HPV regulation of immunity in persistence and regression

Abstract

High-risk human papillomavirus (HPV) infection leads to the development of several human cancers that cause significant morbidity and mortality worldwide. Cervical cancer is the most prevalent type of HPV-caused cancers and is the second leading cause of death in women worldwide. High risk HPV type 16 (HPV16) is responsible for 70% of the cervical intraepithelial neoplasia (CIN) and cervical cancers and the persistent over-expression of oncogene HPV16 E7 (E7) is necessary for carcinogenesis. Currently available vaccines have no therapeutic effect for prevalent infection or already established HPV-associated disease. Besides, due to other factors like poor vaccine uptake, an increase in the global burden imposed by HPV has been forecasted. Additionally, current therapies for HPV caused cervical abnormalities include excision of infected tissue, which can have other health implications like infertility and preterm birth risks in women and therefore need to follow an accurate diagnosis. This necessitates the development of effective immune therapies and of effective biomarkers for HPV associated anomalies. In this study, we aim to, firstly, understand the effects of HPV oncoproteins on immune responses and, secondly, to explore new biomarkers for the prognosis of CIN2. In order to understand the effects of E7 on antigen presentation and downstream T-cell responses in vivo, replication defective lentiviral vectors were used to deliver E7 or E7 cloned in reverse (E7rev), a reporter protein luciferase and a model antigen ovalbumin (OVA) exclusively to the mouse ear epidermis. We found that in comparison to E7rev, co-expression of E7 in Ova-expressing cells strongly suppressed proliferation of lymph node CD8+ T-cells in response towards Ova and reduced the activation of lymph node dendritic cells (DCs). We observed that E7 expression also caused a reduction in migration of and antigen uptake by epidermal Langerhans cells (LCs). Surprisingly, we did not we observe any effect on the reduced T-cell proliferation to OVA following depletion of LCs and langerin positive skin dermal DCs, suggesting that these cells are not involved in activation the CD8 T cell response to OVA. This indicated that other cell populations or cellular factors may be involved in causing immune suppression in lymph nodes. Since E7 was also seen to affect lymph node DC populations, we explored any role keratinocyte-derived exosomes may play in E7 mediated suppression of immune responses. Exosome purification methods were used to purify exosome-like vesicles (ELVs) from E7 and control protein expressing murine keratinocyte cell line. We observed that ELVs from E7 (E7-ELVs) expressing mouse keratinocytes significantly reduced co-stimulatory marker expression on DCs, and proliferation of OVA specific T-cells in vitro when compared to ELVs from control keratinocytes (control ELVs). Preliminary analysis of E7 vs control ELV proteins by mass spectrometry showed differences in relative quantities of proteins associated with immune functions like CD73, 14-3-3 and casein kinase 3. These data indicate that exosomes derived from E7 expressing keratinocytes may have immune-modulatory potential. To address the need for biomarkers that can predict the outcome of HPV caused diseases, we assessed human cervical biopsies from CIN2 patients for the expression of various cellular and immune markers using immunohistochemistry in a retrospective study. A total of 27 women under the age of 25 were involved in the study, 14 of which were persistors and 13 were regressors. Immunohistochemisty for the cellular markers P16, Ki67, E-cadherin, p53 and pRb; and immune markers CD4, Foxp3, CD8, granzyme, langerin and CD3 was performed on formalin fixed paraffin embedded tissues from all participants. Results were stratified by disease outcome. Significant increase in P16 expression was observed in progressors while the immune markers, CD4+ T-cell numbers and percentage of granzyme+CD8+ cell numbers decreased with progression. Thus, we established that P16 and number of helper and cytotoxic T-cells may be used as biomarkers for prediction of CIN2. Overall, the results from this study provided an insight into the cellular and immune-microenvironment of HPV infection, factors that may dictate the progression of regression of infection. This data will have implications in understanding of new mechanisms of antigen presentation, development of therapies and providing new biomarkers for clinical diagnosis of CIN in women.