Background: Hepatocellular carcinoma (HCC) is most common primary liver cancer and the third leading cause of cancer mortality world-wide. Therefore, developing a new strategy to reduce the incidence of liver cancer is urgently needed. Cancer immunotherapy has been revolutionized by introducing an adoptive immune cell transfer (ACT) technique, using mostly T cells from patient or healthy individual to eliminate cancer cells. Recent ACT therapies begin to incorporate other immune cell types, like natural killer (NK) cells and γδ T cells. Many studies have demonstrated that ex vivo expanded γδ T cells exert cytotoxicity against HCC cells, making γδ T cells a feasible candidate for liver cancer. Herein hepatic intrasinusoidal (HI) γδ T cells were investigated as a novel candidate for immune cell therapy.

Methods: HI γδ T cells were isolated from liver perfusate and cultured for 2 weeks in the presence of IL-2, IL-15, and Zoledronate. Lactate dehydrogenase (LDH) assay and CD107a degranulation assay were performed to measure cytotoxicity of γδ T cells from liver perfusate and PBMC. Expression of activating receptors (NKG2D, NKp46, CD16, and DNAM-1), death ligand (TRAIL, and Fas-L), and inhibitory receptors (PD-1, BTLA, and TIGIT) were assessed by flow cytometry. CFSE-labeled HCC cell lines (Huh7, and SNU398) and HI γδ T cells were intraperitoneally injected into NSG mouse. After 48 hours, mice were sacrificed and CFSE+/CFSE- ratio was calculated. Pyrophosphate expression on HCC cell lines and human prostate cancer cell lines were measured. Flow cytometry was used to measure the expression of DNAM-1 ligands, CD112 and CD155.

Results: The results of degranulation assay and LDH assay showed that HI γδ T cells exhibited strong cytotoxicity against HCC cell lines, especially Huh7. HI γδ T cells expressed higher levels of NKG2D and DNAM-1 than PBMC γδ T cells. LDH assay with blocking antibodies revealed that HI γδ T cells relied on an activating receptor, NKG2D, in killing of SNU398. Huh7 cells might activate Vδ2 γδ T cells by expressing high level of pyrophosphate antigen. HI γδ T cells were shown to recognize pyrophosphate antigen on and kill human prostate cancer cells as well.

Conclusion: HI γδ T cells exhibited stronger cytotoxicity against HCC cells than PBMC γδ T cells. Huh7 cells, previously known as an NK-resistant cell line, were killed effectively by HI γδ T cells. HI γδ T cells utilized both pyrophosphate antigen and activating receptor. These results suggest that HI γδ T cells are a promising candidate for cancer immune cell therapy.