CD146 bound to LCK promotes T cell receptor signaling and antitumor immune responses in mice
Hongxia Duan, Lin Jing, Xiaoqing Jiang, Yanbin Ma, Daji Wang, Jianquan Xiang, Xuehui Chen, Zhenzhen Wu, Huiwen Yan, Junying Jia, Zheng Liu, Jing Feng, Mingzhao Zhu, Xiyun Yan
Hongxia Duan, Lin Jing, Xiaoqing Jiang, Yanbin Ma, Daji Wang, Jianquan Xiang, Xuehui Chen, Zhenzhen Wu, Huiwen Yan, Junying Jia, Zheng Liu, Jing Feng, Mingzhao Zhu, Xiyun Yan
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Research Article Cell biology Immunology

CD146 bound to LCK promotes T cell receptor signaling and antitumor immune responses in mice

Abstract

Initiation of T cell receptor (TCR) signaling involves the activation of the tyrosine kinase LCK; however, it is currently unclear how LCK is recruited and activated. Here, we have identified the membrane protein CD146 as an essential member of the TCR network for LCK activation. CD146 deficiency in T cells substantially impaired thymocyte development and peripheral activation, both of which depend on TCR signaling. CD146 was found to directly interact with the SH3 domain of coreceptor-free LCK via its cytoplasmic domain. Interestingly, we found CD146 to be present in both monomeric and dimeric forms in T cells, with the dimerized form increasing after TCR ligation. Increased dimerized CD146 recruited LCK and promoted LCK autophosphorylation. In tumor models, CD146 deficiency dramatically impaired the antitumor response of T cells. Together, our data reveal an LCK activation mechanism for TCR initiation. We also underscore a rational intervention based on CD146 for tumor immunotherapy.

Authors

Hongxia Duan, Lin Jing, Xiaoqing Jiang, Yanbin Ma, Daji Wang, Jianquan Xiang, Xuehui Chen, Zhenzhen Wu, Huiwen Yan, Junying Jia, Zheng Liu, Jing Feng, Mingzhao Zhu, Xiyun Yan

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Figure 6

CD146 deletion damages proximal TCR signaling.

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CD146 deletion damages proximal TCR signaling. (A) Western immunoblot (W...
(A) Western immunoblot (WB) analysis of p-LCK(Y394), LCK, p-ZAP70(Y319), and ZAP70 in splenic naive T cells stimulated with or without anti-CD3 and -CD28 antibodies (1 μg/mL) for 10 minutes. Total LCK and ZAP70 proteins were included as loading controls. Right: Quantification of p-LCK(Y394)/LCK and quantification of p-ZAP70/ZAP70 (n = 3). (B) Staining of p-ZAP70, p-p38, and p-ERK1/2 in splenic naive T cells stimulated with or without antibodies (1 μg/mL) for the indicated times. (C) MFI of p-ZAP70, p-ERK1/2, and p-p38 in the cells described above (n = 3). (D) Left: WB showing the expression of CD146, p-LCK(Y394), and p-ZAP70(Y319) in isolated CD3+ T cells from OT-IItgCD146CD4-WT or OT-IItgCD146CD4-KO mice; total LCK, ZAP70, and actin proteins were used as loading controls. Right: Quantification of p-LCK(Y394)/LCK or p-ZAP70/ZAP70 (n = 3). (E) Left: WB analysis of p-LCK(Y394) and LCK in thymocytes stimulated with or without anti–CD3/CD28 antibodies (1 μg/mL) for the indicated times. Total LCK protein was included as the loading control. Right: Quantification of p-LCK(Y394)/LCK (n = 3). (F) MFI of p-ZAP70, p-p38, and p-ERK1/2 in thymocytes stimulated as above (n = 3). (G) WB showing p-LCK, p-ZAP70, and p-ERK1/2 protein levels in Jurkat cells with CD146 knockdown (CD146 KD), WT (Control), or CD146 overexpression (CD146 OE) left stimulated with or without anti–CD3/CD28 antibodies (1 μg/mL) for 5 minutes. Total LCK, ZAP70, GAPDH, and ERK1/2 proteins were used as loading controls. (H) Quantifications of p-LCK(Y394)/LCK, p-ZAP70/ZAP70, and p-ERK1/2/ERK1/2 (n = 3). Each symbol represents an individual mouse (C, D, and F) or 1 experiment (A, B, E, and H). Data are representative of 3 independent experiments. One-way ANOVA (A and C–F) or 2-way ANOVA with multiple-comparison test (H) was performed. Data are shown as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.