‘Multi-omics’ adds new cell to immune family tree

WEHI researchers have used powerful ‘single cell multi-omics’ technologies to discover a previously unknown ancestor of T and B lymphocytes, which are critical components of our immune system.

Using an approach akin to breaking a sports team’s performance down to the individual player statistics, the researchers looked at multiple aspects of single developing immune cells to define which cells would only give rise to T and B lymphocytes. This revealed a new stage in lymphocyte development, information which could enrich future studies of the immune system. The discovery has also led to new research opportunities, with WEHI establishing of one of Australia’s first dedicated and integrated single cell research platforms in 2018, which is now being used to solve other research questions.

The research, which was published in Nature Immunology today, was led by Dr. Shalin Naik, Dr. Daniela Zalcenstein, Mr Luyi Tian, Mr Jaring Schreuder and Ms Sara Tomei.

Focussing on single cells

Our immune system comprises many different types of cells with different functions, but all immune cells are derived from a single type of cell, a blood stem cell. The development of different immune cell types occurs through a branching ‘family tree’ of immature cells. At earlier stages of immune cell development, individual cells can give rise to several different types of mature cell, but as development progresses, cells become more limited in which final mature cells they can produce.

T and B lymphocytes—which are critical for targeted, specific immune responses—are closely related immune cells, meaning they share many common steps in their development, said Dr. Naik. “Decades of research have defined how T and B lymphocytes develop, and the ‘branch points’ in their family tree when the developing cells lose the capacity to develop into other immune cell types,” he said.

Dr. Zalcenstein said that to gain new insights into questions such as how immune cells develop, the team established Australia’s first ‘single cell multi-omics’ platform, which is now available to all researchers within the Single Cell Open Research Endeavour (SCORE) established by Dr. Naik and Dr. Zalcenstein in collaboration with Dr. Stephen Wilcox of WEHI’s Genomics Hub and Associate Professor Matthew Ritchie.

“Multi-omics technologies combine different biological data sets—such as genomics, transcriptomics and proteomics—to compare different samples in more detail than is possible by looking at one data set. We have applied this approach to study individual cells, in this case developing immune cells, to understand in more detail which cells can give rise to lymphocytes. This approach is called single cell multi-omics,” she said.

“Rather than looking at data combined from many cells in a sample, we focus in on individual cells to understand the differences that exist within a larger population. It’s like looking at a football team—you can average out the number of goals, tackles and kicks per player in a game, but if you look at individual player statistics, you may discover that one player scored lots of goals, while another player was responsible for most of the tackles,” she said.

A new lymphocyte progenitor

SCORE’s study of immune cell precursors revealed a previously unrecognised cell type that could give rise to T and B lymphocytes, but not other immune cells.

“This cell occurred much earlier in lymphocyte development than we had suspected,” Dr. Naik said. “Previous techniques had grouped different immune progenitors together, but by studying individual cells we were able to identify one cell type that was committed to developing into T and B lymphocytes.”

The discovery adds a new layer to the family tree of T and B lymphocytes and could provide a boost to other areas of research.

“Understanding in more detail how T and B lymphocytes develop could lead to better approaches to regenerate these cells as a treatment for certain diseases,” Dr. Naik said. “We also know that many types of leukaemia arise from defects in early stages of immune cell development, so we are curious to know whether this progenitor cell has links to any forms of leukaemia.”

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Immune cell activation in severe COVID-19 resembles lupus

In severe cases of COVID-19, Emory researchers have been observing an exuberant activation of immune cells, resembling acute flares of systemic lupus erythematosus (SLE), an autoimmune disease.

Their findings point towards tests that could separate some COVID-19 patients who need immune-calming therapies from others who may not. They also may begin to explain why some people infected with SARS-CoV-2 produce abundant antibodies against the virus, yet experience poor outcomes.

The results were published online on Oct. 7 in Nature Immunology.

The Emory team’s results converge with recent findings by other investigators, who found that high inflammation in COVID-19 may disrupt the formation of germinal centers, structures in lymph nodes where antibody-producing cells are trained. The Emory group observed that B cell activation is moving ahead along an “extrafollicular” pathway outside germinal centers—looking similar to they had observed in SLE.

B cells represent a library of blueprints for antibodies, which the immune system can tap to fight infection. In severe COVID-19, the immune system is, in effect, pulling library books off the shelves and throwing them into a disorganized heap.

Before the COVID-19 pandemic, co-senior author Ignacio (Iñaki) Sanz, MD and his lab were focused on studying SLE and how the disease perturbs the development of B cells.

Sanz is head of the division of rheumatology in the Department of Medicine, director of the Lowance Center for Human Immunology, and a Georgia Research Alliance Eminent Scholar. Co-senior author Frances Eun-Hyung Lee, MD is associate professor of medicine and director of Emory’s Asthma/Allergy Immunology program.

“We came in pretty unbiased,” Sanz says. “It wasn’t until the third or fourth ICU patient whose cells we analyzed, that we realized that we were seeing patterns highly reminiscent of acute flares in SLE.”

In people with SLE, B cells are abnormally activated and avoid the checks and balances that usually constrain them. That often leads to production of “autoantibodies” that react against cells in the body, causing symptoms such as fatigue, joint pain, skin rashes and kidney problems. Flares are times when the symptoms are worse.

Whether severe COVID-19 leads to autoantibody production with clinical consequences is currently under investigation by the Emory team. Sanz notes that other investigators have observed autoantibodies in the acute phase of the disease, and it will be important to understand whether long-term autoimmune responses may be related to the fatigue, joint pain and other symptoms experienced by some survivors.

“It’s an important question that we need to address through careful long-term follow-up,” he says. “Not all severe infections do this. Sepsis doesn’t look like this.”

In lupus, extrafollicular B cell responses are characteristic of African-American patients with severe disease, he adds. In the new study, the majority of patients with severe infection were African-American. It will be important to understand how underlying conditions and health-related disparities drive the intensity and quality of B cell responses in both autoimmune diseases and COVID-19, Sanz says.

The study compared 10 critically ill COVID-19 patients (4 of whom died) admitted to intensive care units at Emory hospitals to 7 people with COVID-19 who were treated as outpatients and 37 healthy controls.

People in the critically ill group tended to have higher levels of antibody-secreting cells early on their infection. In addition, the B cells and the antibodies they made displayed characteristics suggesting that the cells were being activated in an extrafollicular pathway. In particular, the cells underwent fewer mutations in their antibody genes than seen in a focused immune response, which is typically honed within germinal centers.

The Nature Immunology paper was the result of a collaboration across Emory. The co-first authors are Matthew Woodruff, Ph.D., an instructor in Sanz’s lab, and Richard Ramonell, MD, a fellow in pulmonary and critical care medicine at Emory University Hospital.

Ramonell notes that the patients studied were treated early during the COVID-19 pandemic. It was before the widespread introduction of the anti-inflammatory corticosteroid dexamethasone, which has been shown to reduce mortality.

The team’s findings could inform the debate about which COVID-19 patients should be given immunomodulatory treatments, such as dexamethasone or anti-IL-6 drugs. Patients with a greater expansion of B cells undergoing extrafollicular activation also had higher levels of inflammatory cytokines, such as IL-6.

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A small T cell switch with a big impact

T cells play a key role in the human immune system. They are capable of distinguishing diseased or foreign tissue from the body’s own, healthy tissue with great accuracy; they are capable of triggering the actions necessary to fight off the troublemakers. The details of this immune response are manifold and the individual steps are not yet fully understood.

Scientists of the universities of Würzburg and Mainz have now figured out new details of these processes, showing that tiny point mutations in a gene can modify T cells to be less aggressive. This could be an advantage after stem cell transplantation which includes T-cell transfusion in order to keep a number of severe side effects in check. The researchers have now published the results of their study in the Journal of Experimental Medicine. The study is led by Dr. Friederike Berberich-Siebelt, head of the “Molecular and cellular immunology” research group at the Institute of Pathology of the University of Würzburg.

A protein family with multiple tasks

When T cells detect foreign or altered tissue, such as an infected or tumor tissue, this usually happens through the receptors on their cell surface. These T-cell receptors then send signals into the cell interior, initiating a response. In a first step, they activate a special family of transcription factors—scientifically called NFAT for nuclear factor of activated T-cells. The NFATs then bind to the DNA in the cell nucleus and trigger also the production of cytokines such as interleukin-2.

NFAT is composed of many family members which may have overlapping tasks or assume completely different functions. But that’s not all: Like many other proteins in the cell, they can still be modified after their synthesis to customize their function. The recently published study focuses on one specific modification of the NFATc1 “family member” which is called sumoylation.

Advantageous point mutations

“Sumoylation plays a role in different cellular processes such as nuclear transport, programmed cell death or as an antiviral mechanism,” Friederike Berberich-Siebelt explains. Sumoylation defects have also been observed in various diseases such as cancer and herpesvirus infections.

In the study now published, the scientists worked with laboratory animals that had two actually insignificant point mutations in the NFATc1 gene which, however, prevent sumoylation. This is not necessarily a disadvantage: “The offspring of these animals is perfectly healthy. The modified NFATc1 even mediates specific signals that reduce the clinical symptoms of multiple sclerosis at least in the animal model,” Berberich-Siebelt explains. When using T cells that carry these mutations in stem cell transplantation, they are much less aggressive against the tissues of the host animals than “normal” cells.

Fascinating fundamental research

This effect is due to an increase in interleukin-2 at the beginning of the immune response at the biomolecular level. Interleukin-2 counteracts the differentiation into inflammatory T-cell subtypes and at the same time supports so-called regulatory T cells according to the authors of the study. It is quite possible that this discovery will have consequences for future stem cell transplantation which includes T-cell infusion. When using T cells in which NFATc1 is not sumoylated, this might prevent severe side effects, making the point mutation “a small modification with a big impact” according to Berberich-Siebelt.

To investigate this in more detail, Berberich-Siebelt and her team will continue to research the possibilities of therapeutic implementation within the framework of the Collaborative Research Center/Transregio “Control of graft-versus-host and graft-versus-leukemia immune responses after allogeneic stem cell transplantation” funded by the German Research Foundation (DFG). “We want to find out whether CRISPR/Cas9 gene editing can be applied to human T cells to exhibit just the right amount of activity during hematopoietic stem cell transplantation,” the scientist says.

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Link between calcium, cardiolipin in heart defects discovered

The heart needs the energy to pump blood. So, energy production defects in heart muscles result in a variety of cardiac diseases. In the light of the following information, scientists have now discovered a new link between calcium, heart energy production and cardiolipin, a type of fat.

The discovery helps explain heart defects in the rare genetic disorder Barth syndrome.

The study, published in Proceedings of the National Academy of Sciences, was led by led by Vishal M. Gohil, Ph.D., Department of Biochemistry and Biophysics, Texas A&M College of Agriculture and Life Sciences. Other co-authors were from University of Texas Health Science Center at San Antonio and Massachusetts General Hospital, Boston. The research funding for this study came from the Welch Foundation and the National Institutes of Health.

Heart defects in Barth syndrome

Barth syndrome is a rare genetic disease occurring almost exclusively in boys. The affected children suffer from heart and muscle weakness from early childhood. In this debilitating disease, patients have trouble doing routine activities such as walking and running. Often, their hearts are weak and enlarged.

People with Barth syndrome have a genetic defect that interferes with their body’s ability to make cardiolipin. As the name suggests, cardiolipin is present in large amounts in cardiac — heart — muscles. Cardiolipin belongs to a class of molecules called lipids.

Within muscle cells, cardiolipin is found in mitochondria, which are known as the “powerhouse” of the cell because they produce biological energy from the food we eat. Cardiolipin and other lipids from the membrane “skin” of mitochondria, but cardiolipin seems to be a particularly crucial component. A shortage of cardiolipin undermines mitochondria’s ability to produce energy in the form of adenosine triphosphate, ATP.

A link between cardiolipin, energy and calcium

When cells need a burst of energy, they use calcium as a signal to urge mitochondria to ramp up energy production. Calcium ions enter mitochondria through a special channel in the mitochondrial membrane. Because the calcium channel is present in the same membrane with cardiolipin and other lipids, Gohil and his team wondered what effect the lipids have on the channel.

“We knew this channel sits in the mitochondrial membrane, so we asked, could the lipids in the membrane impact how this channel functions?” said Gohil.

Baker’s yeast helps study energy production in barth syndrome

Gohil’s lab had previously figured out a way to make yeast mitochondria deficient in various lipids, including cardiolipin. Yeasts have mitochondria that closely resemble those of humans in many ways, but they lack the calcium channel.

Sagnika Ghosh, the study’s lead author and a graduate student in Gohil’s lab, genetically modified baker’s yeast mitochondria to include the human calcium channel. She then examined what happens to calcium transport when the membrane’s lipid composition changes.

“We found that the calcium channel was not stable in a mitochondrial membrane with a low amount of cardiolipin, such as the amount seen in Barth syndrome patients,” Gohil said.

Confirmation in patient samples

Next, the team acquired cells and heart tissue samples from Barth syndrome patients. The team confirmed what they saw in their experiments in yeast also happens in the patient samples. Because the mitochondrial calcium channels were unstable, the mitochondria of Barth syndrome patients were much less permeable to calcium than those of healthy cells.

So, when a patient’s cells need a burst of energy, sending a calcium signal to mitochondria may not ramp up energy production as it would in a healthy cell.

“Starting from a fundamental scientific question, our work led to a discovery related to human health,” Gohil said. “In this genetic disease, a defect in calcium uptake in turn could affect energy production. What we observed in yeast was also true in human cells.”

(This story has been published from a wire agency feed without modifications to the text. Only the headline has been changed.)

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