Characterization of Epithelial Progenitors in Normal Human Palatine Tonsils

The palatine tonsils are a collection of lymph nodes overlaid by stratified non-keratinized epithelium that invaginates deep into the tissue, forming tonsillar “crypts” where ingested and inhaled pathogens are collected and initiate an immune response followed by transepithelial lymphocyte infiltration. The dynamic nature of this site suggests the existence of primitive cells responsible for the constant tissue repair and regeneration; however, such cells in the tonsils have not been characterized. Human Papillomavirus (HPV)-associated cancer of oropharynx is a global health concern that is on the rise, with HPV16 oncoproteins E6/E7 frequently detected in the epithelium of tonsillar crypts. It is hypothesized that the long-term self-renewing progenitor cells are the target of HPV-induced malignancy, but the lack of a method to specifically study these critical cells has been a barrier to further investigation. In this study, I have developed and optimized the methodology to identify, isolate and quantitate epithelial progenitors from human palatine tonsil. I show that tonsillar progenitors that form colonies in vitro in 2D colony assays and differentiate into multilayered epithelial tissues in a 3D culture system are CD44+NGFR+ and present in both surface and crypt regions. Transcriptome analysis indicates a high similarity between CD44+NGFR+ cells in both regions, although those isolated from the crypt contained a higher proportion of the most primitive (holo)clonogenic cells. The method was then applied to study effects of HPV infection on purified CD44+NGFR+ cells from both regions. Lentiviral transduction of CD44+NGFR+ cells with HPV16 E6/E7 oncogenes prolonged their growth in 2D cultures and caused aberrant differentiation in 3D cultures. Interestingly, in the presence of the normal cells, the E6/E7-transduced cells proliferated more slowly in 2D cultures and formed uniquely heterogeneous epithelial structures displaying varying degrees of perturbation in 3D cultures suggesting possible inhibitory effects of the cocultured normal cells. The system developed and presented in this thesis allows for a targeted approach to study a specific subset of epithelial cells purified from the tonsillar crypts and their response to E6/E7 infection, setting the stage for addressing many unanswered questions pertaining to the early stages of tonsillar oncogenesis.