Nonclassical action of Ku70 promotes Treg-suppressive function through a FOXP3-dependent mechanism in lung adenocarcinoma
Qianru Huang, … , Qianjun Zhou, Bin Li
Qianru Huang, … , Qianjun Zhou, Bin Li
Published October 24, 2024
Citation Information: J Clin Invest. 2024;134(23):e178079. https://doi.org/10.1172/JCI178079.
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Research Article Immunology Oncology

Nonclassical action of Ku70 promotes Treg-suppressive function through a FOXP3-dependent mechanism in lung adenocarcinoma

Abstract

Ku70, a DNA repair protein, binds to the damaged DNA ends and orchestrates the recruitment of other proteins to facilitate repair of DNA double-strand breaks. Besides its essential role in DNA repair, several studies have highlighted nonclassical functions of Ku70 in cellular processes. However, its function in immune homeostasis and antitumor immunity remains unknown. Here, we discovered a marked association between elevated Ku70 expression and unfavorable prognosis in lung adenocarcinoma, focusing specifically on increased Ku70 levels in tumor-infiltrated Tregs. Using a lung-colonizing tumor model in mice with Treg-specific Ku70 deficiency, we demonstrated that deletion of Ku70 in Tregs led to a stronger antitumor response and slower tumor growth due to impaired immune-suppressive capacity of Tregs. Furthermore, we confirmed that Ku70 played a critical role in sustaining the suppressive function of human Tregs. We found that Ku70 bound to forkhead box protein P3 (FOXP3) and occupied FOXP3-bound genomic sites to support its transcriptional activities. These findings not only unveil a nonhomologous end joining–independent (NHEJ-independent) role of Ku70 crucial for Treg-suppressive function, but also underscore the potential of targeting Ku70 as an effective strategy in cancer therapy, aiming to both restrain cancer cells and enhance pulmonary antitumor immunity.

Authors

Qianru Huang, Na Tian, Jianfeng Zhang, Shiyang Song, Hao Cheng, Xinnan Liu, Wenle Zhang, Youqiong Ye, Yanhua Du, Xueyu Dai, Rui Liang, Dan Li, Sheng-Ming Dai, Chuan Wang, Zhi Chen, Qianjun Zhou, Bin Li

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Figure 5

Human iTreg suppressive function is compromised upon knockdown of Ku70.

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Human iTreg suppressive function is compromised upon knockdown of Ku70. ...
(A and B) Ku70 knockdown assay was performed in human iTregs using shRNA-Ctrl (shCK) or shRNA-Ku70 (shKu70) lentiviruses, and the expression levels of Ku70 and FOXP3 were examined by Western blot (A) and qRT-PCR (B). (C–E) Representative histogram of protein abundance of FOXP3, CD25, and CTLA4 in control and shKu70 knockdown human iTregs and summaries for the MFI. (F and G) IL-10 and IL-2 production from human iTregs were assessed after knockdown of Ku70 by lentiviruses carrying Ku70 shRNA. (H) In vitro suppression assay was performed in human iTregs with or without Ku70 knockdown. Human iTreg and CD8+ T cells (responder) were cocultured for 84 hours at a ratio of 1:1, 1:2, 1:4, or 1:8 with anti-CD3/CD28 beads for activation. The responder groups include 2 control groups: one without anti-CD3/CD28 bead stimulation and one with anti-CD3/CD28 bead stimulation but without coculture with Tregs. The generations of proliferating responder were monitored by dye dilution of CellTrace Violet (CTV). Data are representative of 3 independent experiments. Data are represented as means ± SD. Significance was measured by unpaired 2-tailed Student’s t test.