Overcoming GABA-induced Treg suppression of immunity by ABAT to augment CD8+T cell anti-tumor immune response in liver cancer.

Abstract

Background: ABAT, a key enzyme in GABA catabolism, modulates anti-tumor 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 EMT-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 co-culture. Carboxifluorescein diacetate succinimidyl ester (CFSE) 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.