What is the molcular basis of CTLA-4 trans-endocytosis?

The immune system contains a powerful arsenal of weapons used to fight and destroy different invading organisms that cause disease. One type of weapon is called a T cell. Like most weapons, there can be some collatoral damage to surrounding areas, in this case our bodies. There is therefore a need to regulate T cells and when these controls are lost, diseases such as arthritis and diabetes can occur. We wish to understand how an essential regulator found on a T cell actually functions. The regulator is a protein called CTLA-4 which acts to turn T cells off in some way. If CTLA-4 is missing then death from autoimmunity rapidly results, although we don't know why this occurs. CTLA-4 binds to two proteins, CD80 and CD86, on a different immune cell called a dendritic cell. This proposal will study the mechanisms by which CTLA-4 interacts with these proteins. We have very recently discovered a new mechanism whereby CTLA-4 can literally rip its interacting partner, CD86, from dendritic cells. Since CD86 can stimulate immune responses by binding to another protein (CD28) removing CD86 has the effect of preventing other T cells becoming activated. This results in suppression of immune responses and provides an explanation for why CTLA-4 is continually removed from the surface of T cells and brought inside cells. It also explains why CTLA-4 shares CD86 with the CD28, in order to allow it to steal CD86. In this project we will explore how CTLA-4 achieves this removal of ligands, by seeing which bits of the CTLA-4 protein are required and what these interact with inside the cell. This work is important data since understanding how the CD28/CTLA-4 system works has many implications, for example, medicines are currently being tested which interfere with this pathway. Furthermore if we find new pathways this might be used to make new drugs. Ultimately this project will further our basic biological understanding of one of the most powerful regulators in our immune system which is important in cancer biology, autoimmunity and HIV infection.

CD28 and CTLA-4 are critical regulators of T cell immune responses. Despite a decade of research prompted by the knowledge that lack of CTLA-4 is fatal, we still do not understand what CTLA-4 does. Whilst CTLA-4 is clearly a negative regulator of T cell responses how this is achived is not known, although a large number of theories exist. Increasingly, there is accumulating data indicating a role for CTLA-4 in the function of regulatory T cells. Accordingly, a role for CTLA-4 as an effector molecule on Treg moves away from the widely held view that CTLA-4 is a 'negative signal' although the ideas are not necessarily mutually exclusive. This proposal is based on our recent and novel observation that CTLA-4 can remove the costimulatory molecule CD86 from dendritic cells. This observation is exciting and plausable for several reasons. Firstly, removal of CD86 occurs via trans-endocytosis providing an explanation for the fact that CTLA-4 is very rapidly endocytosed. Secondly, such a mechanism can only be effective if CTLA-4 can bind to the same ligands as the T cell stimulator CD28, thus explaining why ligands are shared. This proposal aims to investigate the importance of CTLA-4 trans-endocytosis by dissecting the molecular basis for this process and then determining the functional significance of disrupting this pathway. By comprehensively investigating the regions of the CTLA-4 cytoplasmic domain that regulate its interactions and promote trans-endocytosis, we hope to define key new players that are essential to the regulation of the healthy immune system.

The research contained in this proposal represents significant progress towards understanding of how the immune system is regulated, as such this work is core to a wide variety of areas of immunology and beyond. Beneficiaries of this research in the short to medium term will include all researchers ans subsequently clincians with an interest in immune function. This research will impact directly on the study and treatment of autoimmune diseases, understanding of tumour immunology, where Treg are thought to play an important role, as well as vaccine design and HIV infection- where it is clear that CTLA-4 is over expressed. Such is the core importance of CTLA-4 to immune regulation, understanding its function is paramount. Thus beneficiaries will include, academic immunologists, clinical immunologists, tumour biologists, vaccine biologists and those working on HIV. In the longer term there is likely to be considerable benefits to patients with a variety of immune related conditions as novel therapies emerge. Because our progress is at the forefront internationally in this area we stand to benefit from significant intellectual property as we unravel the molecular pathways involved in CTLA-4 function. Thus this work has the potential to benefit the UK economy in terms of patents , licencing and pharmaceutical development. We have already taken steps working with the Universitiy's in house technology transfer team to protect our IP and will continue to develop this area as we make progress. In terms of the potential benefits envisaged from this work, it might be possible to develop a series of new compounds that selectively enhance or inhibit CTLA-4 trafficking. These will therefore have the ability to tune down immune responses (by enhancing ligand removal) and could be useful as immune suppressive drugs or enhance responses by stopping ligand removal and therefore be of potential benfits to vaccine design, HIV therapy or tumour therapy. Such approaches may enhance the quality of life form many individuals with a variety of diseases. We believe these are realistic opportunities within the next 10 years. To ensure maximum benefit form this research we are trying to obtain the highest possible impact for our work. This is a lengthy process and we continue to discuss our work with the highest profile Journals. However, we believe that this work is capable of having a wide impact academically which will then facilitate our interactions with various the media, as well as promote interest from relevant companies. The University has a well established media relations office which will be alerted to our work once it is published. Our work has also been funded by and is directly relevant to several medical charities and we will therefore make them aware of the impact of the research and its relevance to their stakeholders.