High-risk human papillomavirus E7 expression reduces cell-surface MHC class I molecules and increases susceptibility to natural killer cells

Abstract

High-risk human papillomavirus (HPV) is a major causative agent of cervical cancer and the E6 and E7 genes encode the major HPV oncoproteins. The E7 protein from high-risk HPV types alters cell cycle progression and represses genes encoding components of the antigenpresentation pathway, suggesting a role for E7 in tumour immune evasion. We show that knockdown of E7 expression in HPV16-and HPV18-transformed cervical carcinoma cells by RNA interference increased expression of major histocompatibility complex (MHC) class I at the cell surface and reduced susceptibility of these cells to natural killer (NK) cells. Tetracycline-regulated induction of HPV16 E7 resulted in reduced expression of cell surface MHC class I molecules and increased NK cell killing. Our results suggest that, for HPV-associated malignancies, reduced MHC class I expression is the result of an active immune evasion strategy that has evolved to assist viral replication. The high-risk human papillomavirus (HPV) types 16 (HPV16) and 18 (HPV18) have a strong association with the incidence of cervical cancer, with at least 90% of cervical adenocarcinomas harbouring HPV16 or HPV18 DNA (Walboomers et al., 1999). The E6 and E7 oncogenes are continuously expressed following cellular transformation and in cervical cancer tissues while expression of the E5 gene contributes to transformation but is not expressed in cervical tumours (Howley and Lowy, 2007). Many viruses and tumours evade the T-cell-mediated immune response, primarily by reducing the levels of surface MHC class I, thus reducing the presentation of viral/tumour antigens (Garcia-Lora et al., 2003;Hewitt, 2003). We have previously shown that the E7 oncoproteins of the high-risk HPV16 and 18 types are able to repress the activity of promoters encoding components of the MHC class I antigen processing and presentation pathway (Georgopoulos et al., 2000) and recent data further suggest that HPV16 E7 interacts with the MHC class I heavy chain promoter in HPV16-transformed human cells (Li et al., 2006). Here, we have used two complementary approaches to evaluate the effect of HPV16 and 18 E7 expression on the levels of cell-surface MHC class I molecules and consequent effects on natural killer (NK) cell recognition.In the first approach, E7 expression was reduced in HPV-transformed cells by transfection of an E7-specific short interfering RNA (siRNA) molecule (Jiang and Milner, 2002;Hall and Alexander, 2003). HPV16-transformed cells (SiHa and CaSki) and HPV18-transformed cells (HeLa) were mock-transfected or transfected with E7-specific or control siRNAs and the level of E7 mRNA analysed 72 h post-transfection by quantitative real-time RT-PCR (Figure 1a). This showed that the E7-specific siRNA reduced E7 mRNA sequences by approximately 90% in the HPV16-and HPV18-transformed cell lines. Treatment of the three HPV-transformed cell lines with E7-specific siRNA induced growth arrest and profound morphological changes in transfected cells, as described previously (Hall and Alexander, 2003); however, these ...

Identification of Key Amino Acid Residues That Determine the Ability of High Risk HPV16-E7 to Dysregulate Major Histocompatibility Complex Class I Expression

“…Both HPV-E7 variants were predominately found in the nucleus and showed retinoblastoma protein (pRb) binding properties reflecting the different interaction affinities of high and low risk HPV-E7 (19). In line with previous findings (14,15,20) we show that HPV16-E7 causes a significant reduction of MHC I expression and surface presentation, whereas no such dysregulation was observed for HPV11-E7. In addition, we observe that stable transfectants expressing HPV11-E7 or HPV16-E7 have comparable steady-state expression levels of TAP and tapasin, indicating that both HPV-E7 proteins apparently do not modulate the synthesis of components of the peptide-loading complex (PLC).…”

Local immune response in the microenvironment of CIN2–3 with and without spontaneous regression

“…To facilitate immune evasion during viral replication, viruses use a wide range of mechanisms to inhibit antigen presentation. HPV-16, and HPV-18, E7 expression decreases the levels of cell-surface MHC-I molecules, 32 thereby reducing the presentation of the viral antigens, which indicates that the balance between cytotoxic T-lymphocytes (CD8 þ ) and T-regulatory lymphocytes (CD25 þ ) has an important role in the development of CIN2-3 lesions. Indeed, the regressive CIN2-3 lesions had higher ratios of CD4 þ /CD25 þ cells both in the stroma and in the epithelium, and higher …”

“…The active strategy adopted by hrHPV E6 and E7 for evading the immune response by reducing the levels of surface MHC class-I molecules is responsible for suppression of the T-cell-mediated immune response and NK cell recognition by reducing the presentation of the viral and tumor antigens. 32 This might lead to chronic disturbance of the tissue homeostasis and an imbalance between cytotoxic T-lymphocytes and suppressor T-cells, as shown by lower ratios of CD8 þ /CD25 þ cells and favoring cancer development.…”

Human papillomavirus entry into NK cells requires CD16 expression and triggers cytotoxic activity and cytokine secretion

“…The presence of L1 seems to be important for dysplasia regression since this phenomenon has been correlated to L1 protein expression [45]. Moreover, E7 protein from high-risk HPVs has been shown to reduce cell surface expression of MHC Class I molecules [46], rendering these cells susceptible to lysis by NK cells. However, viruses have developed mechanisms to avoid the immune response and some of these mechanisms are directed in complete RPMI 1640 with 800 U/ml human recombinant GM-CSF and 40 U/ml IL-4 (ImmunoTools, Friesoythe, Germany).…”