The History Behind The Discovery of toll-like Receptors

Charles A. Janeway Jr.

In so much of science and medicine, breakthroughs begin simply as questions. A provocative one came by chance in January 1989 to Charles A. Janeway Jr., MD, an immunologist and Yale professor. Although the question—about the initial trigger for the body’s immune response—inspired an unorthodox idea, the story might well have ended there had Janeway’s speculative answer not been noticed half a world away.

Janeway did write a paper about his idea—a theory about how a little-studied arm of the immune system alerts the body’s T and B cells to the presence of an invader—but the 1989 paper drew little immediate interest. Three years later, an impoverished graduate student in Moscow stumbled upon it and was galvanized. Launching the kind of quixotic adventure that could have been scripted by Hollywood, the young biochemist followed a circuitous route to Janeway’s lab. There, the novice with no lab experience and the veteran scientist collaborated on research that broke open an important and useful field within immunology. Together, Janeway and Ruslan Medzhitov, PhD, now Sterling Professor of Immunobiology and investigator of the Howard Hughes Medical Institute, elevated Janeway’s theory from obscurity to cutting-edge prominence.

From Medicine to Immunology Research

Janeway wasn’t supposed to have been an immunologist in the first place; as a young man, he seemed destined to fulfill a family tradition of practicing medicine. But during his second year at Harvard Medical School in 1964, Janeway began to realize that, in his words, “the evidence for treating patients the way we were was flimsy.” Rather than admit that they had no effective treatment for a malady, physicians would prescribe something anyway. It bothered Janeway to hear patients thanking their doctors for unproven therapies.

His skepticism prompted a break from his medical studies and a two-year excursion into basic research from 1965 to 1967, first in the lab of immunologist Hugh O. McDevitt, MD, at Harvard for a summer and then with John H. Humphrey, MD, at the National Institute for Medical Research at Mill Hill in London for two years. When a more focused and critical Janeway returned from England for his last two years of medical school and a year of internship at the Peter Bent Brigham Hospital in Boston in 1969-1970, he couldn’t accustom himself to prescribing treatments with so little information to go on. “Clinical medicine,” he said, “was rotting my soul.”

Triggering the Adaptive Immune System

Janeway countered this disillusionment by working for the next five years at the National Institute of Allergy and Infectious Diseases under the tutelage of immunologist William E. Paul, MD, chief of the Laboratory of Immunology. In 1977, he came to Yale as an assistant professor in the Department of Pathology’s immunology division. During the 15 years of research that followed, Janeway gained insights into how T cells originate, develop, and then become activated to pick off specific fragments of specific invaders.

Indeed, the key to T-cell action is specificity. Janeway was at the forefront of efforts to understand how each of the millions of T cells formed in our bone marrow, and later launched into our circulation, has the capacity to respond to one, and only one, unique invader or insult. That pairing of T cell and target takes several days to happen, from the ill person’s first feelings of malaise to the moment when the body has amassed its cellular troops and molecular weaponry. But the delay is necessary, pregnant with potential. Meanwhile, a second immune player, the B cell, produces antibodies, and these two protagonists coordinate an entire arm of human immunity known as the adaptive immune system.

During the 1980s, when the world of immunology revolved around problems of adaptive immunity, there was a basic question that no one gave much thought to: during the initial delay period, how are T and B cells first alerted that an intruder has invaded the body?

That was the question first posed to Janeway by his wife and colleague, H. Kim Bottomly, PhD, a renowned immunobiologist who was at Yale from 1980 to 2007. The couple attended a Keystone Symposia meeting in Steamboat Springs, Colo., in January 1989, and were bantering as usual. “He and I would argue in the car all the time, and then we’d forget what we said,” Bottomly recalled.

“But,” she added, “this time we brought a notebook.”

Janeway’s Hypothesis: Pattern Recognition Receptors

Janeway offered this answer to her question: the alert signal given to T and B cells must come from another arm of the immune system, the one long referred to as innate immunity. Given only a page or two in medical texts, innate immunity is provided by skin, mucous, and other epithelial barriers. It had been proposed that some unknown biochemical component combines with those simple barriers to act as a first line of defense against everyday assaults. Just cut a finger, and the tender redness that develops will demonstrate the workings of the body’s inflammatory process, a function of innate immunity.

As he jotted down Bottomly’s question and its provocations, Janeway began to muse on that long-ignored system. The field of innate immunity had languished in the years since Ilya I. Mechnikov had proposed his revolutionary cell-based theory of immunity in 1883. With subsequent discoveries in the 1940s about the powerful roles of T and B cells in allergic reactions, graft rejection and microbial attack, researchers had relegated innate immunity to a footnote. In fact, Janeway wrote his own terse section about it in his book, Immunobiology, now in its 10th edition.

Janeway began considering the links between the innate immune system and the adaptive system he’d studied over the years. Perhaps innate immunity worked as the intelligence network to tip off the adaptive system, he thought. Perhaps cells within or near the skin, mucous membranes, and intestinal lining bore molecules on their surfaces that could recognize some general aspect of microbes and signal the adaptive immune system that a foreigner had breached security.

Janeway came up with an idea, which he dubbed the “Pattern Recognition Hypothesis.” It goes like this: classes of germs carry molecular patterns, either anchored on their surfaces or secreted from their insides. All multicellular organisms have pattern recognition receptors that can signal, only in vertebrates, the adaptive immune system. Of course, the microbial molecules that carry the patterns would have to be critical to the pathogen in some way; otherwise, it would have evolved a way to do without those molecular patterns centuries ago.

“I couldn’t let go of the idea,” recalled Janeway. In June 1989, he attended a meeting on quantitative biology in Cold Spring Harbor, N.Y., an event at which invited researchers have a chance to discuss ideas in an intimate setting. After the meeting, the immunologist laid out some of the ideas he’d begun developing at Keystone in a paper entitled “Approaching the Asymptote? Revolution and Evolution in Immunology,” published as a chapter in the Cold Spring Harbor Symposia later the same year.

According to Janeway, that paper was “pretty much ignored” by the scientific community. But it intrigued an eager young student at Moscow University who stumbled across it. That student was Ruslan Medzhitov.

Ruslan Medzhitov’s Search for a Lab

Born in 1966 in Tashkent, Uzbekistan, Medzhitov made his way to graduate school in biochemistry at Moscow University in 1990, a time when the Soviet Union was breaking up and science in Russia was in deep trouble. As funding dried up, biochemistry labs lay bare of a single reagent. Entire departments operated on monthly budgets of $20. In fact, only the Academy of Natural Sciences carried the journals Science and Nature, and its lone copy of each made its rounds through every lab before landing, worn and barely legible, on the shelves of the academy’s decaying library. Due to politics between the university and the academy, university students were unilaterally denied access to the library. But Medzhitov, by wit, resourcefulness, and charm, managed to get in. “I sweet-talked the librarians,” he said.

Indeed, in the fall of 1992, the graduate student laid his hands on Janeway’s paper and found his calling. Medzhitov wanted to study immunology, and he wanted to do so with Charles Janeway.

Medzhitov had been working on his doctorate by studying how molecules evolve to recognize each other; how, for example, a receptor and the signaling molecule that binds to it interact. As he read Janeway’s theory about microbial patterns being picked up by human receptors, he was drawn to its logic. “Although I didn’t know much about the subject,” Medzhitov said, “I realized that the way he thinks, I think.”

The problem was that Medzhitov’s department in Moscow had no supplies and, thus, its students gained no lab experience. As a “theoretical” protein biochemist who knew no one with the prestige or resources to back him, Medzhitov almost gave up.

But he didn’t. He photocopied the Janeway paper. This was no simple feat, as it cost half his monthly student stipend of $2. Then Medzhitov began e-mailing Janeway, using the solitary account shared by 400 faculty, staff, and students, each of whom was allotted only 300 words a day in order to control the costs of operating the account.

Janeway, meanwhile, had no idea of what to make of this unknown student who was writing to him. “Charlie told me, ‘I’ve got this fabulously bright guy from Russia who wants to work in my lab,’” Bottomly recalled. Not surprisingly, she was skeptical.

Medzhitov set to work. He won a fellowship given to scientists in developing countries by the United Nations Educational, Scientific and Cultural Organization. That would get him to the United States for three months in 1993 to work in the lab of Russell F. Doolittle, PhD, at the University of California, San Diego. Medzhitov scraped together the plane fare by borrowing from a cousin.

Money in hand, Medzhitov faced another hurdle before he could leave for California: getting a passport. As the Soviet Union was unraveling politically, Medzhitov was considered a citizen of nowhere, having been born in Uzbekistan, but not residing there, and living in Russia, but not having been born there. In the end, the student wended his way through the bureaucracy. He reached California and began working in the burgeoning area of bioinformatics, in which scientists were beginning to write software to comb through and order databases of decoded DNA sequences.

Medzhitov continued to correspond with Janeway. In fact, the Moscow student spoke nonstop about Janeway’s ideas to Doolittle, who in turn arranged a seminar at which Medzhitov could present his work. Attending that presentation, given in halting English, was Richard W. Dutton, PhD, then the president of the American Society of Immunology. When Dutton caught wind of Medzhitov’s ambition of working with Janeway, Dutton immediately called his colleague in New Haven. He told him, “You have to hire Medzhitov.”

And Janeway did. “Dick’s phone call tipped the balance,” he said.

The Discovery of a Human Toll Gene

Medzhitov arrived at Yale in January 1994 as Janeway’s postdoc. He set out to show that the innate immune system could recognize molecules that did not belong in the human body—via alien patterns carried by the invaders. And once the system did its duty, Medzhitov had to show that it could transmit this information to the adaptive immune system. The first task was this: find at least one example of a pattern recognition receptor on some cell in the body that could be linked to innate immunity.

The beginning stages looked grim. Medzhitov said Janeway tried to instill confidence by telling him, “Lab work is a lot like cooking.” Medzhitov answered sheepishly, “I’ve never cooked, either.”

After several unsuccessful attempts to pinpoint the candidate receptor using conventional methods, Medzhitov turned to the experience he had gained in bioinformatics. He began with a template—a DNA sequence for a human gene that encodes the interleukin-1 (IL-1) receptor, a mammalian protein known to trigger inflammation. The receptor itself, however, is triggered by a cytokine, rather than a microbial pattern.

Perhaps humans bear another receptor like IL-1 that could spur the adaptive arm of immunity, the researchers reasoned. With the IL-1 gene sequence and others like it, Medzhitov began searching through warehouses of DNA for sequences for novel genes expressed by all kinds of organisms, from fruit flies to humans. In early 1996, he finally hit pay dirt.

Medzhitov found and decoded a new human gene. It resembled the gene for IL-1 and one other—a fruit fly gene dubbed toll for the German word that means “amazing.” Geneticist Kathryn Anderson, PhD, at Memorial Sloan Kettering Cancer Center, had shown in 1988 that insect toll orchestrates the assembly of flies’ front and back sides during development. At the time of her discovery, however, no one could make a clear connection between fly development and human immunity.

Finding Connections Between Fly Development and Human Immunity

Then in the spring of 1996 in the Cape Ann village of Annisquam, Mass., Medzhitov and Janeway hosted a meeting of scientists who wanted to delve into the connections between microbial defense in insects and human immunity. Fly guru Jules A. Hoffmann, PhD, director of the Institute for Molecular and Cellular Biology at the Centre National de la Recherche Scientifique in Strasbourg, France, had written a Human Frontiers in Science grant and asked Janeway and others to join in. Janeway, in turn, saw the collaboration as an opportunity to advance the use of fly genetics to probe the molecules important in human immunity.

Said Hoffmann, in admiration, “Not many MDs would have been interested in flies.” Especially when scientists had no hard evidence of any insect-mammal connections in this area of immunology.

At that meeting on Cape Ann, Hoffmann announced his surprising discovery: that flies with defects in their toll genes became hypersusceptible to fungal infection. He waved about a photograph of an infected fly covered with a fuzzy fur of fungus. The photo appeared on the cover of the journal Cell later that year.

Janeway and Medzhitov could barely contain their delight. Could their human toll perform the same antifungal tour de force? Immediately, Medzhitov set to work. In essence, he wanted to know if human toll functioned as a sensor—a molecular scout for microbes—as well as a signaler to the adaptive immune system. After a year of experimentation, the answer to both questions was a resounding yes.

“The discovery that toll was part of the recognition pathway gave the field a handle on the most front-line sensor within the circuit,” said immunologist Michael A. Zasloff, MD, PhD, then dean of research and translational science at Georgetown University School of Medicine.

The findings appeared in the July 24, 1997, issue of Nature.

Expanding the Exploration of Toll-like Receptors

Two and a half years later, the idea of innate immunity in humans and its connections to defense in invertebrates had already taken hold. At least 150 scientists gathered at a National Academy of Sciences colloquium in Irvine, Calif., entitled “Virulence and Defense in Host-Pathogen Interactions: Common Features between Plants and Animals.” At the meeting, 12 researchers specifically discussed their work on toll in flies and “toll-like receptors”—as the mammalian versions are now known—and other aspects of innate immunity. Two dozen other scientists focused on patterns common to the insect and mammalian pathogens.

By March 2001, scientists had found 10 other human toll-like receptors, including toll-like receptor 2, which Shizuo Akira, MD, and colleagues at Osaka University showed responds to a particular sequence found in bacterial DNA but not in mammalian DNA. To get an idea of how fast the field has grown since 1997, a literature search for the term “toll-like receptor” in 2022 brought up more than 56,000 abstracts.

The evolutionary connections also awed researchers, as they eventually found toll-like molecules in worms, mice, even plants. Plant geneticist Santosh Misra, PhD, and colleagues at the University of Victoria in British Columbia genetically engineered antimicrobial peptides into potatoes to get the crops to withstand fungal infection. Protective compounds produced by plants could conceivably work as new classes of antibiotics in people as well.

And there are even more fundamental questions being asked about toll and innate immunity. Quite simply, how are plants, flies and people connected in their defense against the microbes that infect them? And, as Janeway first proposed at the Keystone meeting, might the innate arm of immunity—the more ancient system—actually be the means by which the body distinguishes itself from foreign invaders? If so, understanding its mechanisms might have crucial implications for the study of both infectious diseases and autoimmune disorders.

Medzhitov Joins Yale

Indeed, in the aftermath of their breakthrough, Janeway lobbied for Medzhitov’s recruitment for a faculty position at Yale. Medzhitov had seven other offers, including positions at Harvard and MIT, which he turned down. “I knew that at Yale I’d be given a lot of intellectual freedom to pursue riskier ideas,” he said.

Then, Janeway did something unusual: he handed over to Medzhitov the reins of their collaboration. Mentors often give away parts of projects to exiting postdocs. But to do so for “the biggest discovery in immunology in a decade is rare and typical of Charlie’s generosity,” said Richard A. Flavell, PhD, Sterling Professor of Immunobiology and investigator of the Howard Hughes Medical Institute.

The decision had to do with Janeway’s personal circumstances as well as with Yale’s policy of discouraging direct competition between two faculty members working on the same project in the same department. In order to get Medzhitov in, Janeway would have to step aside. “It was an admirable move,” said Alfred L.M. Bothwell, PhD, professor emeritus of immunobiology. The close-knit immunobiology faculty unanimously chose Medzhitov to join them as an assistant professor.

When Medzhitov was first introduced to biochemistry, he was a college student in Tashkent as well as a field laborer required to pick cotton for two to three months each year (“one of the idiotic ramifications of the Soviet political system,” he said.). At 36, he was mapping new territory in an emerging discipline that has had major implications for the treatment of disease. How did he get there?

“There is a hypothesis that if you have to struggle a bit early on, you’ll put more into it later,” said Bottomly.

A Textbook Case

A researcher who spent his career studying the body’s defense system, Janeway eventually fell prey to its limitations. A year after Medzhitov arrived at Yale, Janeway began to complain of fatigue. A series of medical exams and tests over the next several months showed that Janeway had developed B-cell lymphoma, and that it had progressed from the bloodstream to his brain. A relapse in 1999 and several years of treatment drained him.

He reduced his lab personnel and steered back toward his initial interests, working on projects to develop vaccines against diabetes and autoimmune diseases such as multiple sclerosis. In December 2001, he flew to London to work on the next edition of his immunology textbook. And the following February, he traveled to another Keystone meeting to brainstorm with Hoffmann and other colleagues about ancient defense systems.
While cancer limited Janeway in some ways, people about the lab characterized him as much more mild and reflective. Indeed, in talks and conversation, he often referred to his personal history, which was encapsulated on a wall of his cramped office.

There, in a display of photographs, hung a column of personas, a legacy of medical minds. Near the top was a 19th-century photo of Janeway’s great-grandfather, New York City Health Commissioner Edward Gamaliel Janeway, MD, posed in formal attire next to a person on a gurney. He is lecturing a crop of medical students. (Janeway noted that his great-grandfather was a pathologist, making it hard to say if the subject of the lecture was a patient or a cadaver.) Next was his son, professor of medicine Theodore Caldwell Janeway, MD, who died in 1917 after contracting pneumonia from the soldiers he treated in an Army camp. Next there was pediatrician Charles A. Janeway, MD, Janeway’s father and longtime chief of pediatrics at Boston Children’s Hospital, who in 1953 discovered, reported and successfully treated the first cases of gamma globulin deficiency. And there was Janeway himself, wearing a summer suit and boutonniere, standing next to Bottomly at the wedding of their daughter Katherine Anne Janeway in the summer of 1999.

In his presidential address to the American Association of Immunologists in 1997, Janeway referred to Robert Frost’s “The Road Not Taken,” comparing life’s choices to forks in the road. These decisions, sometimes taken on a whim, “have a profound impact on our lives,” he said. By handing the toll project to Medzhitov, he knew he was relinquishing a chance for greater fame, but his satisfaction in seeing his younger colleague thrive made awards seem unimportant. Choosing to mentor Medzhitov, said Janeway, made all the difference: “He basically changed my whole outlook on immunology, on life.”

Janeway died on April 12, 2003.

Originally published Spring 2002; updated May 16, 2022