How a rock 'n' roll scientist built a better mouse

In March 2006, eight healthy volunteers in London received an experimental drug that showed promise for treating rheumatoid arthritis and other autoimmune diseases. In animal tests, the drug, named TGN1412 by its developer, the German biotech company TeGenero, caused T-cells to proliferate, especially a subset known as regulatory T-cells. These cells calmed the immune system, leading TeGenero to believe its candidate molecule would ease symptoms in diseases in which the body is attacked by its own sentinels.

That was the hope. But when TGN1412 was tested in humans, disaster ensued. The six young men who received the drug (two volunteers were given a placebo) became severely ill within 90 minutes, suffering headaches, shivering, searing pain, swelling and nausea. Within 12 hours, one was on a ventilator and all six were admitted to intensive care. Then their organs—kidneys, lungs and livers—began to fail and their T-cell counts, instead of proliferating, dropped to close to zero.

What went wrong? Writing in The New England Journal of Medicine later in 2006, the intensive care team that rescued the six men reported that TGN1412 had launched a “cytokine storm”—the rapid and overwhelming release of toxic immune system molecules. Nothing in the animal studies had suggested this reaction was possible, and the human subjects had received only one-500th of the dose that had proved harmless to mice.

Although Richard A. Flavell, Ph.D., was not involved in this near-fatal clinical trial, its lessons were not lost on him and on other scientists who use animal models to understand human biology and pathology. Studies in lower organisms—from yeast, worms and fruit flies up the chain to nonhuman primates—are powerful precursors to research in people, says Flavell, the department chair and Sterling Professor of Immunobiology at Yale and a Howard Hughes Medical Institute (HHMI) investigator. The vast majority of genes in flies have human homologues, and the genomes of mammals and primates are closer still to our own genome. But what works in mice may not work in humans.

Human molecules in a mouse

Since the 1980s, Flavell had been engineering mice by “knocking out” specific genes or inserting them, with great effect. His group’s work led to the development of a vaccine against Lyme disease and to a more detailed understanding of other disorders, including diabetes, lupus, asthma, Crohn's disease and multiple sclerosis. But Flavell and his colleagues asked whether there might be a better way: “What if there was an experimental animal with a human immune system? How much easier would it be to predict what would happen?”

When the Bill and Melinda Gates Foundation announced its Grand Challenges in Global Health in 2003, with a goal of creating new vaccines for diseases in the developing world, Flavell and several of his collaborators realized that such an experimental mouse would be an invaluable tool. Their proposal was among 1,500 submitted to the Gates Foundation and one of 43 funded in the first round of grants in 2005. Flavell’s group at Yale and his collaborators in Switzerland and New York received $17 million. Their goal is to generate mice in which human immune system genes replace those of the mouse “so that they can support the development and function of human immune cells engrafted into the mice. This should permit reliable assessments of weakened live vaccines prior to human trials,” according to the Gates program.

While this “human immune system mouse” builds on previous advances by other scientists, it represents a major shift in thinking. “Nothing of this kind of grand vision has been attempted before,” says David G. Schatz, Ph.D., a faculty colleague. Flavell’s group is inserting into mice the cytokines and other molecules that immune cells use to summon other cells, the markers that establish identity and mediate transplant acceptance or rejection, and the receptors on which cells rely to recognize viruses and bacteria.

So far, Flavell’s group has successfully altered or inserted nine human immune system genes into the experimental mouse. “I think we’ll have a usable system in 15 months,” he says.

An unusual path

When Flavell was recruited to Yale in 1988 to head the medical school’s newly established Section of Immunobiology, he came with an unconventional pedigree: educated at the University of Hull in the north of England, Flavell had trained and taught at universities in Europe before leaving academia for a job in business. Scientists do that all the time. But after six years running the research division at Biogen, one of the early biotech companies that emerged in the 1980s, Flavell returned to academia. That’s much less common, and often difficult to accomplish after so much time out of the funding stream that supports academic scientists.

Twenty years later, immunobiology at Yale is considered one of the top programs by its peers. It was ranked No. 1 among immunology graduate programs in the Chronicle of Higher Education’s 2006 Faculty Scholarly Productivity Index, which counts scholarly publications, citations, grant dollars, awards and honors in its assessment of the best departments in the United States. Flavell has authored a number of highly cited papers and has been recognized with science’s top honors: he was made a member of the Royal Society in 1984 and elected to the National Academy of Sciences in 2002 and the Institute of Medicine in 2006.

Immunobiology’s rise at Yale may have resulted in part because of—rather than in spite of—Flavell’s unconventional approach to his career and to science. For example, he leads one of the larger university-based lab groups in the world, but while he is a scientist with significant clout, he is anything but a top-down leader. “Richard couldn’t care less where a good idea comes from, as long as it’s a good idea. He’s very good at gathering information, building consensus and then making a decision,” says Schatz, professor of immunobiology and an HHMI investigator. “It’s very inclusive.”

No prima donnas

This congenial atmosphere may stem from a decision Flavell and his Yale departmental co-founders—colleague Alfred Bothwell, Ph.D.; former professor H. Kim Bottomly, Ph.D. (now the president of Wellesley College); and Bottomly’s late husband, Charles Janeway Jr., M.D.—made at the beginning. ”We wanted to have outstanding people working in complementary areas using different approaches,” Flavell says. “But there was one other thing that was really essential—no prima donnas. So we hired people who were outstanding but were all easy to get along with.”

The department now numbers 13 primary faculty and close to 200 researchers overall, including faculty with secondary appointments as well as research scientists and postdocs. Flavell’s own lab has more than 40 people working on a combination of projects funded by HHMI, the National Institutes of Health and the Gates Foundation.

Flavell has trained several dozen young scientists from around the world who rotated through his lab as postdocs. In selecting them he has looked for “a combination of creativity and the ability to get something done.” Most have gone on to highly productive careers in the United States and abroad. For example, Lena Alexopoulou, Ph.D., who earned her doctorate in Greece before doing her postdoc at Yale, is now a group leader at the Centre d’Immunologie de Marseille-Luminy in France. In 2001 she and Flavell published a paper in Nature showing for the first time that toll-like receptors (TLRs, molecules at the core of the innate immune response) play a role in the recognition of viruses. With more than 1,000 citations, the paper is one of the most highly referenced in the rapidly expanding TLR field.

Alexopoulou says that Flavell’s mentoring launched her career and gave her an appreciation for how to manage people in her own lab. “He knows how to motivate people,” she says. “He gives people a chance to develop their own ideas and projects. He provides you with everything you need that he can offer you—connections, money, scientific information—and he’s very open to new ideas.”

As a result, says Deputy Dean Carolyn W. Slayman, Ph.D., “any wonderful young postdoc … would just die” for a slot in Flavell’s lab. “He’s got a terrific knack for doing translational research and training people in it. And that’s led to an amplification factor—there are more scientists doing this kind of extraordinary work by virtue of Richard training others.”

By translational work, Slayman means conducting fundamental scientific studies and applying that knowledge to clinical questions. Flavell explains: “I would say a high proportion of it is curiosity-driven, pursuing such basic questions as ‘How does the body work?’ But we always study this using diseases that matter. And what we try to do is to force ourselves to ask the question, ‘Is this an important problem?’

“We want to study only important problems,” he says, meaning problems that are fundamental or relevant to human disease. “You can spend your life doing unimportant things and in both cases you will spend just as much energy and get just as tired and as frustrated—and you’ll be a lot less productive.”

Finding motivation

In the late 1950s, when he was a rock ‘n’ roll-crazed teenager in the English county of Norfolk, no one would have mistaken Flavell for a budding academic. “I was a totally unmotivated student, except for the one thing I was interested in at the moment,” he says. “First it was French, then it was history. Anything I was obsessed about, I was the best student in the class. For the rest I was in the bottom 25 percent.”

That changed when he nearly failed his O-level exams, an essential step on the road to a university education in Britain. Raised in a family of teachers (his father was a school principal and a pilot during World War II), Flavell says the shock of a poor grade roused him from his scholarly indifference. Around the same time, thanks to an exceptional teacher, he discovered chemistry. After earning bachelor’s and doctoral degrees in biochemistry at Hull, Flavell did postdoctoral work in the Netherlands and Switzerland and then joined the faculty of the University of Amsterdam. From the Netherlands, he moved to London, where he was head of the Laboratory of Gene Structure and Expression at the National Institute for Medical Research at Mill Hill.

Then came an offer to join a commercial enterprise, the biotechnology startup Biogen, in Cambridge, Mass. “It was a very difficult decision, because I was totally satisfied with what I was doing. … And along comes this crazy opportunity, which was also great because it was at a time when a new industry was being created.” It was exciting, Flavell says—“You’re alternately up in the clouds or on the precipice”—but when then-Yale Dean Leon E. Rosenberg, M.D., came looking for someone to head the medical school’s new program in immunobiology, he decided he was ready for a return to academia. Biogen’s next phase of growth, in which it would bring its first drugs to market, was a decade-long proposition. “I thought, ‘Do I want to commit the next 10 years to doing that?’” he says. “I decided that I wanted to be more of a scientist and less of a manager.”

At Yale, Flavell has struck a balance between the pursuit of basic biological questions and the pursuit of solutions to the causes of human disease. During its 20 years, the immunobiology department has opened major new areas of understanding, in particular discoveries establishing that the long-ignored innate immune system plays a much more significant role in the body’s defenses than previously thought. Overturning assumptions that had stood for decades, department members Janeway and Ruslan Medzhitov, Ph.D., demonstrated in Nature in 1997 that the innate response is the activating factor for the adaptive immune system’s release of T- and B-cells.

“It was like saying there are only four planets in the solar system and then one day somebody comes along and says, no, there are eight,” says Schatz. In the dozen years since the Medzhitov-Janeway paper appeared in Nature, a new branch of immunology research has been established, with hundreds of scientists looking for drug and vaccine targets among TLRs.

To help speed the clinical application of its basic immunology findings, the department launched a new program in 2007 called Human Translational Immunology (HTI), bringing together scientists from across the medical school and university to focus exclusively on research that could lead to better treatment of disease. The group studies the immune components of a wide range of disorders, including autoimmune diseases, cancer and the immune rejection of transplanted organs.

A better mouse

In the three years since the Gates project began, Flavell’s group has made steady progress toward developing a mouse with a functional human immune system for the purposes of testing vaccines. His collaborator in Switzerland, Marcus Manz, M.D., of the Institute for Research in Biomedicine in Bellinzona, established the basis for the project in 2004, when his group created a mouse with human T-cells, B-cells and natural killer cells (NK cells target and destroy other cells that are infected with viruses or bacteria or are cancerous). To do this, they began with an immunodeficient mouse lacking RAG1 or RAG2—genes needed for generation of B-cells and T-cells—and lacking the receptor required for the growth of the NK cells. When they injected human stem cells from umbilical cord blood into the liver of the mouse, those stem cells found their way to the bone marrow and began to develop into mature human immune cells.

“It is hard to believe that a human system may settle down in the organism of a mouse, yet the evidence pointsthat way,” Manz’s colleague Elisabetta Traggiai, Ph.D., said in 2004. “Two months since the transplant into the mouse, we have observed fully fledged human cells capable of reacting to human viruses and vaccines.”

One problem that emerged, says Elizabeth E. Eynon, Ph.D., who manages the Gates project as a research scientist in Flavell’s group, was that the human cells did not mature properly. They did not have the expected longevity, they did not multiply in sufficient numbers and they did not interact with other cells as expected.

Eynon says those problems can be attributed to the lack of human growth factors and other molecules required by the T-cells and B-cells to develop correctly but not present in the mouse. “We tested to see how much alike a number of different factors were in the mouse and human and found about a dozen with significant differences,” she says. Thus began the current effort to “knock in” the genes for those molecules and create the support system the human immune cells will need to thrive in the mouse.

In that work, the project has drawn on the expertise of Sean Stevens, Ph.D., and others at Regeneron Pharmaceuticals in Tarrytown, N.Y. Regeneron created a highly efficient technology for knocking out genes and inserting human replacements into mouse DNA. Eynon says they are now working on introducing genes for the human major histocompatibility complex, which the body relies on to recognize foreign cells.

“What we hope to have [by July 2010] is a mouse that pretty nearly recapitulates the basic, fundamental immune responses that are human. All the appropriate cells will be there in the appropriate amounts, the cells will live a long time and the immune response will be functional,” she says. “It certainly won’t be the perfect mouse, but we think that it will make enough of an improvement to make this a useful model, not only for our lab but for other groups as well.” YM