (B–D) Bar graphs showing (B) MFI CD69, (C) proportion of CD69+, or (D) proportion of TNF+ MAIT cells from PBMCs coincubated with titrated doses of RF or RF catabolites in the presence of 5-OP-RU. (E and F) Bar graphs showing (E) proportion of CD69+ or (F) proportion of TNF+ MAIT cells from PBMCs coincubated with titrated doses of RF or RF catabolites in the presence of 5-A-RU. (G–J) Bar graphs showing (G) the proportion of CD69+ MAIT cells, (H) the proportion of CD69+ TRAV1-2−ve MR1-5-OP-RU tetramer−ve cells, (I) the proportion of TNF+ MAIT cells, or (J) the proportion of TNF+ TRAV1-2−ve MR1-5-OP-RU tetramer−ve cells from PBMCs preincubated with titrated doses of RF or RF catabolites followed by stimulation with PMA and ionomycin. (K and L) Bar graphs showing the proportion of CD69+ (K) MAIT cells or (L) TRAV1-2−ve MR1-5-OP-RU tetramer−ve cells, from PBMCs preincubated with titrated doses of RF or RF catabolites followed by stimulation with αCD3/αCD28-coated beads. Data from all graphs represent the average of three independent experiments performed in duplicate on PBMCs from three different donors. The error bars represent the standard error of the mean (SEM). One-way ANOVA statistical analysis was performed for all samples with Dunnett’s multiple comparisons performed using vehicle, 5-OP-RU, or 5-A-RU as controls (ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

RF catabolites selectively inhibit ex vivo human MAIT cell...

in The antigen-presenting molecule MR1 binds host-generated riboflavin catabolites > Journal of Experimental Medicine
Published: 26 November 2025
Figure 4. RF catabolites selectively inhibit ex vivo human MAIT cell activity. (A) Bar graph showing the proportion of CTVlow MR1-5-OP-RU tetramer+ MAIT cells as a proportion of total MAIT cells in PBMC after 7-day culture in the presence of More about this image found in RF catabolites selectively inhibit ex vivo human MAIT cell...
Images
Overall docking and molecular interactions of RF and RF catabolites within MR1 A′-pocket. (A) Superposition of the TCR-MR1-RF catabolite crystal structures showing the RF and its catabolites within the MR1-binding cleft of the MR1-RF structure interacting with MR1-Lys43. (B–E) Electron density omit maps (green mesh) of (B) RF, (C) FMF, (D) lumiflavin, and (E) lumichrome contoured at 2σ. (F–J) Molecular contacts of (F) RF, (G) FMF, (H) lumiflavin, and (I) lumichrome with the residues of MR1-A′-pocket in the MR1-Ag structures. Shown in J is the superposition of RF (cyan) with 5-OP-RU (yellow; PDB: 6PUC), which both have ribityl tail that extends toward the α2 helix. (K–L) Superposition of lumichrome (green) with (K) Ac-6-FP (pink; PDB: 4PJ5) and (L) DB28 (salmon; PDB: 6PVC) within MR1 ligand-binding cleft. (M) Superposition of lumichrome within the MR1 ligand-binding pocket of MR1R9H structure (PDB: 6W9V) showing the MR1 residues from MR1–lumichrome structure in green and MR1R9H residues in yellow. MR1 and β2m are colored white and marine, respectively. Here, the cutoff for hydrogen bonds, salt bridges, and VDW interactions was set at 3.5 Å, 4 Å, and 4 Å, respectively. Ligands are colored as follows: RF, cyan; FMF, orange; lumiflavin, magenta; and lumichrome, green. The α and β chains of the AF-7 TCR are colored yellow and pale green, respectively.

Overall docking and molecular interactions of RF and RF catabolites within ...

in The antigen-presenting molecule MR1 binds host-generated riboflavin catabolites > Journal of Experimental Medicine
Published: 26 November 2025
Figure 5. Overall docking and molecular interactions of RF and RF catabolites within MR1 A′-pocket. (A) Superposition of the TCR-MR1-RF catabolite crystal structures showing the RF and its catabolites within the MR1-binding cleft of the MR1-RF More about this image found in Overall docking and molecular interactions of RF and RF catabolites within ...
Images
CCL19-expressing FRCs and VSMCs in murine lymph nodes. (A and B) Confocal microscopy images showing cross sections of inguinal lymph nodes and the mesenteric lymph node chain from Ccl19-iEYFP+ mice. Boxed areas indicate regions of higher magnification micrographs. Microscopy images are stitched tile scans representative for four inguinal and four mesenteric lymph nodes from three independent experiments. Scale bars: 200 µm (A) and 500 µm (B). (C–E) Confocal microscopy images showing the blood vessels in the inguinal and mesenteric lymph nodes at higher magnification. Arrows and arrowheads highlight the localization of VSMC and PRC around the vessel. High-resolution images are representative for three inguinal and three mesenteric lymph nodes from three independent experiments. Scale bars: 10 µm (C and D, left panels), 3 µm (C and D, right panels), and 5 µm (E). (F–L) Flow cytometric analysis of non-hematopoietic cells in peripheral (inguinal, axillary, and brachial) and mesenteric lymph nodes. (F and I) Phenograph clustering projected on UMAP showing CD31− cells from pooled lymph nodes. (G and J) Expression of surface markers used to identify different FRC and VSMC populations projected on the UMAP. (H and K) Quantification of Ccl19-iEYFP+ cells gated according to the gating strategy shown in Fig. S2, A and C with pre-gating on CD31− cells. Data are shown as the mean and SEM from n = 11 mice from three independent experiments. (L) Quantification of the relative abundance of different FRC and VSMC populations. Relative abundances were calculated according to the gating strategy shown in Fig. S2, A and C, and data are shown as the mean and SEM from n = 15 mice from four independent experiments. P values were calculated with unpaired Student’s t test.

CCL19-expressing FRCs and VSMCs in murine lymph nodes. (A and B) Confocal ...

in A common progenitor gives rise to fibroblastic reticular cells and vascular smooth muscle cells in murine lymph nodes > Journal of Experimental Medicine
Published: 26 November 2025
Figure 1. CCL19-expressing FRCs and VSMCs in murine lymph nodes. (A and B) Confocal microscopy images showing cross sections of inguinal lymph nodes and the mesenteric lymph node chain from Ccl19-iEYFP+ mice. Boxed areas indicate regions of More about this image found in CCL19-expressing FRCs and VSMCs in murine lymph nodes. (A and B) Confocal ...
Images
Molecular characterization of lymph node FRCs and VSMCs. (A and B) scRNA-seq data of VSMCs and FRCs from peripheral and mesenteric lymph nodes of 8-wk-old Ccl19-iEYFP mice. (A) UMAP representation split by lymph node location and colored by subset identity. (B) Relative abundance of FRC subsets and VSMCs in peripheral and mesenteric murine lymph nodes of Ccl19-iEYFP mice. (C and D) scRNA-seq data of VSMCs and FRCs from peripheral and mesenteric lymph nodes of 8-wk-old Cxcl13-EYFP mice. (C) UMAP representation split by lymph node location and colored by subset identity. (D) Relative abundance of FRC subsets and VSMCs in peripheral and mesenteric murine lymph nodes of Cxcl13-EYFP mice. (E) Dot plot indicating the average expression of signature genes across VSMCs and FRC subsets in lymph nodes of Ccl19-iEYFP mice. (F) Dot plot indicating the average expression of signature genes across VSMCs and FRC subsets in lymph nodes of Cxcl13-EYFP mice. (G and H) Differentially expressed gene analysis between FRCs and VSMCs isolated from peripheral and mesenteric lymph nodes. (G) Enriched gene sets based on differentially expressed genes in peripheral and mesenteric lymph nodes. (H) Violin plots showing gene expression profiles of selected differentially expressed genes. Lymph node scRNA-seq data of Ccl19-iEYFP mice are representative of n = 15 mice from four independent experiments; 52,188 cells in total. Lymph node scRNA-seq data of Cxcl13-EYFP mice are representative of n = 10 mice from two independent experiments; 22,288 cells in total.

Molecular characterization of lymph node FRCs and VSMCs. (A and B) scRNA-s...

in A common progenitor gives rise to fibroblastic reticular cells and vascular smooth muscle cells in murine lymph nodes > Journal of Experimental Medicine
Published: 26 November 2025
Figure 2. Molecular characterization of lymph node FRCs and VSMCs. (A and B) scRNA-seq data of VSMCs and FRCs from peripheral and mesenteric lymph nodes of 8-wk-old Ccl19-iEYFP mice. (A) UMAP representation split by lymph node location and More about this image found in Molecular characterization of lymph node FRCs and VSMCs. (A and B) scRNA-s...
Images
Characterization of Ccl19-iEYFP+progenitors in lymph node anlagen and cell fate analysis in peripheral and mesenteric lymph nodes. (A) Schematic representation for the analysis of inguinal and mesenteric lymph node anlagen from Ccl19-iEYFP embryos at the indicated time points. (B–D) Whole-mount confocal microscopy analysis of mesenteric lymph node anlagen from Ccl19-iEYFP+ embryos at E15 (B), E16 (C), and E18 (D). Boxed areas indicate regions of higher magnification. Arrows and arrowheads indicate the localization of Ccl19-tTA+ cells. High-resolution images are representative for three inguinal and three mesenteric lymph node anlagen from three independent experiments. Scale bars: 80 and 40 µm (B), 150 µm (C, upper panels) and 15 µm (C, lower panels), and 200 µm (D, upper panels) and 15 µm (D, lower panels). (E) Schematic of cell fate analysis of inguinal and mesenteric lymph nodes from Ccl19-iEYFP+ mice. (F and G) Fate-mapping analysis of EYFP+ cells in inguinal (F) and mesenteric (G) lymph nodes harvested from adult Ccl19-iEYFP+ mice after Dox administration starting at E18. Microscopy images are representative for three inguinal and three mesenteric lymph nodes from three independent experiments. Scale bars: 200 µm (F) and 1,000 µm (G). (H) Localization and appearance of FRC subsets and VSMCs in cross sections of mesenteric lymph nodes. High-resolution microscopy images are representative for three mesenteric lymph nodes from three independent experiments. Scale bar: 20 µm (H). Figure was complemented with elements from https://BioRender.com.

Characterization of Ccl19-iEYFP + progenitors in lymph node an...

in A common progenitor gives rise to fibroblastic reticular cells and vascular smooth muscle cells in murine lymph nodes > Journal of Experimental Medicine
Published: 26 November 2025
Figure 3. Characterization of Ccl19-iEYFP + progenitors in lymph node anlagen and cell fate analysis in peripheral and mesenteric lymph nodes. (A) Schematic representation for the analysis of inguinal and mesenteric lymph node anlagen from More about this image found in Characterization of Ccl19-iEYFP + progenitors in lymph node an...
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Molecular characterization of Ccl19-tTA+and Cxcl13-Cre+FRC and VSMC progenitors in inguinal and mesenteric lymph node anlagen. (A) Schematic representation for the analysis of inguinal and mesenteric lymph node anlagen from Ccl19-iEYFP+ and Cxcl13-EYFP+ embryos at E18. (B and C) Whole-mount confocal microscopy analysis of inguinal (B) and mesenteric (C) lymph node anlagen from Ccl19-iEYFP embryos at E18. Arrows highlight the localization of Ccl19-iEYFP+ cells inside the lymph node anlage. Microscopy images are representative for three inguinal and three mesenteric lymph node anlagen from three independent experiments. Scale bars: 100 µm (B) and 200 µm (C). (D and E) Whole-mount confocal microscopy images showing inguinal (D) and mesenteric (E) lymph node anlagen from Cxcl13-EYFP+ embryos at E18. Arrows highlight the localization of Cxcl13-EYFP+ cells in the lymph node anlage. Arrowhead highlights the appearance of Cxcl13+ cells in the mesenchyme around the lymph node anlage. Microscopy images are representative for three inguinal and three mesenteric lymph node anlagen from three independent experiments. Scale bars: 100 µm (D) and 200 µm (E). (F) scRNA-seq analysis of Ccl19-iEYFP+ cells from inguinal and mesenteric lymph node anlagen at E18. UMAP representation of Ccl19-iEYFP+ cell clusters colored by lymph node entity (left panel) and Ccl19 expression (right panel). (G) scRNA-seq analysis of Cxcl13-EYFP+ cells from inguinal and mesenteric lymph node anlagen at E18. UMAP representation of Cxcl13-EYFP+ cell clusters colored by lymph node entity (left panel) and Ccl19 expression (right panel). (H) scRNA-seq analysis of Cxcl13-EYFP+ cells filtered for Ccl19 expression colored by cluster identity. (I–K) UMAP representation of Cxcl13-EYFP+Ccl19+ cells and projection of genes associated with cell proliferation (I), perivascular/mural (J), and FRC (K) signatures on the scRNA-seq dataset. (L–S) scRNA-seq analysis of Ccl19-iEYFP+ cells isolated from inguinal and mesenteric lymph node anlagen at E18. (L) Dot plot indicating the average expression of signature genes across embryonic Ccl19-iEYFP+ cell populations. (M) UMAP representation of Ccl19-iEYFP+ cell clusters. (N–P) Projection of proliferation (N), perivascular/mural (O), and FRC (P) signatures consisting of the indicated genes on the scRNA-seq dataset. (Q and R) Projection of selected genes associated with stem cell proliferation (Q) and stem cell population maintenance (R) on the scRNA-seq dataset. (S) Differentiation trajectory analysis of Ccl19-iEYFP+ cells from mesenteric lymph nodes using slingshot colored by the inferred slingshot pseudotime. Lymph node anlagen scRNA-seq data are representative of n = 15 embryos from two independent experiments; 2,699 cells in total from Ccl19-iEYFP+ embryos and 8,787 cells from Cxcl13-EYFP+ embryos.

Molecular characterization of Ccl19-tTA + and Cxcl13-Cre ...

in A common progenitor gives rise to fibroblastic reticular cells and vascular smooth muscle cells in murine lymph nodes > Journal of Experimental Medicine
Published: 26 November 2025
Figure 4. Molecular characterization of Ccl19-tTA + and Cxcl13-Cre + FRC and VSMC progenitors in inguinal and mesenteric lymph node anlagen. (A) Schematic representation for the analysis of inguinal and mesenteric lymph node anlagen from More about this image found in Molecular characterization of Ccl19-tTA + and Cxcl13-Cre ...
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Differentiation trajectories of FRCs and VSMCs in murine lymph nodes. (A) Schematic representation of the workflow for transcriptome analysis of FRCs from mesenteric lymph nodes of Ccl19-iEYFP mice using droplet-based scRNA-seq. (B and C) UMAP visualizing Ccl19-iEYFP+ cells from mesenteric lymph nodes colored by (B) age group and (C) FRC subset identity derived from the collective analysis of all adult FRCs. (D and E) UMAP visualizing Ccl19-iEYFP+ cells from mesenteric lymph nodes with the inferred differentiation lineages from slingshot analysis (D) and cells colored by the inferred slingshot pseudotime (E). (F) Expression fits of the assigned genes along the pseudotime for each of the inferred slingshot lineages. Genes that have similar expression patterns along all lineages were clustered, and clusters with >7 genes are shown. (G) Selected differentially expressed genes in the slingshot TRC/BRC/MedRC lineages along the pseudotime. (H) Selected differentially expressed genes in the slingshot PRC/VSMC lineages along the pseudotime. (I) UMAP visualizing Ccl19-iEYFP+ cells from mesenteric lymph nodes colored by cluster identity inferred from unbiased clustering. (J) Heatmap showing the scaled average activity of transcription factors (TFs) across clusters of Ccl19-iEYFP+ cells from mesenteric lymph nodes. For each adult FRC/VSMC cluster the top five transcription factors with the highest averages activity are shown. (K) Scaled activity in each cell of the most active transcription factors for adult FRC/VSMC clusters projected on the UMAP of Ccl19-iEYFP+ cells from mesenteric lymph nodes. Mesenteric lymph node scRNA-seq data are representative of n = 10–15 mice per time point; 33,903 cells from E18; 10,005 cells from P7; 12,291 cells from 3-wk-old mice; 52,188 cells from 8-wk-old mice.

Differentiation trajectories of FRCs and VSMCs in murine lymph nodes. (A) ...

in A common progenitor gives rise to fibroblastic reticular cells and vascular smooth muscle cells in murine lymph nodes > Journal of Experimental Medicine
Published: 26 November 2025
Figure 5. Differentiation trajectories of FRCs and VSMCs in murine lymph nodes. (A) Schematic representation of the workflow for transcriptome analysis of FRCs from mesenteric lymph nodes of Ccl19-iEYFP mice using droplet-based scRNA-seq. (B More about this image found in Differentiation trajectories of FRCs and VSMCs in murine lymph nodes. (A) ...
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Molecular characterization of Ltbr-deficient FRCs in murine lymph nodes. (A–C) scRNA-seq data of FRCs and VSMCs from mesenteric lymph nodes of Ccl19-iEYFP and Ccl19-iEYFP Ltbrfl/fl mice. (A) UMAP representation colored according to cluster identity. (B) Dot plot showing the average expression of signature genes in VSMCs and FRC subsets of EYFP+ cells isolated from mesenteric lymph nodes of Ccl19-iEYFP and Ccl19-iEYFP Ltbrfl/fl mice. (C) Pie chart showing the relative abundance of FRC subsets and VSMCs in mesenteric lymph nodes from Ccl19-iEYFP and Ccl19-iEYFP Ltbrfl/fl mice. (D and E) Confocal microscopy images showing cross sections of mesenteric lymph nodes from Ccl19-iEYFP Ltbrfl/fl mice. Boxed areas indicate regions of higher magnification. Arrows indicate appearance of Ccl19-tTA+ cells in perivascular areas. Microscopy images are representative for three mesenteric lymph nodes from three independent experiments. Scale bars: 500 µm (D) and 30 µm (E). (F and G) Fate analysis of EYFP+ cells in mesenteric lymph nodes harvested from adult Ccl19-iEYFP+ (F) and Ccl19-iEYFP Ltbrfl/fl (G) mice after Dox administration from E18. Microscopy images are representative for four mesenteric lymph nodes per condition from three independent experiments. Scale bars: 40 µm (F–G). (H and I) Quantification of perivascular Ccl19-iEYFP+ cells using histology of cross sections from mesenteric lymph nodes of fate mapped Ccl19-iEYFP and Ccl19-iEYFP Ltbrfl/fl mice. (H) Average pixel intensity of Ccl19-iEYFP signal with distance from CD31+ blood vessels (BV). (I) Quantification of perivascular grey values of EYFP signal in Ccl19-iEYFP and Ccl19-iEYFP Ltbrfl/fl mice. Data are representative of four mesenteric lymph nodes from three independent experiments (H and I). (J) Schematic depiction of differentiation trajectories of CCL19-expressing LTo cells in murine lymph nodes. Figure was complemented with elements from https://BioRender.com.

Molecular characterization of Ltbr-deficient FRCs in murin...

in A common progenitor gives rise to fibroblastic reticular cells and vascular smooth muscle cells in murine lymph nodes > Journal of Experimental Medicine
Published: 26 November 2025
Figure 6. Molecular characterization of Ltbr-deficient FRCs in murine lymph nodes. (A–C) scRNA-seq data of FRCs and VSMCs from mesenteric lymph nodes of Ccl19-iEYFP and Ccl19-iEYFP Ltbrfl/fl mice. (A) UMAP representation colored according to More about this image found in Molecular characterization of Ltbr-deficient FRCs in murin...
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in Correction: Neonatal microbiota colonization primes maturation of goblet cell–mediated protection in the pre-weaning colon > Journal of Experimental Medicine
Published: 26 November 2025
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Microbiota-dependent induction of senGC function. (A) Total bacterial load in the colon of rats at different postnatal days quantified by 16S qPCR of stool DNA. (B) Principal coordinate analysis of postnatal rat microbiota beta diversity (unweighted UNIFRAC) based on metataxonomic 16S sequencing of DNA from stool samples. (C) Linear discriminant analysis (LDA) size effect analysis of bacterial taxa significantly enriched in stool from rats at different ages. Taxa enrichment in specific age groups is indicated. (D) Ex vivo mucus growth in adult conventionalized (ConvD) and B. fragilis monoassociated mouse colon stimulated with P3CSK4 in the presence or absence of senGC activation inhibitors targeting endocytosis (Dynasore) or inflammasome activation (Casp IP). (E) Total bacterial load in colon of conventionally raised (CR), ConvD, and monoassociated mice quantified by 16S qPCR of stool DNA. (F) Ex vivo mucus growth in adult MyD88+/+ and MyD88−/− ConvR, GF, and 4-wk (w) ConvD mouse colon stimulated with flagellin in the presence or absence of a senGC activation inhibitor targeting inflammasome activation (Casp IP). (G) Ex vivo mucus growth in adult Nlrp6+/+ and Nlrp6−/− ConvR, GF, and 4-wk ConvD mouse colon stimulated with P3CKS4 in the presence or absence of a senGC activation inhibitor targeting inflammasome activation (Casp IP). (H) Principal coordinate analysis of microbiota beta diversity (Bray–Curtis dissimilarity) based on metataxonomic 16S sequencing of DNA from ConvR and ConvD stool samples. (I) Linear discriminant size effect analysis of bacterial taxa significantly enriched in stool from mice with the senGC− or senGC+ phenotype. (J) Principal coordinate analysis of microbiota beta diversity (Bray–Curtis dissimilarity) based on metataxonomic 16S sequencing of DNA from ConvR WT, ConvD WT, and ConvD MyD88−/− and Nlrp6−/− stool samples. (K) Relative abundance (RA) of the genus Mucispirillum in ConvD Nlrp6+/+ and Nlrp6−/− mice determined by metataxonomic 16S sequencing of stool DNA. (L) Standardized abundance (z-score) of bacterial taxa identified in F in 16S sequencing data from ConvD WT and ConvD MyD88−/− and Nlrp6−/− stool samples. Data represent n = 4–9 animals per group, as indicated. All data are pooled from at least two independent experiments or litters. Where relevant (A–C, E, and H–K) experimental groups are color coded by the absence (senGC−; blue) or presence (senGC+; brown) of the senGC-dependent secretory response. All error-bar graphs show median and interquartile range. Statistical comparisons between groups by two-way ANOVA and Fisher’s LSD (D, F, and G), Kruskal–Wallis and Dunn’s multiple comparison (A and E), PERMANOVA (B, H, and J), or Mann–Whitney test (K); P < 0.05 (*), <0.01 (**), <0.001 (***), <0.0001 (****).

Microbiota-dependent induction of senGC function. (A) Total bacterial load...

in Correction: Neonatal microbiota colonization primes maturation of goblet cell–mediated protection in the pre-weaning colon > Journal of Experimental Medicine
Published: 26 November 2025
Figure 5. Microbiota-dependent induction of senGC function. (A) Total bacterial load in the colon of rats at different postnatal days quantified by 16S qPCR of stool DNA. (B) Principal coordinate analysis of postnatal rat microbiota beta More about this image found in Microbiota-dependent induction of senGC function. (A) Total bacterial load...