Towards an integrated understanding of the CD28/CTLA4 immune checkpoint in the regulation of autoimmunity

The immune system contains a number of powerful weapons that defend us from constant attacks by microbes that threaten our health. Unfortunately, like any complex system it sometimes goes wrong and instead of defending us, can attack our own body and cause damage. Such "collateral damage" is what causes autoimmune diseases like type 1 diabetes, rheumatoid arthritis and multiple sclerosis: the weapons responsible are part of our immune army performing their normal jobs, just in the wrong place at the wrong time. The key weapon that starts the autoimmune attack in these diseases is called a T cell. When scientists explored which genes are responsible for autoimmune diseases, they identified a strong bias towards genes that tell our T cells to fire or affect their ability to stop firing. Careful experiments using animal models have also shown that it is possible to cause autoimmune diseases just by transferring T cells from one mouse to another. This provides strong evidence that T cells are critical players in determining whether a person develops an autoimmune disease.

Properly understanding the signals that tell T cells to fire or cease firing is therefore critical to understanding and treating many autoimmune diseases. A precise system involving 4 molecular switches is used to make this decision. Two of these switches (CD28 and CTLA4) are receptors expressed on the T cell and these interact with 2 binding partners (CD80 and CD86) expressed on other cells. Correct use of these 4 molecules can mean the difference between life and death. Without CD28 immune responses cannot be mounted properly and without CTLA4 the immune response fires indiscriminately causing lethal inflammation. Whilst it is very clear that CD28 promotes immune responses and CTLA4 inhibits them, why these opposing switches share the same triggers (CD80 and CD86) is a complete mystery. At face value, it would seem more intuitive to have one binding partner to engage CD28 (the on switch) and another to engage CTLA4 (the off switch). However both CD80 and CD86 can bind to both CD28 and CTLA4. How then does the immune system decide whether CD28 or CTLA4 wins out?

Recent work from our groups has made substantial progress in understanding this system. We discovered that CTLA4 works by hoovering up CD80 and CD86 thereby preventing CD28 from being triggered. Such a model is fundamentally different to previous ideas and explains many features of the system that until now did not make sense. Viewing the system in this way prompts us to ask different questions: Why are there 2 binding partners? Are they both equivalently hoovered up by CTLA4? Do both take the same amount of time to be re-expressed after removal? What does this mean for the control of immune responses? In this proposal we will address these and other questions to generate a detailed understanding of this important system and use this knowledge to come up with new ideas for immune therapies. What we learn in this project will be of direct relevance to a wide variety of immune mediated conditions from autoimmunity to cancer and vaccination to organ transplantation.

This programme is focused on understanding how the T cell molecules CD28 and CTLA4 dictate control of tolerance versus immunity, an area of profound importance in immunology. Whilst their involvement is understood in broad principle, the mechanistic details of how the system actually works have not been established and will be crucial for sophisticated manipulation of the immune response. We recently identified a mechanism of action for CTLA4 (transendocytosis) where its inhibitory function is linked to physical destruction of its ligands (CD80 and CD86). Since CD28 binds to the same ligands, CTLA4 controls CD28 engagement. This model generates an integrated view in which the function of each individual molecule must be understood in the context of the other three players. This also leads to an emphasis on the regulation of CD80/CD86 expression and re-expression as the arbiters of immune outcome. Surprisingly, despite the fundamental importance of these ligands in driving immune responses, regulation of their expression is still poorly understood. Furthermore, the rationale for having two biophysically distinct ligands is entirely unclear.

It is our contention that a transendocytosis perspective holds the key to understanding why two ligands exist. We will therefore carefully evaluate the differences in the control of their expression, and establish the capacity of each ligand to stimulate responses via CD28 as well as be regulated via CTLA4 transendocytosis. We will use novel mouse models to dissect whether differences between ligand function in vivo reflect inherent functional properties or distinct expression patterns. By utilising a combination of molecular and cellular in vitro and in vivo approaches we will provide new insights into an area of fundamental importance to immunology. This will result in a more complete and predictive understanding of this crucial immune checkpoint enabling us to conceive and test novel therapeutic strategies.

In addition to serving the academic community, there are several wider benefits of our research.

Benefits to industry
The ability to modulate the immune system in a predictable way is a major goal of the pharmaceutical industry. The molecules which are the focus of this research programme (CD28, CTLA4, CD80, CD86) are widely recognized to play pivotal roles in the initiation and suppression of immune responses and as such have attracted considerable interest from pharmaceutical companies. Drugs to enhance the immune response can be used to fight cancer while drugs that suppress immunity can be used to treat autoimmune diseases such as type 1 diabetes, rheumatoid arthritis and multiple sclerosis. However, effective use of these drugs depends on an accurate understanding of the biological function of each of these molecules. The results from this research are therefore likely to be of high value to the pharmaceutical industry and may lead to new drugs to target the CD28/CTLA4 pathway or to existing ones being used in different ways and in novel combinations.

Benefits to patients
A better understanding of how to control the immune system through the CD28/CTLA4 pathway promises to offer significant benefits for patient groups, particularly those with autoimmunity. Our recent work has shown that CTLA4 mutations in humans are associated with defective regulatory T cell function and an autoimmune syndrome (Nature Medicine, 2014) providing direct evidence of the critical importance of this pathway in humans. This prompts consideration of new therapeutic approaches in these patients and has already informed clinical decision making for some of these individuals. Further understanding of how this pathway works will therefore improve our ability to regulate autoimmunity. Given that the CD28/CTLA4 pathway is a major control point for immunity, relevant to a wide range of diseases, our research has the potential to bring health benefits to a large number of individuals.

Impact on Treg research and therapy
The study of regulatory T cell (Treg) biology is major area of biomedicine. Treg therapies hold promise for treatment of many autoimmune diseases. Nonetheless, meaningful assays for studying Treg function in vitro are lacking. As one example, the commercially available "Treg inspector kit" is widely used yet relies on antibody-mediated engagement of CD28. This process cannot be regulated by CTLA4-mediated control of ligand expression, meaning that arguably the most significant mechanism of Treg function is therefore not "inspected". By developing a refined understanding of how CTLA4 interacts functionally with its two ligands, our work has the ability to impact on research practice and assay development in a very significant area of therapeutic immunology.

Training and career development
This work will support the career development of two postdoctoral scientists. The collaborative aspects of the programme will offer them opportunities to enhance their skill sets as well as disseminate understanding from our work. The proposed work is highly translational and will leave these individuals well-placed for careers in academia, the health sector or in the pharmaceutical industry.