The Role of Large Extracellular Vesicles in Human Papillomavirus-Associated Immune Modulation of Langerhans Cells

Human papillomavirus type 16 (HPV16) is an oncogenic virus that infects basal keratinocytes of stratified squamous epithelium. Langerhans cells (LCs) reside within the stratified squamous epithelium and initiate immune responses against invading pathogens. The suppression of LC function is one of the many mechanisms of HPV immune evasion, which can contribute to viral persistence and cancer development. Extracellular vesicles (EVs) are small, cell-derived particles that are key players to carcinogenesis, immunity and viral spread. Previous work in the Hibma laboratory has attributed impairment of LC activation and suppression of the cytotoxic T cell response to interactions with large EVs (lEVs) shed by keratinocytes expressing HPV16 E7. The present study aimed to understand the effects of both HPV16 E6 and E7 (E6/E7) genes on the production of lEVs by keratinocytes and investigate the regulatory roles of lEVs produced by keratinocytes expressing HPV16 E6/E7 on the activation of and cross-presentation by LCs. Mouse (C57Bl/6) immortalised keratinocyte (PDV) cell lines expressing HPV16 E6/E7 or empty vector control were established following lentivirus transduction. Cell lines were validated, and lEVs from the cell culture supernatants of PDV cells expressing HPV16 E6/E7 (E6/E7 lEVs) or empty vector control (Ctrl lEVs) were purified by differential centrifugation. The purification step was followed by a number of methods to characterise the protein profile, morphology, size and numbers of lEVs. In addition, co-culture experiments were conducted to investigate the effects of E6/E7 lEVs on the activation of LC-like cells (LCLCs) differentiated from the bone marrow progenitors of C57Bl/6 mice. We found that the overall amounts of lEVs determined by counts and total protein were increased from PDV cells expressing HPV16 E6/E7 compared with empty vector control. Following co-culture with E6/E7 lEVs, LCLCs expressing intracellular IL-12 were markedly downregulated when compared with Ctrl lEVs. In contrast, expression of cell surface co- stimulatory molecules, CD40, CD80 and CD86, on LCLCs was not affected. Furthermore, the effect of E6/E7 lEVs on cross-presentation by LCLCs was examined by measuring the proliferation of CD8+ T cells in response to LCLC-presented ovalbumin. Co-incubations with E6/E7 lEVs resulted to a significantly reduced proliferation of CD8+ T cells when compared with Ctrl lEVs. This effect was ablated when LCLCs derived from Transporter associated with Antigen Processing (TAP1) knockout mice were used, suggesting the involvement of TAP-dependent cross-presentation pathways. In conclusion, lEVs produced by keratinocytes expressing HPV16 E6/E7 impair LCLC activation and function. A better understanding of E6/E7 lEVs may translate to the production of drugs to inhibit their release and synergise with therapeutic vaccines in development to improve their efficacy in treating HPV-associated malignancies