NADPH oxidase deficiency underlies dysfunction of aged CD8+ Tregs
Zhenke Wen, Yasuhiro Shimojima, Tsuyoshi Shirai, Yinyin Li, Jihang Ju, Zhen Yang, Lu Tian, Jörg J. Goronzy, Cornelia M. Weyand
Zhenke Wen, Yasuhiro Shimojima, Tsuyoshi Shirai, Yinyin Li, Jihang Ju, Zhen Yang, Lu Tian, Jörg J. Goronzy, Cornelia M. Weyand
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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 5

CD8 Treg-mediated suppression requires ROS production and NOX2 function.

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CD8 Treg-mediated suppression requires ROS production and NOX2 function....
(A and B) CD8 Tregs were loaded with oxidation-sensitive CellROX and analyzed by flow cytometry to detect ROS levels. Fresh CD8 T cells from the same donor were used as controls. (A) Representative histograms and (B) a summary of 5 independent experiments (mean ± SD) are shown. (C and D) CD8 Tregs were pretreated with or without the ROS scavenger tempol, mixed with naive CD4 T cells, and stimulated with anti-CD3/CD28 beads. pZAP70 in CD4 T cells was analyzed. (C) One representative dot blot and (D) results (mean ± SD) from 3 independent experiments are shown. (E) CD8 Tregs were pretreated with the mitochondrial complex I inhibitor rotenone (ROT) or (F) the mitochondrial ROS scavenger mitoTEMPO (mito-Temp) and tested for their suppressive activity by mixing them with naive CD4 T cells and quantifying pZAP70 after anti-CD3/CD28 stimulation. Frequencies of pZAP70+ CD4 T cells (mean ± SD) in 5 independent experiments are shown. (G) CD8 Tregs were pretreated with the NADPH oxidase inhibitor DPI or (H) gp91ds-tat, an inhibitor of the NADPH oxidase complex assembly, or (I) were transfected with NOX2 shRNA or control shRNA. CD8 Tregs were tested for their suppressive activity in a CD4-CD8 coculture assay by quantifying pZAP70 in CD4 T cells. Results are from (G and H) 4 and (I) 5 independent experiments and are presented as mean ± SD. (J and K) CD8 Tregs were pretreated with DPI, gp91ds-tat, or NOX2 knockdown, as above, and their suppressive activity was assessed in CD4-CD8 coculture assays. CD4 activation was quantified by measuring the phosphorylation of the signaling molecule LAT. Results are from 3 independent experiments (mean ± SD). Unpaired 2-tailed Student’s t test was used for comparisons.