HPV induced cervical neoplasia and cancer: Characterization of local immune infiltration and mapping of T cell recognition towards HPV

Human Papilloma Virus (HPV) is the primary cause of cervical cancer. It is evident that an impaired immune system plays an important role in the persistence of the viral infection, oncogenic transformation, and cancer development. Patients with advanced, recurrent, or metastatic cervical cancer still have poor prognosis and improved treatment strategies are needed.

Recognition and elimination of cervical neoplasia and cancer is a multifactored interplay of the immune system, which can both promote and reject tumor growth. Immune therapy has shown immense potentials and is now a major contributor in cancer treatment where immune checkpoint inhibitors (ICI) presents the most advanced therapy available.

ICI treatments are capable of reinvigorating the functional capacity of exhausted T cells to kill the affected cells. With cancer immune therapy, the ambition is to achieve a long-lasting ability to detect and eliminate foreign tumor antigens. However, despite the promising developments within immune therapy, little is known in the tumor microenvironment about characteristics of immune infiltration which governs peptide-MHC T cell recognition and immune activation.

The overall goal of the research presented in this PhD thesis, was to characterize local and systemic immune infiltration, phenotype characteristics, state of activation, signs of T cell exhaustion in patients with high-grade intraepithelial neoplasia (CIN3) and cervical cancer compared to healthy individuals.

The main observation was detection of a late differentiated immune profile among CD8 and CD4 T cells in the cancer group. The frequency of terminally activated or even exhausted CD8 T cells was more abundant in CIN3 lesions and even further increased in the cancer patients, compared to the healthy individuals.

Cells from biopsy and cytology were evaluated and strikingly, these specimens displayed identical signatures, hence suggesting cytology as a useful alternative to biopsies for evaluation of immune signature in cervical neoplasia and cancer. The analysis of blood demonstrated unique immune phenotypic characteristics associated with cancer, but different from those signatures found in cytology and biopsies.

For the investigated myeloid compartment we observed lower levels of classical antigen presenting cells, while myeloid populations in general expressed higher levels of PD-L1, compared to the same subsets of cells in the healthy individuals. All together, data suggest that immune recognition plays an active role in shaping the neoplastic development, and that immune inhibitory mechanisms emerge during cancer development.

Research presented in this thesis also included mapping of HPV-restricted T cell recognition. 685 potential distinct human leucocyte antigen (HLA)-binding peptides were evaluated covering E2, E6 and E7 genes of both HPV 16 and HPV 18. This was done to examine CD8 T cell recognition of Human Papilloma Virus.

The cells were analyzed using DNA-barcoded peptide-MHC complex multimers, and we were thereby able to detect 127 immunogenic epitopes recognized by CD8 T cells. The majority of the predicted epitopes came from the E2 protein, and this was also where most epitopes were recognized. Conclusively, the E2 gene must be understood as a very immunogenic region of the HPV genome.

Our results were validated using tetramer staining assays on selected CD8 T cells and the recognized peptides were confirmed. Among the three study groups, a higher number of recognitions to HPV derived peptides were found in both the neoplasia and cancer group compared to the healthy individuals. The HLA-C05:01 allele turned out to be very dominant in the total number of identified epitopes but some skewing due to cross-reactivity is likely the case.

These results provide insight into the CD8 T cell recognition and the immunogenic hotspots of interest, and this can hopefully be of use in the future, when designing immune therapy and deciding the coveted targets of Human Papilloma Virus.