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Interaction of the host immune system with tumor cells in human papillomavirus associated diseases

Sauer, Madeleine

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Human papillomaviruses (HPV) are very common in the sexually active population and contribute to 610,000 cancers per year occurring at different locations. The initial step of HPV-related carcinogenesis is the induction of transforming processes in the host cells mediated by the viral oncoproteins E6 and E7 that interfere with critical host cell pathways. The transforming infection is highlighted by overexpression of the tumor suppressor protein p16INK4a. Only a small number of precancerous lesions progress while the majority can be controlled by the host’s immune system and undergo regression. Progressing lesions under the immunoselective pressure seem to acquire characteristics that enable them to circumvent the host’s immune attack and promote disease progression. Immune evasion might be mediated by the immune microenvironment of the tumor as well as by tumor cell intrinsic features. The here presented thesis addressed different questions and strategies with regard to the role of the immune system in HPV-associated diseases and can be subdivided in two main parts: In the first part immunologic characteristics of precancerous lesions and cancers are investigated to gain insight into possible immune evasion mechanisms developed during disease progression. In the second part treatment options to positively influence the balance between immune evasion and anti-tumoral immune responses are evaluated. In the first part a) the immunohistochemical characterization of cervical precancers and cancers for infiltration with different T cell phenotypes revealed that generally increasing T cell densities occur late in carcinogenesis – and not yet with the onset of early transforming infection - and are accompanied by immunosuppressive regulatory T cells (Tregs). Mean cell densities for Tregs in the stroma significantly increased from 121.6 cells/mm2 (range: 24-286.8 cells/mm2) in low-grade lesions to 308.8 cells/mm2 (24-724.8 cells/mm2) in high-grade lesions and 673.6 cells/mm2 (52.8-1564.8 cells/mm2) in cancer which points to their immunosuppressive role during carcinogenesis. The demonstrated large variances in T cell densities within one diagnostic category, however, point to a remarkable heterogeneity of the immune control with potential interesting prognostic implications. On keratinocytes themselves b) a selective loss for human leukocyte antigen (HLA) class I heavy chain A expression was observed in about 55% high-grade cervical intra-epithelial neoplasia (CIN) and 65% of cervical cancers. HLA class II de novo expression was found in 50% of low-grade CIN and in about 85% of high-grade CIN and cervical cancers. These alterations could represent another fundamental mechanism contributing to immune evasion. A c) longitudinal analysis of immune infiltrates in patients treated with imiquimod, an immuno-modulatory Toll-like receptor (TLR) agonist, revealed that the patient’s local immune constitution might be decisive for a possible response to immune-enhancing treatment strategies. Importantly, in patients responding to imiquimod immune cell densities increased during the treatment as epithelial CD3+ T cell counts (from 160.8 to 371.1 cells/mm2) and CD8+ T cell counts (from 113.8 to 174.1 cells/mm2) demonstrated. The d) development and establishment of an automated cell quantification tool for high-throughput analysis allows the search for immune evasion markers and strategies to be continued in an objective, standardized and faster way. In consideration of the clinical efficacy of imiquimod and the observed stimulatory effects on the immune infiltrate density in part one of this thesis e) a new second generation TLR-agonist (TMX-202) potentially having less side-effects than imiquimod was tested for the first time in an in vitro T cell stimulation model in part two of this thesis. Its potential to stimulate innate and adaptive immunity was demonstrated by an enhanced killing capacity of T cells that were stimulated with HPV-related antigens loaded on dendritic cells and then co-incubated with HPV16-positive CaSki cells. Based on the dense infiltration with Tregs observed in part one of the presented thesis the f) immune stimulating effects of Treg depletion was tested in an autologous in vitro model. In this regard, one major aim of the thesis was the generation of a new HPV-positive tumor cell line derived from an oropharyngeal squamous cell carcinoma that serves as model system for HPV-associated tumors. In combination with peripheral blood lymphocytes obtained from the same patient this autologous system allowed to address Treg depletion as an immunotherapeutic approach. The results demonstrated that this strategy might enhance the cell-mediated immune response against tumor cells and emphasize the role that this particular T cell phenotype is obviously playing in the carcinogenesis of HPV-associated tumors. Based on the results obtained in the first part of the thesis it is well conceivable that the combination of different immunologic markers contributes to the definition of a prognostic biomarker tool for progression and regression of precancerous lesions. Such a prognostic “immune score” has a high clinical relevance and allows risk-adapted treatment decisions minimizing the costs and long-term sequelae of surgical interventions. In particular the newly developed microscopy based method in this work allowing for the automated histological high-throughput quantification of infiltrating immune cells in cervical intraepithelial neoplasia provides an important methodical tool to realize this long term goal. The immuno-stimulating effects of the novel TLR7-agonist TMX-202 and Treg depletion demonstrated in the second part of this thesis by in vitro models indicate that immunomodulatory approaches could play an important role for the treatment of HPV-associated cancers in the future. In this regard, the established novel tumor cell line in combination with autologous immune cells provides a valuable in vitro model system for HPV-associated cancers that can be used to investigate further immunotherapeutic intervention and treatment strategies.

Item Type: Dissertation
Supervisor: Müller, Prof. Dr. Martin
Date of thesis defense: 29 January 2015
Date Deposited: 16 Feb 2016 11:21
Date: 2016
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Medizinische Fakultät Heidelberg > Pathologisches Institut
Subjects: 570 Life sciences
610 Medical sciences Medicine
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