For ten years Suzanne Ildstad trained to be a pediatric transplant surgeon, and she doesn’t underestimate the seductions of her calling. I like using my hands, and I like taking care of patients, she says, and it’s nice to see immediate results. Parts of surgery are really exciting. For example, she says, you always know right away when you’ve got a successful kidney transplant: We always hook the vessels up first to restore the blood supply, Ildstad says. Then if the kidney is healthy, you know immediately because it makes urine and squirts it out. That’s one of the greatest feelings I know as a surgeon: the urine popping out as soon as the blood starts profusing through. Not a knockout image, perhaps. But it is a sign of new life for a patient who 50 years ago would have been given up for dead.
Yet despite the adrenaline charge and the rewards of transplant surgery, Ildstad has largely forsworn them. Last June she decided to take a detour, a temporary turn away from her clinical practice at the University of Pittsburgh School of Medicine and into the research lab. The move, she says, came because miracles in the operating room have their limits. By the 1980s we’d developed operations to treat almost any surgical problem, Ildstad remarks. Yet the patients who undergo transplants often suffer potentially devastating complications.
Kidneys, for example, are the most compliantly transplantable of major organs, yet one in every five kidney transplants from cadavers--the most common source--fails within a year. The kidney is rejected by the transplant recipient’s immune system, a vast, intricate array of cells, antibodies, and proteins designed to seek out and destroy anything in the body that’s foreign. Usually the foreign interloper is a harmful virus or bacterium; in a transplant, unfortunately, it’s a donated vital organ. And this happens even though surgeons carefully select the organs, using only those whose cells barely provoke the recipient’s immune system in preoperative tests. The search for such an organ can be long and desperate. There aren’t a lot available. Some people undergo kidney dialysis for years. Others die waiting.
Drugs have been developed to help combat rejection, but they do so by undermining the immune system, exposing patients to the very infections and malignancies that the system is designed to attack. And these immunosuppressive drugs don’t work forever: the four out of every five transplanted cadaver kidneys that aren’t immediately rejected typically succumb within ten years.
That, Ildstad argues, is simply not good enough. Our goal is to have everything be perfect: permanent graft acceptance without drugs, no complications, no transplant rejection. The reason I went into research is that I think our next major advances--the breakthroughs that will let us solve the clinical limitations of transplant surgery--won’t come from the operating room but from the lab, from research at the cellular and molecular level.
So while Ildstad still spends some time on call as a surgeon, these days she’s more often found amid a large population of experimental rodents at the university’s labyrinthine animal research facility. When you visit her mice, snuffling about contentedly in their clear plastic cages, they don’t look like pioneers on the transplant surgery frontier. They appear to be utterly normal specimens: stubby, black-furred, companionably beady-eyed. Inside, though, they’re made of different stuff. They’re part rat.
These animals have been reoutfitted as hybrids, harboring a mixed collection of rat and mouse immune system cells. On their backs and sides some of these mice sport thumbtack-size, healthy patches of skin transplanted from the same rats whose immune cells they carry. I’ve got pictures of some that’ve been in place for up to a hundred and eighty days, Ildstad reports. That’s nearly forever for a mouse, which has a life span of at most a year and a half. And they look just beautiful, she adds. Skin grafts, Ildstad explains, particularly interest transplant researchers because transplanted skin provokes an extremely strong immune response. It’s always been used as a standard for graft tolerance.
Ildstad and her colleagues have given these creatures a new biological identity, reshuffling the body’s sense of self to enable it to accept a new organ as its own yet retain a vigilant defense force against invading diseases.
They may also have created--if this success can be repeated in humans--a new future for organ transplants, without exasperating, often futile hunts for donors with tissue types similar enough to the recipient’s. This is a future in which children won’t die of liver or kidney disease while waiting for a suitable organ, a future in which organs can move freely between people and even between species without being rejected.
It is also a future ripe with the promise of radical new treatments for old diseases. Skin isn’t the only body tissue that Ildstad and her co-workers have transplanted. We’ve done thyroids, parathyroids, adrenal medullas, cortex; we did some animals where we put multiple endocrine tissues in, she notes with some pride. Recently her lab successfully transplanted clusters of insulin-making cells into diabetic mice.
Ildstad is not the first person to think about turning one creature partway into another. Every day on her way to work she drives past a particularly fine, if also slightly unnerving, reminder of the long hold this notion has had on the human imagination. A few blocks east of her lab is a Shriner temple that is adorned by four magnificent statues. Each has the reclining body of a lion topped by a preternaturally tranquil-looking human head. Such sphinxes are merely the most famous of the mythical creatures called chimeras. In ancient Greek legends chimera denoted a fire- breathing she-monster--part lion, part goat, part snake. But eventually the term came to apply to all sorts of imagined hybrids, such as the centaur, with its human torso and equine extremities, and the griffin, with an eagle’s head and wings and a lion’s body. Today the image has become reality, in the form of rodents that are not quite themselves.
These modern chimeras came into Ildstad’s life in the early 1980s. She’d completed her medical training at the Mayo Medical School in Rochester, Minnesota, in 1978, and had then gone on to Massachusetts General Hospital for a residency in general surgery (where she was the seventh woman to complete the program). Surgical residents, Ildstad recalls, spend a lot of time with senior surgeons, talking about this and that while waiting for a patient to wake up.
I’d been on a rotation with Paul Russell, one of the fathers of transplant surgery, Ildstad says. Talking with him, I got interested in immunology. He pointed out that transplant surgery and immunology have gone in parallel; immunology has helped transplantation to advance. The more biologists learn about the immune system, in other words, the easier it becomes to trick it into accepting a donated organ. Ildstad decided she had to learn more about the arcane cellular mechanisms that either permit gracious acceptance by a host or goad it into rejection.
That realization led her in 1982 to a research fellowship at the National Institutes of Health in Bethesda, where she joined David Sachs, a transplant immunologist. The two began collaborating on ways to subdue, or even eliminate, the immune system’s inveterate hostility to grafts without resorting to immunosuppressive drugs. Their search began, as does the immune system itself, with bones.
Biologists had known for some time that all immune cells originate in bone marrow. The cells that do the immune system’s scut work-- engulfing and devouring--travel directly to various body tissues, where they mature and proliferate. But in humans and mice (and many other animals as well), the immature cells that will eventually control this activity-- deciding when to engulf and devour--first migrate into the thymus gland, which lies under the breastbone. The thymus is a finishing school of sorts: there the cells learn what’s self and what isn’t, and they mature and begin circulating through the bloodstream.
The most important of these cells, for Ildstad’s work, are the T cells. From the moment a surgeon implants a donated organ and it makes a first, tentative acquaintance with your arteries and veins, your T cells bolt into a frenzy of activity, drafting other kinds of immune cells into action and launching an ingenious, coordinated, and all-too-often victorious campaign to exterminate the intruder. To complicate matters further, any of the organ donor’s T cells that are left lurking in the transplanted organ will spring into action against the recipient--a response called graft-versus-host disease.
More than three decades earlier, Nobel Prize winner Peter Medawar and other researchers had transplanted bone marrow cells from one animal into a newborn animal of the same species and shown that the recipient would afterward tend to accept any additional tissue transplanted from the donor. To Sachs and Ildstad that seemed a promising lead. But there was a big snag with these bone marrow transplants: they didn’t work in adult animals, which rejected the grafts. There appears to be a privileged time before an immune system matures when it can be retrained to accept foreign marrow grafts. In people the window of opportunity is the first 16 weeks of gestation--not a very useful opening.
Other researchers tried to work around this obstacle by completely obliterating the recipient’s immune system with toxic chemicals or radiation. Their idea was to let the donor marrow then provide the recipient with a whole new immune system--the donor’s--that wouldn’t attack the graft. And indeed, the experimental animals tolerated grafts well. But they developed another, more serious problem. Their immune systems became incompetent--vulnerable to all sorts of infections and malignancies.
The problem, the researchers would eventually learn, was a matter of improper protocol. Like any large bureaucracy, the immune system has procedures that must be followed, and these experiments seriously breached them. T cells can’t recognize invaders on their own. An invader must first be grabbed by another type of immune cell called a macrophage and then hustled into the presence of a T cell. The T cell will react to the invader only if it recognizes that the macrophage making the introduction is an ally from the same body. However, in the experimental animals that recognition wasn’t forthcoming.
As it turned out, the destruction of the recipient’s marrow had set the stage for a tragicomedy of immunological errors. In act 1 the immature donor T cells traveled to the recipient’s thymus, where they were taught that self equals the recipient. In act 2 they moved through the body, looking for pieces of an invader presented by recipient macrophages. But this was a total bone marrow replacement, and the recipient’s macrophages had been replaced by donor mac-rophages. So in act 3--the final act for the poor recipient animals--the T cells couldn’t find what they were looking for, and the two types of immune cells sailed past each other like ships in the night.
To make matters worse, those animals that didn’t die off quickly began to develop graft-versus-host disease. The donor bone marrow contained not only immature T cells but also some mature ones, and to those cells their new host was foreign, a prime target for attack. A slow but insidious battle ensued between the donor marrow and the recipient; in the end the marrow transplant failed.
Sachs and Ildstad realized that no one had tried to solve this problem by creating true chimeras, with brand-new immune systems that essentially came of age in a recipient. They could, they thought, reoutfit a transplant recipient with a hybrid immune system, one that contained both some of the recipient’s own cells, to prevent the tragicomedy of immune system failure, and donor cells, to produce future transplant tolerance. And, the researchers further reasoned, if they removed mature T cells from the donor marrow, they could keep the graft from attacking the host.
The duo used mice as recipients and either rats or other mice as donors. First they removed some marrow cells from a mouse destined to be a transplant recipient. Then they did the same to the donor animal. Afterward they filtered mature T cells out of both types of marrow, leaving only the immature cells to be educated in the recipient’s thymus. Next they mixed the two types of marrow together. Then they irradiated the recipient mouse to destroy its native immune system, making it a blank slate, immunologically speaking. Finally they transplanted the hybrid bone marrow back into the irradiated mouse.
What Sachs and Ildstad hoped would happen was that as various kinds of immune system cells from both animals matured, they would travel to the thymus along with the maturing T cells. In the thymus the T cells would bump into mouse-derived cells and learn that those cells are part of self. They would also bump into donor-derived cells, which would teach them a similar lesson. Any T cell that didn’t learn both lessons--that attacked any of the other cells--would get destroyed in the thymus; it would flunk out of immune school. Any T cell that made it through, however, would recognize both donor- and recipient-derived immune system cells--such as the all-important macrophages--as allies and would combine with them for an assault on anything else traveling around the body, which would appear foreign.
And that’s pretty much what happened, at least with Sachs and Ildstad’s mouse-mouse chimeras. Some of them accepted later tissue transplants from their bone marrow donors while vigorously rejecting grafts from third parties. There was also no sign of a graft-versus-host reaction. It was a breakthrough, Ildstad says.
Yet the rat-mouse transplants didn’t work. For some reason the hybrid marrow grafts never fully took in the mice, and as a result the mice rarely reconstituted immune systems containing rat-derived cells. The greater the degree of genetic difference between two animals, Ildstad says, the harder it is to get engraftment. My major goal at the NIH was a permanent, stable graft between species, and I never achieved it.
For Ildstad this was a major disappointment. There’s a critical shortage of organs. I had a lot of patients who died while they were on the waiting list for a liver transplant. And with children, in addition to the shortage of donors, there’s a size limitation that makes it difficult to find livers and hearts. But if a cross-species transplant were possible, she knew, it would make available a whole new source of lifesaving donors-- pigs and sheep, for example. With other animals to choose from, the chances of finding an organ that fits increase tremendously. However, the poor results Ildstad achieved made these chances look like a very long shot indeed.
Curiously, though, despite the apparent failure of the marrow graft, some rat-skin transplants did stay on a recipient mouse for brief periods before being rejected. Yet Sachs and Ildstad couldn’t figure out why. To do so, they had to track any rat-derived immune cells that might be roaming around inside the mouse, making up some kind of chimerical immune system allowing that animal to accept such skin transplants, if only for a while. To find these cells, they were using monoclonal antibodies, which are like molecular dog tags that clip on to and identify a particular class of cells; Ildstad would take antibodies that attach to rat-derived immune cells and stain them with a fluorescent dye. If any rat cells were surviving in the mouse, she would see them light up. But few of the cells turned up in these tests, and thus the researchers could not tell if the moderately successful skin transplants were the result of a moderate case of chimerism. We couldn’t track the cells, so we just stopped, says Ildstad.
In 1985 she returned to her surgery residency at Mass General. A year later she moved on to Children’s Hospital Medical Center in Cincinnati, where she established her specialty as a pediatric surgeon. But the conundrum of the chimeras was never far from her mind. In 1988, when Ildstad came to the University of Pittsburgh, she saw a chance to pick up where she had left off.
Ildstad decided to begin again using essentially the same procedure she and Sachs had developed, with rats as the donors and mice as the recipients. But this time, as she was setting up her new lab, she experienced a stroke of luck that has provided momentum for her research ever since. It was, she recalls, total serendipity. Because the lab was still unpacking its scientific luggage and settling into quarters, we didn’t have an agent to deplete T cells from the rat marrow. So we decided we’d transplant it anyway, just to see what would happen.
In theory not much should have: it remained the conventional view, remember, that you had to remove all the mature T cells from donated bone marrow or else they’d attack the recipient and the marrow graft would fail. But Ildstad was flabbergasted to find her T cell-saturated rat marrow cells growing like crabgrass in her chimerical mice--much better than they’d ever done when the T cells were removed. We found unbelievable levels of detectable rat cells, she recalls.
Still more exciting was that marrow transplants also improved the later acceptance of rat tissue. Skin grafts took root with extraordinary alacrity in three-month-old mice, and they lasted until the rodents died a natural death at the ripe old mouse age of a year and a half. We use tail skin because it’s very easy to see, Ildstad explains. A healthy tail-skin graft is nice and smooth and soft, doesn’t have any scabbed-over areas, and doesn’t have any petechiae, which are like bruises. If there is rejection, you start seeing areas of redness. Then, if it’s acute rejection, over a few days it becomes scabbed over, dried up, and crusted.
Ildstad put three grafts on each animal--one from the donor rat, another from a genetically different rat, and a third from a different mouse. The donor rat’s skin survived, while skin taken from third-party rats and even from other mice came off immediately. The mice also showed no sign of immunoincompetence, and they valiantly fought off infections.
What exotic ingredient in this transplanted T cell-rich rat bone marrow could suddenly render a mouse so receptive to the donor rat’s tissue? Presumably it couldn’t be a T cell; there was no reason to think a cell designed to react against foreign tissue would suddenly work to ensure a warm welcome for it. Yet whatever it was, this cell, chemically speaking, had to look very much like a T cell; otherwise it wouldn’t have been eliminated by treatments designed to recognize and remove T cells. Ildstad suspected the existence of a cell that somehow both helps the rat’s bone marrow adapt to the mouse and makes the mouse tolerant of rat tissue as well.
It took nearly two years to confirm those suspicions, but five months ago Sherry Wren, a postdoctoral student working in Ildstad’s lab, finally put a face on this mystery cell: it was a dendritic cell. These cells are distributed throughout the body and ingest invaders that float by. Dendritic cells originate in the bone marrow, like the T cell, and share many of the T cell’s chemical markers. What’s more important is that dendritic cells release proteins called cytokines, which, among other things, stimulate colonies of cells to grow. Ildstad isn’t sure exactly what happens, but she thinks the cytokines floating around the immediate area of the graft stimulate the marrow cells to take root.
Flushed with this success, Ildstad began to turn different animals into chimeras. In addition to mice transformed partway into rats, she reversed the process and gave rats part of the im-mune systems of mice. She’s also used hamsters as donors, grafting part of the hamster immune system into mice.
Ultimately, Ildstad believes, the dendritic cell could become a powerful and invaluable aid in transplants. Indeed, this spring doctors at the M. D. Anderson Cancer Center in Houston are going to use Ildstad’s mix- and-match technique to transplant bone marrow into leukemia patients. But as Ildstad points out, there is a wide range of transplants that could be helped by such a procedure. After her stunning success with skin, Ildstad began to explore other types of transplants with the same technique, moving various rat glands into her chimeric mice without rejection.
Last year she turned her attention to the pancreas, where damage leads to juvenile-onset diabetes. Nearly one and a half million people in the United States have diabetes. Half of them go blind, lose their limbs, or suffer kidney failure and need a transplant, Ildstad says. All this devastation is the result of the failure of a small number of pancreatic cells, called islet cells, which produce the hormone insulin to regulate sugar levels in the body. If we could find a benign way to modify the immune system of people prone to diabetes, we might be able to induce tolerance to either pancreas or islet cell grafts. Insulin-producing islets represent less than two percent of the pancreatic tissue anyway, so that’s all you’d need to cure diabetes and prevent further complications.
At least in Ildstad’s chimeric mice, that’s already a reality. Our rat islets have survived in mice for ten months now, she says. And they don’t merely survive, they work, controlling sugar levels even in mice with previously induced diabetes. We’ve given glucose-tolerance tests to these animals; within fifteen minutes their blood sugar peaks, and within thirty minutes they’re back down to normal.
The human applications of chimeras for diabetes or any other disease are still years away. Research needs to be conducted on larger, more human-size animals, with organs that might prove practical in an interspecies transplant but with immune systems that might behave very differently from those of rats and mice. And ethical issues need to be settled, such as the moral implications of sacrificing animals for transplants. Ildstad points out that no one is considering primates for the purpose; the likely candidates are pigs or sheep, already used for human consumption. Beyond that, a few ethicists are troubled by the prospect of people infiltrated by inhuman cells and solid organs; they wonder if that could constitute a subtle metaphysical threat to our humanity.
Though Ildstad acknowledges the importance of these issues, the metaphysics involved in being two things at once doesn’t trouble her to excess. After all, she’s something of a chimera herself, part surgeon and part immunologist. And that, she thinks, is the reason for her success. People question whether surgeons can do research or whether it’s an oxymoron, she says. But I really think we get a unique perspective through interacting with our patients and seeing the suffering that people sometimes go through. That in turn brings new ideas into the lab with the hope that they can be applied clinically. There are some things we can’t fix in surgery. But I think we’re close to making a major contribution through research.