Clathrin-mediated regulation of T cell activation and intercellular communication

By using components from the bacterial immune system known as CRISPR/Cas9, one can relatively easily and specifically modify human DNA. This technique has been coined genetic surgery because it can be used to repair disease-causing DNA sequences in humans. The challenge is to maximize this potential without inducing lasting alterations in the hereditary genetic makeup of future generations. The cells of the immune system are optimal in this regard: They are experts in finding and fighting disease, and we can alter them outside the body (ex vivo) to improve their disease-fighting properties before returning them to patients. This can be done without affecting the genetic expression of other cells than those in question, and the changes will disappear when those cells die. This strategy has proved to be very promising in the development of new cancer treatments within immunotherapy. In this project we are using CRISPR/Cas9-mediated gene editing to modify T cell activation and intercellular communication, specifically regarding the role of clathrin and the ESCRT machinery in immunological synapse formation. We also recently identified a novel compartment in the immunological synapse which we have termed the corolla which among other things seems to be important for the function of tumor infiltrating lymphocytes (Demetriou et al., Nature Immunology 2020). We are now following up on that work by identifying the most important components involved in T cell activation in the immunological synapse. Furthermore, we are now in the process of submitting a paper describing the interplay between clathrin and the ESCRT complex during T cell activation.

This project has lead to a strong international collaboration between Oslo University Hospital and the University of Oxford which is likely to last for many years to come. It has also been instrumental in the establishment of new technologies in Oslo important for analyzing T cell activation ex vivo.

The underlying molecular interactions responsible for regulating the adaptive immune response occur within a nanoscale gap between T cells and APCs, termed the immunological synapse. Upon T cell activation, the components of the synapse rearrange into a characteristic bullseye pattern, with activated TCR microclusters moving centripetally in the membrane to eventually form the centre of the bullseye. Here, the TCRs are released from the synapse in microvesicles through a process requiring the ESCRT machinery. Such microvesicles can subsequently be internalised by interacting APCs. We have preliminary data showing that clathrin is required for formation of TCR microclusters and that clathrin and TCRs move centripetally together during synapse formation. Clathrin-positive TCR microclusters eventually coalesce in the centre of the immunological synapse, likely during release of TCR-loaded microvesicles. Based on these data, and in light of what is already known, we hypothesize that clathrin is a key regulator of TCR microcluster formation, TCR endocytosis and ESCRT-mediated microvesicle release at the immunological synapse. We will use CRISPR/Cas9-mediated genome-editing to induce expression of endogenous fluorescent fusion proteins of interest. This will enable us to study these processes with minimal perturbation by cutting-edge imaging techniques such as TIRF-SIM (total internal reflection fluorescence-structured illumination microscopy) and LLSM (lattice light-sheet microscopy) at the resolution of single molecules in live, interacting cells. We will adapt custom-written Matlab detection and tracking algorithms to analyse the data. This work will enhance our understanding of how T cell activation is regulated and how this process in turn regulates intercellular communication between T cells and APCs. Furthermore, it will form the basis for a novel strategy of directly evaluating the effects of modifying the T cell genome at the molecular level.