The role of natural killer cell killer immunoglobulin-like receptors in the development and treatment of hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is the fourth most common cause of cancer-related death worldwide, and incidence is increasing related to increasing prevalence of chronic liver disease. Survival is poor, and systemic treatments are limited. Immunotherapies are increasingly important in the treatment of many cancer types. NK cells are innate lymphocytes with an important role in tumour surveillance and are an attractive target for immunotherapy. Their function is regulated the balance of activating and inhibitory receptors, an important component of which is the killer immunoglobulin-like receptor (KIR) family. KIR genotype has been associated with the outcome of various diseases. The activating KIR2DS2 receptor recognises flavivirus peptides in the context of HLA-C. This is a potential therapeutic target. Exosomes are extracellular vesicles with a role in cellular communication, which are a potential vehicle for cancer immunotherapies. I aimed to investigate the relationship between KIR genotype and hepatocellular carcinoma development in patients with hepatitis C. I then aimed to produce exosomes expressing HLA-C and the Dengue virus peptide DP1, and examine their potential to activate NK cells, and enhance killing cancer cell lines. In a cohort of 169 patients with hepatitis C, no patients with the KIR2DS3 gene developed HCC (p=0.04). Increasing age was significantly associated with the development of cirrhosis (p=0.007) and HCC(p=0.02). Exosomes were successfully purified from a 721:221 cell line expressing HLA-C + DP1. This was confirmed with electron microscopy, nanoparticle tracking analysis and Western blotting. In-vitro co-culture of exosomes with peripheral blood mononuclear cells (PBMCs) from KIR2DS2 positive donors did not produce KIR2DS2 positive NK cell expansion, increased degranulation, or produce enhanced killing of HCC cell lines. Injection of DP1 exosomes into a KIR transgenic mouse model produced significantly increased total NK frequency (p=0.014), and increased frequency of mature CD27-/CD11b+ NK cells compared to controls. This effect was not peptide specific. KIR2DS2 positive NK cells exhibited significantly greater frequency of the liver residence and memory marker CXCR6 and activation marker CD69 than KIR2DS2 negative NK cells in C01 control and DP1 exosome injected mice. A heterotopic syngeneic murine cancer model using the RMA-S murine lymphoma cell line did not find any difference in survival, tumour volume, or NK cell tumour infiltration in DP1 exosome injected mice versus controls. In this cohort of patients with hepatitis C from the UK, KIR2DS3 positivity was significantly associated with reduced incidence of HCC. In-vitro, DP1 exosomes did not impact NK cell proliferation or function. In a mouse model DP1 exosomes were significantly associated with increased NK cell frequency and maturity, which were not peptide specific. A tumour model did not demonstrate any reduction in tumour growth in exosome injected mice.

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ORCID for Salim Khakoo: orcid.org/0000-0002-4057-9091

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