Trials in autoimmune disease

By Susan Williamson
Wednesday, 21 August, 2013

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Giving children their own cord blood to prevent them getting type 1 diabetes and the development of a new immunotherapy are part of a more tailored approach to treating autoimmune disease.

Preventing diabetes with the right cells

Having a first- or second-degree relative with type 1 diabetes increases your chance of getting this lifelong disease, which results from an imbalance in the immune system. This knowledge has led Associate Professor Maria Craig, a paediatric endocrinologist at the Children’s Hospital at Westmead, to investigate whether umbilical cord blood may restore immune balance and prevent or delay the development of type 1 diabetes in children in this high-risk group.

“There is an increased risk of developing type 1 diabetes if your father, mother or a sibling has the disease,” said Craig. “In fact, the risk is higher if your father has the disease, although we do not know why. It is probably related to the immune protection provided by the mother during pregnancy.”

Craig is lead investigator on the national study, called cord blood reinfusion in diabetes (CoRD). The study’s aim is to determine whether immune cells present in cord blood can stop and potentially reverse the immune destruction of the insulin-producing beta cells in the pancreas.

“This is a new direction for stem cell research,” Craig said of the pilot study. “We will be looking at whether giving children their own cord blood can prevent them progressing from pre-type 1 diabetes to full-blown type 1 diabetes. If successful, it will also validate the storage of cord blood for those at risk.”

Restoring immunomodulation

In type 1 diabetes the breakdown of immune tolerance to self-antigens, such as insulin, results in the expansion of autoreactive T cells and the consequent destruction of the beta cells of the pancreas. There is an average of two new cases of type 1 diabetes diagnosed per day in Australian children.

Although an intervention that successfully prevents type 1 diabetes remains elusive, increasing evidence suggests that immune therapies, particularly those based on restoration of peripheral immune tolerance, may help thwart this autoimmune disease.

Regulatory T cells (Tregs) play a key role in inducing and maintaining immune tolerance to self and non-self antigens - that is they make sure the body does not attack itself.

Abnormalities in the function and/or number of Tregs have been found in people with type 1 diabetes and infusion of Tregs in the non-obese diabetic mouse model of type 1 diabetes prevents the development of the disease.

Umbilical cord blood contains large numbers of highly functional Tregs, as well as other stem cells that can develop into the different blood cell types. Cord blood infusions have been given to children who have already developed type 1 diabetes, but no studies have examined whether cord blood may have therapeutic potential in preventing type 1 diabetes in high-risk children.

This is where the CoRD study comes in.


The presence of multiple islet autoantibodies in the blood is a known risk marker for type 1 diabetes. These autoantibodies indicate that an immune attack directed towards the beta cells of the pancreas has already begun.

“There are four markers that can predict with significant accuracy whether you are on the pathway to developing type 1 diabetes,” Craig explained. “If you have two or more of these then you have about a 50% risk of developing the disease.”

Phase one of the two-phase CoRD study will screen children for autoantibodies to the four islet antigens - insulin, glutamic acid decarboxylase, insulinoma-associated-2 and a beta-cell-specific zinc transporter called ZnT8. Those who test positive for two or more of these antigens will be eligible to take part in the second phase of the study, where they will be given treatment and follow-up.

Treatment will involve a single infusion of autologous (their own) cord blood. The children will be tested before and after treatment to make sure they don’t have diabetes. And, their Tregs will also be counted before and after treatment, using flow cytometry, to see whether their cord blood infusion has led to an increase in these cells.

“We expect to screen 600-800 children and of these we expect about 5% to have two or more of these autoantibodies,” said Craig. “These 20 children will be at high risk of developing type 1 diabetes over the next two years. The maths doesn’t quite add up because we expect about half of the 40 eligible to participate.”

The study is currently in the screening phase, having received ethics approval last year.

Balancing the immune system

Cord blood contains a large proportion of Tregs compared to blood from adults or children. And children who develop diabetes seem to be deficient in these T cells.

“We will be looking at whether there are enough regulatory T cells in cord blood to prevent these children progressing to type 1 diabetes,” Craig explained. “There are other factors that generate immune benefits in cord blood, the immune tolerance of the mother during pregnancy indicates this.”

The study will use cord blood from Cell Care Australia, a large private cord blood bank based in Melbourne that has provided funding for the study.

“It can be difficult to use public cord blood banks,” Craig explained. “This is because it is an altruistic donation to the public bank and donors cannot get their cord blood back under the current legislation - it would be a concern if people started accessing cord blood for their child when it is used for transplant in childhood leukemia.”

Children, or the parents of children, who have their cord blood stored in private banks and who have first- or second-degree relatives with type 1 diabetes will be invited to take part in the CoRD study.

Craig said the disproportionately higher rate of cord blood stored by people with relatives who have type 1 diabetes compared with the general population suggests there is awareness in the community that cord blood may be useful.

Associate Professor Maria Craig is a paediatric endocrinologist at the Children’s Hospital at Westmead and St George Hospitals and an NHMRC practitioner Fellow at the University of Sydney. She completed her Bachelor of Medicine and Bachelor of Surgery (MBBS) at the University of Melbourne in 1989 and subsequently specialised in paediatric endocrinology. Since completing a PhD investigating the association between enterovirus infection and onset of type 1 diabetes at the University of New South Wales, in 2002, her major research focus has been the link between viruses and childhood diabetes. She is principal investigator of the CoRD study and lead investigator for NSW on the ENDIA (Environmental Determinants of Islet Autoimmunity) study, which is looking into what contributes to the development of type 1 diabetes in early childhood.

Immunotherapy as a treatment

One of Associate Professor Simon Barry’s goals is to develop a routine procedure for the transplantation of regulatory T cells (Tregs) to induce immune tolerance.

Barry is based at the Women’s and Children’s Hospital in Adelaide, where he is chief hospital scientist and head of the Molecular Immunology Laboratory. He also works at the University of Adelaide and the Women’s and Children’s Health Research Institute, making him well placed to pursue this ambitious task.

Tregs are a subpopulation of immune cells that play a key role in modulating the immune system. They maintain a balance between the immune system’s tolerance of normal cells, tissues and organs (known as self-tolerance) and reactivity to harmful foreign bacteria and pathogens.

“Autoimmune diseases involve the breakdown of this immune balance and if there is too much reactivity and not enough tolerance, the system becomes reactive to self,” explained Barry. “In contrast, cancer involves too little immune reactivity and too much immune tolerance so the cancer cells are able to grow undetected.”

In type 1 diabetes, the immune system overreacts and breaks down the beta cells of the pancreas. Defects in Treg function are increasingly being linked to autoimmune diseases like type 1 diabetes and multiple sclerosis.

There are 25,000,000 people with autoimmune disease worldwide, 100,000 people per year receive an organ transplant and 30,000 people suffer from graft versus host disease (GVHD) because of bone marrow transplants each year. Thus, a therapy that helps restore tolerance would benefit many people.

Biomarkers for bait

White blood cells make up 10-30% of cells in adult blood and 1% of these are the Tregs. One of the challenges Barry faces in getting these cells into the clinic is generating enough of them for therapeutic use.

To put Tregs to use in clinical work, the researchers need to be able to identify the cells and extract them while retaining their function.

The forkhead transcription factor p3 (Foxp3), a key regulator in the development and function of Tregs, is a specific marker for these cells. Along with the cell surface markers CD4, CD25 and CD127, it is used in flow cytometry to detect Treg cells.

“But staining for Foxp3 kills the cells,” Barry explained. “Therefore, we went on a search for biomarkers to use as bait to pull live cells out and enable us to purify them for use in humans.”

For the last five years, Barry’s team has been mining gene array data to identify genes that are essential to Treg function. Using diseased and non-diseased human cohorts, they have identified many of the genes necessary for immune tolerance and regulatory T cell function.

“By looking at genes that were turned on or off in these different cohorts, we could work out which genes were important in Treg function and confirm a role for these genes,” explained Barry.

Their search for genes expressing cell surface proteins resulted in the discovery of PI-16, now called CD359, which is highly expressed on the surface of Treg cells. In a partnership with the Cooperative Research Centre for Biomarker Translation, PI16 is showing promise as a novel human Treg biomarker.

“We made an antibody to PI-16, patented it and then used it to purify cells from cord or peripheral blood,” said Barry.

Putting their new antibody to work, the researchers retrieved cells that were functional and expressed high levels of PI-16 and Foxp3. But they also found a population of Tregs that did not express PI-16.

“We were able to distinguish two subsets of Treg cells,” said Barry. “Natural Tregs that expressed PI-16 and inducible Tregs that did not.”

Application in the clinic

In a normal healthy gut the ratio of Tregs to T helper (Th 17) cells is in balance. In diseased guts, such as those of people with ulcerative colitis and Crohn’s, there are more Th17 cells than Treg cells.

Th17 cells are thought to play a role in autoimmune diseases; however, they also serve an important function in antimicrobial immunity at epithelial mucosal barriers such as the lining of the gut.

Proinflammatory cytokines, such as interleukin 6 and interleukin-1 beta, are also elevated in the guts of patients with ulcerative colitis and Crohn’s disease.

“We looked at purified Tregs and their differences in function and found that in an environment high in inflammatory cytokines the iTregs lose function but the nTregs do not,” Barry explained. “So, if the two subsets of Treg cells are separated out there may be an increased therapeutic benefit.”

Thus, the aim is to use PI-16 to pull nTreg cells out of cord blood and adult blood and ultimately generate a scalable technique that can be taken into trials with hundreds of patients.

“Feasibility was an issue,” said Barry. “We didn’t know whether we could make enough of them to readily expand for treatment.”

But now that they have the PI-16 biomarker to apply in a technique that generates highly purified cells, and these cells demonstrate robust function in assays, Barry sees them able to generate enough cells needed for cellular therapy. Being part of the new $59 million Cooperative Research Centre for Cellular Therapy Manufacturing, which will open in July this year, will help facilitate this process.

But Barry is keen to generate more data in humans before taking the work into the clinic. Efficacy tests in mouse models of human GVHD are underway and clinical studies are ongoing to determine whether there is an imbalance in PI-16 Tregs in human diseases including type 1 diabetes and irritable bowel disease.

Tregs have broad applications in the clinical context such as correcting immune imbalances in people with autoimmune disease, eliminating the chance of rejection after organ transplant and negating the need for immunosuppressant drugs.

“Lab data is not always a good predictor of how a therapy will work in real patients and we want to be more sophisticated in our testing before we go into a clinical trial in humans, “ Barry said, adding that he expects the work to enter into feasibility studies in humans within three years.

Associate Professor Simon Barry is currently chief hospital scientist and head of the Molecular Immunology Laboratory at the Women’s and Children’s Hospital in Adelaide. Barry is also leader of the Autoimmune Diseases Stream at the Children's Research Centre in the Discipline of Paediatrics at the University of Adelaide, and Senior Research Fellow at the Women’s and Children’s Health Research Institute. Barry completed his PhD at the National Institute for Medical Research Mill Hill in London before pursuing a postdoc at the Hanson Institute in Adelaide. He then continued his research career at the University of Washington before working with Immunex and Amgen. At the University of Adelaide since 2004, Barry’s work is focused on the behaviour of Tregs and the genes that drive their formation and function, with a view to identifying new and better ways of diagnosing and treating autoimmune disease.

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