Policing the Immune System

Science magazine, 22 October 2004

From blood samples, pediatric immunologist Hans Ochs has diagnosed five infant boys who all had the same devastating problems. Their immune systems had gone haywire, attacking their gastrointestinal tracts within a few weeks of birth and causing severe, intractable diarrhea. Wayward immune cells also laid siege to each boy’s pancreas, producing diabetes around 3 months of age. Within months of their births, several of the infants became depleted of red and white blood cells from the immune onslaught.

No cure exists for this rare and frequently deadly immune disease dubbed immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX). But thanks in large part to recent work by Ochs at the University of Washington School of Medicine in Seattle and his colleagues, its cause in most cases is now known. A genetic defect severely impairs, if not abolishes, the body’s ability to produce regulatory T cells, a mysterious class of immune cells apparently designed to squelch dangerous immune responses.

Once dismissed as artifacts of misguided research, regulatory T cells—originally called suppressor T cells—are now white hot among immunologists, thanks to a body of research that over the past 8 years clarified their existence. Hundreds of researchers are flocking to the field, which could lead to novel treatments for an array of immune disorders, such as type I diabetes, multiple sclerosis, graft-versus-host disease, and allergy, that are far more common than IPEX. Studying regulatory T cells may also provide clues to the treatment of cancer; the cells actually seem to protect tumors against immune attack.

“All anyone is talking about these days is regulatory T cells,” says Ethan Shevach, a cellular immunologist at the National Institute of Allergy and Infectious Diseases (NIAID) in Bethesda, Maryland. Nevertheless, fundamental mysteries remain about this newfound class of immune cells. Their mechanism of action is almost totally opaque. Also unclear is to what extent they play roles in more ordinary human autoimmune diseases that develop later in life, such as diabetes and multiple sclerosis. “Regulatory T cell research is very intriguing but is not yet ready for mass consumption,” Ochs says. “There are still a lot of puzzles.”

Even so, many researchers are optimistic that studying regulatory T cells will lead to new therapies. Drugs that seem to target these cells are already being tested in people with cancer and diabetes, and pharmaceutical companies are trying to develop drugs that augment or suppress regulatory T cells for other disorders. “Within 5 years, some clinical application of these cells will be here,” predicts Shimon Sakaguchi, an immunologist at Kyoto University in Japan and a pioneer in the field.

From fantasy to reality

The human body makes several types of T cells, including killer T cells, which eradicate infected cells, and helper T cells, which arouse killer T cells and various other immune cells to fight invaders. And for decades, researchers have kicked around the idea that the body also makes a class of T cells that act like a police department’s internal affairs unit, keeping tabs on the immune system’s cellular cops and cracking down on them if they threaten to spiral out of control. In the early 1970s, the late Yale immunologist Richard Gershon formally proposed the existence of these suppressor cells to explain a form of immune tolerance he observed in a mouse. The idea caught the fancy of immunologists, and numerous teams rushed to identify and characterize these cells.

The entire concept of suppressor T cells fell into disrepute, however, when no one could verify reports of molecules that supposedly characterized the cells. “It was a kind of fantasy not supported by modern genetics and biochemistry,” recalls immunologist Alexander Rudensky of the University of Washington, Seattle.

By the mid-1980s, virtually everybody had abandoned the idea of suppressor T cells except Sakaguchi, who continued his quest for the elusive cells. Extending earlier work, Sakaguchi and his colleagues showed, in the 1980s, that removing the thymus of a mouse on day 3 of its life—which depletes the animal of most of its T cells—causes various autoimmune diseases to develop. Inoculating the thymus-free mice with a mixed population of T cells from another mouse prevents those diseases, the researchers found.

Sakaguchi felt that the autoimmune diseases resulted from a deficit in putative suppressor T cells that were made in the thymus on or after day 3; their absence left unchecked any T cells that had developed earlier. But critics contended that infections could have triggered the autoimmune reaction. Without pinpointing the suppressors, the Kyoto team could not prove its case.

Finally, in 1995, Sakaguchi and his colleagues reported that they had identified suppressor T cells by the presence on them of a newly identified cell surface protein called CD25 as well as the more ubiquitous surface protein CD4. When they infused a batch of T cells devoid of ones with these markers into mice that lacked their own T cells, the mice developed autoimmune disease. But if they infused the suppressors along with the other T cells, no autoimmune disease appeared. These experiments convincingly showed that a small, specific population of T cells worked to dampen autoimmune reactions.

Virtually no immunologists read the paper, however, because hardly anybody was interested in suppressor T cells anymore. But NIAID’s Shevach did. He was so struck by the finding that he rushed to repeat it in his own laboratory—and succeeded. “I had a religious conversion to believe in regulatory T cells,” he says. That brought others into the fold, as Shevach had been an outspoken skeptic of the idea. Rudensky credits Sakaguchi’s 1995 paper as the turning point: “The field took off.”

In 1998, Shevach’s and Sakaguchi’s groups independently developed cell culture systems that enabled others to study the suppressive activity of the rodent cells in dishes. In 2001, several research teams, including Don Mason and Fiona Powry’s at the University of Oxford, plucked out CD4+ CD25+ cells in human blood and determined that they halted the proliferation of other T cells, showing that the rodent data had some relevance to humans.

And last year, the cells were shown to underlie human disease. Three teams of investigators reported that Foxp3, the protein that Ochs and others found to be missing or defective in IPEX patients in 2001, is specifically expressed in regulatory T cells and is essential to their development.

Mice engineered with a defective Foxp3 gene have a deficit in CD4+ CD25+ T cells and suffer from an IPEX-like disease called scurfy that can be blocked by treatment with regulatory T cells at 1 day old, Rudensky and his colleagues revealed in Nature Immunology. In the same journal, a team led by Fred Ramsdell, formerly at Celltech Research & Development in Bothell, Washington, reported that the expression of Foxp3 by T cells in mice correlates with their ability to suppress immune responses. And transferring Foxp3 into naïve T cells converts them into regulatory cells, the Sakaguchi group showed the same year (Science, 14 February 2003, p. 1057). Together, the studies indicated that a deficiency of regulatory T cells in humans can lead to severe immune dysfunction.

How suppressor T cells do their job remains a mystery, however. In the test tube, natural regulatory T cells—the type that express CD25 and are made during immune system development—seem to suppress other T cells through direct contact. In living animals, they also may release anti-inflammatory cytokines such as interleukin-10 or transforming growth factor β. So-called adaptive regulatory T cells, which become regulatory only after being stimulated by an antigen and respond only to immune cells targeting that antigen, seem to exert their influence solely by means of cytokines. If that’s not complicated enough, Shevach recently reported that regulatory T cells may directly kill the B cells that generate antibodies (Science, 6 August, p. 772).

Good cop, bad cop

Despite basic gaps in their understanding of regulatory T cells, researchers are tracking down potential roles for the cells in human disease. David Hafler and his team at Harvard Medical School in Boston reported in April in the Journal of Experimental Medicinethat patients with multiple sclerosis seem to have defective regulatory T cells.

Impotent regulatory T cells may also play a role in allergy and asthma. Allergist Douglas Robinson of Imperial College London and his colleagues isolated natural regulatory T cells from the blood of people with and without allergies. They then exposed the remaining T cells to an allergen. In all of the samples, the allergen (from grass pollen) triggered T cell proliferation and a release of inflammatory molecules, or cytokines, from immune cells. Adding back regulatory T cells from the nonallergic people completely suppressed this inflammatory response, whereas the regulatory T cells from the allergic individuals were far less effective in doing so. The suppression was weaker still from regulatory T cells from patients with hay fever during the pollen season, the researchers reported in February in The Lancet.

“One implication is that people who get allergic disease do so because their regulatory T cells don’t respond,” Robinson says. Boosting the response of these cells, he adds, might help prevent or treat their disease. Boosting the adaptive class of regulatory T cells may also be important. Two years ago, for example, Stanford’s Dale Umetsu and his colleagues identified adaptive regulatory T cells that protect against asthma and also inhibit allergic airway inflammation in mice.

Although regulatory T cells seem to be protective in autoimmune disorders and allergy, they may have a darker side. In the March issue of Immunity, a team led by immunologist Kim Hasenkrug of NIAID’s Rocky Mountain Laboratories in Hamilton, Montana, suggests that some viruses, such as those that cause hepatitis and AIDS, may exploit regulatory T cells to dampen the body’s antiviral response and allow chronic infections.

Similarly, regulatory T cells may protect tumors from immune attack. Researchers have shown, for example, that removing such T cells from a cancer-afflicted mouse can cause the rodent to reject a tumor. High levels of regulatory T cells have also been found in samples from several types of human tumors. More recently, tumor immunologist Weiping Zou of Tulane University Health Science Center in New Orleans, Louisiana, and his colleagues linked the quantity of regulatory T cells associated with a tumor to disease severity in cancer patients.

Zou’s team isolated and counted the T cells in tumor tissue from 104 ovarian cancer patients and noted that the higher the ratio of regulatory T cells to total T cells in the tumor, the farther the cancer had progressed. Regulatory T cells were also associated with a higher risk of death: The more tumor-associated regulatory T cells, the worse the prognosis. Zou and his colleagues further showed that the regulatory cells recovered from tumor tissue protected tumors in a mouse model of ovarian cancer by inhibiting both the proliferation and potency of tumor-attacking T cells.

Zou’s team also discovered that, as the cancer progressed, a patient’s regulatory T cells appeared to migrate progressively away from their normal home in the lymph nodes to the tumor. The investigators determined that tumor cells secrete a chemical, dubbed CCL22, that attracts regulatory T cells. Blocking CCL22 with an antibody stopped regulatory T cells from migrating to the tumor in the mouse model, the team reported in the September 2004 issue of Nature Medicine.

By extension, disarming these regulatory cells or preventing their migration to the tumor could leave the tumor vulnerable to immune destruction. The Tulane researchers and others are testing a regulatory T cell-killer called Ontak in advanced cancer patients. The drug binds to CD25 and kills the cells with diphtheria toxin. The results so far, Zou says, are “encouraging.”

Immunologist Steven Rosenberg of the National Cancer Institute in Bethesda has tested another regulatory T cell-blocker in patients with metastatic melanoma. The treatment—an antibody to an essential molecule on the surface of regulatory T cells, called cytotoxic T lymphocyte-associated antigen 4— induced cancer remission in three of 14 treated patients who had end-stage cancer, Rosenberg’s team reported last year. More than 2 years later, the patients are still in remission, and Rosenberg’s team has now seen a similar remission rate among almost 100 patients.

“The fact that inhibiting regulatory T cells enabled three patients to undergo cancer regression was very strong evidence that regulatory T cells are inhibiting the immune response against tumors,” says Rosenberg. “This is the first time that getting rid of this brake on the immune system has been shown to have any impact in humans.”

The study also suggests how tricky it may be to interfere safely with the regulatory T cell system, however. Six of the initial 14 cancer patients, including the three who went into remission, developed autoimmune diseases affecting the intestines, liver, skin, or pituitary gland, although these were all reversible with short-term steroid treatment.

Expansion plans

Even in disorders such as type I diabetes, in which regulatory T cells have not been consistently shown to be abnormal in function or number, researchers are exploring them as potential therapy. “Traditionally, immunotherapy is designed to block effector cells or their activities. Now there is the entirely new possibility that we could treat the disease by expanding suppressors,” says Ralph Steinman, an immunologist at Rockefeller University in New York City.

In June, Steinman’s team and, separately, a team led by Jeffrey Bluestone at the University of California, San Francisco (UCSF), reported mouse studies in the Journal of Experimental Medicine that illustrated how this might work. Both research teams plucked out natural regulatory T cells from diabetes-prone mice that made only one type of T cell, one that responds to an antigen on the islet cells of the pancreas. Each team then used different methods to expand the mouse regulatory T cells in lab dishes and found that they could prevent or reverse diabetes when infused into other diabetes-prone mice.

A diabetes treatment that is thought to boost T cell regulation has already reached human trials. The treatment is an antibody to CD3, a cell-surface protein tightly associated with the T cell receptor. The antibody was first found to induce long-term remission of diabetes in mice a decade ago. That surprising result contradicted the idea that the CD3 antibody—which was then used to treat organ rejection—worked by inactivating destructive T cells, because the treatment’s effects far outlasted the depletion of activated T cells.

Last year, immunologists Jean-François Bach and Lucienne Chatenoud and their colleagues at Necker Hospital in Paris, along with UCSF’s Bluestone, reported in Nature Medicine that the antibody appeared to activate natural regulatory T cells in mice. When diabetes-prone mice were treated with the antibody a month after diabetes onset, they became nondiabetic. But if the mice were also treated with drugs that block regulatory T cells, the diabetes remained. “It’s a nice story indicating that, in the mouse, immunoregulation explains the long-term effect of the antibody,” Bach says.

After initial tests of this antibody approach proved encouraging in a small number of diabetics, Kevan Herold, an endocrinologist at Columbia University School of Medicine in New York City, and his colleagues recently launched a six-center trial of the therapy in 81 diabetic patients. Meanwhile, Chatenoud and her colleagues are about to unveil the results of a multicenter, 80-patient, placebo-controlled trial of the CD3-targeting antibody conducted in Belgium and Germany.

Boosting regulatory T cell activity might someday also induce drug-free immune tolerance to donor organs. In July, Sakaguchi’s team reported removing natural regulatory T cells from a normal mouse and expanding them in cell culture with interleukin-2, a growth promoter, along with an antigen from a donor mouse of a different strain. This generated a population of antigen-specific regulatory cells, which they then infused into so-called nude mice, which lack T cells. The regulatory cell infusion enabled those rodents to accept skin grafts from the donor strain even though they were simultaneously infused with killer and helper T cells. By contrast, nude mice that received only killer and helper T cells—but no regulatory cells—quickly rejected the grafts. “With just a one-time injection of regulatory T cells, we can induce graft-specific tolerance without drugs,” Sakaguchi says.

In cases in which organ donors—such as living donors—are known in advance, Sakaguchi envisions generating antigen-specific regulatory T cells prior to transplantation of human organs. If the therapy works, he says, it could replace the use of immunosuppressive drugs, which come with a significant risk of infection and cancer.

A boost from bugs

The growing understanding of regulatory T cells may eventually shed some light on an immunology-based theory called the hygiene hypothesis. According to this controversial idea, the rise in allergic disorders in recent decades in developed countries results from those countries’ increasing cleanliness, which reduces children’s exposure to protective microbes. A number of researchers have shown that exposure to parasitic worms called helminths may protect against allergy and asthma, among other immune disorders, largely through the induction of regulatory T cells (Science, 9 July, p. 170).

Some strains of bacteria have also been shown to be protective—and again regulatory T cells may be involved. Immunologist Christoph Walker of the Novartis Institutes for Biomedical Research in Horsham, U.K., and his colleagues demonstrated that treating mice with killed Mycobacterium vaccae before sensitizing them to egg-white allergen significantly reduced the rodents’ inflammatory responses to the allergen, as compared to mice that did not receive the bacteria. Regulatory T cells isolated from bacteria- treated mice could transfer the protection to untreated mice sensitized to the same allergen, demonstrating that the cells mediated the bacteria’s protective effects, the team reported in 2002.

The suppressive response was allergen-specific: The regulatory T cells generated in the egg white- sensitized mice could not dampen the response to cockroach allergen in mice made allergic to cockroaches. “Regulatory T cells generated by mycobacteria treatment may have an essential role in restoring the balance of the immune system to prevent and treat allergic diseases,” the authors wrote in Nature Medicine. Walker’s team is now trying to mimic the bacteria’s effects with a chemical that stimulates the same receptors on regulatory T cells that the bacteria stimulate.

But some researchers note that opportunities for rational drug design may be limited by the paucity of knowledge about how regulatory T cells suppress their immune system colleagues. Says Shevach: “We won’t know how to enhance the response until we know what it is.”

Nevertheless, he, Sakaguchi, and others have succeeded in a vital first step. They’ve at long last convinced fellow immunologists that regulatory T cells exist and are important. “So many people are working on regulatory T cells,” Sakaguchi says. “It’s been a pleasant surprise.”