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Investigation of the effect of hypoxia on antigen presentation and T cell-based cytotoxicity of HPV16-transformed cells.

Mohan, Nitya

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Abstract

Human papillomavirus (HPV)-induced malignancies have long been considered the ideal scenario for the development of a therapeutic cancer vaccine, as viral proteins can serve as immune targets. However, the attempts to develop a therapeutic vaccine against HPV-induced malignancies have not been clinically successful to date. One possible reason may be the hypoxic microenvironment present in most tumors, including cervical cancer. Recent studies have shown that hypoxia leads to decreased levels of E6 and E7 proteins in HPV-transformed cells. Hypoxia is also known to dysregulate the levels of HLA I (human leukocyte antigen class I) molecules in the context of different tumors. Furthermore, hypoxia has been reported to both enhance and suppress cytotoxic CD8+ T cell functions. Hence, the aim of this project is to investigate the effect of hypoxia on antigen presentation in HPV16-transformed cells as well as their targeting by CD8+ T cells. In this study, it was observed that hypoxia led to downregulation of the protein levels of HPV16 oncoproteins E6 and E7 in several cervical cancer cells transformed with different HPV16 variant lineages. More than 24 hours of hypoxia were needed for complete loss of expression of the E7 oncoprotein in HPV16-transformed CaSki cells. Presentation of E6 and E7-derived epitopes on the cell surface, in context of HLA class I (HLA-I) molecules, is essential for recognition and targeting of these cells by cytotoxic T cells. Peptide presentation occurs with the help of a repertoire of proteins (proteasome, transporters, chaperones and enzymes) known as the antigen processing and presentation machinery (APM). Any perturbations in the APM could result in an altered epitope repertoire on the cell surface. Thus, the effect of hypoxia on the APM was investigated in different HPV16-transformed cells, as well as two HPV negative control cells, using label free quantitation (LFQ) mass spectrometry. Next, using flow cytometry, the effect of hypoxia on the surface expression of the HLA-I (HLA-A2) molecule was assessed. Interestingly, no significant change was observed in the expression of any of the APM proteins. The surface levels of HLA-A2 were also not significantly affected by hypoxia in any of these cells. No effect of hypoxia on the APM is promising from the context of therapeutic vaccine design. However, for the success of the vaccines, it is essential that the HPV16-transformed cells are actually targeted by cytotoxic CD8+ T cells. Interestingly, enhanced killing of CaSki cells by E6 and E7-derived peptide-specific CD8+ T cells was observed in a majority of the cases, with the exception of one CD8+ T cell line, which showed suppressed killing upon hypoxia. The contradictory results are however consistent with the contradictory results published about the effect of hypoxia on CD8+ T cells, in the context of different tumors. Enhanced cytotoxicity in most cases was surprising given the results that the target proteins are decreased in hypoxia. Using hypoxia- VIII preincubated targets, it was demonstrated that the enhanced killing of CaSki cells under hypoxia is suppressed, with the likely reason being the decreased expression of the source oncoprotein under hypoxia. Thus, the inhibitory effect of hypoxia on the target cells seems to supersede the positive effect of hypoxia on the CD8+ T cells. In summary, the results obtained in this thesis provide important insights into the effect of hypoxia on cervical cancer cells and their targeting by CD8+ T cells. The study highlights the importance of considering the effect of the tumor microenvironment (TME), particularly hypoxia, while developing immunotherapies. Incorporating these insights into developing combination therapies will hopefully pave the way to making immunotherapies a mainstream standard of care for HPV-induced malignancies.

Item Type: Dissertation
Supervisor: Riemer, PD Dr. Dr. Angelika
Place of Publication: Heidelberg
Date of thesis defense: 31 August 2021
Date Deposited: 01 Oct 2021 07:30
Date: 2022
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 500 Natural sciences and mathematics
Controlled Keywords: Hypoxia, Antigen presentation, Cytotoxic T cells, HPV16
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