Internalization and presentation of myelin antigens by the brain endothelium guides antigen-specific T cell migration
- Vrije Universiteit Medical Center, The Netherlands
Figures
Figure 1 with 2 supplements
Human brain endothelial cells internalize myelin particles.
Confluent monolayers of brain endothelial cells (BECs) were stimulated with 5 ng/ml TNFα for 24 hr. (A) Expression of MHC-I, MHC-II, CD40, PD-L1 and VCAM−1 was determined by flow cytometry. …
Figure 1—figure supplement 1
Human brain endothelial cells increase MHC and costimulatory molecule expression upon activation by IFN-γ.
Confluent monolayers of brain endothelial cells (BECs) were stimulated with 10 ng/ml IFN-γ for 24 hr. Expression of MHC-I, MHC-II, CD40 and VCAM−1 was determined by flow cytometry. Histograms depict …
Figure 1—figure supplement 2
Brain endothelial cells internalize myelin particles.
BECs were loaded with fluorescently labeled human myelin and internalization of myelin particles was assessed by imaging flow cytometry. To determine internalization scores, a mask was designed …
Figure 2
Myelin particles are preferably routed to the endo-lysosomes.
Resting or activated BECs were loaded with Atto-633 labeled myelin for 4 hr or 24 hr. Uptake of myelin particles and their co-localization with early endosomal (EEA1) or endosomal/lysosomal (LAMP1) …
Figure 3
Migration of myelin-specific T-cells depends on presentation of myelin-antigens in MHC-II by BECs.
(A) Th1 and Th17 subsets were generated in-vitro from naive CD4+CD62Lhigh 2D2 T cells. Expression of IFN-γ, Il−17, IL−10, T-bet and RORγT was determined using qRT-PCR. Data are the means of …
Author response image 1
Confluent human brain endothelial cells form an impermeable barrier.
The human brain endothelial cells (cell line hCMEC/D3) were cultured on Ibidi-slides. After reaching confluence, cells were washed, fixated with 4% formaldehyde and stained with FITC-labeled …
Author response image 2
Induction of T cell activation but not proliferation by brain endothelial cells.
(A) Brain endothelial cells were loaded with myelin or MOG35-55 in the presence of TNF and subsequently co-cultured with in vitro generated myelin-specific Th1 or Th17 cells. As a control, none …
Author response image 3
OVA-specific T-cells only transmigrate upon encounter of BECs presenting OVA-antigens in MHC-II.
Th1 and Th17 subsets were generated in-vitro from naive CD4+CD62Lhigh OT-II T cells. mBECs were seeded onto trans-wells, activated with TNF and loaded with OVA for 24h. As a control, BECs loaded …
Author response image 4
Myelin is internalized and routed to lysosomal compartments by brain endothelial cells.
(A) BECs were loaded with Atto 633-labeled human myelin for 24h and internalization of myelin particles was assessed by imaging flow cytometry. To determine internalization scores, a mask was …
Author response image 5
Human brain endothelial cells increase MHC and costimulatory molecule expression upon activation by IFN-γ.
Confluent monolayers of brain endothelial cells (BECs) were stimulated with 10 ng/ml IFN-γ for 24 hr. Expression of MHC-I, MHC-II, CD40 and VCAM-1 was determined by flow cytometry. Histograms depict …
Author response image 6
Confluency of cultured brain endothelial cells shown using F-actin, Claudin-5 and VE-Cadherin staining on samples shown in Figure 2 of the manuscript.
https://doi.org/10.7554/eLife.13149.013
Author response image 7
Co-localization of green and red channels.
(A) co-localization of high intensity green and high intensity red image shows a yellow merge image. Histogram shows overlap of intensities. (B) Co-localization of low intensity green dot with …
Author response image 8
Average size of lysosomes in myelin-loaded brain endothelial cells.
The diameter of lysosomes was determined using ImageJ software and shown as histograms. The average size of 5 lysosomes per cell of a total of 10 cells that contain myelin is compared with that of …
