NADPH oxidase deficiency underlies dysfunction of aged CD8+ Tregs
Zhenke Wen, … , Jörg J. Goronzy, Cornelia M. Weyand
Zhenke Wen, … , Jörg J. Goronzy, Cornelia M. Weyand
Published April 18, 2016
Citation Information: J Clin Invest. 2016;126(5):1953-1967. https://doi.org/10.1172/JCI84181.
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Research Article Aging Immunology

NADPH oxidase deficiency underlies dysfunction of aged CD8+ Tregs

Abstract

Immune aging results in progressive loss of both protective immunity and T cell–mediated suppression, thereby conferring susceptibility to a combination of immunodeficiency and chronic inflammatory disease. Here, we determined that older individuals fail to generate immunosuppressive CD8+CCR7+ Tregs, a defect that is even more pronounced in the age-related vasculitic syndrome giant cell arteritis. In young, healthy individuals, CD8+CCR7+ Tregs are localized in T cell zones of secondary lymphoid organs, suppress activation and expansion of CD4 T cells by inhibiting the phosphorylation of membrane-proximal signaling molecules, and effectively inhibit proliferative expansion of CD4 T cells in vitro and in vivo. We identified deficiency of NADPH oxidase 2 (NOX2) as the molecular underpinning of CD8 Treg failure in the older individuals and in patients with giant cell arteritis. CD8 Tregs suppress by releasing exosomes that carry preassembled NOX2 membrane clusters and are taken up by CD4 T cells. Overexpression of NOX2 in aged CD8 Tregs promptly restored suppressive function. Together, our data support NOX2 as a critical component of the suppressive machinery of CD8 Tregs and suggest that repairing NOX2 deficiency in these cells may protect older individuals from tissue-destructive inflammatory disease, such as large-vessel vasculitis.

Authors

Zhenke Wen, Yasuhiro Shimojima, Tsuyoshi Shirai, Yinyin Li, Jihang Ju, Zhen Yang, Lu Tian, Jörg J. Goronzy, Cornelia M. Weyand

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Figure 1

CD8 Tregs express CCR7, home to secondary lymphoid tissues, and suppress CD4 T cell activation.

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CD8 Tregs express CCR7, home to secondary lymphoid tissues, and suppress...
(A) Expression of FoxP3 and CCR7 was analyzed within gated peripheral blood CD8 T cells. One representative dot blot from 7 healthy donors. (B) Frozen sections from human tonsils were stained with mouse anti-human FoxP3 and rabbit anti-human CD8 antibodies. CD8 T cells were visualized with Alexa Fluor 488 fluorescence-labeled goat anti-rabbit antibodies. FoxP3 was detected with peroxidase-labeled goat anti-mouse antibodies. Merged images demonstrate CD8+FoxP3+ T cells in T cell–rich zones. Images are representative for 4 different tissues. Scale bar: 20 μm. (C–G) CD8 Tregs were isolated directly from PBMCs or generated ex vivo. Noncultured, fresh CD8 T cells served as controls. Naive CD4 T cells were mixed with CD8 T cells (1:1 ratio) and activated with anti-CD3/CD28 beads. (C) pZAP70 in CD4 T cells was measured by flow cytometry. A representative example of pZAP70 expression after 5 minutes of stimulation in the presence and absence of CD8 Tregs. (D) Frequencies of pZAP70+ CD4 T cells were monitored over a time span of 30 minutes following activation. Results are shown as mean ± SD from 4 independent experiments. (E) Frequencies of pZAP70+ CD4 T cells were measured after 5 minutes of stimulation in 19 independent experiments (mean ± SD). (F) CD4 T cells were mixed with CD8 Tregs at different ratios, and pZAP70 in CD4 T cells was measured by flow cytometry. Date are mean ± SD from 4 independent experiments. (G) CD8+CD39+CD26– Tregs were sorted from peripheral blood and immediately tested for suppressive function by mixing them with CD4 T cells. Percentages of pZAP70+ CD4 T cells (mean ± SD) were measured by flow cytometry in 3 independent experiments. Unpaired 2-tailed Student’s t test was used for comparisons.