通过ABAT克服GABA诱导的调节性T细胞对免疫的抑制作用以增强肝癌中CD8 + T细胞的抗肿瘤免疫反应

Overcoming GABA-Induced Treg Suppression of Immunity by ABAT to Augment CD8+T Cell Antitumor Immune Response in Liver Cancer.

作者信息

Han Hui, Lu Yandi, Xu Suwen, Zhang Weirui, Lin Weisen, Zhan Jianwen, Chen Gengzhen, Gu Binbin

机构信息

Digestive Disease Center, People's Hospital of Chenghai, Shantou, China.

Departments of Gastroenterology, Taizhou Hospital of Zhejiang Province Affiliated to Wenzhou Medical University, Linhai, China.

出版信息

Int Arch Allergy Immunol. 2024 Nov 25:1-11. doi: 10.1159/000542404.

DOI:10.1159/000542404

PMID:39586256

Abstract

INTRODUCTION

ABAT, a key enzyme in GABA catabolism, modulates antitumor immune activity across various cancers. However, the molecular mechanisms by which the ABAT/GABA axis exerts immune regulation in the liver cancer microenvironment remain unclear.

METHODS

ABAT expression in liver cancer tissues was scrutinized via the TCGA-LIHC database, and the 5-year survival rates of liver cancer patients were appraised through Kaplan-Meier survival analyses. The mRNA levels of ABAT in liver cancer cell lines were quantified by qRT-PCR. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), colony formation, and Transwell assays were employed to gauge the influence of ABAT overexpression on liver cancer cell growth, proliferation, migration, and invasion, respectively. Western blot evaluated epithelial-mesenchymal transition-related protein expression. ELISA quantified GABA, IL-10, TGF-β1, Granzyme B, and IFN-γ in the culture medium. Flow cytometry was used to measure the frequency of CD25+FOXP3+ cells and the expression of CD25, CD69, and PD-1 on CD8+T cells in coculture. Carboxifluorescein diacetate succinimidyl ester dilution assays were performed to assess the proliferative activity of CD8+T cells.

RESULTS

ABAT was found to be underexpressed in liver cancer tissues and cells, and such underexpression is indicative of a poorer prognosis for patients, while its overexpression was shown to curb the malignancy of liver cancer cells. Upon overexpression of ABAT, there is a decrease in GABA levels in the cell supernatant, coupled with an increase in IL-10 and TGF-β1 cell number, and an upsurge in the CD25+FOXP3+ cell ratios, all of which were restored by the addition of exogenous GABA. Furthermore, the depletion of ABAT led to a reduction in the proliferation and tumor-killing ability of CD8+T cells, effects that were reversed by the application of GABAA receptor inhibitors.

CONCLUSION

ABAT functions to inhibit Treg differentiation in liver cancer through downregulation of GABA, thus promoting the antitumor activity of CD8+T cells.

摘要

引言

ABAT是γ-氨基丁酸（GABA）分解代谢中的关键酶，可调节多种癌症的抗肿瘤免疫活性。然而，ABAT/GABA轴在肝癌微环境中发挥免疫调节作用的分子机制仍不清楚。

方法

通过TCGA-LIHC数据库仔细研究肝癌组织中ABAT的表达情况，并通过Kaplan-Meier生存分析评估肝癌患者的5年生存率。采用qRT-PCR定量肝癌细胞系中ABAT的mRNA水平。分别采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐（MTT）法、集落形成实验和Transwell实验来检测ABAT过表达对肝癌细胞生长、增殖、迁移和侵袭的影响。蛋白质免疫印迹法评估上皮-间质转化相关蛋白的表达。酶联免疫吸附测定法（ELISA）定量培养基中GABA、白细胞介素-10（IL-10）、转化生长因子-β1（TGF-β1）、颗粒酶B和干扰素-γ（IFN-γ）的含量。流式细胞术用于检测共培养体系中CD25+FOXP3+细胞的频率以及CD8+T细胞上CD25、CD69和程序性死亡受体1（PD-1）的表达。进行羧基荧光素二乙酸琥珀酰亚胺酯稀释实验以评估CD8+T细胞的增殖活性。

结果

研究发现ABAT在肝癌组织和细胞中表达下调，这种低表达表明患者预后较差，而其过表达则可抑制肝癌细胞的恶性程度。ABAT过表达后，细胞上清液中GABA水平降低，同时IL-10和TGF-β1细胞数量增加，CD25+FOXP3+细胞比例升高，而添加外源性GABA后这些变化均得以恢复。此外，ABAT的缺失导致CD8+T细胞的增殖和肿瘤杀伤能力降低，而应用GABAA受体抑制剂可逆转这些作用。