Dissection of the tonsillar cancer immune microenvironment : Perspectives of the myeloid APC – T-cell axis

Sammanfattning: Tonsillar cancer (TC) is a subset of head and neck cancer (HNC). TC incidence is sharply rising due to an increased prevalence of human papillomavirus (HPV) infection in the western world. Currently HPV status is considered during clinical staging due to its positive association to patient prognosis. Presumably, the relation between HPV and patient survival is dependent on the generation of an effective anti-viral immuneresponse. However, there is a lack of knowledge of the immune-phenotypes of TC, and of the activity of thecancer-immunity cycle in these lessions. Such knowledge is essential to develop predictive biomarkers of prognosis and treatment response. In the papers included in this thesis we aim to characterize the immunephenotypes of HPV+ TC, with special attention to the myeloid antigen presenting cell (APC)-T-cell axis, to broaden our understanding of the tumor immune-microenvironment and its clinical implications.In paper I, we described an increased infiltration of CD8+ T-cells, dendritic cell (DC), and macrophages in HPV+ TC compared to paired healthy tonsil. Intra-tumoral levels of CD8+ T-cells and DC were correlated to one another in HPV+ lesions, indicative of a cross-talk between these populations. In paper II, we investigated the distribution and targeted protein expression of CD8+ cells, using spatial proteomics in acohort of 105 TC patients. We observed that increased CD8 infiltration was associated with higher 5-year overall survival, independently of TNM7 classification, age at diagnosis and HPV status. HPV+ TC patients with low degree of CD8+ cell infiltration displayed a similar 5-year overall survival as HPV- patients, highlighting interpatient variations. Upon segmentation of CD8+ regions according to their location in the tumor, we observed an upregulation of immune checkpoint and effector molecules in CD8+ cells infiltrating cancer-cell nests, compared to CD8+ cells in the stroma. In addition, CD8+ T-cell density in cancer-cell nests, but not in stromal regions, was associated with a higher 5-year overall survival. Together, these results suggest an effective anti-tumoral function of CD8+ T-cells in cancer-cell islets. In paper III, we explored myeloid APC diversity in HPV+ TC and healthy tonsil using single-cell RNA-seq and flow cytometry. We identified four DC lineages, two macrophage polarization events, and described their maturation processess.Among the findings with clinical implications, we described that HPV+ TC featured higher levels of cDC1, and that high score of a cDC1-specific 3-gene signature was associated with higher 5-year overall survival in TC as well as HNC in general. Finally, in paper IV we developed an in vitro 3D holographyc imaging protocol to study TC-derived organoid biology. Using this protocol, we described that CD44+NGFR- cells displayed higher organoid forming capacity and higher motility than CD44-NGFR- and CD44+NGFR+ cells.Taken together, the research included in the present thesis highlights the interplay between myeloid APC and T-cells, and suggests several biomarkers that may be applied in clinical settings to assess prognosis.Our data also illustrate mecahnisms involved in immunosuppression as well as in succesful immunosurveillance related to HPV+ TC biology.

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