Human papillomavirus E7 oncoprotein expression by keratinocytes alters the cytotoxic mechanisms used by CD8 T cells

// Purnima Bhat 1, 2 , Anne-Sophie Bergot 1 , Nigel Waterhouse 3 and Ian Hector Frazer 1 1 University of Queensland Diamantina Institute, University of Queensland, Translational Research Institute, Brisbane, Qld, Australia 2 Medical School, Australian National University, Canberra, Act, Australia 3 QIMR Berghofer Medical Research Institute, Brisbane, Qld, Australia Correspondence to: Ian Hector Frazer, email: i.frazer@uq.edu.au Keywords: cervical cancer; head and neck cancer; immunotherapy; cell-mediated killing; time-lapse microscopy Abbreviations: HPV, human papillomavirus; KC, keratinocyte; OVA, ovalbumin; EGFP, enhanced green fluorescent protein Received: November 30, 2016     Accepted: April 17, 2017     Published: December 14, 2017 ABSTRACT Cervical cancer is a malignant transformation of keratinocytes initiated by the E7 oncoprotein of human papillomavirus (HPV). These tumors are characterized by keratinocyte hyperproliferation and are often infiltrated with activated CD8 T cells. HPV infection confers changes to gain immunological advantage to promote chronic infection, and these persist with malignant transformation. We investigated the relative importance of the many redundant mechanisms of cytotoxicity used by CD8 T cells to kill keratinocytes expressing HPV E7 oncoprotein using extended-duration time-lapse microscopy that allows examination of cell-to-cell interactions during killing. E7 expression by keratinocytes increased susceptibility to cell-mediated killing. However, while killing of non-transgenic keratinocytes was traditional, perforin-mediated, and caspase-dependent, E7-expression favored killing by perforin-independent, caspase-independent mechanisms. The roles of perforin, TNFα, IFNγ, Fas/FasL and PD1/PD-L1 were graded according to target cell survival to produce a hierarchy of killing mechanisms utilized in killing E7-expressing cells. TNFα was essential for perforin-mediated killing of E7-expressing cells, but not perforin-independent killing. IFNγ facilitated killing by Fas/FasL interaction, especially in the absence of perforin. Additionally, expression of E7 offered protection from killing by up regulation of PD-L1, Fas and FasL expression on keratinocytes promoting fight-back by target cells, resulting in effector cell death. This study shows that keratinocytes expressing E7 are highly susceptible to killing by CD8 T cells, but utilizing different armamentarium. Down-regulation of CD8 T cell cytotoxicity in HPV-related tumors may be due to suppression by E7-expressing keratinocytes. Immunotherapy for HPV-related cancers may be improved by suppression of PD-L1, or by suppression of FasL.