LC3-dependent intercellular transfer of phosphorylated STAT1/2 elicits CXCL9+ macrophages and enhances radiation-induced antitumor immunity
Jun-Yan Li, Ying-Qing Li, Jia-Hao Dai, Sha Gong, Qing-Mei He, Jie-Wen Bai, Sai-Wei Huang, Ying-Qi Lu, Yu-Fei Duan, Sen-Yu Feng, Xi-Rong Tan, Xiao-Yu Liang, Jun Ma, Rui Guo, Na Liu
Jun-Yan Li, Ying-Qing Li, Jia-Hao Dai, Sha Gong, Qing-Mei He, Jie-Wen Bai, Sai-Wei Huang, Ying-Qi Lu, Yu-Fei Duan, Sen-Yu Feng, Xi-Rong Tan, Xiao-Yu Liang, Jun Ma, Rui Guo, Na Liu
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Research Article Cell biology Immunology Oncology

LC3-dependent intercellular transfer of phosphorylated STAT1/2 elicits CXCL9+ macrophages and enhances radiation-induced antitumor immunity

Abstract

The efficacy of anticancer treatments, including radiotherapy, depends on the activation of type I IFN signaling. However, its regulatory networks and mechanisms remain to be elucidated. Here, we report that tumor cell–intrinsic type I IFN signaling can be transferred to macrophages via secretory autophagy, inducing CXCL9hi macrophages and enhancing CD8+ T cell–mediated antitumor immunity. Mechanistically, K63-linked ubiquitination at the K167 site of phosphorylated STAT2 (p-STAT2) facilitates its binding to LC3B, promoting the loading of p-STAT1 and p-STAT2 into extracellular vesicles and intercellular transference from tumor cells to macrophages, which, however, is suppressed by USP5-mediated STAT2 deubiquitination. Genetic depletion or pharmacological inhibition of USP5 promotes autophagy-dependent unconventional protein secretion of p-STAT1 and p-STAT2, leading to the induction of CXCL9+ macrophages. This process promotes the expression of T cell chemokines and upregulates the antigen presentation machinery, thereby enhancing radiation-induced CD8+ T cell antitumor immunity and radiotherapy efficacy. Our findings reveal a critical role of USP5 in type I IFN–induced antitumor immunity, providing potential targets for improving the efficacy of radiotherapy.

Authors

Jun-Yan Li, Ying-Qing Li, Jia-Hao Dai, Sha Gong, Qing-Mei He, Jie-Wen Bai, Sai-Wei Huang, Ying-Qi Lu, Yu-Fei Duan, Sen-Yu Feng, Xi-Rong Tan, Xiao-Yu Liang, Jun Ma, Rui Guo, Na Liu

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Figure 5

USP5 deficiency promotes LC3-dependent secretory autophagy of p-STAT1/2.

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USP5 deficiency promotes LC3-dependent secretory autophagy of p-STAT1/2....
(A) The cytosolic and nuclear p-STAT1 and p-STAT2 levels in WT and USP5-deficient SUNE1 cells. (B) Representative confocal microscopy images and line scan analysis showing the different localization of p-STAT2 in WT or USP5-deficient SUNE1 cells. Scale bar: 10 μm. (C) Bubble diagram shows the enriched biological process of anti-STAT2 immunoprecipitated proteins in USP5-deficient SUNE1 cells after IFN-β treatment. (D) Volcano plot shows 1,353 upregulated and 566 downregulated proteins (FC ≥ 1.5 and P < 0.05) in anti-STAT2 immunoprecipitates from USP5-deficient compared with control SUNE1 cells. (E and F) Co-IP shows the interaction between STAT2 and LC3B-II in USP5-deficient SUNE1 cells (E) and HK1 cells (F) after IFN-β treatment. (G and H) Representative immunofluorescence assay images (G) showing the colocalization of p-STAT2 and LC3B in USP5-deficient SUNE1 cells after IFN-β treatment. Scale bar: 10 μm. (H) The Manders’ colocalization coefficients are reported. (I) The decreased p-STAT2 and p-STAT1 in large extracellular vesicles (LEVs) from the culture medium of USP5-deficient SUNE1 after LC3B knockdown. (J) LC3B knockdown in STAT2-EGFP SUNE1 cells decreased the EGFP+ population in coculture with THP1-MΦ. (K and L) Control and USP5-deficient SUNE1 cells were transfected with si-LC3B and treated with IFN-β for 48 hours. LEVs were collected from the culture medium and used to treat the THP1-MΦ. The relative CXCL9 and CXCL10 expression in macrophages was measured by RT-qPCR (K); the CD86 and HLA-DR expression on macrophages was measured by flow cytometry (L). The results are representative of 3 independent experiments (A, B, and E–L). The data are presented as mean ± SD (H and J–L). Comparisons were performed using 1-way ANOVA (H) or 2-way ANOVA (J–L) with Bonferroni’s test for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.