Determining Regulatory Pathways That Maintain FOXP3 in Human T regulatory Cells
Lead Research Organisation:
Newcastle University
Department Name: Translational and Clinical Res Institute
Abstract
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
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
Technical Summary
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.
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.
Planned Impact
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.
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.
People |
ORCID iD |
Shoba Amarnath (Principal Investigator) |
Publications
Amarnath S
(2020)
Harnessing proteases for T regulatory cell immunotherapy.
in European journal of immunology
Amarnath S
(2020)
Protocols for Innate Lymphoid Cell Phenotypic and Functional Characterization: An Overview.
in Methods in molecular biology (Clifton, N.J.)
Laba S
(2022)
The depths of PD-1 function within the tumor microenvironment beyond CD8+ T cells.
in Seminars in cancer biology
Lim JX
(2023)
Programmed cell death-1 receptor-mediated regulation of Tbet+NK1.1- innate lymphoid cells within the tumor microenvironment.
in Proceedings of the National Academy of Sciences of the United States of America
Mallett G
(2020)
Isolation and Characterization of Innate Lymphoid Cells within the Murine Tumor Microenvironment.
in Methods in molecular biology (Clifton, N.J.)
Smith AL
(2020)
Age-associated mitochondrial DNA mutations cause metabolic remodelling that contributes to accelerated intestinal tumorigenesis.
in Nature cancer
Smith K
(2023)
Twenty-One Flavors of Type 1 Innate Lymphoid Cells with PD-1 (Programmed Cell Death-1 Receptor) Sprinkles
in Discovery Immunology
Description | Efficacy of artificial imprinted antibodies in driving unwarranted immune responses |
Amount | £90,000 (GBP) |
Organisation | National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) |
Sector | Public |
Country | United Kingdom |
Start | 01/2022 |
End | 12/2025 |
Description | Novel Blood Based Biomarkers for Checkpoint inhibitor therapies in Melanoma |
Amount | £45,000 (GBP) |
Organisation | United Kingdom Research and Innovation |
Department | Northern Accelerator |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 08/2021 |
End | 07/2022 |
Description | Translating coreceptor biology into immunotherapeutics in cancer |
Amount | £250,000 (GBP) |
Organisation | Lister Institute of Preventive Medicine |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 09/2022 |
End | 08/2027 |
Description | Cutaneous Squamous Cell Cancer Collaboration |
Organisation | University of Southampton |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | This collaboration resulted in understanding the existence of a novel immune cell subset in cSCC patients pre treatment. |
Collaborator Contribution | The collaborators recruited and analysed immune cell subsets in the blood and tumour of cutaneous squamous cell cancer. |
Impact | No outputs yet but a manuscript is under preparation. |
Start Year | 2020 |
Description | 2020-COVID-19 Winter Scenarios Policy Working Group Academy of Medical Sciences |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Policymakers/politicians |
Results and Impact | I was part of the working group that developed the policy for handling the UK government regulations for the country during winter 2020. The policy working group consisted of senior academics and early/mid career researchers. This policy document was co-opted by SAGE and informed the government working model for UK shutdown, hospital management etc for winter 2020. |
Year(s) Of Engagement Activity | 2020 |
Description | COVID-19 Winter Scenarios Policy Working Group Academy of Medical Sciences |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Policymakers/politicians |
Results and Impact | I was part of the working group that developed the policy for handling the UK government regulations for winter 2021. The policy working group consisted of senior academics and early/mid career researchers. This policy document was co-opted by SAGE and will inform the government working model for winter 2021. My contribution was part of the policy document on post-COVID symptoms. |
Year(s) Of Engagement Activity | 2021 |
Description | COVID-19 research career support working group Academy of Medical Sciences |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Postgraduate students |
Results and Impact | I was part of the working group that developed key supportive materials during COVID for early career researchers. We organised a number of helpful websites, videos and other supportive outlets for Early Career Researchers within the UK. The panel consisted of senior researchers and early/mid career researchers. One of the outcomes of the career support working group was the workshop on COVID-19 Impact on funders and researchers which I participated in. |
Year(s) Of Engagement Activity | 2020 |
Description | DNA Technologies in Life Sciences, Institute of Genetic Medicine, Newcastle University |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | We prepared a slide show presentation describing how murine lines are genotyped in the laboratory using simple techniques such as PCR and gel blots. This programme allowed an insight into the methodologies used for genotyping within cancer laboratories and their relevance in clinical medicine. |
Year(s) Of Engagement Activity | 2021 |
Description | Funding Barriers for Early Career Researchers Pre and Post COVID |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | Invited to give a talk on funding barriers for early career researchers during and post- COVID for the Academy of Medical Sciences. Research Funding workshop on Impact of COVID-19 on biomedical and clinical research careers. Only n=2 ECRs where invited, with one basic researcher and one clinical researcher. Impact: My presentation led to the development of "COVID Memory" statement for funders that was drafted and adopted by the funders namely MRC, Wellcome Trust, CRUK etc. |
Year(s) Of Engagement Activity | 2020 |
Description | Interview |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Media (as a channel to the public) |
Results and Impact | This engagement activity was done as part of an Invitation from Science Media Centre to comment on an article describing COVID immunity in cancer patients authored by Prof. Adrian Hayday. My comments were covered and broadcasted by multiple media outlets including CNN. Impact: My summary of the work predicted that cancer patients would require two shots of COVID-19 vaccination for full immune system protection. |
Year(s) Of Engagement Activity | 2021 |
URL | https://edition.cnn.com/2021/03/11/health/pfizer-vaccine-cancer-patients-intl-gbr/index.html |
Description | Invited Case study on Murine Research for the Academy of Medical Sciences |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Policymakers/politicians |
Results and Impact | The Academy of Medical sciences is a signatory of the 2014 Concordat on Openness in Animal Research and approximately 1 in 7 of their grant awardees use animals in research. The Academy invited my group to feature our research as a case study. Only 5-6 case studies were selected from all the researchers funded by the Academy. This case study demonstrated how we incorporate the NC3Rs within our work and also highlighted the team science that is currently being performed within Newcastle with respect to animal research. |
Year(s) Of Engagement Activity | 2021 |
Description | Leadership During Covid ECR workshop |
Form Of Engagement Activity | A broadcast e.g. TV/radio/film/podcast (other than news/press) |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Other audiences |
Results and Impact | This video helped early career researchers who lead research programmes in fundamental science to identify various paths that can result in the success of their research programme. |
Year(s) Of Engagement Activity | 2020 |
URL | https://www.youtube.com/watch?v=ZmsJGv4bnq0 |