For Betty Lewis, 1979 was a big year: she got married, was diagnosed with a progressive liver disease, and had a liver transplant. But two years later, she had a new baby, was getting divorced, and hated the way her immunosuppressive drugs made her feel. So, against her doctors' advice, she ditched the drugs. Today, she has confounded medical opinion and is still alive and well.

Immunologists are working to understand why some patients naturally achieve this state of tolerance, in which the immune system learns to accept a target that it would ordinarily attack. It's an important endeavour, because if doctors could induce tolerance at will, the prospects for transplant patients would greatly improve — immunosuppressive drugs may prevent organ rejection, but by damping down the entire immune system, they render patients susceptible to infections and cancer. Selective immune tolerance might also be used to combat debilitating allergies, or autoimmune conditions such as type 1 diabetes and multiple sclerosis.

Making such treatments part of standard clinical practice is the goal of the Immune Tolerance Network (ITN), a seven-year, $165-million project launched by the US National Institutes of Health in 1999. The network has 15 clinical trials in progress, and is gathering huge quantities of immunological data from the enrolled patients in the hope of elucidating the basic mechanisms of tolerance. But the ITN faces some tough challenges. Our understanding of the immune system is incomplete — which means that attempts to manipulate it can lead to unpredictable side-effects. And even if a tolerance therapy proves to be safe and effective, can the pharmaceutical industry be persuaded to invest in it?

“We would be incredibly excited if we could show that a very expensive drug worked in a disease that just three people in the world have, if it helped us to understand how tolerance works,” observes Jeffrey Bluestone, who directs the ITN. “But a drug company would not find that a very attractive trial to run.” Nevertheless, Bluestone, an immunologist at the University of California, San Francisco, is optimistic that the ITN will identify treatments that are both clinically and commercially viable.

The key to achieving tolerance is to tame the specific cells responsible for undesired immune responses. The foot-soldiers of the immune system are B cells, which produce antibodies that recognize a particular molecular signature, or antigen; and T cells, which likewise respond to individual antigens and attack cells bearing that signature.

Immature B and T cells are created all the time in our bone marrow, and they circulate in our blood until they encounter their specific antigen and become activated. T cells, for instance, bear receptors that allow them to lock onto 'antigen-presenting cells' (APCs) that process foreign antigens and carry them on their surface. The T cell and the APC then send each other a series of biochemical signals in a process called co-stimulation. If the T cell receives all the proper co-stimulatory signals, it becomes an activated killing machine targeted against the antigen.

The immune system needs to prevent immune cells reacting to the body's own proteins, and this is achieved through mechanisms of central and peripheral tolerance. In central tolerance, immature T cells that react with 'self' proteins are destroyed in the thymus gland in the neck. Some self-reactive T cells escape this central control, but their activation is normally blocked by one of the peripheral tolerance mechanisms. In some cases, the T cells recognize their antigens on APCs, but do not receive the correct co-stimulatory signals. Other self-reactive T cells express receptors that cannot lock onto APCs. And some immunologists believe that a special population of regulatory T cells helps to keep self-reactive T cells in check.

Immune-tolerance therapies aim to co-opt these natural mechanisms. The most advanced work has been done in organ transplantation, where surgeons are trying to promote central tolerance by dosing transplant recipients with bone marrow from the same donors as their new organs. By establishing 'chimaerism' — a state in which the recipient's bone marrow is made up of both their own cells and those from the donor — the immune system should begin to treat the transplanted organ as self rather than foreign.

Intolerant attitudes

Reading the signs: Angus Thomson and Adriana Zeevi have identified signals that aid tolerance. Credit: UNIV. PITTSBURGH

Autoimmune disease occurs when the mechanisms of tolerance break down. Here, researchers are trying to restore peripheral tolerance by interfering with T-cell receptors and associated molecules, or by muddling the process of co-stimulation. And to treat allergy, immunologists are trying to shift the immune response away from debilitating inflammatory reactions.

Transplant surgeons were interested in tolerance long before the ITN got under way, hoping that it might be possible for their patients to avoid a lifetime of immunosuppressive treatment. At the University of Pittsburgh's Thomas E. Starzl Transplantation Institute, surgeons have been systematically weaning liver-transplant recipients off their immunosuppressive drugs for a decade — essentially a controlled version of Betty Lewis's sudden eschewal of her drugs. Pittsburgh immunologists Angus Thomson and Adriana Zeevi are using ITN funding to run a battery of blood tests on patients who have successfully been removed from immunosuppression. They hope to find out what makes these patients different from others who have to keep taking the drugs.

Thomson, Zeevi and transplant surgeon George Mazariegos already have some clues about how to predict which patients will become tolerant. In April, they reported a difference in the production of two cytokines — biochemical signals that help to coordinate immune responses — between 12 children given liver transplants who had been weaned off immunosuppression and 37 patients who had to stay on the drugs1. The tolerant children were genetically predisposed to make low levels of tumour-necrosis factor-α, and high levels of interleukin-10. Such work could help doctors to define cytokine profiles to predict which patients will respond well to the withdrawal of immunosuppression.

While the Pittsburgh group examines patients who have naturally achieved tolerance, other researchers are trying to give nature a little help by using transplants of bone marrow cells from the same donor as a donated kidney. By temporarily suppressing the recipient's own bone marrow using drugs and targeted radiation, these scientists hope that the transplanted cells will establish themselves and create a chimaeric immune system.

Samuel Strober, an immunologist who is leading an ITN-backed trial at Stanford University in California, already has promising results from a preliminary study of patients given kidney transplants by Stanford surgeon Maria Millan. His patients' blood became chimaeric, with cells from the donor accounting for up to 16% of those in their circulation. Three out of four of the patients are responding well to the phased withdrawal of immunosuppressive drugs2.

Meanwhile, a group at Harvard University led by immunologist Megan Sykes and surgeon Ben Cosimi is coordinating a similar trial at multiple centres in patients with multiple myeloma, a bone-marrow cancer that leads to kidney failure in about one in five cases. This builds on a pilot study in which two such patients were completely weaned off immunosuppressive drugs3.

Surgeons are optimistic about these preliminary results — Strober's initial trial, which involved patients who were not perfectly matched to their donors, is particularly encouraging. But there is still a long way to go before bone-marrow chimaerism moves into the transplantation mainstream. First there are safety concerns — temporarily suppressing the patient's bone marrow is a harsh procedure, and there is a risk that the introduced marrow will attack the recipient's body in a potentially fatal reaction called graft-versus-host disease. Immunologists also warn that chimaerism may not prevent chronic transplant rejection, a hard-to-treat complication that can take about a decade to emerge. “The problem comes down the road,” says Hugh McDevitt of Stanford University, who is not involved in Strober's trial.

Cracking chronic rejection

Immunologists are beginning to shed some light on chronic rejection, however. The T cells that attack a transplanted organ may recognize its foreign antigens directly, or require the involvement of APCs. But last year, a team led by Robert Lechler of Imperial College London reported that patients who develop chronic rejection have a stronger APC-mediated response than those who don't develop the condition4. Lechler now has funding from the ITN to investigate how some patients manage to keep this response in check — he suspects that regulatory T cells may be involved.

But Sykes believes the risks from chronic rejection are minimal. She points to experiments on chimaerism and kidney transplants in monkeys5, in which the animals have survived for seven years with healthy kidneys and no signs of chronic rejection. One of the two patients from the Harvard pilot study is also still healthy nearly five years after the transplant. “Although the caveat of chronic rejection can be raised legitimately, I personally do not believe it is a major concern,” Sykes says.

The ITN is also trying to develop new therapies for autoimmune diseases. In type 1 diabetes, for instance, T cells destroy the body's β-cells, which make insulin in the pancreas. In an attempt to halt this reaction, Bluestone and Kevan Herold of Columbia University in New York are working with an antibody to a molecule called CD3, which associates with the T-cell receptor and transmits the signal that activates the T cell.

In mice, the anti-CD3 antibody reversed symptoms of diabetes6, although the mechanism remains unclear. The antibody seems to reverse the activation of recently activated T cells — including those that are targeting β-cell antigens — while leaving the rest of the immune system intact.

Herold has also tested injections of the anti-CD3 antibody in a small group of newly diagnosed diabetics. First, he measured how much insulin 24 patients produced at diagnosis, then he split them into a treatment group that received the antibody, and a control group that did not. One year later, 9 of the 12 diabetics who received the antibody were still producing at least as much insulin as they did when they entered the study; but in 10 of the 12 control patients, insulin production had dropped7.

Fighting back

Lloyd Kasper and Randy Noelle of the Dartmouth Medical School in Lebanon, New Hampshire, hope to use a different antibody to treat multiple sclerosis — a condition in which the immune system attacks the protein myelin that coats neurons within the central nervous system. This antibody targets a molecule found on the surface of T cells called CD154, which binds to another cell-surface molecule called CD40, carried by many immune-system cells, in an interaction that is an important component of co-stimulation. In a mouse model of multiple sclerosis, the anti-CD154 antibody has already achieved promising results8.

But in June, Kasper and Noelle's plans hit problems when a trial they were conducting with IDEC Pharmaceuticals of San Diego was halted after concerns about one patient who seemed to develop blood clots. An earlier trial of a similar antibody to treat lupus, another autoimmune disease, was also abandoned by Biogen of Cambridge, Massachusetts, in 1999 after three out of 19 patients developed symptoms of clots.

Such problems serve as a stark reminder that immune-tolerance therapies remain poorly understood, and so are potentially prone to unexpected side-effects. This inherent riskiness, combined with doubts about the potential of immune tolerance to yield real money-spinners, explains the drug industry's reluctance to throw itself behind the ITN's efforts. “A lot of these therapies are not going to rate as a high priority for pharmaceutical companies,” warns Mark Feinberg, an immunologist and HIV researcher at Emory University in Atlanta, Georgia.

Jeffrey Bluestone hopes to interest drug firms in immune tolerance.

Bluestone admits that it is difficult to get drug companies to expand their interests beyond obvious blockbusters. For instance, the company that owns the rights to the anti-CD3 antibody — pharmaceuticals giant Johnson & Johnson — has not yet sponsored a clinical trial. So Bluestone's own lab has had to manufacture the antibody for use in the trials he has been doing. “I wish I could say on every level that people are knocking down our door, but sometimes we need to push them a bit,” he says.

Firm potential

Nevertheless, Bluestone points to a recent ITN success as an example of what can be achieved if a company does climb enthusiastically on board. In March, at the annual meeting of the American Academy of Allergy, Asthma and Immunology in New York, Dynavax Technologies of Emeryville, California, reported promising results on a trial of a 'tolerizing' vaccine to treat people allergic to ragweed pollen.

Allergies are caused by inflammatory cytokines released when the immune system responds to an allergen with a 'TH2' response, which is supposed to quell parasitic infections. Doctors have long realized that TH2 responses can be toned down by raising an alternative TH1 response — normally deployed against viruses and bacteria — to the same antigen. But Dynavax has managed to boost the efficiency of this approach by linking the ragweed pollen allergen to a short stretch of single-stranded DNA that helps to stimulate the desired TH1 response. As a result, it should be possible to desensitize allergy sufferers to ragweed over six weeks, rather than six months or more.

Dynavax's clinical trials were the first to be funded by the ITN. And next year, the company plans to begin large-scale trials aimed at gaining permission to market its vaccine. With 35 million people in the United States alone being afflicted by seasonal allergies, there is a huge potential market.

If Dynavax's experience can be repeated in other aspects of immune tolerance, Bluestone believes it will begin to draw drug companies into the field. If so, future generations of patients may be able to look forward to the robust health currently enjoyed by Betty Lewis — but by clinical design, rather than sheer good fortune.

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