A LEGACY IN TRANSPLANTATION
My Father’s Work
An essay by Mark Terasaki
The barrier to transplantation is rejection. Beginning around 1960, tremendous advances by many clinicians and researchers around the world resulted in the partial conquest of this barrier. Transplantation now extends the lives of thousands of seriously ill people, and my father, Paul Terasaki, was an important figure in its development. This essay attempts to explain his work and to give an overview of the Terasaki Innovation Center and where future developments may lie.
Paul I. Terasaki’s middle name was Ichiro (which means “first born” in Japanese). He often signed lab communications as “PIT”. In this essay, I have the problem of whether to call him “Paul Terasaki”, “Dr. Terasaki”, “my dad”, “my father”, etc … I will use “PIT”!
PIT's first major achievement was the invention of the "microcytotoxicity" test for HLA typing. Every person has an HLA type, something like the ABO blood cell type except quite a bit more complicated. The microcytotoxicity test was reliable and robust enough to make transplantation possible.
The very first HLA typing involved clumping of white cells instead of red cells. When blood is centrifuged at moderate speed, the red blood cells sink to the bottom, the serum / plasma is on the top, with a thin white layer (buffy coat) between the two which contains the leukocytes (lymphocytes, neutrophils, monocytes). Just after World War II, when he as a member of the French resistance, Jean Dausset came across a patient who had an antibody that clumped leukocytes. By studying this antibody and its reactions, and investigating other patients, Dausset came to the conclusion that an inherited characteristic, akin to the red blood cell type, was involved in transplant rejection. This idea was successfully tested on human volunteers. Dausset worked with Felix Rapaport, a surgeon who transplanted miniature circles of skin between human volunteers and looked directly at transplant rejection. The first HLA antigen was established in this way. In 1980, Dausset won the Nobel Prize for this work.
But HLA is a much more complicated system than the ABO red blood cell antigens. We now know that there are two HLA class I loci and hundreds of alleles. Jan van Rood first realized the nature of this problem and developed experimental techniques and strategies to work out the HLA system. Ruggero Ceppellini and Walter Bodmer also made important contributions to the genetics of HLA. But it all depended on antibodies to identify HLA antigens. How to get these antibodies? Rose Payne discovered that during pregnancy, mothers make antibodies to the HLA type of their children. It is complicated by the fact that the mother can make antibodies to more than one type of HLA, and antibodies can cross react with different HLA types, which made the identification and analysis more challenging.
The leukocyte clumping or skin transplant approaches were simply not reliable or reproducible enough for systematic screening required to define the whole HLA system. There was a period of a few years where many labs were trying to develop a practical, robust method. PIT was a young assistant professor with a small lab at UCLA. For several years, he worked long days on this problem with his technician, John McClelland (I have a strange affinity for this period. I was 6-9 years at the time and saw him leave at 7 am and return at 7 pm every day!).
In the end, PIT developed the microcytotoxicity test, in which the target was lymphocytes (a subpopulation isolated from the leukocytes), and the assay was cell killing instead of clumping. The killing depends on adding complement, a component of blood that perforates cells that have bound an antibody. The results couldn’t be read by eye, instead needed a microscope. People recognized that the microcytoxicity test had the required reliability and reproducibility and it was used world-wide by laboratories during the period when most of the HLA class I system was characterized (late 1960's, early 1970's). It became possible to identify the HLA type of individuals (potentially 4 class I alleles because of the two loci), and the process of determining this became known as tissue typing. It was also the basis for starting the company One Lambda in 1984. PCR based methods eventually took over in the 1990's, and HLA typing is now done frequently by DNA sequencing. But it all started with antibodies which were the only tools available at the time. For the invention of the microcytotoxicity test, PIT was elected posthumously to the National Inventor's Hall of Fame, Washington D.C in 2018 - https://www.invent.org/inductees/paul-terasaki
When a kidney is removed from a deceased donor, such as from a car accident, it is a race against time. The kidney must be kept alive until the surgery, often it must be transported to another hospital where the transplant will be performed. In the early days, a kidney could only be kept alive for a few hours, and this limited the number of potential recipients. PIT worked first with some surgeons to extend this time by transporting them at cold temperatures. In 1969, while working in PIT’s laboratory, Dr. Geoffrey Collins of Australia replaced the standard saline solution with a solution mimicking the intracellular concentration of ions, i.e., high potassium, low sodium. This became known as the Collins solution, and extended the survival time for up to 30 hours. For many years, transport on ice in the Collins solution was the standard method for transporting kidneys. Because a kidney could be transported at greater distance, this dramatically increased the potential pool of recipients. It then made sense to match the donor HLA against the HLA of people in this pool, and to coordinate this over a large number of participating hospitals in a large geographic region. The Collins solution led directly to the creation of ROPA (Regional Organ Procurement Agency) in 1968, which eventually became part of UNOS (United Network for Organ Sharing).
With advances in surgical techniques, and progress in testing HLA, the number of kidney transplants increased tremendously in the late 1960's. HLA was used to try to match donor and recipient as closely as possible. It was particularly successful in identifying siblings for transplants – there is a 25% chance of one sibling being HLA identical to another. However, one of the biggest problems was the possibility of acute rejection. Occasionally there was an acute, massive rejection as the new kidney was sutured into place in the operating room. Why did this happen, and could it be prevented? It was not merely caused by an HLA mismatch, because many successful transplants had already been done with HLA mismatches. It became apparent that the recipient already has antibodies against the donor kidney. This is known as pre-sensitization. How can this be detected before the surgery is attempted? PIT and his colleague Raman Patel developed the crossmatch test to do this. In this test, the recipient's serum is mixed with lymphocytes of the donor. If the lymphocytes are killed, the recipient has been pre-sensitized to the donor and will lead to an immediate rejection. Furthermore, and very importantly, they demonstrated that the pre-sensitization antibodies are specifically against one of the HLA types of the donor.
To avoid the disastrous acute rejection, the crossmatch test was immediately adopted by kidney transplant centers around the world. It is used in modified form even today, and the fact that antibodies to HLA involved was the key starting point for single antigen bead assay which was developed 30 years later. It is such an important advance that a symposium in July 2019 was held on the 50th anniversary of the publication of the 1969 paper by Patel and Terasaki - https://www.terasakisymposium.org
Actually another episode in PIT's career was not a success story but does show how determined he was to advance science. As the data accumulated regarding the relevance of HLA to transplant rejection, PIT saw that HLA matching would probably not solve transplantation in the same sense that ABO typing solves blood transfusion. It was very successful in predicting sibling transplants (children have a 25% chance of being identical, HLA testing identifies which siblings are perfectly matched). But for unrelated donors, the results were not as positive. HLA matching had a significant effect, mismatched were rejected sooner, but not as dramatically as would be expected if it were the main factor. He announced this at a major meeting and was met by the disapproval of the HLA community. The reaction was noted by an NIH administrator who withdrew PIT’s NIH contract, a very serious consequence.
Yet, it turns out that PIT was right. The practical success of kidney transplantation was not due to HLA typing but instead was due to the use of immunosuppressive drugs, the first being cyclosporin and later FK-506 / tacrolimus, both pioneered by the surgeon Dr. Tom Starzl. Even today, kidneys are routinely transplanted with HLA mismatches. So what is HLA typing good for? The answer to that came in around the year 2000 and is described in the last part of this essay.
In 1986, the melt down of the nuclear plant in Chernobyl caused many workers at the plant to be exposed to radiation in doses high enough known to destroy their bone marrow. To save their lives, a bone marrow transplant would be required. In this procedure, the hematopoietic stem cells of a donor repopulate the bone marrow and make a new immune system for the recipient. But a peculiar problem arises. This new immune system can recognize the recipient as foreign and attack it - this is known as graft versus host disease. This reaction can be tamped down by immunosuppression but this is often accompanied by serious even fatal side effects. In the same way as kidney transplants, bone marrow transplants are more successful if the recipient and donor are matched. The CEO of Occidental Petroleum, Dr. Armand Hammer, who had strong relations with the Russian Government, organized a group to travel to Chernobyl to perform bone marrow transplants. He recruited UCLA surgeon, Dr. Robert Gale to perform the bone marrow transplants and PIT to do the HLA typing to match donors.
The bone marrow transplant project at Chernobyl was ultimately not successful because the Russian technology / equipment was not very modern and there was too little time to perform the transplants before the victims became sick. However, this dramatic incident raised awareness of bone marrow transplants and how it is facilitated if potential donors are tissue typed ahead of time.
Prior to this, a legendary admiral in the US Navy, Elmo Zumwalt had a son with lymphoma. He had been lobbying US Congress to start a registry, and the Chernobyl incident provided the extra impetus to establish in 1986 the National Bone Marrow Registry under the sponsorship of the U.S. Navy - https://bethematch.org
While PIT accomplished many things in his career, it was the work he did from 2000-2003 while in his 70’s that is the most relevant today. It caused a major change in how transplants are allocated and monitored. Many research labs are actively pursuing the ramifications because future progress could eliminate transplant rejection.
PIT's key insight was that antibodies have a bigger role in rejection than cells (more specifically, T lymphocytes). In fact, there is no question that T cells are involved in transplant rejection, the issue is whether it is clinically more useful to monitor antibodies. He stuck to this idea from early in his career, and for many years it was contrary to prevailing ideas. To convince people of his idea, his group developed an accurate and reliable clinical test to detect antibodies to HLA in the blood. With the advance in recombinant DNA technology in the 1990’s, PIT had decided to invest major resources to create a panel of common HLA antigens using recombinant DNA technology in 2000. The Single Antigen Bead (SAB) assay was introduced to the HLA community by One Lambda in 2003. Within a few years of its introduction the test was adopted world-wide. It is widely used at present and is still One Lambda’s flagship product line.
The SAB assay measures the recipient's antibodies to HLA. There are several hundred HLA variants in the human population. The problem is how to detect whether the patient’s serum contains antibodies to HLA, and which HLA’s in particular. The SAB technology involves coupling HLA variants to labeled beads, mixing them with the recipient's blood, and detecting binding using a special variant of a flow cytometer called a Luminex machine.
How is the SAB assay used in practice? A person who needs a transplant may already have antibodies to HLA, due to transfusions or for women, pregnancy. The SAB assay is used to help select a donor which the patient won’t reject immediately. The second major use of the SAB assay is after the transplant. The transplant recipient may develop antibodies to HLA, it has been shown that this is predictive of eventual rejection / transplant failure (reference, Wiebe and Nickerson?). Periodically, the recipient’s serum is tested with the SAB assay to see whether antibodies to the donor HLA are appearing.
The SAB approach, and generally the focus on antibodies to HLA is an active area of research, and the Terasaki Innovation Center is devoted to this topic.
Why is it important to work on transplant rejection?
In 2019, ~100,000 people were on the waiting list for kidney transplants. 23,000 received one. This illustrates the biggest problem in transplantation, the relative scarcity of kidneys for people who need them.
The situation has improved over the last 10-20 years. Due to three factors, approximately twice as many transplants are performed as before. The first factor is an increase in living donors. Originally almost all kidneys were transplanted from deceased donors, but now 30-40% come from living donors. In large part, this is due to development of a minimally invasive (laparoscopic) surgical procedure which lessened the ordeal for kidney donors. The second factor is "paired donations". Often, a recipient has a relative willing to donate but is an incompatible HLA match. If a number of recipients are pooled together, it is possible to find matches so that every recipient gets a transplant, albeit from a non-family member. Thirdly, the adaptation of procedures to use organs from hepatitis C-positive individuals who have died from opiate overdose. Remarkably, one surgeon, Robert Montgomery, has had a hand in all three of these advances.
How long does a kidney transplant last? in the 1980's / 1990’s, the survival rate increased dramatically to 10 years by the use of new immunosuppression drugs. Surprisingly, there has been little improvement in survival rate since then. Transplants fail because the recipient’s immune system attacks the capillaries of the kidney. PIT’s big discovery was that a central feature of this attack is that the recipient makes antibodies to HLA molecules of the donated kidney.
To study the process by which the recipient starts to make these antibodies and to devise procedures to slow or prevent this would have enormous benefits. First of all, the kidney would last longer. Secondly, the need for immunosuppression drugs would be lessened, which would improve the quality of life as well as life-shortening side effects on the recipient. In fact, a number of research labs are working on this general subject, including the Terasaki Innovations Center.
In the future, it is thought that xenotransplants (such as kidneys from pigs), kidneys grown from the recipient's stem cells, or portable / implantable dialysis machines will address the problem of organ availability. It is hard to predict when that may happen. For various reasons is thought that both approaches will be able to outskirt the problem of chronic rejection.
So, is there a long-term value in understanding transplant rejection? In my mind, yes. Anyone who studies immunology quickly finds out that HLA is right at the center of the immune system. T cells are the orchestrators of the immune system. For instance, they tell the B cells to make antibodies. T cells are in large part activated by foreign peptides that are "presented" by HLA molecules on other cells. Auto-immunity, which is responsible for arthritis and other diseases, is thought to be misidentification by T cells of self-proteins. Allergies are an over-reaction to harmless molecules. It is thought that T cells normally kill abnormal dividing cells, but that under-activity allows them to survive as cancer cells. It is still something of a mystery why HLA is so polymorphic, and the existence of many puzzling aspects of immune response shows that there are fundamental principles / processes that remain to be discovered. Because HLA molecules are involved in the most important interactions tells me, at least, that HLA is part of the story. In the end, the enormous amount of data and experience with human transplant rejection may have hidden clues as to how the immune system works.
Terasaki Innovation Center, Inc.
Terasaki Innovation Center, Inc. (TIC) is a newly established private non-profit organization. It is supported by a donation from the Paul Terasaki Research Fund which I set up, and a grant from Thermo Fisher Scientific.
TIC is partly the result of a promise I made a few years ago. I actually didn’t get along with my father very well after I left home in 1975. For instance, I was a Macintosh fanatic and he hated them – I’m sure most people are familiar with how this can cause problems! But before my father passed away, I told him that I would do what I could to move his legacy forward and in the few years since he passed away, I have become more familiar with what he did and the transplant community.
The work of TIC will be done by Dr. Jar-How Lee and Ricardo Ordoñez. There cannot be two people more suited to help move my father's legacy forward. Dr. Lee worked with my father for over 30 years. He is a tremendous innovator and problem solver who is very familiar with the intricacies of HLA testing. For most of his career, Dr. Lee worked with my father to develop commercial products for One Lambda and the HLA community. The innovative technologies he and his team introduced for the HLA community transformed the way transplant professionals managed their patients. What we are embarking on now is to allow him to work directly with scientific collaborators on research projects that were of interest to my father and projects independent of the profit line.
Ricardo Ordonez was one of the 7 employees from my father’s HLA Laboratory that help start One Lambda in 1984. He has been in charge of many aspects of customer relations, has organized many meetings and workshops, and was most recently Senior Director of Marketing for the transplant diagnostics business (One Lambda) of Thermo Fisher Scientific. He will serve as President of TIC and will be responsible for the day to day operations of the foundation. He has the reputation of knowing many people in the field of transplantation. With time, he will develop projects related to the transplantation community.
Jar-How Lee, Ricardo Ordonez, and myself are members of the governing board. The other members of the board and officers are internationally known, leading transplant scientists. They are Dr. Peter Nickerson, University of Manitoba, Dr. Robert Bray, Emory University, Dr. Cathi Murphey, Southwest Immunodiagnostics, and Dr. Mayra Lopez-Cepero, LifeLink Foundation. I envision serving as chairman for a year or so and then to remain in an advisory role. It is my hope that Terasaki Innovation Center, Inc. will continue my father’s work with the introduction of new technologies and innovations that improve the quality of life of transplant patients.
Department of Cell Biology
University of Connecticut Health Center
Farmington, CT 06030