Manipulating brain Tregs to protect against neuropathology

From the GlobalImmuno Talks 20222:

New understanding of cell stability with potential to improve immune cell therapies

Researchers identify the origin of potentially dangerous unstable cells

Key points:

• Researchers have identified the origin of unstable cells, with potential to improve the safety of immune cell therapies.
• When using immune cells to treat disease, there is a risk that the cells switch from protective to destructive behaviour.
• Studies in mice have allowed researchers to identify the cells most at risk of becoming harmful.

By purifying cells using markers of instability, or following a two-step purification process, the researchers are able to produce a robust set of protective cells. Research in mice, published today by researchers at the Babraham Institute, UK and VIB-KU Leuven, Belgium, provides two solutions with potential to overcome a key clinical limitation of immune cell therapies. Cell therapy is based on purifying cells from a patient, growing them up in cell culture to improve their properties, and then reinfusing them into the patient. Professor Adrian Liston, Immunology group leader at the Babraham Institute, explained: “The leading use of cell therapy is to improve T cells so that they can attack and kill a patient’s cancer, however the incredible versatility of the immune system means that, in principle, we could treat almost any immune disorder with the right cell type. Regulatory T cells are particularly promising, with their ability to shut down autoimmune disease, inflammatory disease and transplantation rejection. A key limitation in their clinical use, however, comes from the instability of regulatory T cells – we just can’t use them in cell therapy until we make ensure that they stay protective”. By identifying the unstable regulatory T cells, and understanding how they can be purged from a cell population, the authors highlight a path forward for regulatory T cell transfer therapy. The study is published today in Science Immunology.

T cells come in a large variety of types, each with unique functions in our immune system. “While most T cells are inflammatory, ready to attack pathogens or infected cells, regulatory T cells are potent anti-inflammatory mediators”, Professor Susan Schlenner, University of Leuven, explains. “Unfortunately this cell type is not entirely stable, and sometimes regulatory T cells convert into inflammatory cells, called effector T cells. Crucially, the converted cells inherit both inflammatory behaviour and the ability to identify our own cells, and so pose a significant risk of damage to the system they are meant to protect.”

The first key finding of this research shows that once regulatory T cells switch to becoming inflammatory, they are resistant to returning to their useful former state. Therefore, scientists need to find a way to remove the risky cells from any therapeutic cell populations, leaving behind the stable regulatory T cells. By comparing stable and unstable cells the researchers identified molecular markers that indicate which cells are at risk of switching from regulatory to inflammatory. These markers can be used to purify cell populations before they are used as a treatment.

In addition to this method of cell purification, the researchers found that exposing regulatory T cells to a destabilising environment purges the unstable cells from the mixture. Under these conditions, the unstable cells are triggered to convert into inflammatory cells, allowing the researchers to purify the stable cells that are left. “The work needs to be translated into human cell therapies, but it suggests that we might be best off treating the cells mean”, says Professor Adrian Liston. “Currently, cell culture conditions for cell therapy aim to keep all the cells in optimal conditions, which may actually be masking the unstable cells. By treating the cultures rougher, we may be able to identify and eliminate the unstable cells and create a safer mix of cells for therapeutic transfer”. Dr Steffie Junius, lead author on the paper, commented: “The next stage in the research is to take the lessons learned in mice and translate them into optimal protocols for patients. I hope that our research contributes to the improved design and allows the development of effective regulatory T cell therapy."

Establishing a thorough process to improve cell population stability in mice helps to lay the groundwork for improved immune cell therapies in humans, although the methods described in this work would require validation in humans before they were used in cell therapy trials. Tim Newton, CEO of Reflection Therapeutics, a Babraham Research Campus-based company designing cell therapies against neuro-inflammation and independent from the research, commented on the translational potential of the study: "This research makes a significant impact on regulatory T cell therapeutic development by characterising unstable subsets of regulatory T cells that are likely to lose their desirable therapeutic qualities and become pro-inflammatory. The successful identification of these cells is of great importance when designing manufacturing strategies required to turn potential T cell therapeutics into practical treatments for patients of a wide range of inflammatory disorders."

Read the full paper here.

Go with the flow – a new algorithm streamlines and improves flow cytometry analysis

Key points:

• A new algorithm developed by researchers at the Babraham Institute provides a fast and effective way to reduce errors in flow cytometry data analysis, overcoming a major restriction on harnessing the full potential of the power of flow cytometry in cell analysis.
• The tool, called AutoSpill, addresses the problem of overlapping signals and autofluoresence, which can be misinterpreted as genuine results.
• Researchers can use the tool, available online and through the software package FlowJo, to easily reduce compensation errors in their flow cytometry data.

Flow cytometry is a key investigative tool used in biomedical research, allowing researchers to identify, separate and study cells according to their characteristics, often working with cell samples containing millions of cells at an analysis pace of a million cells per minute. Cell identification is achieved by labelling cells with fluorescent tags. As with personal gadgets and devices, innovation in molecular biology technologies isn’t standing still. Advances in flow cytometry have allowed scientists to gather data on a growing number of parameters, simultaneously detecting over 30 different tags at a time to allow more sophisticated analyses and much deeper levels of insight. However, while flow cytometry equipment has been updated, the accompanying computational requirements have received less attention, until now. AutoSpill, an algorithm developed by researchers at the Babraham Institute and the VIB Center for Brain Research, brings data processing in line with state-of-the-art machines, simplifying data analysis and increasing accuracy. The new technique is published in Nature Communications today.

Immunology programme senior group leader Prof. Adrian Liston, explained: "Flow cytometry is a foundational technology across many different biomedical research areas, and is a key diagnostic tool in immunology, haematology and oncology. Despite the technical progress over the past decades, the technology has been held back by the mathematical processing of the data. Our new approach reduces error by 100,000-fold, making research and diagnostics more accurate. The collaboration with FlowJo has enabled us to instantly reach 80,000 users. It is very gratifying to see computational biology have a direct and real impact on research and diagnostics."

Using multiple fluorescent signals raises a key issue in flow cytometry called spillover. Spillover occurs because each tag, called a fluorophore, emits light within a range of wavelengths, giving it a unique colour. When multiple fluorophores are used, the signals begin to overlap. To accurately distinguish between two distinct fluorophore signals, researchers must process their data to compensate. Because flow cytometry uses so many different colour tags on each cell, the spillover between colours quickly accumulates, limiting scientists’ power to draw reliable conclusions from their results. The processing of data to remove the spillover between the different colours, known as compensation, is necessary for all flow cytometry experiments. Current methods require many hours of manual work, but AutoSpill reduces the process to minutes.

Dr Rachael Walker, Head of the Institute Flow Cytometry facility, commented: “The new AutoSpill Fluorescence Compensation algorithm is a great tool for quick, simple and accurate compensation. It allows compensation to be accurately calculated on samples where the traditional algorithm is difficult to use. AutoSpill’s integration into the FlowJo post-acquisition software highlights the importance of this new compensation method.”

Another limitation of flow cytometry is autofluoresence, fluorescence produced naturally by cells. The removal of these artefacts by AutoSpill is particularly useful for cancer biologists as tumour cells are high in autofluorescence, which can confuse identification of the type of tumour cell present. By solving these sources of error, AutoSpill can help remove false positives from cell analyses, ensuring more accurate data interpretations.

AutoSpill is available through open source code and a freely-available web service. AutoSpill, and a complementary related tool, AutoSpread, are also available in FlowJo v.10.7. Dr John Quinn, Director of Science and Product Development, FlowJo added: “AutoSpill & AutoSpread have been a revelation for FlowJo users. Compensation has long been one of the most perplexing aspects of cytometry, with the most critical requirement being pristine compensation controls collected for each and every parameter in an experiment. Overall, the combination of these two tools makes compensation both easier and more robust. As an indicator of the popularity of this new approach, the webinar held in conjunction with Nature to introduce AutoSpill / AutoSpread in FlowJo has been viewed over 400 times after the initial live event. We at FlowJo believe the AutoSpill / AutoSpread approach will be the primary means of approaching compensation moving forward.”

Battle Robots of the Blood reading

Me and Hayden read "Battle Robots of the Blood" together.

European “ImmunAID” project for the diagnosis of rare autoinflammatory systemic diseases launched in Belgium

The project wishes to diagnose rare autoinflammatory systemic diseases through the identification of biomarkers

In December 2020 a new project has been launched in the University Hospitals Leuven. The ImmunAID project aims to identify new tools for the diagnosis of systemic auto-inflammatory diseases (SAID). SAID are a complex and evolving group of rare diseases characterised by extensive clinical and biological inflammation. These conditions are caused by a dysregulation of the innate immune system leading to a release of immune cells and mediators provoking fevers, tissue and organ inflammation and damage.

Sometimes it is difficult for the physicians to make a correct diagnosis, since the main symptoms of these diseases (such as fever, rash, joint pain, etc.) are also present in many other conditions. Thus, a patient may have received on average up to 5 inappropriate or ineffective treatments before being properly diagnosed, having a great impact on their health and quality of life. The aim of ImmunAid is to understand the mechanisms that drive the pathology in order to provide better diagnosis and care for patients with these rare but potentially devastating diseases.

An unprecedented body of clinical and biological data in the field of SAID

This new project aims to find new and more effective ways to diagnose SAID. While it is already known that some SAID are due to specific genetic mutations, a large number of SAID can only be detected by a set of clinical signs and symptoms and after other diagnostic possibilities have been excluded. Since SAID are rare conditions, a large group of patients suffering from various SAID is being recruited throughout Europe. As such, the ImmunAID cohort represents a very important tool for researchers defining biological fingerprints, or biomarkers, specific to distinct SAID.

The team expects to find a set of biological features common to all SAID, which will allow to quickly confirm or refute the diagnosis of suspected autoinflammatory syndrome. In addition, for each SAID, a list of characteristic biomarkers and an algorithm will be generated to allow the physician to make an appropriate diagnostic assessment.

In order to achieve the project's objectives, biological samples collected from the patients will be analysed in a European-wide research network by set of state-of-the-art technologies and will generate an unprecedented amount of data (genomics, transcriptomics, proteomics and microbiome). Simultaneously, other analyses will focus on immune cells, molecular mechanisms and specific agents of the immune system (cytokines, etc.). All data generated will be subjected to artificial intelligence and modelling analysis.

Prof. Carine Wouters, paediatric rheumatologist at the University Hospitals Leuven, is highly committed to the success of the project "We are delighted and proud to be able to work with ImmunAID partners as it represents a unique opportunity for the European scientific community to advance research in an important field of rare diseases that can only be tackled at large scale. We will do our best to come up with meaningful results that will improve patients’ diagnosis and medical care.”

Leuven teams are the forefront of the project

The teams of the Leuven University Projects are at the forefront of the project. The activities carried out in the Belgian centre will be two-fold. First, the team from professor Carine Wouters and professor Steven Vanderschueren will be in charge of recruiting patients suffering from monogenic SAID (FMF, CAPS, TRAPS, MKD) or genetically-undiagnosed SAID (Still disease, neutrophilic dermatosis, Schnitzler syndrome, Takayasu arteritis, Kawasaki disease, Behçet disease, chronic osteitis, recurrent pericarditis and chronic systemic inflammation of unknown origin).

Second, professor Wouters, professor Patrick Matthys and professor Paul Proost from the Rega Institute and KU Leuven department for Microbiology, Immunology and Transplantation will be involved in the biochemical and biological analysis of the samples. The team of Carine Wouters and Patrick Matthys will apply their extensive knowledge on Natural Killer cells to identify and characterize their possible altered activity in SAID patients. On the other hand, the team of Paul Proost will study whether modifications of messengers of the immune system (cytokines and chemokines) in patients play a role in regulation of the inflammation processes. The team of professor Stephanie Humblet-Baron and professor Adrian Liston will analyse in-depth the immune cellular compartment of the blood of affected patients in addition to genetic investigation in order to identify new genes responsible for SAID.

These activities are intended to gain insight into the mechanisms triggering the aberrant behaviour of the autoinflammation process. The results will be pooled with other analyses from other European research laboratories to help identify biomarkers of the diseases and possible therapeutic interventions.   

Regarding the ImmunAID project: ImmunAID is a research project (www.immunaid.eu), which aims to identify a set of disease-specific biomarkers to confirm the diagnosis of SAID. ImmunAID is implemented by a large consortium (25 partners in 12 European countries) and has been funded with € 15.8 million by the European Commission. The ImmunAID project has received funding from the European Union's Horizon 2020 research and innovation programme under Grant Agreement No. 779295.

IL-2 cytokine networks

If anyone is interested in our lab's work on IL-2 cytokine networks, I just gave a seminar on the topic, which I am putting up here:

It is a new talk for me, and was an interesting one to write. I started to work on IL-2 right at the start of my PhD. I was very keen to return to the topic when I opened my own lab in Belgium (2009), with one of my first PhD students (Dr Wim Pierson) working on the niche-sensing and niche-filling negative feedback loop that provides a stable number of Tregs in the system. (An excellent collaboration with one of my favourite immunologists, Prof Daniel Gray from WEHI, Australia).

Then Prof Stephanie Humblet-Baron joined my lab for a post-doc, wanting to work on a disease known as Familial hemophagocytic lymphohistiocytosis (FHL). At the time, this was thought to be a disease of CD8 hyper-activation and IFN-gamma. Thanks to great work by Stephanie, in mouse and human, we now know that FHL is only partly driven by IFN-gamma, and instead a key part of pathogenesis comes from flipping the negative feedback loop between IL-2 and Tregs into a postivie feedback loop between IL-2 and CD8 T cells.

Right back in 2009 we started to work on a new genetic switch that would let us turn IL-2 on in different cell types. At first I just wanted to see what would happen if Tregs could make their own IL-2. By breaking that dependency on exogenous IL-2 do you get a run-away Treg reaction? (answer: yes, yes you do). Once we finally made the mice, however, it just opened so many different doors. What happens if CD8 T cells make their own IL-2? How about NK cells, dendritic cells, B cells? What if we turn it on in different organs? It has really been a phenomenal mouse that just kept on delivering interesting results. Dr James Dooley led a team working on the mouse, and more recently Dr Carly Whyte drove the project to publication. Or, at least, pre-publication - you can see the paper here on BioRxiv. So many interesting aspects of IL-2 biology were illuminated by this work - easiest to show in a circuit diagram:

I hope you enjoy the seminar. Keep an ear out for the muffled bang at the 29 minute mark. It doesn't sound like much on the audio feed, but across Cambridge we all jumped up as the windows rattled and the building shuddered. I fumbled the graph on this slide, calling Tregs Tconv by mistake, wondering if an explosion had gone off downstairs. Fortunately it was just a sonic boom as fighter jets scrambled overhead.

Top 10 health innovations of 2020

Great to see our recent Cell paper on brain T cells licensing microglia listed as one of the top 10 health innovations of 2020!

Dissecting the immune characteristics of severe COVID-19 responses

• Researchers have analysed immune cell types and numbers from the blood of healthy volunteers, COVID-19 patients experiencing mild-to-moderate effects and patients classified as severe to understand whether particular characteristics of their immune system response can identify treatment targets or indicate disease severity.
• After comparing the T cell immune response, the researchers noted the surprising absence of a strong anti-viral response in the blood of COVID-19 patients.
• The study identified an elevated presence of anti-inflammatory-producing regulatory T cells in the severely affected patients. If confirmed by larger studies, this could be used as a marker for identifying worsening cases and could provide an insight into the mechanism of disease pathology.

A team of immunology experts from Belgium and the UK research organisations have come together to apply their pioneering research methods to put individuals’ COVID-19 response under the microscope. Published today in the journal Clinical and Translational Immunology, their research adds to the developing picture of the immune system response and our understanding of the immunological features associated with the development of severe and life-threatening disease following COVID-19. This understanding is crucial to guide the development of effective healthcare and ‘early-warning’ systems to identify and treat those at risk of a severe response.  

One of the most puzzling questions about the global COVID-19 pandemic is why individuals show such a diverse response. Some people don’t show any symptoms, termed ‘silent spreaders’, whereas some COVID-19 patients require intensive care support as their immune response becomes extreme. Age and underlying health conditions are known to increase the risk of a severe response but the underlying reasons for the hyperactive immune response seen in some individuals is unexplained, although likely to be due to many factors contributing together.

To investigate the immune system variations that might explain the spectrum of responses, teams of researchers from the VIB Centre for Brain and Disease Research and KU Leuven in Belgium and the Babraham Institute in the UK worked with members of the CONTAGIOUS consortium to compare the immune system response to COVID-19 in patients showing mild-moderate or severe effects, using healthy individuals as a control group.

Professor Adrian Liston, senior group leader at the Babraham Institute in the UK, explained: “One of our main motivations for undertaking this research was to understand the complexities of the immune system response occurring in COVID-19 and identify what the hallmarks of severe illness are. We believe that the open sharing of data is key to beating this challenge and so established this data set to allow others to probe and analyse the data independently.”

The researchers specifically looked at the presence of T cells – immune cells with a diverse set of functions depending on their sub-type, with ‘cytotoxic’ T cells able to kill virus-infected cells directly, while other ‘helper’ T cell types modulate the action of other immune cells. The researchers used flow cytometry to separate out the cells of interest from the participants’ blood, based on T cell identification markers, cell activation markers and cytokine cell signalling molecules.

Surprisingly, the T cell response in the blood of COVID-19 patients classified as severe showed few differences from the healthy volunteers. This is in contrast to what would usually be seen after a viral infection, such as the ‘flu. However, the researchers identified an increase in T cells producing a suppressor of cell inflammation called interleukin 10 (IL-10). IL-10 production is a hallmark of activated regulatory T cells present in tissues such as the lungs. While rare in healthy individuals, the researchers were able to detect a large increase in the number of these cells in severe COVID-19 patients.

Potentially, monitoring the level of IL-10 could provide a warning light of disease progression, but the researchers state that larger-scale studies are required to confirm these findings.

“We’ve made progress in identifying the differences between a helpful and a harmful immune response in COVID-19 patients. The way forward requires an expanded study, looking at much larger numbers of patients, and also a longitudinal study, following up patients after illness. This work is already underway, and the data will be available within months,” says Professor Stephanie Humblet-Baron, at the KU Leuven in Belgium.

“This is part of an unprecedented push to understand the immunology of COVID-19”, concludes Professor Liston. “Our understanding of the immunology of this infection has progressed faster than for any other virus in human history – and it is making a real difference in treatment. Clinical strategies, such as switching to dexamethasone, have arisen from a better understanding of the immune pathology of the virus, and survival rates are increasing because of it”.  

Professor Liston and Professor Humblet-Baron both emphasized the importance of the scientific team that led the study. "This work happened during a period of incredible stress. When much of our laboratory was shut down due to the pandemic, Dr Teresa Prezzemolo and Silke Janssens were in the hospital day-after-day, preparing blood samples that were critical not just for this study but for a whole host of clinical trials on COVID-19 based in Leuven. Julika Neumann and Dr Mathijs Willemsen put their PhD research on hold to run samples, and Dr Carlos Roca and Dr Oliver Burton provided the computational support to turn the data into biological understanding. We are both incredibly proud of the entire team."

Neumann, J., Prezzemolo, T., Vanderbeke, L. & Roca, C.P. et al. Increased IL-10-producing regulatory T cells are characteristic of severe cases of COVID-19. Clinical and Translational Immunology

The CrispR revolution

Emmanuelle Charpentier and Jennifer Doudna have just won the Nobel Prize for Chemistry. They have been my picks for the prize for years now. Nobel Prizes are often awarded decades after the fact, but CrispR has been such an obvious winner that it is a surprise it took until 2020 to be awarded. (Largely, I guess, due to the politics of several competing claims and patents, that have been going through the courts). 

This Noble is a well-deserved recognition of one of the seminal breakthroughs in biology of the last several decades. The award recognises elegant basic biological experiments that identified a novel immune mechanism that bacteria use to fight off viruses. The key insight is that the chemistry of this system allowed simple modifications to rewire this bacterial system into a tool to edit the genome of essentially any living being. A striking example of blue-skies research on basic science having an incredible translational effect. The CrispR system ranks up there with identifying the structure of DNA or the sequencing of the human genome - indeed, for the first time it allows us to really use the information gained by these earlier revolutions. CrispR tools are used daily across the globe to create new vaccines, generate gene therapy, design bacteria to help industrial processes. Essentially, the discovery of CrispR as a genome-modification tool has put biology on steroids - dramatically accelerating the pace of both basic research and translational applications

New role for white blood cells in the developing brain

Whether white blood cells can be found in the brain has been controversial, and what they might be doing used to be complete mystery. In a seminal study published in Cell, an international team of scientists led by Prof. Adrian Liston (VIB-KU Leuven, Belgium & Babraham Institute, UK) describe a population of specialized brain-resident immune cells discovered in the mouse and human brain, and show that the presence of white blood cells is essential for normal brain development in mice.

Like a highly fortified headquarters, our brain enjoys special protection from what is circulating in the rest of our body through the blood-brain barrier. This highly selective border makes sure that passage from the blood to the brain is tightly regulated.

The blood-brain barrier also separates the brain from our body’s immune system, which is why it has its own resident immune cells, called microglia, which trigger inflammation and tissue repair. Microglia arrive in the brain during embryonic development, and later on, the population becomes self-renewing.

Yet, white blood cells—which are part of our immune system—have been found to play a role in different brain diseases, including multiple sclerosis, Alzheimer’s and Parkinson’s disease or stroke. Whether or not white blood cells can be found in healthy brains as well, and what they might be doing there, has been subject of intense debate. An interdisciplinary team of scientists led by Prof. Adrian Liston (VIB-KU Leuven, Babraham Institute) set out to find the answers.

White blood cells in the brain

"A misconception about white blood cells comes from their name,” explains Dr. Oliver Burton (Babraham Institute). “These 'immune cells' are not just present in the blood. They are constantly circulating around our body and enter all of our organs, including—as it turns out—the brain. We are only just starting to discover what white blood cells do when they leave the blood. This research indicates that they act as a go-between, transferring information from the rest of the body to the brain environment"

The team quantified and characterized a small but distinct population of brain-resident T helper cells present in mouse and human brain tissue. T cells are a specific type of white blood cells specialized for scanning cell surfaces for evidence of infection and triggering an appropriate immune response. New technologies allowed the researchers to study the cells in great detail, including the processes by which circulating T cells entered the brain and began to develop the features of brain-resident T cells.

Dr. Carlos Roca (Babraham Institute): “Science is becoming increasingly multidisciplinary. Here, we didn't just bring in expertise from immunology, neuroscience and microbiology, but also from computer science and applied mathematics. New approaches for data analysis allow us to reach a much deeper level of understanding of the biology of the white blood cells we found in the brain.”

An evolutionary role

When T helper cells are absent from the brain, the scientists found that the resident immune cells – microglia – in the mouse brain remained suspended between a fetal and adult developmental state. Observationally, mice lacking brain T cells showed multiple changes in their behavior. The analysis points to an important role for brain-resident T cells in brain development. If T cells participate in normal brain development in mice, could the same be true in humans?

“In mice, the wave of entry of immune cells at birth triggers a switch in brain development,” says Liston. “Humans have a much longer gestation than mice though, and we don't know about the timing of immune cell entry into the brain. Does this occur before birth? Is it delayed until after birth? Did a change in timing of entry contribute to the evolution of enhanced cognitive capacity in humans?”

The findings open up a whole new range of questions about how the brain and our immune system interact. "It has been really exciting to work on this project. We are learning so much about how our immune system can alter our brain, and how our brain modifies our immune system. The two are far more interconnected than we previously thought," says Dr. Emanuela Pasciuto (VIB-KU Leuven).

The study also brings in a connection with the gut microbiome, says Liston: “There are now multiple links between the bacteria in our gut and different neurological conditions, but without any convincing explanations for what connects them. We show that white blood cells are modified by gut bacteria, and then take that information with them into the brain. This could be the route by which our gut microbiome influences the brain.” 

Taken together, the results contribute towards the increasing recognition of the role of immune cells in the brain and shed new light on its involvement in a range of neurological diseases.

Check out the full article here