Tissue Tregs seminar

Recording of a seminar I gave for the Cytek seminar series, on tissue-resident regulatory T cells.

Designer IL2 mutations for clinical use

We have a new pre-print out, on novel designer IL2 mutations!

IL2 is a powerful immunomodulator, but the dual roles make it complex to use, and many designer mutations reduce bioactivity or result in poor production. This makes it much harder to move these muteins to the clinic.

Rob van der Kant, Joost Schymkowitz and Frederic Rousseau from the VIB Switch lab took up the challenge of designing new IL2 mutants that not only improve the specificity for Tregs or CD8 T cells, but also maintain bioactivity and actually improve production capacity. They sent the designs over to us to test!

Great work from Amy Dashwood screened these mutations designs in vitro, with in vivo testing by Ntombizodwa Makuyana resulting in a set of mouse and human IL2 muteins with the desired biological properties. In particular we solve some of the common issues with IL2 muteins by considering the bound and unbound structures. For example, to make IL2 specific for Tregs, the approach is to allow binding to IL2RA, the high affinity receptor sub-unit used by Tregs, while block binding to the IL2RB used by CD8 T cells. The problem is that Tregs also need the full IL2RA-IL2RB-IL2RG trimer to assemble for optimal signal. So the typical Treg mutein is more specific, but also has poorer bioactivity. We solved this by creating a block between IL2 and IL2RB that moves out of the way after IL2 binds IL2RA, allowing the full trimer to form. These muteins are not only superior to the original IL2 in terms of cellular specificity, but by removing the aggregation-prone regions. By identifiying the aggregation gateway residues and changing them to be aggregation-resistant, we can further improve these muteins by making them aggregation resistance. The net effect is that the IL2 muteins we made are more specific for either Tregs or CD8 T cells, and will also be cheaper and easier to produce - the perfect combo for biologic drugs!

Our take-home message: if you are engineering proteins for therapeutic use, remember to take into account production, aggregation and bioactivity. These factors count when it comes to making a drug!

Take a look at the full paper on BioRxiv.

Discovery of ‘new rules of the immune system’ could improve treatment of inflammatory diseases, say scientists.

Scientists at the University of Cambridge have discovered that a type of white blood cell - called a regulatory T cell - exists as a single large population of cells that constantly move throughout the body looking for, and repairing, damaged tissue.

This overturns the traditional thinking that regulatory T cells exist as multiple specialist populations that are restricted to specific parts of the body. The finding has implications for the treatment of many different diseases – because almost all diseases and injuries trigger the body’s immune system.

Current anti-inflammatory drugs treat the whole body, rather than just the part needing treatment. The researchers say their findings mean it could be possible to shut down the body’s immune response and repair damage in any specific part of the body, without affecting the rest of it. This means that higher, more targeted doses of drugs could be used to treat disease – potentially with rapid results.

It's difficult to think of a disease, injury or infection that doesn’t involve some kind of immune response, and our finding really changes the way we could control this response.

Adrian Liston

“We’ve uncovered new rules of the immune system. This ‘unified healer army’ can do everything - repair injured muscle, make your fat cells respond better to insulin, regrow hair follicles.  To think that we could use it in such an enormous range of diseases is fantastic: it’s got the potential to be used for almost everything,” said Professor Adrian Liston in the University of Cambridge’s Department of Pathology, senior author of the paper.

To reach this discovery, the researchers analysed the regulatory T cells present in 48 different tissues in the bodies of mice. This revealed that the cells are not specialised or static, but move through the body to where they’re needed. The results are published today in the journal Immunity.

“It's difficult to think of a disease, injury or infection that doesn’t involve some kind of immune response, and our finding really changes the way we could control this response,” said Liston.

He added: “Now that we know these regulatory T cells are present everywhere in the body, in principle we can start to make immune suppression and tissue regeneration treatments that are targeted against a single organ – a vast improvement on current treatments that are like hitting the body with a sledgehammer.”

Using a drug they have already designed, the researchers have shown - in mice - that it’s possible to attract regulatory T cells to a specific part of the body, increase their number, and activate them to turn off the immune response and promote healing in just one organ or tissue.

“By boosting the number of regulatory T cells in targeted areas of the body, we can help the body do a better job of repairing itself, or managing immune responses,” said Liston.

He added: “There are so many different diseases where we’d like to shut down an immune response and start a repair response, for example autoimmune diseases like multiple sclerosis, and even many infectious diseases.”

Most symptoms of infections such as COVID are not from the virus itself, but from the body’s immune system attacking the virus. Once the virus is past its peak, regulatory T cells should switch off the body’s immune response, but in some people the process isn’t very efficient and can result in ongoing problems. The new finding means it could be possible to use a drug to shut down the immune response in the patient’s lungs, while letting the immune system in the rest of the body continue to function normally.

In another example, people who receive organ transplants must take immuno-suppressant drugs for the rest of their lives to prevent organ rejection, because the body mounts a severe immune response against the transplanted organ. But this makes them highly vulnerable to infections. The new finding helps the design of new drugs to shut down the body’s immune response against only the transplanted organ but keep the rest of the body working normally, enabling the patient to lead a normal life.

Most white blood cells attack infections in the body by triggering an immune response. In contrast, regulatory T cells act like a ‘unified healer army’ whose purpose is to shut down this immune response once it has done its job - and repair the tissue damage caused by it.

The researchers are now fundraising to set up a spin-out company, with the aim of running clinical trials to test their findings in humans within the next few years.

The research was funded by the European Research Council (ERC), Wellcome, and the Biotechnology and Biological Sciences Research Council (BBSRC).

Reference: Liston, A. ‘The tissue-resident regulatory T cell pool is shaped by transient multi-tissue migration and a conserved residency program.’ Immunity, June 2024. DOI: 10.1016/j.immuni.2024.05.023

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In brief

• A single large population of healer cells, called regulatory T cells, is whizzing around our body - not multiple specialist populations restricted to specific parts of the body as previously thought.
• These cells shut down inflammation and repair the collateral damage to cells caused after our immune system has responded to injury or illness.
• Tests, in mice, of a drug developed by the researchers showed that regulatory T cells can be attracted to specific body parts, boosted in number, and activated to suppress immune response and rebuild tissue.
• Current anti-inflammatory drugs used for this purpose suppress the body’s whole immune system, making patients more vulnerable to infection.
• The discovery could lead to more targeted treatments, with fewer side-effects, for issues from lengthy COVID infections to autoimmune diseases like multiple sclerosis. Clinical trials in humans are now planned.

After the Smoke Clears: Scars on the Immune System

Check out this article written in The Scientist by Danielle Gerhard, on the epigenetic marks of smoking on the immune system. A few quote from me in the piece:

“There is no human immune system; there is everyone's individual immune system,” said Adrian Liston, an immunologist at the University of Cambridge who was not involved in the study. “We really need to study things at the scale that the group in Paris is studying if we want to understand what's going to be relevant to human health at a population level.” 

Overturning how we think about immune regulation across the body

by Kat Steer

A Fellow at St Catharine’s College has led extensive new research that looks set to overturn the established model about how one type of white blood cell regulates immune responses in tissues – what was assumed to be a static part of the immune system is actually dynamic, opening the door to new treatments for a range of conditions.

Professor Adrian Liston (2023) is Professor of Pathology at the University of Cambridge and has run a research laboratory with Dr James Dooley since 2009, which relocated to Cambridge’s Department of Pathology earlier this year.

Professor Liston explains, “All of us are familiar with immune activation in our daily lives – the body’s response to injury or infection – but immune regulation is just as important to our health. A poorly regulated or hyperactive immune system can be hugely damaging, as we see in cases of flu, COVID-19, autoimmune diseases and inflammatory diseases. The team based at our laboratory have been conducting a broad range of research to learn more about the different processes and cells that are key to immune system regulation.”

Their latest findings are focused on a group of white blood cells known as regulatory T cells (Tregs) which have a role in regulating or suppressing other cells in the immune system. Over the last 10 years, scientists have established that Tregs are found not just in our blood but also throughout different tissues in the body. It is also known that Tregs play a significant role beyond controlling the immune system by enabling the body to return normal (homeostasis) and orchestrating repair and rejuvenation after an immune response.

“A decade of research has begun to establish the importance of Tregs but there is still so much we don’t know for sure. Like so many other scientists, we accepted the prevailing wisdom that Tregs travel into tissues where they remained as a static part of the immune system and specialised to their surroundings – an idea borrowed from evidence about another type of white blood cell (macrophages). We never set out to challenge this model, but our new findings indicate that these Tregs are really different from what we all thought.”

After an earlier successful study into Treg cells in the brain, the Liston-Dooley laboratory set out to complete an ambitious systematic analysis of the Tregs throughout the body, which has involved studying tissues from 48 different tissues in mice.

“It was only once we took global look at the body as a whole that it was possible to see for the first time that the Tregs in tissues are not specialised or static. In fact, they are highly dynamic and percolate throughout the body to serve different organs, moving from one place to the next as needed.

“It is exciting to know more about these important cells and potentially open up a new avenue for treating diseases – if we can find a way of boosting the number of Tregs in targeted areas of the body, then we can help the body do a better job of repairing itself or managing immune responses. We are in the early stages of planning a clinical trial to understand more about the benefits of boosting the levels of Tregs and look forward to sharing what we find.”

This work was supported by the European Research Council, the Wellcome Trust, and the Biotechnology and Biological Sciences Research Council.

Pre-print details

Burton O, et al. The tissue-resident regulatory T cell pool is shaped by transient multi-tissue migration and a conserved residency program. bioRxiv 2023.08.14.553196; doi: https://doi.org/10.1101/2023.08.14.553196

The Tissue Treg project

Biggest paper yet from the lab now a preprint on BioRxiv. A massive open science resource on tissue Tregs, and what makes Tregs tick in the tissues.

This project started back when we thought that tissue Tregs formed by seeding tissues and differentiating into unique terminal cells. We had examples of fat Tregs and muscle Tregs becoming unique permanent residents, and wanted to look at Tregs across the tissues.

We undertook a massive project to look at Tregs across 48 different tissues. At first glance, tissue Tregs looked special. Take a tissue and compare it against lymphoid/blood Tregs and the differences are huge. But the more tissues you add, the more they look the same. The only three distinct phenotypes were gut, lymphoid and bulk non-lymphoid. (Try our interactive web-browser resource). They have the same phenotypes, they use the same genetic triggers to differentiate and they only stay in the tissues for around 3 weeks. In short, the "seeding & specialisation" model doesn't fit the data.

Instead we came up with the "pan tissue" model, where tissue Tregs slowly percolate between different tissues. We've spent years testing this model in every possible way. We used the TCR as genetic barcodes, showing that the same Treg #clones are found in different tissues. We used ProCode technology to make #retrogenics for the tissue Treg TCRs, formally demonstrating that they impart a multi-tissue Treg fate. We extracted cells from tissues and reintroduced them, showing that they are tissue-agnostic on rehoming. By every test, the "pan-tissue" model holds strong.

What is amazing is that tissue Tregs have so many key functions in tissue repair and homeostasis, and now we find that it is the same cells that are able to restore the balance across all of these different tissues. Tissue Tregs are global homeostatic police. They are regulatory cells with a pan-tissue beat. A truly amazing cell type.

Could only have happened due to an amazing team - lead by Oliver, Burton, Orian Bricard and James Dooley. 

A New Hope for IPEX patients

A new paper from our lab suggests a novel approach to treating IPEX patients. IPEX is a rare severe primary immunodeficiency, caused by a genetic deficiency in the gene FOXP3, which results in a lack of anti-inflammatory regulatory T cells.

IPEX is usually fatal in childhood if left untreated. The only cure is a haematopoietic stem cell transplantation, however patients are often so sick from autoimmunity that they are in poor condition to receive a transplant. The patients are put on symptomatic support (hormonal and nutritional supplements to compensate for the damaged organs) and immunosuppressive drugs to reduce further damage. These immunosuppressive drugs are typically combinations of cyclosporine A, tacrolimus, rapamycin and corticosteroids, although recently biologics such as orthoclone have been suggested. Unfortunately the patient cohort has been too small and heterogeneous to allow a proper clinical trials as to which immunosuppression regimen works best. 

We sought to answer this by turning to the mouse model - also with a genetic deficiency in Foxp3 and a lack of regulatory T cells. We developed a comprehensive pathology scoring system for the model that takes into account the multiple different autoimmune symptoms, and then tested in a side-by-side comparison rapamycin (the most common standard treatment), anti-CD4 antibody (analgous to orthoclone in its proposed approach) and CTLA4-Ig (based on our prior work on CTLA4-Ig compensating well for Treg-deficiency). 

The results were striking. As seen in patients, rapamcyin cleared up some of the skin pathology, but otherwise it had little impact on the course of pathology in the mice. Anti-CD4 antibody prevented many of the immunology symptoms, but again, didn't actually improve the aggregate health outcomes of the mice. CTLA4-Ig, by contrast, improved essentially every parameter - the mice started gaining weight like normal, improved their serology, skin pathology and organ histology - and had greatly improved life-spans. Most importantly, the overall condition of the CTLA4-Ig-treated mice improved to such an extent that they were capable of supporting curative bone-marrow transplants: survival improved from 50% to 100% in mice given CTLA4-Ig prior to transplantation. 

There are caveats to every disease model, however we believe this is sufficient evidence to strongly consider a clinical trial of CTLA4-Ig (abatacept) in IPEX patients. The genetic and cellular defects are entirely conserved between mouse and human in this case, and the drug is in widespread use in patients for other autoimmune conditions (such as arthritis). We know that there are IPEX patients who respond poorly to the current standard treatments and need to improve their condition before receiving a bone-marrow transplant. CTLA4-Ig treatment could be the bridge that these patients need to the curative transplantation!

Thanks to the Jeffrey Modell Foundation for sponsoring this study, which was done in collaboration with lab alumni Prof Stephanie Humblet-Baron at the University of Leuven in Belgium. Check out the full paper at the Journal of Clinical Immunology!

The key to healthy aging brains

As people age, they often experience problems with their memory and cognitive abilities. This happens in part because their brains become mildly inflamed. But there may be a solution: a small group of special T cells, called regulatory T cells, could help reduce this inflammation in aging brains. Administering a protein called interleukin-2 (IL2) can help these special T cells grow and prevent inflammation. Now, researchers at VIB, KU Leuven, Babraham Institute, and i3S have tested this approach in mice and found that it can prevent neurological decline. Their findings, published in EMBO Molecular Medicine suggest that targeting the immune system might keep people’s brains healthy as they age.

Emanuela Pasciuto, co-first author of the study: “Our goal was to see whether we could slow down the aging process of the brain by changing its immune system through the delivery of IL2. We know that inflammation plays a significant role in various aging processes, and IL2 could help us tilt the balance back in our favor.”

Inflammation in the aging brain

Aging is a degenerative process that affects the whole body, including the brain. As we age, our brains may experience cognitive decline, affecting our memory and ability to think clearly. Increasing evidence suggests that inflammation in the brain, called “inflammaging,” can worsen this decline. Inflammaging is caused by immune cells entering the brain as we age. This inflammation can activate microglia, the resident immune cells in the brain, and induce neuroinflammation, leading to cognitive decline and dementia.

However, researchers have found a way to reduce inflammation in the brain by targeting a small group of special immune cells in the brain called regulatory T cells. Previously, the team of Adrian Liston (VIB-KU Leuven, Babraham Institute) and Matthew Holt (VIB-KU Leuven, i3S Porto) showed that administering a protein called Interleukin-2 (IL2), which helps regulate the immune response, increased the number of regulatory T cells in the brain. This treatment has been successful in mouse models of traumatic brain injury and neuroinflammation.

Now, the researchers want to see if delivering IL2 directly to the brain can help reduce age-induced inflammation and cognitive decline.

Gene therapy improves brain aging

In their latest study, the team discovered that delivering IL2 to the brain improved brain function in aging mice. The research showed that the treatment restored cognitive performance in spatial memory tests, allowing older mice to form new memories almost as well as young mice. The mice given IL2 treatment were better at remembering visual cues than those that did not receive the treatment. Additionally, some of the changes in cellular aging in the brain were reversed, especially among several types of glial cells, which are critical to support overall brain function and health.

Pierre Lemaitre, co-first author of the study: “Our approach was to harness the body’s own system to regulate inflammation and to boost it precisely where it was needed.”

IL2 was delivered to the brain using a gene therapy vector, which is a tool that provides genetic material to specific cells. The additional dose of IL2 allowed the regulatory T cells to survive and create an anti-inflammatory environment. Matthew Holt and Lidia Yshii, co-senior authors of the study: “This reinforces our belief that viral vector-based systems are the way forward for the delivery of therapeutics to combat chronic neurodegenerative diseases and preventing cognitive decline in aging populations.”

Adrian Liston, senior author of the study: “The most important part of this study is the high potential for translation into patients. Inflammation is a process that is conserved in both mice and humans, and regulatory T cells can respond to IL2 in both species. However, there are still regulatory hurdles to clear, and it’s crucial to ensure safety before testing it in patients. Nonetheless, we see a clear path to conducting clinical trials.”

The laboratory is working through spin-off company Aila Biotech to drive entry of this therapeutic into clinical trials. Read the full study here. 

Overnight staining of flow cytometry samples │ Oliver Burton

Doing more with less: Improving flow cytometry staining

Our latest paper isn't going to cure cancer. But it may just making research into curing cancer 10-fold cheaper. A major cost for immunology, oncology and haematology labs is antibodies, especially with the fancy (and expensive!) new dyes coming on the market, which let us hit the 40-50 parameter range in flow cytometry. Fortunately, Dr Oliver Burton in our lab has developed a simple and easy approach to reduce those costs by 10-fold, while also improving the quality of the data: shifting to overnight staining. You can read the full paper here, describing the protocol, fixatives and optimisation approaches, but essentially, lower antibody concentrations give less background, and if left overnight to stain are able to give even better signal detection. It is also a game-changer for wet-lab staff: rather than having all day dissections and staining, and then cueing for flow cytometer hours in the evening, you can set everything up, put on the staining and get an early night. Next morning your cells are ready to go! 

Out now at Current Protocols!

Update on Thursday, November 17, 2022 at 4:54PM by Adrian Liston

We have had multiple requests for a detailed staining protocol, so here it is!