Journal: bioRxiv

Article Title: Immunomodulatory endothelial cells contribute to T cell recruitment and activation through antigen presentation on MHC class II

doi: 10.1101/2025.07.17.665432

Figure Lengend Snippet: A , Expression of HLA-DR, CD80, CD86 on the surface of human endothelial cells after stimulation with IL-1β (50 ng/mL), IFN-γ (50 ng/mL) and TGF-β2 (5 mg/mL) for three days; n=6 independent cell batches (one-way ANOVA and Tukey multiple comparisons test). B , Representative images showing MHC class II and VE-cadherin in human endothelial cells (EC) and IMEC; bar = 20 µm. Similar images were obtained in 5 independent cell batches.

Article Snippet: Lysates were incubated overnight with an antibody against MHC class II (2 μg antibody/1 mg of protein lysate, Cat. #NBP3-08644H, Novus Biologicals, Wiesbaden Nordenstadt, Germany) or isotype control IgG (2 μg antibody/1 mg of protein lysate, Cat. #NI01-100UG, Merck, Darmstadt, Germany) and protein A/G-sepharose 4B conjugate (30 μL slurry/1 mg protein lysate; Cat. #17061801; Cytiva, Dreieich, Germany).

Techniques: Expressing

Journal: bioRxiv

Article Title: Immunomodulatory endothelial cells contribute to T cell recruitment and activation through antigen presentation on MHC class II

doi: 10.1101/2025.07.17.665432

Figure Lengend Snippet: A, Expression of transcripts for MHC class II and co-stimulatory molecules in endothelial cells from ligated left (L) or right (R) murine carotid arteries 2 days (2D) or 2 weeks (2W) after partial carotid artery ligation. Bubble size indicates the percentage of cells expressing a given transcript and the colour scale represents average expression level. Carotid arteries were pooled from 10 mice to obtain single cells. B, Expression (RiboTag RNA sequencing) of MHC class II and Cd86 in the endothelium of ligated left (L) compared to control right (R) carotid arteries from hypercholesterolemic endothelial cell-specific RiboTag mice (EC RiboTag) 7 days after partial carotid artery ligation; n=5 mice. C, UMAP showing endothelial cell clusters identified by the expression of CDH5 and/or KDR and lack of Prox1 and Lyve1. The IMEC population is highlighted in red. n=12 patients with atherosclerosis. D , Expression of transcripts for MHC class II and co-stimulatory molecules in IMEC compared to other endothelial cells in human atheromas. Bubble size indicates the percentage of cells expressing a given transcript and the colour scale represents average expression level.

Article Snippet: Lysates were incubated overnight with an antibody against MHC class II (2 μg antibody/1 mg of protein lysate, Cat. #NBP3-08644H, Novus Biologicals, Wiesbaden Nordenstadt, Germany) or isotype control IgG (2 μg antibody/1 mg of protein lysate, Cat. #NI01-100UG, Merck, Darmstadt, Germany) and protein A/G-sepharose 4B conjugate (30 μL slurry/1 mg protein lysate; Cat. #17061801; Cytiva, Dreieich, Germany).

Techniques: Expressing, Ligation, RNA Sequencing, Control

Journal: bioRxiv

Article Title: Immunomodulatory endothelial cells contribute to T cell recruitment and activation through antigen presentation on MHC class II

doi: 10.1101/2025.07.17.665432

Figure Lengend Snippet: A, Representative immunoblot showing the efficiency of the immunoprecipitation (IP) of MHC class II from whole cell lysate (WCL) of human IMEC. IgG was included as a control. Similar results were obtained in 3 independent cell batches. B, Volcano plot showing mass spectrometry-detected peptides enriched in MHC class II immunoprecipitates compared to control IgG from human IMEC under basal conditions. C , Volcano plot showing mass spectrometry-detected peptides enriched in MHC class II immunoprecipitates compared to control IgG from human IMEC treated with monocyte lysate. D , Volcano plot showing mass spectrometry-detected peptides enriched in MHC class II immunoprecipitates from IMEC treated with monocyte lysate versus IMEC under basal conditions. B-D, n=3 independent cell batches/condition. The dashed lines mark the significance threshold (Pvalue: 0.05). E, Expression of CD69 in CD4+ T cells (CD45+, CD3+, CD4+, CD25-,CD127+/-); γδ T cells (CD45+, CD3+, CD4-, CD8-, gdTCR+), Tregs (CD45+, CD3+, CD4+, CD25+CD12low) and CD8 + T cells (CD45+, CD3+, CD8+) three days after incubation of PB-MNC with endothelial cells or IMEC. Dashed lines indicate basal activation level of CD69 in PB-MNC alone. n=4 independent cell batches (unpaired Student’s t-test). F, Expression of CD279 in CD4 + T cells, γδ T cells, Tregs, and CD8 + T cells three days after incubation of PB-MNC with endothelial cells or IMEC, both treated with THP-1 protein lysate. Dashed lines indicate basal activation level of CD279 in PB-MNC alone. only n=4 independent cell batches (unpaired Student’s t-test).

Article Snippet: Lysates were incubated overnight with an antibody against MHC class II (2 μg antibody/1 mg of protein lysate, Cat. #NBP3-08644H, Novus Biologicals, Wiesbaden Nordenstadt, Germany) or isotype control IgG (2 μg antibody/1 mg of protein lysate, Cat. #NI01-100UG, Merck, Darmstadt, Germany) and protein A/G-sepharose 4B conjugate (30 μL slurry/1 mg protein lysate; Cat. #17061801; Cytiva, Dreieich, Germany).

Techniques: Western Blot, Immunoprecipitation, Control, Mass Spectrometry, Expressing, Incubation, Activation Assay