Systematically defining the T cell signalling network of the phosphatase CD45

The recent pandemic has brought the importance of T cells in fighting viral infections to the the public sphere. CD8+ T cells in particular protect us from infections, such as SARS-CoV-2, by killing virally infected cells. They are also important in mounting successful responses to vaccinations, which are critical for the prevention of infectious diseases. Vaccination efficacy declines with age, with older people generally less protected from infections and related mortality. This is due to decreased T cell numbers as well as weaker responses to antigens. Antigens are recognised by proteins that span the cell membrane of T cells, known as the T cell receptor complex. While T cells must respond rapidly to foreign antigens, this must be balanced against aberrant recognition of self-generated antigens, which otherwise can lead to autoimmune responses and health challenges such as arthritis. Determining how different signal strengths are sensed by the T cell receptor and translated into changes in T cell behaviour will give a greater understanding of CD8+ T cell function in fighting infection to maintain health across the lifecourse.

CD45 is amongst the most abundant proteins spanning the cell surface membrane of T cells. It has a sugary domain presented to the outside of the cell linked to a domain with enzymatic function on the inside of cells, known as a phosphatase. This enzyme domain removes phosphate groups from substrate proteins to control their functions, and, importantly, is required for correct responses of T cell receptors to antigens. We have identified new CD45-interacting proteins and this research project will define how they are regulated by CD45. This will enable us to dissect how CD45 directly controls T cell activation in response to antigen. To this end, we have built a new research tool that enables us to measure CD45 proximity to other proteins in live unmanipulated T cells from mice. This research will deliver a better understanding of how immune cells are activated and how signalling is propagated to elicit correct responses. This will be important for understanding responses to infections, but could also open up avenues to create drugs targeting CD45 or its interactors to control the extension of T cell activation, which could be useful when T cell activation is aberrantly high or low.

CD8+ T cells mediate immune responses against infectious pathogens. They are initiated when antigen, presented by major histocompatibility complexes, binds to the T cell receptor leading to downstream signalling. This drives T cell activation, proliferation and differentiation to recognise and kill pathogen-infected cells. While activation is critical, the signalling cascades must also be tightly regulated to avoid aberrant activation and autoimmunity. The strength of T cell activation is determined by antigen affinity as well as co-stimulatory interactions and cell adhesions. The receptor protein tyrosine phosphatase CD45 is amongst the most abundant proteins on the surface of T cells. It has been implicated in the first steps of TCR activation, and is required for correct T cell development, differentiation and activation. Despite its importance, the precise substrates and regulation of CD45 remain to be determined. CD45 is also a therapeutic target and its forced recruitment to, and presumptive dephosphorylation of, negative regulators of TCR signalling, such as PD1, has been proposed as a strategy to reactivate T cells in cancer. Our new data, presented in this proposal, identifies specific binding partners of CD45. This proposal combines biochemistry, cell biology, quantitative proteomics and leverages a novel mouse model to understand CD45 interactions, substrates and signalling in response to T cell stimulation. This work will provide fundamental knowledge related to T cell activation and insights into how manipulation of CD45 or its targets could be leveraged therapeutically.