Background

More effective immunotherapeutic approaches are urgently needed for hepatocellular carcinoma (HCC). Gamma delta (γδ) T-cells are promising candidates for cancer immunotherapy, due to their potent cytotoxicity, tissue localisation and HLA-unrestricted tumour reactivity. We characterised liver and tumour infiltrating γδT-cells in HCC, and explored whether modulating features of tissue-residency could provide a novel immunotherapeutic approach.

Method

Lymphocytes isolated from paired blood, liver, and tumoural tissue from patients with HCC (n=29) in comparison to colorectal cancer liver metastases (n=30) were analysed by flow cytometry. γδT-cell counts were determined by immunostaining. Long-lived persistence of intrahepatic γδT-cells was examined using donor/recipient HLA-mismatched liver allografts (7-11 years post liver transplantation). Aminobisphosphonate (Zoledronate) and IL-2 expanded blood Vγ9Vδ2T-cells, intrahepatic lymphocytes, and tumour-infiltrating lymphocytes, were co-cultured with human hepatoma cell-lines pre-treated with Zoledronate to promote tumour-cell phosphoantigen accumulation for Vγ9Vδ2T-cell activation.

Results

Higher intratumoural γδT-cell counts associated with greater HCC patient survival (p<0.01). γδT-cells exhibited a tissue-resident memory (T_RM) phenotype (CD69⁺CD49a⁺) in human liver and HCC, with superior anti-tumour cytokine production and long-lived persistence in the liver (>10 years), an attractive profile to recapitulate with immunotherapy. A subset of γδT-cells, Vγ9Vδ2 T-cells, were selectively depleted within HCC but displayed the highest γδT_RM phenotype. Expansion of blood Vγ9Vδ2 T-cells in vitro using clinically approved Zoledronate and IL-2 induced a de novo T_RM phenotype with increased liver-homing chemokine receptor expression (CXCR6⁺CXCR3⁺) and improved cytotoxicity. Furthermore, direct sensitisation of hepatoma cell-lines with Zoledronate enhanced the anti-tumour function (IFNγ,TNFα) of co-cultured expanded Vγ9Vδ2 T-cells and Vγ9Vδ2 T-cells isolated from HCC livers and tumours, with increased tumour-cell lysis.

Conclusion

γδT_RM cells demonstrate long-lived immunotherapeutic properties for HCC. We show that aminobisphosphonates such as Zoledronate can enhance the efficacy of γδT-cell immunotherapy for HCC via two mechanisms: direct delivery to the tumour increases Vγ9Vδ2 T-cells anti-tumour function, whilst in vitro expansion of blood Vγ9Vδ2 T-cells for adoptive cell transfer confers de novo tissue residency to achieve long-lived local tumour immunosurveillance.

Impact statement

Our findings indicate a novel immunotherapeutic strategy for HCC; future trials could combine use of aminobisphosphonates to induce Vγ9Vδ2T_RM capable of replenishing the depleted pool, with intra-tumoural delivery to sensitise HCC to Vγ9Vδ2 T-cell based targeting.