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Abstract

The development of a therapeutic human papillomavirus (HPV) vaccine is important to cure people that are already infected with HPV. However, multiple immune evasion mechanisms have been described for HPV-induced malignancies, including altered expression of antigen processing machinery (APM) components. These changes are able to directly influence epitope presentation and thus hamper CD8 T cell responses and are therefore regarded as considerable challenges in the development of a therapeutic vaccine. Until now, no systematic analysis of the APM had been performed in a large array of different HPV-positive tumor samples. Therefore, a systematic analysis of APM component expression on the mRNA level was performed in a comprehensive collection of HPV16-positive tumor cell lines in this study. The experiments revealed an upregulation of several APM components, which were subsequently investigated on the protein level. This analysis showed that the only component that was consistently upregulated in HPV16-positive tumor cells was the endoplasmic reticulum aminopeptidase 1 (ERAP1). Immunohistochemistry revealed that ERAP1 was more highly expressed in cervical intraepithelial lesions and cervical cancer than in nonmalignant cervical epithelium. Mechanisms that could account for ERAP1 overexpression were investigated. Single nucleotide polymorphisms (SNPs) of ERAP1 were analyzed in selected HPV-negative and HPV16-positive cell lines. Further, knockdown of HPV16 E6 and E7 was performed to investigate whether HPV oncogenes may influence ERAP1 expression levels. However, no influence of SNPs or the HPV oncogenes could be observed. ERAP1 plays a key role in editing the human leukocyte antigen (HLA) class I epitope repertoire. Therefore, we further focused on the question whether ERAP1 overexpression influences HPV16 E6 and E7 epitope presentation. To do so, an ERAP1 siRNA-mediated knockdown approach was established. HPV16-positive cells lines with attenuated ERAP1 expression or normal (over)expression were compared. To assess epitope presentation, a panel of HPV16-derived epitopes was analyzed using mass spectrometry. Additionally, T cell lines were generated from healthy donors, who showed memory responses against HPV16 epitopes. T cell lines were used to investigate the influence of ERAP1 expression levels in target cells on HPV16-specific CD8 T cell-mediated cytotoxicity. Mass spectrometry revealed that ERAP1 attenuation had no influence on presentation of the selected epitopes. Conversely, T cell assays showed that cytotoxicity against some HPV16-derived epitopes is influenced by ERAP1 expression levels, including one T cell line that showed enhanced killing towards HPV16-positive cells upon ERAP1 attenuation. Taken together, ERAP1 was observed to be highly expressed in HPV-induced malignancies. It was shown that ERAP1 influences the presentation of some, but not all, HPV16 E6 and E7-derived epitopes in an epitope-specific and tumor-specific way. In conclusion, ERAP1 overexpression can inhibit some HPV16-specific CD8 T cell responses and thus possibly represents a novel immune evasion mechanism of HPV-induced malignancies.