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

Lead Research Organisation: University of Birmingham
Department Name: Immunity and Infection

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Related Projects

Project Reference Relationship Related To Start End Award Value
BB/H013598/1 01/05/2010 28/02/2013 £519,090
BB/H013598/2 Transfer BB/H013598/1 28/05/2013 27/10/2014 £156,442
 
Description CTLA-4 is an essential protein that prevents the immune system attacking our own bodies. Mice which genetically lack CTLA-4 die very early beacuse components of their immune system (T cells) recognise and destroy a number of organs as if they were foreign invaders.

Whilst the importance of CTLA-4 has been known for more than a decade, exactly how CTLA-4 worked was not understood and is a controversial issue in immunology.

During the course of this work we have identified and established a new mecahnism by which CTLA-4 works. We have shown that CTLA-4 has the unusual capacity to capture and "hoover up" proteins from other cells. Importantly, the proteins it removes are responsible for stimulating T cells to attack. thus by removing stimulating proteins CTLA-4 can keep T cells under control. We call this process of hoovering up stimulatory proteins "trans-endocytosis".

These finding provide a new perspective and new ideas in a key area of immunology which is important in diseases such as Arthritis and Diabetes, immune deficiency and inflammatory bowel disorders as well as transplant rejection and Cancer therapy.
Exploitation Route We are currently working to understand the transendocytosis process and have filed patents to facilitate exploitation of our work.

Our work will be of interest to the pharmaceutical industry in designing new modulators of the immune system. By understanding clearly the mechanism of transendocytosis we open up new approaches to immune therapies for a number of important diseases. Most recently we have used our knowledge generated from BBSRc funded work identify and study individuals who are suffering from CTLA-4 deficiency. Our BBSRC funded work has provided a powerful platform with which to study, understand and suggest treatments for this immune condition.

this work has leveraged further funding and will continue to be developed.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description The work funded by BBSRC has resulted in the identification of a new mechanism of CTLA-4 function. This has changed the way the field thinks about and understands this pathway. This has resulted in our ideas being widely cited, changing perspectives in undergraduate texts so that new graduates are provided with a more cohesive foundation in this area of immunology. The work has underpinned our identification and understanding of a new medical condition which results from CTLA-4 deficiency and has been helpful in assessing a second condition LRBA deficiency The work will influence our understanding of new anti-cancer therapies which are currently having a major impact in cancer therapy
First Year Of Impact 2011
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Description Talk at European Society For Immune Deficiency meeting (Edinburgh)
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
Impact The biology and understanding we have described has led to the design and implementation of new tests, which inform clinical diagnosis. We are currently discussing how to transfer these to an approved clinical diagnostic setting. presentations such as those above inform the wider clinical audience of such developments.
 
Description Is CTLA-4 -dependent Treg function compromised in T1D
Amount £207,509 (GBP)
Funding ID 12/0004477 
Organisation Diabetes UK 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2012 
End 08/2015
 
Description Understanding the relationship between clathrin-mediated endocytosis and transendocytosis of CTLA-4: cell biology at the heart of immune regulation.
Amount £632,285 (GBP)
Funding ID BB/M009203/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2015 
End 06/2018
 
Title Transendocytosis assays 
Description we invented and designed assays to detect CTLA-4 uptake of its ligands using GFP-tagged CHO cell lines. these have been used to assess the capacity of CTLA-4 mutants to function properly. They have also been used to confirm a CTLA-4 defect in LRBA deficient samples. These tools have been used by others in the assessment of clinically significant CTLA-4 mutations. 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact helped our lab assess and identify CTLA-4 deficiency in human patients. Has aided las at the NIH to evaluate patients with LRBA deficiency 
 
Title Application for patent relating to CTLA-4 trafficking and its use 
Description Patent application 1104950.9 
IP Reference GB1104950.9 
Protection Patent application published
Year Protection Granted
Licensed No
Impact the patent has not been pursued further
 
Description Public engagement at open days 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact discussions with members of public relating to immunology and its disorders

discussions with interested lay public and patient groups
Year(s) Of Engagement Activity 2013,2014