Regulation of Antigen Presenting Cells by Human Papillomavirus Small Extracellular Vesicles

Abstract

Human Papillomavirus (HPV) causes over 99% of cervical cancer cases in the world, and of these, over 70% of cases can be attributed to high-risk HPV. There are prophylactic vaccines for HPV, that specifically target high-risk HPV, however, these are not therapeutic and are ineffecive for those already infected with the virus. Further research into HPV and potential therapies is essential to provide treatments pathways to those living with HPV infection. E6 and E7 are oncoproteins present in the HPV genome, and are highly expressed in high-risk HPV type genomes. Persistent expression of these oncoproteins, combined with subversion of the immune system is what causes carcinogenesis to progress. Previous work in the Hibma laboratory has shown a potential way HPV subverts the immune system is by release of large extracellular vesicles (EVs), termed microparticles. This was attributed to HPV oncoprotein- expressing microparticles causing downregulation of co-stimulatory molecule CD40 and cytokine IL-12 expressed by langerhans cells (LCs). Our aim for this research was to determine if a cell line expressing the E6/E7 oncoproteins was able to express small EVs, then, to co-culture these small EVs with mouse bone marrow derived dendritic cells (BMDCs), the professional antigen presenting cell (APC) of the epidermal layer of skin. We were aiming to see the effects that these small EVs had on antigen presenting funtions of BMDCs. Small EVs were purified from an HPV E6/E7 expressing cell line (TC-1), and an HPV negative cell line (PDV) using differential centrifugation methods, followed by a sucrose cushion centrifugation method. EVs were characterised by presence of protein markers, CD9 and CD81, via flow cytometry, and by visual imaging/ sizing using negative staining and cryogenic electron microscopy. Bone marrow were extracted from mice and differentiated into (BMDCs)via granulocyte-macrophage colony-stimulating factor, then purified small EVs were co- cultured with BMDCs for 24 hours. Effects on co-stimulatory molecle expression and cytokine expression was analysed via flow cytometry. It was found that small EVs from the TC-1 HPV E6/E7 expressing cell line caused an increase in co-stimulatory molecule CD40 expression, and cytokine IL-12 expression from BMDCs, when compared to small EVs from the PDV HPV negative cell line. These findings suggest small EVs from this particular cell line may regulate antigen presenting functions and have immunostimulatory effects on the immune system.