Understanding the relationship between clathrin-mediated endocytosis and transendocytosis of CTLA-4: cell biology at the heart of immune regulation.

We rely on our immune systems to keep us alive in the face of constant challenges from infections. Because we never know what each infection will look like we generate an immune system that contains many millions of different cells each capable of recognising different infections. Whilst this solves the problem of being able to recognise a vast array of different bacteria and viruses, it creates a second problem which is that those same immune cells can now also recognise bits of our bodies causing disease. Controlling our immune system so that it only attacks invaders and not our bodies is critical and getting it wrong can be fatal. Understanding how the immune system achieves this balance is relevant to most areas of our health. Understanding the mechanism underpinning this balance is the basis of this proposal.

To understand this process we need to focus on proteins found at the interface between two types of immune cell, the T cell and the dendritic cell which are responsible for initiating a response. The T cell expresses two proteins, CD28 which acts as a "go" signal and CTLA-4 which inhibits this process. Surprisingly, both CD28 and CTLA-4 interact with the same proteins called CD80 and CD86. We recently discovered that CTLA-4 acts like a tiny hoover and can remove CD80 and CD86 molecules from the surface of dendritic cells using an unusual process called transendocytosis. By hoovering up CD80 and CD86 this means that there is effectively a competition between CD28 and CTLA-4. Removing all the ligands prevents CD28 from receiving a "go" signal keeping our immune system switched off. In this proposal we are seeking to understand exactly how the CTLA-4 hoover functions and by doing this better understand situations where the process goes wrong.

To perform this work we will use several approaches:

1) We will take apart the CTLA-4 protein in order to figure out which bits are necessary for it to work.
2) We will find our which parts of the cell interact with CTLA-4 in order to promote transendocytosis.
3) We will used advanced microscopy to visualise CTLA-4 interacting with its cellular partners to understand the time and place where transendocytosis occurs.
4) We will test our predictions to see whether interfering with selected parts of the CTLA-4 hoover mechanism cause defects in our immune system as we expect.

Together these experiments will provide a complete picture of both an unusual process in biology and a better understanding of how an essential part of our immune system works.

The CD28/CTLA-4 pathway controls the key decision point in T cell activation. It is clear from knockout mice that CD28 is critical for generating effective T cell responses, immune memory and for providing T cell help needed for class switched high affinity antibody responses. In contrast, CTLA-4 is an essential inhibitor of T cell responses and its loss causes fatal multi-organ autoimmune destruction. These two opposing receptors are governed via interactions with a pair of ligands, CD80 and CD86, to which both receptors bind. Understanding how CTLA-4 works is fundamental to our understanding of adaptive immunity but is largely incomplete.

We recently identified a novel mechanism of action for CTLA-4 where we observed that it acts as a ligand depleting mechanism, which we term transendocytosis(TE). In this process CTLA-4 utilises its unusual cell biology to capture, internalise and degrade entire trans-membrane ligands expressed on antigen presenting cells. The molecular mechanism underpinning transendocytosis is still unclear. We have also established that there are clear differences in the requirements for CTLA-4 to undergo clathrin-mediated endocytosis (which happens in the absence of its ligands) compared with TE. Here we propose to carry out experiments to understand the differences between clathrin-mediated endocytosis and TE of CTLA-4 in order to better define the nature of TE and the pathways affecting this process. We will carry out mutagenesis of CTLA-4 to define regions that are required for TE as well as using a variety of knockdown and dominant interfering mutants to establish the cellular pathways involved. We will apply high resolution imaging technologies to define the spatial and structural organisation of components of the TE pathways and finally, we will test the significance of the molecular interactions identified via these approaches for their impact on immune function.

As detailed above there will be wide ranging benefit to academia at an international level as we gain a greater understanding of this important pathway. Accordingly, this work will directly contribute to the knowledge economy by providing new insights which will impact at the level of academic research but also at graduate level immunology. For example, our BBSRC funded work identifying CTLA-4 TE as a ligand removal mechanism now appears in graduate texts such as Cellular and Molecular Immunology by Abbas. This work provides a new immunological paradigm and is therefore capable of influencing the thinking of new generations of immunologists.

In addition, there is very significant potential for the work to impact on the the pharmaceutical industry. There are currently a number of companies who have active development programmes based in this area of work which target cancer, autoimmunity, transplantation. The knowledge gained from our studies can be readily translated into new approaches for drug development. Given the large number of immunologically related areas affected by this area of work there is enormous potential for significant economic impact.The long term outcome of this work will therefore be in the potential for better drugs and vaccines with greater efficacy and fewer side effects.

Given our leading position in the study of trans-endocytosis as a process, there is also significant potential for developing technological advances in methods of studying this immune process which may have impact on areas of cell biology outside of immunology. This work will therefore build research capacity and contribute to training of individuals in this emerging field.

Ultimately, the sophisticated knowledge gained from enhanced understanding of the CD28 /CTLA-4 pathways has the potential to impact on individuals with immunological diseases and groups who support them. Despite the basic nature of the work in this proposal the fundamental understanding gained from our approach is already being applied in the context of immunological disorders. Based on our expertise, our lab is able to provide support for the clinical immunology laboratory at the Royal Free in terms of measuring CTLA-4 function. Arguably, our lab is one of very few worldwide with the tools (generated with BBSRC support) capable of measuring CTLA-4 behaviour and its role in Treg function and this is now identifying defects in patients with immune deficiency and autoimmunity. Strikingly, and of relevance to this proposal, it is emerging that defects in pathways relating to intracellular protein trafficking also affect CTLA-4 function and are likely disease causative. This provides a clear example of an immediate impact on health generated entirely from BBSRC sponsored studies on basic mechanisms of immune regulation

Together the above demonstrates the potential for significant impacts of this research area on lifelong health and wellbeing.