NOTCH-induced rerouting of endosomal trafficking disables regulatory T cells in vasculitis
Ke Jin, … , Jorg J. Goronzy, Cornelia M. Weyand
Ke Jin, … , Jorg J. Goronzy, Cornelia M. Weyand
Published September 22, 2020
Citation Information: J Clin Invest. 2021;131(1):e136042. https://doi.org/10.1172/JCI136042.
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NOTCH-induced rerouting of endosomal trafficking disables regulatory T cells in vasculitis

Abstract

The aorta and the large conductive arteries are immunoprivileged tissues and are protected against inflammatory attack. A breakdown of immunoprivilege leads to autoimmune vasculitis, such as giant cell arteritis, in which CD8+ Treg cells fail to contain CD4+ T cells and macrophages, resulting in the formation of tissue-destructive granulomatous lesions. Here, we report that the molecular defect of malfunctioning CD8+ Treg cells lies in aberrant NOTCH4 signaling that deviates endosomal trafficking and minimizes exosome production. By transcriptionally controlling the profile of RAB GTPases, NOTCH4 signaling restricted vesicular secretion of the enzyme NADPH oxidase 2 (NOX2). Specifically, NOTCH4hiCD8+ Treg cells increased RAB5A and RAB11A expression and suppressed RAB7A, culminating in the accumulation of early and recycling endosomes and sequestering of NOX2 in an intracellular compartment. RAB7AloCD8+ Treg cells failed in the surface translocation and exosomal release of NOX2. NOTCH4hiRAB5AhiRAB7AloRAB11AhiCD8+ Treg cells left adaptive immunity unopposed, enabling a breakdown in tissue tolerance and aggressive vessel wall inflammation. Inhibiting NOTCH4 signaling corrected the defect and protected arteries from inflammatory insult. This study implicates NOTCH4-dependent transcriptional control of RAB proteins and intracellular vesicle trafficking in autoimmune disease and in vascular inflammation.

Authors

Ke Jin, Zhenke Wen, Bowen Wu, Hui Zhang, Jingtao Qiu, Yanan Wang, Kenneth J. Warrington, Gerald J. Berry, Jorg J. Goronzy, Cornelia M. Weyand

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Figure 4

NOTCH4 signaling regulates the surface translocation of NOX2.

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NOTCH4 signaling regulates the surface translocation of NOX2. (A) Surfac...
(A) Surface expression of NOTCH4 and NOX2 on CD8+ Tregs determined by flow cytometry. Each dot represents 1 sample. Linear regression curve generated by GraphPad Prism software. P value calculated by F test. (B) CD8+ Tregs were treated with the NOTCH signal inhibitor DAPT or vehicle. Cell surface NOX2 expression determined by flow cytometry. Result from 8 controls and 7 patients. (C and D) CD8+ Tregs were transfected as indicated. (C) NOTCH4 or control siRNA. (D) N4ICD-containing or control vector. Cell surface NOX2 measured by flow cytometry. Representative histograms and results from 10–12 samples. (E) Healthy CD8+ Tregs were transfected with an N4ICD-containing vector and treated with actinomycin D or vehicle. Cell surface NOX2 was measured at the indicated times. Representative histograms and results from 4 samples. (F) NOX2 transcript concentrations in control and GCA CD8+ Tregs (RT-PCR, n = 7 samples each). (G) NOX2 protein determined by immunoblots in isolated cell membranes and in whole cell lysate from control and GCA CD8+ Tregs. CD8 was used as a loading control for cell membranes and β-actin for whole cell lysates. Data from 5 samples. (H–J) CD8+ Tregs were treated or transfected as indicated. NOX2 protein was quantified in isolated cell membranes and in whole cell lysates as above. CD8+ Treg cells were induced ex vivo. Data are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 by unpaired Mann-Whitney-Wilcoxon rank test (F and G), paired Mann-Whitney-Wilcoxon rank test (C and D), or 1-way ANOVA and post-ANOVA pairwise 2-group comparisons conducted with Tukey’s method (B).