Hepatocellular carcinoma-infiltrating γδ T cells are functionally defected and allogenic Vδ2⁺ γδ T cell can be a promising complement

In hepatocellular carcinoma (HCC), γδ T cells participate in mediating the anti-tumour response and are linked with a positive prognosis. However, these cells can become pro-tumoural in the tumour microenvironment (TME). We aimed to decipher the immune landscape and functional states of HCC-infiltrating γδ T cells to provide fundamental evidence for the adoptive transfer of allogeneic Vδ2⁺ γδ T cells in HCC immunotherapy. We performed single-cell RNA sequencing (scRNA-seq) on γδ T cells derived from HCC tumours and healthy donor livers. Confocal microscopy, flow cytometry and a Luminex assay were applied to validate the scRNA-seq findings. The γδ T cells in the HCC TME entered G2/M cell cycle arrest, and expressed cytotoxic molecules such as interferon-gamma and granzyme B, but were functionally exhausted as indicated by upregulated gene and protein LAG3 expression. The γδ T cells in the HCC TME were dominated by the LAG3⁺ Vδ1⁺ population, whereas the Vδ2⁺ γδ T population was greatly depleted. Moreover, glutamine metabolism of γδ T cells was markedly upregulated in the glutamine-deficient TME. Both in vitro and in vivo experiments showed that glutamine deficiency upregulated LAG3 expression. Finally, our results indicated that ex vivo-expanded Vδ2⁺ γδ T cells from healthy donor could complement the loss of T cell receptor clonality and effector functions of HCC-derived γδ T cells. This work deciphered the dysfunctional signatures of HCC-infiltrating γδ T cells in the HCC TME, providing scientific support for the use of allogeneic Vδ2⁺ γδ T cells in HCC cellular therapy.