"The immunology is still the main stumbling block in transplantation," says Sir Roy Calne of the University of Cambridge. "I would predict that tolerance, or 'almost tolerance,' would be the next major advance."

   As recently as 40 years ago, organ transplantation was still a distant dream. Since then it has been transformed into a major branch of surgery and a valuable form of treatment.

   One of the figures most responsible for this transformation is Sir Roy Calne, Professor of Surgery at the University of Cambridge. Calne began his research on organ transplantation in 1959 at the Royal Free Hospital, and described the first effective immunosuppression for experimental kidney transplantation. He developed this approach further while working as a Harkness Fellow at the Peter Bent Brigham Hospital in Boston, Massachusetts, where it was applied to the treatment of patients in 1962. In 1977, Calne developed the immunosuppressive agent cyclosporine A and introduced it into clinical practice in 1978. This breakthrough is reflected in his highly cited papers--most notably, a 1984 report discussing cyclosporine in renal transplantation. (See R.M. Merion et al., "Cyclosporine: Five years experience in cadaveric renal transplantation," New Engl. J. Med., 310[3]:148-54, 1984. This paper has been cited nearly 300 times since its publication.)

   In 1968 Calne performed the first liver transplant in Europe; in 1987, the world's first liver, heart, and lung transplant; in 1992, the first intestinal transplant in the U.K.; and in 1994, the first successful combined stomach, intestine, pancreas, liver, and kidney cluster transplant.

   In addition to his duties at the University of Cambridge, Calne is also an Honorary Consultant Surgeon at Addenbrooke's Hospital, Cambridge, and is Immediate Past President of the Transplantation Society. Aside from his research, he has other, wide-ranging interests. For example, he is an artist whose paintings, depicting images of his clinical work, have been exhibited in many countries to help promote awareness of transplantation. In 1994, adding to his roster of textbooks on surgery and transplantation, he wrote a book entitled Too Many People, which warns of the dangers of the continuing, rapid growth in the world's population.

   Calne, 64, was educated at Lancing College and received his medical training at Guy's Hospital, London. He was elected a Fellow of the Royal Society in 1973, and was knighted in 1986.

Science Watch's European correspondent,
Amir Amirani, met with Calne at his office in Cambridge.

Many of your most-cited papers have been concerned with cyclosporine. What is the significance of this research?

   Calne: The use of cyclosporine was a watershed in transplantation. I have always felt that my own most interesting work was the introduction of 6-mercaptopurine and Imuran as immunosuppressants in 1959 and 1960. But it was only when Imuran was used together with steroids that it was possible to develop clinical transplantation as a useful therapeutic option. We had to wait 20 years for cyclosporine. The experimental work on cyclosporine with organ grafts in animals was done in Cambridge in my department. We applied our findings in the clinic for the treatment of patients with organ grafts. When cyclosporine was perceived as an important advance in immunosuppression, the whole attitude of the medical profession towards transplantation changed. Before that, it was regarded as an enterprise for mad surgeons ignorant of immunology who really didn't know what they were doing and who obtained unpredictable results. Subsequently, however, the image changed to that of an extremely valuable form of treatment for a majority of patients--but not all. In the early 1980s there was an important consensus meeting on liver transplantation held by the American National Institutes of Health. It was decided that the procedure was no longer experimental and was the preferred treatment for most forms of liver disease.

What is the current status of cyclosporine among the other immunosuppressants?

   Calne: Cyclosporine is probably still the pivotal drug of immunosuppression for all organ grafts, except perhaps intestinal grafts, where FK506 seems to have better results. Everything that comes along has to be assessed in relation to cyclosporine, and if a new drug is developed that is clearly much better, cyclosporine will be displaced. But it has not been superseded so far and does not seem likely to be in the immediate future.

Is there any discernible trend in the development of drugs in this area?

   Calne: Cell biologists have been analyzing the mode of action of cyclosporine. This has led to an unraveling of important intracellular mechanisms of signal transduction, from the antigen appearing on the surface of a lymphocyte to the signal for the lymphocyte to synthesize the powerful cytokine interleukin-2, which causes the clonal proliferation of lymphocytes.
   There has been intense activity searching for other molecules, and analogues of cyclosporine. FK506, a macrolide with action similar to cyclosporine, has now been registered, and rapamycin, which is chemically very similar to FK506 but has a different action, is undergoing trials in the laboratory. There are a number of agents in phase II trials. FK506 has recently been licensed for liver and kidney transplantation in the U.K. Mycophenalate, the Syntex drug, which is a purine antagonist, seems to be superior to azathioprine, and I believe the definitive phase III trial on this is soon to be published.
   So there is going to be a plethora of agents, which will make it extremely difficult for the clinician to know what to do and how best to treat the patient. I think we will need to train pharmacologists in immunosuppression--a special breed of physician/clinical pharmacologists who will be expert in the use of these drugs in all transplant patients.

You've said that the surgical problems of transplantation have essentially been solved, and only immunological problems remain. Could you say what the next major development in the field may be?

   Calne: There is a reasonable consensus among surgeons as to the techniques for transplanting a heart, lungs, kidney, and liver. It's true: the immunology is still the main stumbling block. If I were to crystal-ball gaze, I would predict that tolerance, or "almost tolerance," would be the next major advance.
   There are a number of different models to produce tolerance. They have in common the use of extra donor material, particularly donor bone-marrow-derived cells, and immunosuppression--hopefully for a short period of time--with the aim of manipulating the immune system to be able to accept the graft without any continuous immunosuppression, or with a minimal dose.
   The classical Medawar-type tolerance is applicable only to the neonate or to the unborn fetus, but nevertheless it demonstrated that it is possible to manipulate the immune system--at least when it is not fully developed. The question arises, can we render the immune system to a similar pliable state as in the neonate or the embryo? There have been many different approaches to this pioneered by Dr. Anthony Monaco. The idea is to give extra bone-marrow-derived cells and immunosuppression therapy for a short period of time. Dr. David Sacks in Boston has been one of the most active and successful and has produced what's called "mixed chimerism," in which the bone marrow becomes populated with cells of both recipient and donor origin.
   Dr. Thomas Starzl and his group in Pittsburgh have published a number of papers on "microchimerism," in which donor-derived bone marrow cells are scattered throughout the body, even in the skin of patients, many years after transplantation. Microchimerism is prominent in the case of livers but also occurs with other organs. Dr. Starzl believes that this microchimerism is the cause of graft acceptance, but most of these patients are still on immunosuppression, so they are not really tolerant. The Pittsburgh group is in the process of a very extensive trial using large amounts of bone marrow from the donor along with a very high dosage of immunosuppression at the time of organ transplantation. Tolerance has not yet been reported in these patients.

What is the main focus of your research at the moment?

   Calne: For about 25 years, we have been intrigued by the fact that the liver can sometimes produce tolerance in animals without any immunosuppression at all. The tolerance produced by the liver is interesting in that the liver undergoes a rejection crisis and recovers spontaneously, and then the animal will accept another organ from the same donor. From a whole variety of experiments, it would seem that the liver induces tolerance by two mechanisms: 1) the bone-marrow-derived cells in the liver, which include a special population of Kupffer cells, probably establish themselves in the recipient and may be involved in some kind of immunological engagement or conflict that leads to tolerance; and, 2) the liver produces something that maintains tolerance--probably Class-I antigen. This is true in humans as well. In a patient who has had a liver graft, about half of the Class-I antigen in the blood is from the donor.
   In man, if a liver is transplanted together with another organ, such as a kidney, the kidney graft is protected from rejection. The longest survivor in the world with a liver transplant (a patient of Dr. Starzl's), after 24 years, has had no immunosuppression for 14 years.
   The liver effect has been reproduced in many different laboratories. We have been struggling with the obvious question: could we mimic the liver effect without having to actually transplant the liver? Can we use, say, ground-up liver cells?
   Our hypothesis, and we're not alone in this, is that there should be a chance for a dynamic immunological engagement to occur between the host and graft--host against graft and graft against host--without destruction of the graft. If we could only inhibit aggressive T-cell activity for a period of time, we might establish tolerance. And the period of time could be very critical, because if we miss that opportunity, we might then be stuck with continuous high-dose immunosuppression as the only way of keeping an organ in place.
   We have done a number of experiments in which the strategy has been to leave a window of opportunity--I call it "WOOFIE," for "Window of Opportunity For Immunological Engagement." The experiments are simple, and the results are straightforward. I do not know if the interpretations are correct, but the principle is that we give one large dose of immunosuppression--we've been using a very large dose of intravenous cyclosporine in the pig--and either donor spleen cells or donor blood. We then leave a space of two or three days without any more immunosuppression, followed by six more daily doses of immunosuppression. The model is a kidney graft between grossly mismatched strains of pigs.
   Control subjects reject renal allografts after seven to ten days if we don't give any immunosuppression. If we give live spleen cells or fresh blood, three out of four animals go on to long-term tolerance beyond a year without any rejection and no chronic rejection. If the spleen cells were irradiated, there were no long-term survivors, so living cells in the spleen would seem to be important. If we eliminated the window and gave continuous immunosuppression, three out of four grafts were rejected. So this proposed window of opportunity does seem to be important. Without any spleen cells there was one long-term survivor out of four. So it's not an all-or-none thing, but the phenomenon is fairly clear. We now have a protocol that could be used in the clinic, although such a dose of cyclosporine would be too high to give to man. A modified protocol would be needed with a gradual reduction to either minimal dosage or nil. That's our hypothesis.

What's actually going on in this window of opportunity?

   Calne: Imagine an analogy with a football match: you want the match to take place and, at the end of it, for the two teams to shake hands and be friends. Unfortunately, there'll be football hooligans who'll cause a great deal of trouble and maybe destroy the match so it cannot take place. The initial dose of immunosuppression would either kill or imprison the football hooligans, allow the match to occur, and permit the shaking of hands--which is analogous to tolerance. The maintenance of tolerance will also be precarious, because there will be recruitment of new hooligans, or they'll be let out of jail, and that's the reason for giving another six doses although in man you'd probably need to give it longer, I would think, because human beings are much more complicated. They have alternate strategies of immunity. As to the exact molecular mechanism involved in this window of opportunity: the short answer is that we don't know. Some kind of contact between donor marrow-derived cells and those of the recipient seems to be necessary in every kind of tolerance. We have experiments that work but we do not know why they work. So that's my main research interest at the moment: to try and adapt the experiments to a clinical protocol in patients.

Aside from the immunological problems, what about the issue of shortage of organs?

   Calne: No matter how much propaganda we have and how ever much political pressure is put on people, there will not be enough organs because the indications for transplantation are widening rather then contracting. So the dream of a xenograft becomes more and more attractive.
   One would have thought that a xenograft from a close relative such as a nonhuman primate would be much better than a discordant species such as a pig, but the pig is more acceptable on ethical grounds. Also, one can get pig organs of any size. However, the history of xenografting in humans has been depressing.
   The theoretical hurdle to be overcome first in a discordant graft is complement activation, which causes an almost immediate graft destruction. David White in my department is producing transgenic pigs with the anticomplementary human gene. I hope that this approach will be successful in stopping complement activation. We will then be in a position to see what the next barrier is.
   There could be a whole variety of immunological obstacles and physiological considerations. We do not know if xenograft rejection without complement can be controlled with currently available drugs.
   I think it would be facile to assume that there won't be important physiological barriers, because every mammalian cell produces 1,000 or more proteins, and every protein in the pig is different from the equivalent protein in man; some are only slightly different, others are very dissimilar. These differences are likely to be important at least in the long term, even if they are compensated for in the short term. I don't think this has been appreciated fully. I would regard this as very interesting science, but still a long way from practical application. Optimists say it's going to be this year, but I believe that there is much difficult and painstaking work to be done before we get near to using pig organs as a "piggy bank" for humans.