Determining Regulatory Pathways That Maintain FOXP3 in Human T regulatory Cells

Autoimmune diseases such as rheumatoid arthritis (RA) and Type I diabetes are driven by over-activated immune cells. A type of immune cell called regulatory T cell (Tregs) can efficiently stop uncontrolled immune system activation and prevent autoimmune diseases. Pilot clinical trials using Tregs as a therapy for diseases such as colitis, type 1 diabetes and other immune related diseases have shown promising results. Furthermore, evidence of Treg cell function is apparent in patients with mutation in Treg specific gene called FoxP3. Foxp3 mutation results in severe autoimmune disease in humans which is known as IPEX (immunodysregulation polyendocrinopathy enteropathy X-linked) syndrome. Therefor all these observations in humans clearly demonstrate that Tregs are indispensable for controlling over-activation of the immune system.

However, how Foxp3 expression in Tregs are controlled within the human body is still not fully understood. Therefore, the goal of this grant proposal is to understand how human Foxp3 is regulated.

We have discovered a new regulatory molecule (asparaginyl endopeptidase; AEP) that can control Treg cell function in mice. AEP directly cleaved and degraded FoxP3 protein in Tregs and abolished their suppressive function. In addition to this observation, we also found that AEP expression in Tregs can be significantly down-regulated by a receptor on Tregs called programmed cell death receptor-1 (PD-1).

By extending our mouse studies in humans, we have discovered the expression of AEP in human Tregs but its function and importance in human Treg cells is unknown.

In this grant proposal, we aim to understand the function and importance of AEP in human Tregs and other T helper cell subsets. We will study whether:

1. AEP controls FoxP3 in human Treg cells and whether blocking AEP increases Treg cell function

2. If PD-1 and other similar coreceptors can block AEP expression in Tregs

3. Determine if AEP is expressed in other T helper cell subsets in humans and test its function within these subsets.

We propose that if AEP has a similar function in human Tregs, then it would be possible to target AEP in order to generate engineered Treg cell therapies that can efficiently control a variety of autoimmune disease. Such targeted therapies will enable a better quality of life for those patients who suffer from chronic autoimmune diseases. The current standard of care for these patients include global immune suppressive drugs such as corticosteroids; the long-term use of which can result in unwanted side effects. Engineered cell therapies can not only control autoimmunity but can be deleted from the body on reaching immune cell control. Success of these therapies is already apparent in the cancer field therefore providing a proof of principle for generating similar cell therapies for treating autoimmuntiy

CD4+ T-regulatory cells (Tregs) are important in maintaining immune tolerance and are characterised by Foxp3 expression. In particular, Tregs are efficient at inhibiting autoimmune diseases but regulatory processes that determine Foxp3 expression and stability in human Tregs is yet to be fully understood.

My research on Treg biology has defined important molecular mechanisms that can maintain Treg cell function. Recently, I have uncovered a previously unknown regulatory mechanism involving post-translational regulation of Foxp3 in murine Treg cells. We found that Foxp3 can be degraded by a protease known as asparaginyl endopeptidase (AEP) in murine Tregs and PD-1 receptor signalling can significantly inhibit this process and maintain Foxp3 expression and stabilise Treg cell phenotype.

Although previously AEP gene expression has been noted in Tregs, my work is the first to identify AEP protein, enzyme activity and functional role in murine Tregs. I will extend this work to study the importance of AEP in human Tregs and T helper cell subsets.

The major goal of this project is to understand (a) the importance of AEP in human Treg cell function (b) identify coreceptor driven signalling pathways that induce AEP in human Tregs and (c) determine the function of AEP in human Th17 helper cell subsets.

These research questions will provide a new in-sight into the post-translational regulation of human Tregs and demonstrate the role of proteases in regulating Treg and Th17 cell function.

This proposal will have overreaching impact in several aspects:

PATIENT POPULATION
The research set forth here will result in better cell therapy treatments for patients suffering from steroid resistant autoimmunity and patients who have undergone stem cell transplantation who then develop GvHD. This will prevent the patients from being treated with complex immune suppressive drugs, which while not curing the underlying disease ultimately results in infection related deaths. Rejection of organs or bone marrow in organ transplants and bone marrow transplants is a significant problem where T regulatory cell therapy has shown to be useful in these settings.

NHS HEALTHCARE
Allogeneic bone marrow transplantation (BMT) as a therapy for cancers, such as Leukaemia and primary immune-deficiency, is the only currently available treatment option. This expensive treatment is an increasing concern, with severe implications in healthcare systems. Complications such as GvHD add further pressure to the health care system whereby "treating the treatment" results in long-term hospitalisation followed by lifelong immune-suppression. In addition to the BMT patient population, in inflammatory autoimmune diseases, new methods of treatment and prevention are required in order to provide long term cure and enhance the quality of life of patients. Our work will provide insights into a new regulatory axis that maintains Treg cell function thereby identifying potential targets for development of novel Treg cell based therapeutics.

ECONOMIC
In recent years, a number of drugs have been developed by pharmaceutical industries that are targets of immune checkpoints. A large number of these drugs require continuous administration and do not provide long-term remission. Hence, it is important to develop therapies that can cure the disease rather than manage it. T cells can persist throughout a person's lifetime due to their capacity to proliferate. Therefore, T cells that can maintain their phenotype in vivo can provide lasting cure to diseases without continuos administration. Treg cells may fill this gap given their ability to proliferate and propagate in the body over a period of time. Proliferation is directly associated with loss of Treg function, hence research elucidating the expansion of Tregs by blocking specific targets (AEP in this case) may provide better Treg cellular therapeutics for use in the clinic. This will minimise the cost associated with other currently available treatment strategies for patients suffering from autoimmunity and cancer. This work therefore has the potential to result in economic benefit through a reduction in the healthcare systems budgetary burden while providing an increased quality of life and health to patients and families.


OUTREACH
An important aspect of any publicly funded research is its potential impact to the wider public. This work will translate into better treatment for patients with debilitating inflammatory diseases. The research has the potential to be translated into clinical medicine and will ensure a better quality of life not just for patients but also their families. In addition, I am a strong advocate of engagement with the general public in my research and outputs. Towards this goal, I am in the process of getting involved in a programme for sixth formers from minority, underprivileged backgrounds to be exposed to cutting edge medical research. Such programmes are already set up at Newcastle University and I am closely working with the coordinator Ms. Janet Lewis to promote these activities within my laboratory. I will also engage in scientific communication with adults and children in order to develop and enhance the awareness of different cancer therapies and immunology. This knowledge of science and research will form the basis for a better understanding of medical problems and treatment and an overall awareness of the societal benefits of scientific research.