The immune system protects us from outside pathogens like viruses and bacteria, but also from internal treats like cancerous cells. Central to the function of the immune system is its capability to discriminate these treats from the vast number of harmless cells of our own bodies. Two distinct cell types of the immune system, termed dendritic cells (DCs) and T cells, are crucial to this process. DCs act as sentinels and messengers. They constantly take samples of their surroundings, harmless cells and pathogens alike, digest them and present these digested parts on their surface to identify potential threats. During this process, cues and signals from their environment lead DCs to become either mature or tolerogenic. Both, mature and tolerogenic DCs, communicate with T cells that recognise the identifier on the DC's surface by forming a close cell-cell contact, termed an immunological synapse. T cells that interact with mature DCs will become activated, start to multiply and finally attack and eliminate the treat, a process termed immunity. In contrast, tolerogenic DCs instruct T cells to become non-responsive, leading our immune system to ignore the source of the identified entity, which is deemed harmless, in a process termed tolerance. Importantly, many diseases in humans are caused by a mis-regulation of immunity and tolerance. Cancers for example, are able to instruct DCs to become tolerogenic, leading to escape from our immune system and metastasis. Conversely, the accidental identification of a healthy cell as a threat by mature DCs, leading to an immune response, is the cause for many autoimmune diseases where the immune system attacks our own body. Therefore, a comprehensive understanding of how the different properties of mature DCs and tolerogenic DCs lead to T cell responses or non-responses is an important topic in developing effective vaccines, immunotherapies and cures for autoimmune diseases. The different properties of mature and tolerogenic DCs are in part explained by the particular set of protein types that they carry on their surface to communicate and instruct T cells. Each of these proteins is recognised by its counterpart on T cells, triggering internal signals in the T cell that either contribute to immunity or tolerance. However, previous research suggests that it is not only the mere presence or absence of particular proteins that matters in this process. DCs also seem to be able to control the movement of these proteins on their surface, either allowing them to move freely or fixing them to certain positions. This in turn, changes how individual types of proteins are recognised by their counterparts on T cells and influences their signals. Consequently, the aim of this project is to study how protein mobility on DCs influences the decision between immunity and tolerance in a detailed, comprehensive and comparative manner. To this end we will employ a novel, artificial substrate that we developed to specifically control the mobility of different types of proteins i.e. some will be mobile while others will be immobile. This will allow us to emulate the surface of DCs in a fully controlled manner and to study the effect of mobility of a broad selection of specific types of proteins. In addition, we will measure protein mobility on the surface of mature and tolerogenic DCs and characterize their immunological synapses with T cells in a three-dimensional setup that mimics the complexity of human tissue. Additionally, and to complement our results from the artificial substrates, we will alter the mobility of specific types of proteins in mature and tolerogenic DCs by genetic engineering and measure how this affects the interaction and instruction of T cells. Together, this will give us an extensive understanding of the role of protein mobility in the decision between immunity and tolerance, potentially opening up new avenues for therapeutic interventions.
The aims of this project are to study (1) how the lateral mobility of dendritic cell-expressed ligands for T cell-expressed co-stimulatory receptors promotes or impedes T cell activation at the molecular level and (2) how co-stimulatory ligand lateral mobility is differentially regulated between mature and tolerogenic dendritic cells to promote either immunity or tolerance at the immunological synapse. To achieve these aims we are using two different, complementary approaches together with fixed-cell and live imaging, flow cytometry and comparative proteomics. In the first approach we are using a novel, advanced supported lipid bilayers system that allows for the simultaneous presentation of mobile and immobile co-stimulatory ligands. This emulates the surface of dendritic cells while providing superior spatiotemporal resolution for the study of T cell activation at the molecular level. In the second approach, we are measuring the mobility of co-stimulatory ligands on the surface of dendritic cells and employ a 3D dendritic cell-T cell co-culture system to characterize the immunological synapse of mature or tolerogenic dendritic cells and T cells within a physiological setting. Furthermore, we are using targeted genetic engineering to alter the lateral mobility of co-stimulatory ligands on the surface of mature and tolerogenic dendritic cells and study the ensuing effects on T cell activation in order to verify our results from the bilayer system in. In particular, we are focusing on the ICAM1-LFA-1 axis, CD80 and PD-L1 and the interaction of their signalling pathways, and barrier proteins like CD43, CD44 and CD45. We anticipate that the results of this project will greatly expand our knowledge of the basic principles that make dendritic cells uniquely suited for the induction of immunity and tolerance, potentially opening up new avenues for immunomodulatory therapies.
