Effects of hypoxia on antigen presentation and T cell-based immune recognition of HPV16-transformed cells.

Attempts to develop a therapeutic vaccine against human papillomavirus (HPV)-induced malignancies have mostly not been clinically successful to date. One reason may be the hypoxic microenvironment present in most tumors, including cervical cancer. Hypoxia dysregulates the levels of human leukocyte antigen (HLA) class I molecules in different tumor entities, impacts the function of cytotoxic T cells, and leads to decreased protein levels of the oncoproteins E6 and E7 in HPV-transformed cells. Therefore, we investigated the effect of hypoxia on the presentation of HPV16 E6- and E7-derived epitopes in cervical cancer cells and its effect on epitope-specific T cell cytotoxicity. Hypoxia induced downregulation of E7 protein levels in all analyzed cell lines, as assessed by Western blotting. However, contrary to previous reports, no perturbation of antigen processing and presentation machinery (APM) components and HLA-A2 surface expression upon hypoxia treatment was detected by mass spectrometry and flow cytometry, respectively. Cytotoxicity assays performed in hypoxic conditions showed differential effects on the specific killing of HPV16-positive cervical cancer cells by epitope-specific CD8+ T cell lines in a donor- and peptide-specific manner. Effects of hypoxia on the expression of PD-L1 were ruled out by flow cytometry analysis. Altogether, our results under hypoxia show a decreased expression of E6 and E7, but an intact APM, and epitope- and donor-dependent effects on T cell cytotoxicity towards HPV16-positive target cells. This suggests that successful immunotherapies can be developed for hypoxic HPV-induced cervical cancer, with careful choice of target epitopes, and ideally in combination with hypoxia-alleviating measures.

A Clathrin light chain A reporter mouse for in vivo imaging of endocytosis.

Clathrin-mediated endocytosis (CME) is one of the best studied cellular uptake pathways and its contributions to nutrient uptake, receptor signaling, and maintenance of the lipid membrane homeostasis have been already elucidated. Today, we still have a lack of understanding how the different components of this pathway cooperate dynamically in vivo. Therefore, we generated a reporter mouse model for CME by fusing eGFP endogenously in frame to clathrin light chain a (Clta) to track endocytosis in living mice. The fusion protein is expressed in all tissues, but in a cell specific manner, and can be visualized using fluorescence microscopy. Recruitment to nanobeads recorded by TIRF microscopy validated the functionality of the Clta-eGFP reporter. With this reporter model we were able to track the dynamics of Alexa594-BSA uptake in kidneys of anesthetized mice using intravital 2-photon microscopy. This reporter mouse model is not only a suitable and powerful tool to track CME in vivo in genetic or disease mouse models it can also help to shed light into the differential roles of the two clathrin light chain isoforms in health and disease.

Epithelial cell cluster size affects force distribution in response to EGF-induced collective contractility.

Several factors present in the extracellular environment regulate epithelial cell adhesion and dynamics. Among them, growth factors such as EGF, upon binding to their receptors at the cell surface, get internalized and directly activate the acto-myosin machinery. In this study we present the effects of EGF on the contractility of epithelial cancer cell colonies in confined geometry of different sizes. We show that the extent to which EGF triggers contractility scales with the cluster size and thus the number of cells. Moreover, the collective contractility results in a radial distribution of traction forces, which are dependent on integrin ß1 peripheral adhesions and transmitted to neighboring cells through adherens junctions. Taken together, EGF-induced contractility acts on the mechanical crosstalk and linkage between the cell-cell and cell-matrix compartments, regulating collective responses.

ASPP2 maintains the integrity of mechanically stressed pseudostratified epithelia during morphogenesis.

During development, pseudostratified epithelia undergo large scale morphogenetic events associated with increased mechanical stress. Using a variety of genetic and imaging approaches, we uncover that in the mouse E6.5 epiblast, where apical tension is highest, ASPP2 safeguards tissue integrity. It achieves this by preventing the most apical daughter cells from delaminating apically following division events. In this context, ASPP2 maintains the integrity and organisation of the filamentous actin cytoskeleton at apical junctions. ASPP2 is also essential during gastrulation in the primitive streak, in somites and in the head fold region, suggesting that it is required across a wide range of pseudostratified epithelia during morphogenetic events that are accompanied by intense tissue remodelling. Finally, our study also suggests that the interaction between ASPP2 and PP1 is essential to the tumour suppressor function of ASPP2, which may be particularly relevant in the context of tissues that are subject to increased mechanical stress.