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The nuclear warfare at the end of World War II inspired medical-radiobiologist Dick van Bekkum to begin his study of total-body irradiation.
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Van Bekkum's multidisciplinary team defined the variables that control the outcome of a bone marrow transplant and established protocols for bone marrow transplantation in diseases of the hematopoietic system.
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The history of graft-versus-host disease, including transfusion-associated graft-versus-host disease, is provided.
Nuclear warfare at the end of World War II inspired Dick W. van Bekkum to study total-body irradiation (TBI) in animal models. After high-dose TBI, mice died from “primary disease” or bone marrow (BM) aplasia. Intravenous administration of allogeneic BM cells delayed mortality but did not prevent it. Initially the delayed deaths were said to be caused by “secondary disease,” which was later renamed graft-versus-host disease (GvHD). GvHD is caused by donor T lymphocytes that destroy recipient cells in skin, intestinal mucosa, bile ducts, and lymph nodes. GvHD is opposed by host-versus-graft disease (HvGD), in which host T lymphocytes destroy the administered allogeneic BM cells, including the administered T lymphocytes of the BM donor. In 1960, van Bekkum became the director of the Radiobiological Institute of the Dutch Organization for Applied Scientific Research TNO, Rijswijk, The Netherlands, where he built a multidisciplinary team that defined the variables controlling the outcome of a BM transplant. The team published their early results in the Journal of Experimental Hematology [1981;9:904–916 and 1956;4:482–488]. Later, protocols were established for BM transplantation (BMT) in patients with severe combined immunodeficiency disease, leukemia, lymphoma, and other diseases of the hematopoietic system. This review honors the scientific contributions made by Dick van Bekkum and his team in defining the four dominant variables for improving the therapeutic ratio of allogeneic BMT and in fostering the international collaboration necessary to translate this knowledge into current clinical practice.
Dick van Bekkum was born in the Dutch Indies but spent his teenage years in The Netherlands, where he attended high school. Growing up in a Nazi-occupied country and learning of the atrocities committed by the Japanese in the Dutch Indies and Japan's surrender after the 1945 nuclear warfare that destroyed Hiroshima and Nagasaki informed van Bekkum's decision to finish medical school and study total-body irradiation (TBI) using animal models. Like the citizens of Hiroshima and Nagasaki, after exposure to high-dose TBI, mice die from “primary disease,” or bone marrow (BM) aplasia. Intravenous administration of allogeneic BM cells delays mortality but does not prevent it. Initially, the delayed deaths were said to be caused by “secondary disease,” which was later renamed graft-versus-host disease (GvHD). GvHD is caused by donor T lymphocytes in the intravenously administered allogeneic BM cell suspension or by donor-type T lymphocytes produced in the new host by the hematopoietic stem cells (HSCs) of the BM donor. These T lymphocytes destroy recipient cells in skin, intestinal mucosa, bile ducts, and lymph nodes.
Graft-versus-host disease is opposed by host-versus-graft disease (HvGD), in which host T lymphocytes attempt to destroy the administered allogeneic BM cells, including the administered T lymphocytes of the BM donor. The best outcome of the interaction between host and donor T lymphocytes is to let donor BM cells engraft, while inactivating enough donor T lymphocytes to prevent lethal GvHD. As director of the Radiobiological Institute of the Dutch Organization for Applied Scientific Research TNO in Rijswijk, The Netherlands, van Bekkum assembled a multidisciplinary team that was the first to describe GvHD under a different name, secondary disease. This team defined the four variables that control the outcome of a BM transplant using four animal models: mice, rats, dogs, and rhesus monkeys. The four variables are as follows.
1.
Number of donor lymphocytes administered per kilogram body weight of the recipient.
2.
Number of recipient lymphocytes that survive TBI, chemotherapy, and immunotherapy administered to the recipient before the donor BM cells.
3.
Administration of immunosuppressive therapy to the recipient/host after intravenous administration of BM cells.
4.
Gastrointestinal microflora of the host.
This review honors the role van Bekkum played in the discovery of GvHD. Here, we recognize van Bekkum for fostering international collaboration by founding the International Society of Experimental Hematology and for publishing his pioneering work in Experimental Hematology [
In 1956, Vos, Davids, Weijzen, and van Bekkum compared the survival of mice receiving supralethal TBI alone with mice receiving the same dose of TBI followed by intravenously administered isologous or allogeneic BM cells [
]. Mice that received only TBI died before post-TBI day 20 from BM aplasia: the “primary” or BM syndrome. Mice treated with sufficient isologous BM cells survived. Mice treated with allogeneic BM marrow cells died after post-TBI day 20 from a “secondary” syndrome.
Histologic studies of mice dying from the secondary syndrome confirm that these mice regenerated enough red cells, leukocytes, and platelets to survive. Instead, their cause of death was multiorgan failure in the skin, intestinal tract, liver, and lymphoid tissues, as documented in histologic studies of experimental animals and human patients by van Bekkum and de Vries in their classic 1967 textbook Radiation Chimeras [
What motivated van Bekkum to study primary and secondary disease?
A young student in The Netherlands in World War II
On May 10, 1940, Germany invaded The Netherlands without a preceding declaration of war. That very day, Mr. and Mrs. van Bekkum and their son Dick were driving to Amsterdam. Mr. and Mrs. van Bekkum had reached the end of their leave and needed to return to their home on Java in the Dutch Indies by boat. They missed their boat because the captain decided to leave Amsterdam early to reach open water before the German Army and Air Force advanced westward. The van Bekkums noticed that their boat was still in the harbor of Ijmuiden but could not manage to get on board. The German invasion forced the van Bekkums to remain in The Netherlands, and they never managed to return to the Dutch Indies to reclaim their home and possessions (Fig. 1) [
Vriesendorp HM. Interviews with Dick and Ada van Bekkum in Emmalaan 4, Rotterdam, The Netherlands, for a lecture titled “Dick van Bekkum, Past, Present and Future,” presented at the International Panorama Stem Cell Conference in the World Trade Center, Rotterdam, The Netherlands. [Dick van Bekkum is interviewed in his office seated between his percussion set and his computer. Ada van Bekkum is answering questions at the living room dining table while serving tea and cookies.]
At 15 years old, Dick van Bekkum graduated from high school 2 years earlier than his classmates. For the rest of his life, he refused to consume Dutch bananas, which he felt did not taste as good as bananas from Java. In the Dutch Indies, van Bekkum enjoyed roaming through the jungles of Java with his local Boy Scout troop. In 1941, he enrolled in the University of Wageningen in the Netherlands to study tropical forestry.
In the first year of the German Occupation, the Dutch Directors at the University Hospital of Leiden refuse to treat wounded German soldiers, because the Germans had placed a new sign at the gate of the hospital: “No Jews allowed.” Though the sign was eventually removed, in 1942, an overwhelming majority of Dutch physicians adamantly refused to join a medical association run by Dutch Nazis/physicians, empowered by German administrators to take over the pre-war Dutch Medical Association. Eventually, the Germans gave in and allowed the Dutch physicians to run their own association without interference from the Dutch Nazis. Still, the Germans dictated that Jewish physicians could treat Aryan, non-Jewish patients only in emergencies.
Inspired by the anti-German attitude at Leiden University, van Bekkum left the University of Wageningen and went to Medical School in Leiden, where he met his future wife Ada Kylstra. In April 1943, the Germans ordered all Dutch students to sign a Loyalty Declaration, by which they would pledge to follow the directions of the German Occupation. On the advice of his father, van Bekkum refused to do so and went into hiding to avoid arrest and deportation to Germany, where he would have been made to perform slave labor in the German war industry.
In Leiden, van Bekkum and some of his fellow medical students hid in the attic of the Anatomy Pavilion of the University Hospital. The Anatomy Pavilion featured a central atrium that was located directly under the skylights of the three-story building. Each morning, a German officer on the ground floor thundered the day's orders to his soldiers. The Dutch students, hiding three floors above the German officer, wrote down the orders and relayed the information to the Dutch Resistance in Leiden.
Dick van Bekkum becomes a physician/scientist
In 1950, van Bekkum completed his medical studies and served his military conscription in the Medical Biological Institute of the Dutch Research Organization TNO. He started to investigate the radiobiology of TBI because many citizens of Hiroshima and Nagasaki were dying from a mysterious “radiation syndrome.”
In 1960, van Bekkum became the director of a brand new laboratory: the Radiobiological Institute of TNO, in Rijswijk, The Netherlands (Fig. 2). Van Bekkum assembled a multidisciplinary team with expertise in physics, internal medicine, chemistry, radiobiology, microbiology, pathology, immunogenetics, and animal husbandry to help define the therapeutic options for humans exposed to TBI. The faculty generated a complete “translational chain” to human patients by way of mouse, rat, dog, and rhesus monkey models. Each animal model served a different purpose: Mice and rats were used to explore the basic biological principles of BM transplantation (BMT) [
]. Rhesus monkeys are the best animal model for the study of human GvHD because BM aspirates of rhesus monkeys contain 2 × 108 lymphocytes per milliliter, similar to the high lymphocyte concentration in BM aspirates of human donors. Unmodified mouse, rat, and dog BM cell suspensions used for transplantation contain less than 5 × 107 lymphocytes per milliliter [
Figure 2The man who discovered that secondary disease = GvHD, Dick van Bekkum, in his new office in the Radiobiological Institute of TNO, Rijswijk, The Netherlands.
Beagles provide large litters and have a short generation time of 1 year. Donor–recipient combinations identical for DLA, the major histocompatibility complex (MHC) of the dog and similar to the human MHC HLA are readily available [
]. Moreover, adult beagles with an average body weight of 12 kg are larger than small rodents and rhesus monkeys. TBI provides a homogeneous radiation dose to mice and rats, but only a moderately homogeneous TBI dose to human patients and larger experimental animals. The biology of the inevitable, only moderately homogeneous TBI dose distribution in human patients (with a highest dose/lowest dose ratio in the body between 1.10 and 1.25) is best studied in dogs. The larger size of the dogs allows for modifying the TBI dose distribution to let the higher doses fall in the hemopoietic system and the lower doses in radiation sensitive normal tissues such as lungs [
Van Bekkum and his colleagues organized many international workshops and symposia at The Radiobiological Institute of TNO (Fig. 3).
Figure 3Group photo of one of the workshops on “Response of Different Species to Total Body Irradiation,” organized by the Radiobiological Institute TNO and the American Armed Forces Radiobiological Institute in July 1983
More than a complication of BMT: Transfusion-associated GvHD
In 1955, a Japanese cardiac surgeon, Shimoda, reported on 12 patients who received donor blood transfusions during cardiac surgery and developed postoperative erythroderma. Six of the 12 died. Five of these 6 developed leukopenia before they died. Shimoda's article, written in Japanese, did not attract a lot of attention in Europe or North America. In it, Shimoda called this unexpected complication postoperative erythroderma (POE) [
]. In 1990, Israeli cardiac surgeons reported on their patients with POE, but they renamed it transfusion-associated graft-versus-host disease (TaGvHD) because the histology of the skin lesions found on the their patients was identical to the GvHD histology described in 1967 and 1985 by van Bekkum [
In Israel and Japan, family members of patients usually donate blood for relatives in need. Because of founder effects on the islands of Japan and intermarriage among Ashkenazi Jews in Israel, more individuals in Japan and Israel are homozygous for HLA antigens. A blood donor homozygous for HLA will recognize an HLA heterozygous family member as HLA incompatible, whereas in the reverse direction, the recipient who carries the same HLA determinants as the blood donor will not: A/A versus A/B. Lymphocytes of the HLA homozygous blood donor A/A kill cells of the heterozygote recipient A/B by reactivity to the HLA B haplotype. In 1986, other Japanese investigators reported that after a blood transfusion from an HLA homozygous donor, the heterozygous HLA phenotype of the recipient (A/B) changed to an A/A HLA phenotype [
]. Van Bekkum and his colleagues reported in 1956—1 year after Shimoda's report, which they had not read—that F1 mice of AA and BB parents will switch from their own heterozygous phenotype for hematopoietic cells to the homozygous phenotype of the parent BM cells [
In Japan a registry of POE patients was started. After the administration of blood transfusions irradiated with 15 Gy, no new POE = TaGvHD cases were noted. From that moment on, all blood to be transfused in Japan was irradiated [
]. Irradiated erythrocytes, granulocytes, macrophages, and platelets remain functional and survive in circulation as long as nonirradiated blood cells. The only radiation-sensitive cells in donor blood and donor bone marrow are lymphocytes and HSCs.
New terminology: Chimera, GvHD, host HvGD, and reciprocal interference
Ford et al. are probably the first to use the term chimeras to describe animals carrying a different genotype in their hematopoietic system and their regular genotype in all other tissues. They borrowed this term from Homer's description of a mythical animal that is part goat, part snake, and part lion [
]. Later, when van Bekkum wanted to stimulate “investigator-driven” research in human patients, he encouraged physicians to become “chimeric” physicians, experienced in two different disciplines: (1) delivering clinical care and (2) being well-trained investigators. Van Bekkum himself, who did not pursue a license as a family physician or a board-certified medical specialist, never became a chimera.
Barnes et al. are probably the first to use the term graft-versus-host disease instead of secondary disease [
]. An advantage of the new acronym GvHD and its mirror term, HvGD, is that both emphasize that BMT is a special form of transplantation. The outcome of BMT is determined by the battle between two competing immune attacks: GvH and HvG reactions. In 1981, Vriesendorp et al. proposed a new name for the battle between host and donor lymphocytes: reciprocal interference [
First bone marrow transplants in human radiation accident victims
In November 1958, six staff members working at an experimental nuclear reactor in Vinca, Serbia were exposed to inhomogeneous TBI at doses ranging from 4.36 to 2.07 Gy. (Gray [Gy] is the SI unit for absorbed radiation energy, 1 Gy = 1 Joule/kg tissue.) A poorly controlled experiment made the reactor critical for approximately 8–10 min before the physicist supervising the experiment smelled ozone (O3) and stopped the reactor by inserting moderators into its core.
The LD50 for humans for homogeneous gamma TBI, extrapolated in 1984 at the Radiobiological Institute TNO from experimental animal data and human radiation accident data, was 3.5 Gy [
]. For inhomogeneous TBI, the LD50 was higher. When the blood counts of five of the six Serbians began to drop, they were flown to Paris. Mathé et al. performed the first human BM/fetal liver transplants. The sixth Serbian, who received only 2.07 Gy because he made a trip to the bathroom while the reactor was critical, remained at home [
The BM donors were French volunteers unrelated to the victims. An anonymous liver from a spontaneous late abortion provided a source of fetal liver cells—liver and spleen being the major hematopoietic organs of the fetus [
]. Four victims manifested temporary evidence of donor-type hematopoiesis. They survived after rejecting the transplanted allogeneic cells by HvGD and were able to regenerate their own hematopoietic system to live out their regular life span without developing “late” side effects of radiation. The physicist, who received the highest TBI dose, received BM as well as fetal liver cells. He died on posttransplantation day 25 from multiorgan failure. In retrospect, his death was probably caused by GvHD, but if it was, it was ignored at that time for two reasons: (1) Most BMT research was being performed in small rodents. Results in mice were not considered directly translatable to human patients. (2) van Bekkum et al.'s studies of the pathogenesis of GvHD had not yet appeared in print, and the influence of HLA mismatches between donor and recipient on survival after TBI and BMT were yet to be defined.
In retrospect, hindsight usually being 20/20, all five Serbians would have survived if they had received state-of-the-art intensive care instead of BM transplants.
More human BM transplants
The French doctors, encouraged by their experience with the Vinca victims, used allogeneic BMT to treat leukemia patients in relapse, that is, patients without other treatment options. The patients received more homogeneous 4-Gy “deliberate” TBI and intravenously administered BM cells from a healthy donor; almost none of the patients survived beyond posttransplantation day 50, underlining the need for more translational animal research.
Results in human BMT improved dramatically when in 1979 Thomas and his BMT group in Seattle, Washington courageously and wisely introduced four changes to human BMT for patients with acute leukemia [
]: (1) They used a MHC/HLA-identical sibling of the patient as the BM donor. (2) They performed BMT after regular-dose chemotherapy brought the leukemia of the patient into remission. (3) They added high-dose intravenous cyclophosphamide (Cy) chemotherapy to TBI to prepare (“condition”) the patients for BMT. Last, (4) they used chemical immunosuppression, intravenous methotrexate starting on posttransplant day 3 to suppress GvHD.
Approximately one half of the patients with acute myelogenous leukemia treated in first remission survived long-term without leukemia recurrence. Thirty-five percent of patients with an acute lymphoblastic leukemia treated in second remission survived long-term without a leukemia recurrence.
The success of the Seattle group was based on their translational BMT studies in a single experimental animal model: dogs. The enormous steps forward taken by Thomas et al. convinced more experimentalists and clinicians to become involved in clinical BMT. Most did not engage in their own preclinical animal experiments; they simply duplicated the protocols of Dr. Thomas. The weakest link in Dr. Thomas's protocols was the collection of the maximum amount of BM cells that can be obtained from a dog or human BM donor. This leads to the administration of large numbers of BM donor lymphocytes, which cause hard-to-treat GvHD, because large volumes of BM obtained by aspiration will be contaminated with large numbers of peripheral blood lymphocytes. The results obtained by van Bekkum's team using mice, rats, dogs, and rhesus monkeys illuminate the importance of lymphocyte dose per kilogram of recipient; yet, at the time, these results were ignored.
The radiation setup for TBI by Thomas et al. is unique: A patient in the middle of the crossfire of two opposing cobalt-60 sources received a prescribed dose to the midline of 10 Gy in a single fraction. When 10 Gy TBI was administered in other centers, large percentages of patients died from interstitial pneumonitis, whereas the incidence of interstitial pneumonitis in Seattle was much lower. In 1980, Lam et al. identified a calibration mistake in Seattle [
]. The Seattle patients were receiving not 10 Gy, but 9.2 Gy. Lowering the lung dose after TBI in other centers to a single low-dose rate TBI fraction of 9.0-Gy fraction or 4 × 3-Gy fractions at a high-dose rate with lung shielding for one fraction [
]. The latter TBI schedule also decreased the incidence of radiation-induced vomiting during the delivery of TBI. Both TBI schedules decreased the incidence of radiation pneumonitis to rates seen in Seattle in 1979 [
Preparation of bone marrow cells for transplantation
In Seattle, human BM cell suspensions were prepared with the method Dr. Thomas developed in the dog colony of the Mary Imogene Basset Hospital in Cooperstown, New York. BM was filtered over a series of metal sieves of increasingly finer mesh. The final suspension used for transplantation still contained visible fat and small bone spicules. Most new human BMT centers copied Dr. Thomas' BM collection method. Patients treated on protocol received different amounts of BM cells per kilogram of body weight. In contrast, in the Radiobiological Institute in Rijswijk, all large-animal BM cell suspensions were collected by repeated small BM aspirations before centrifugation. The buffy coat was collected for transplantation and counted. The cell dose was adjusted to the prescribed amount of BM cells per kilogram of body weight.
Other investigators tried to improve the engraftment of BM cells with intraosseal administration. As expected, intraosseal administration is a more painful method for administering fluids, but is otherwise equivalent to intravenous administration, because the fluids are pressed through a needle ending in a BM cavity, which creates extra pressure in the bone cavity that can be released only by letting the administered fluids escape from the bone cavity into the venous system. Intraosseal administration of fluids or BM cells is needed only in situations in which venous access is difficult to obtain, as in critically ill small children and critically wounded soldiers [
Intravenously administered HSCs cross the blood–BM barrier by an incompletely defined, selective, transfer mechanism. Within the bone marrow cavity, HSCs settle in their “niche” close to the endosteal membrane [
To create “space” for the administered blood cells, as in the game of musical chairs, where a contestant is out of the game if there is no chair left in which to sit when the music stops. If hematopoietic stem cells cannot sit in an empty endosteal BM niche, BMT fails.
2.
To immunosuppress the recipient, to prevent an HvG reaction.
3.
To eradicate the malignancy of the BMT recipient if he or she is a cancer patient.
Conditioning aims 1 and 2 are readily accomplished with high-dose TBI alone. In dogs, a single fraction of 5.0 Gy TBI is sufficient for the permanent engraftment of 4 × 108 DLA-identical BM cells per kilogram of body weight.
Chemotherapy is added to TBI to accomplish aim 3, eradication of the malignancy. This is the most difficult aim to achieve and limits curative BMT to oncology patients with liquid tumors, that is, leukemias and lymphomas, because of their higher sensitivity to radiation and chemotherapy. Allogeneic or autologous BMT is not recommended for adult patients with radiation- and chemotherapy-resistant solid tumors.
Targets for T lymphocytes
T lymphocytes, educated in the thymus, attack “foreign”/non–self-structures. In tissue transplantation procedures, this amounts to attacking cells carrying major or minor histocompatibility complex (MHC and MiHC) antigens on their outer cell membrane. Of all cells in the body, lymphocytes have the highest density of MHC antigens on their cell membrane. Most tissue-typing techniques use peripheral blood lymphocytes as the antigen source. Platelets and red cells do not have a nucleus, but platelets do carry MHC antigens on their membranes, whereas red cells do not. If human red cells had carried MHC antigens on their cell membrane, blood transfusions would have become a logistic nightmare because of the low population frequency of a given HLA phenotype, <0.1%. Patients with chronic low platelet levels become rapidly sensitized against the HLA antigens of the platelet donor. It is difficult to find HLA-matched platelet donors for such patients. Frequently, family members of the patient are recruited for platelet donation [
In all species examined for an MHC, only one MHC is found. MHCs in all species examined in sufficient detail consist of a closely linked group of loci containing genes programmed for different immune functions, antibody production, cellular immunity, and proteins for the complement cascades [
]. In inbred mice, more than 40 different MiHCs have been identified. The immunologic identification of MiHC antigens is difficult because the rejection of an MiHC graft does not lead to the production of antibodies, tissue-typing reagents, recognizing MiHC antigens. Most, if not all, mammalian species have a monomorphic MiHC on the Y chromosome and a polymorphic MiHC on the X chromosome [
In 1960, Sir Frank Macfarlane Burnet and Sir Peter Medawar received the Nobel Prize in Physiology or Medicine for their work on cellular immunity and acquired immune nonreactivity, respectively [
]. Burnet postulated that lymphocytes carry a “dictionary” that describes which structures are “self” and, by exclusion, recognizes all other cells, structures, and molecules that are “nonself”. Recipient cells will identify membrane structures as nonself and reject/destroy them by a specific cellular immune response and by the secretion of aspecific cytocidal biological response modifiers (BRMs). Structures recognized as self will not be rejected; in other words, autoimmunity does not occur.
In 1961, Crick, Barnett, Brenner, and Watts-Tobin discovered the lengths of the words in Burnet's dictionary: three-letter words, permutations of A, T, G, and C, the first letters of the DNA bases, 4 × 4 × 4 = 64 triplet codes, for 20 different amino acids. Therefore, most amino acids have more than one code, and three codes are “stop reading” codes [
Sir Peter Medawar received the Nobel prize for a hypothesis overlapping with Burnet's postulate: that foreign spleen cells administered to fetal or neonatal mice would not induce an immune response but would lead to incorporation of the MHC and MiHC antigens carried by the spleen cell membranes into the dictionary of the recipient's lymphocytes under the heading of self. When later in life, these mice received a skin graft from the spleen cell donor, they would not reject the skin graft because the skin allograft was considered self.
Owen, a Wisconsin veterinarian, alerted Medawar and co-workers to his studies of free martins, “feminized” bulls called free because they are not able to breed and martin because they have a male genotype [
]. Free martins always have a female twin. Owen inspected the placentas of the bovine twins. He noticed that probably because of the crowded conditions of the cow's uterus, the placentas of the twin bull and cow fetuses had fused and made vascular connections. The bull fetus could not break down the estrogens produced by his female partner and became feminized. The bovine twins exchanged HSCs in utero as well. They became permanent, mixed chimeras, carrying each other's blood groups and histocompatibility antigens in different, fluctuating levels for life. Kidney grafts and skin grafts between the cow and her free martin twin partner survived more than 100 days. This appears to confirm the hypotheses of both Burnet and Medawar regarding the cellular recognition of self and nonself and acquired immunotolerance. However, only one of the two hypotheses has survived.
Other battles between HvG and GvH reactions
Confrontations between allogeneic T lymphocytes occur after allogeneic BMT, during all pregnancies, and after blood donor transfusions. There are three possible outcomes of the confrontation:
1.
HvG wins and kills all donor (GvH-causing) lymphocytes. If conditioned for BMT with high-dose TBI and chemotherapy, the host most likely will die from infections and/or bleeding: the primary “bone marrow syndrome.” There is little to no evidence that spontaneous abortions before week 20 (incidence of 8%–20%, according to UpToDate, accessed in 2016) are due to the rejection of the fetus by the mother's immune system. Successful deliveries are more common and exemplify a draw between maternal and offspring lymphocytes and/or a strong immunologic barrier between mother and child in the form of the placenta. The placental barrier can be breached in either direction. Fetal blood carrying the MHC antigens of the sperm donor will find its way to the circulatory system of the mother and immunize her. Rose Payne and Jon van Rood discovered such anti-sperm donor antibodies. This led to the discovery of the class I antigens of the HLA system [
The immunoglobulin G (IgG) of the mother is also present in her breast milk. Maternal IgG will cross the placenta into the fetal circulation by way of the Brambell receptor, and after breastfeeding, breast milk IgG will cross the lining of the small intestinal mucosa of the newborn baby using the same receptor [
Rarely, placental cells of fetal origin, including paternal histocompatibility antigens, migrate through the placenta into the maternal circulation to settle in/metastasize to maternal tissues to grow into tumors. These tumors are called choriocarcinomas. Tumor cells produce human chorionic gonadotropin serum levels that correlate with tumor load. Because of the eminent sensitivity of choriocarcinomas to chemo- and radiotherapy, all choriocarcinomas in women are curable by physicians experienced in treating these rare tumors [
]. The human testicle can be the source of choriocarcinomas in men. They have a poor prognosis, possibly because they do not contain allogeneic MHC antigens foreign to the host and do not elicit “spontaneous/natural” immunotherapy [
GvH wins and kills all the lymphocytes of the host. The host will develop GvHD in one of its three forms: acute, delayed, or chronic. POE and the death of severe combined immune deficiencies (SCID) patients after blood donor transfusions are examples of acute lethal GvHD [
]. SCID patients cannot mount an HvG reaction. Therefore, SCID patients do not need to be conditioned with chemo- or radiotherapy before BMT. After HLA-identical BMT, most SCID patients become “split” chimeras, with T lymphocytes from the BM donor and their own B lymphocytes, red cells, granulocytes, and platelets [
The fierce battle ends in a draw. The erythroid, granuloid, and thromboid lines of both parties produce blood cells in sufficient numbers. This results in a so-called “mixed chimera” with cells with the two different genotypes in all four differentiation lines of hematopoiesis [
]. This is illustrated in Figure 4 by the red and green snakes, which are eating away at each other, like GvH and HvG reactions destroy/eat each other. Eventually the meal ends when GvHD or HvGD wins or the surviving parts of the respective immune systems learn to respect each other. Stopping to eat commands, armistice conditions, might exist but have not yet been uncovered. Under all armistice conditions, a mixed chimera will be less immunocompetent, unless the chimera can regenerate the parts of his or her immune system that were eaten by the partner.
Regeneration of an adult immune system
Regeneration of a larger, more functional immune system by the surviving T and B lymphocytes and HSCs in mixed chimeras will take time because of the three or four different cloning events that need to take place between HSCs and functional, antigen-specific T or B lymphocytes. This is difficult to achieve in patients older than 6 years of age. The “educational” organs for T and B cells, the thymus and the bursa of Fabricius equivalent, respectively disappear/atrophy spontaneously before the age of 6 and are not likely to return after allogeneic BMT in adult patients. If this is indeed the case, dictionary T- and memory T- and B-lymphocyte deficiencies will be chronic if not permanent and help to explain the autoimmune diseases and immunocompromise in patients suffering from chronic GvHD.
Inducing acquired immunotolerance in adult recipients
Transplantation surgeons have tried to apply Medawar's acquired immunotolerance concept to patients in need of a new kidney or a liver transplant. Their hope was that the prolonged presence of organ donor hematopoietic cells in the recipient, creating a mixed chimera, would lead to the incorporation of donor antigens into the self-dictionary of the recipient, resulting in acquired immune nonreactivity and eliminating the need for long-term use of toxic, immunosuppressive, medications to prevent rejection.
The induction of mixed chimerism in experimental animals or human patients is a complicated process with many variables. In mouse experiments, Wood et al. [
] achieved prolonged survival of skin allografts after treatment with antilymphocyte serum (ALS) and the administration of skin donor BM cells. However, they disproved Medawar's hypothesis that recipient mice do not acquire immune nonreactivity to the skin donor. Instead, the murine recipients develop cellular antiskin donor immune reactivity. The prolonged survival of mouse skin allografts in this model might be due to the immune suppression induced by competing GvH and HvG reactions.
Another strike against Medawar's theory is that the free martin cows eventually rejected skin or kidney grafts from their sister. They, too, do not acquire immunotolerance [
Investigators at the Dana-Farber Center conditioned 43 patients with six or seven fractions of TBI and high-dose Cy for 2 days. They applied GvH prophylaxis by incubating donor BM cells with an anti–T-cell antibody and complement. One half of the patients experienced a slowly increasing percentage of recipient cells in their bone marrow. The incidence of GvHD was significantly lower in patients with “mixed” chimerism. Early immune recovery after transplantation correlated positively with complete donor-type chimerism. Patients with stable mixed chimerism experienced more relapses of their hematologic malignancy than patients with complete chimerism. Complete and mixed chimeras have similar long-term, progression-free survival [
]. Surprisingly, Walter et al. were able to restore memory lymphocytes from the BM donor for cytomegalovirus (CMV) in complete BMT chimeras without inducing serious GvHD [
In 1977, Obertop, Bijnen, and van Urk, surgical residents at Erasmus Medical Center, Rotterdam, The Netherlands, performed kidney allografts from a DLA-identical BM donor to recipients that had accepted, quickly rejected, or slowly rejected transplanted BM cells at the Radiobiological Institute of TNO in Rijswijk. Results (illustrated in Figure 5, Figure 6) were so confusing to the experimenters that they were never published. The writing of this article, almost 40 years later, provides better insights into the underlying mechanisms believed to provide a logical and correct explanation for these older, unpublished observations. There are two important issues. First, the kidneys of the dogs receiving a new kidney were removed on the day of transplantation, and second, the recipients did not receive any posttransplant immunosuppression. Their survival was dependent on the survival of the single transplanted kidney allograft in the three different experimental protocols described below.
Figure 5Survival of dogs (N = 80) TBI and BM and lymphocytes of DLA-mismatched dogs
Figure 6Survival of dogs after 7.5 Gy of TBI and different amounts of intravenously administered BM cells and lymphocytes. DLA = Dog MHC; PB = peripheral blood containing zillions of lymphocytes. = indicates identity for MHC; ≠ indicates mismatch for MHC
The relative role of the DLA complex in bone marrow transplantation.
in: Zaleski M.B. Abeyounis C.J. Kano K. Immunobiology of the Major Histocompatibility Complex. 7th International Convention on Immunology. Karger,
Basel1981: 214-223
Dogs DLA-identical with the littermate BM donor were conditioned for DLA-identical BMT with a single TBI fraction between 5.0 and 9.0 Gy. On day 1 after TBI, recipients were administered 4 × 108 BM cells/kg of body weight intravenously. Eight of eight recipients completed DLA-identical BM chimeras, as determined by karyotypes, blood groups, and polymorphic enzyme markers [
] and accepted the kidney of the BM donor for more than 400 days.
Conclusion
The immune system of the radiation chimera did not reject a kidney of the BM donor.
Experiment 2
After a lower dose of 3.5 Gy TBI and 1 × 108 BM cells/kg, donor BM cells were rejected. The recipient dogs survived by gradually recovering their own BM, comparable to what happened to the Vinca victims flown to Paris. Two of two dogs rejected a subsequent kidney allograft of the DLA-identical BM donor in an accelerated fashion, surviving only 10 days instead of the medium survival time of 42 days for a DLA-identical kidney in dogs not receiving BMT or chemical immunosuppression. There was no reason to reject Medawar's hypothesis at this point.
Conclusion
The dogs' immune system had been sensitized and, as expected, rejected the kidney of the BM donor in an accelerated fashion.
Experiment 3
Five dogs rejected BM from a DLA-identical sibling after less intensive TBI conditioning between 4.25 and 7.5 Gy and lower BM cell doses of 2, 1, or 0.5 × 108 BM cells/kg. These dogs rejected the donor BM in approximately 18 days, after which a slower regeneration of their own autologous BM occurred, as in experiment 2. Five of the five dogs accepted a kidney from the rejected BM donor for more than 400 days without any chemical immunosuppression.
Conclusion
Medawar's hypothesis of acquired immunotolerance did not apply: Dogs that rejected DLA-matched BM cells because of differences in minor histocompatibility antigens were expected to reject in an accelerated manner a DLA-identical kidney from the BM donor. The new hypothesis/explanation was that prolonged interactions between lymphocytes of two DLA-identical littermates would eliminate/kill each other's lymphocytes by reciprocal interference, by eating each other's lymphocytes, and in doing so, destroying the “dictionaries” for most or at least some minor histocompatibility antigens expressed on kidney cell membranes, which in turn prolong the survival of dog kidney allografts to >400 days. No histologic signs of rejection were observed in the long-surviving kidney allograft.
Acquired immunotolerance in patients with osteogenesis imperfecta
Non-MHC–matched first-trimester fetal cells were used to isolate mesenchymal stem cells (MSCs) that can differentiate in bone, fat, and/or cartilage. Osteogenesis imperfecta (OI) causes broken bones in utero because of the poor-quality collagen made by those with OI. The first injection of MSCs was performed in two fetuses at week 31 of a 40-week pregnancy. After birth, both babies did better than an untreated OI control baby, but they remained below the third percentile of body length. Both babies relapsed but responded again to the same source of MSCs at 1.5 years for one child and at 8 years for the other child. The MSCs administered the second time were again able to support the production of better collagen for a while.
Conclusion
Although a promising lead for improving the lives of children with OI, both administrations of MSCs did not support Medawar's postulate. The first in utero administration did not induce acquired immunotolerance because the allogeneic MSCs, carrying class 1 but not class 2 MHC antigens, did not survive as “self,” as postulated by Medawar. The second administration of MSCs, after the babies were born, was rejected slowly again, leaving the door open for another improvement with a third administration of MSCs [
Medawar wrote eloquently about the importance of formulating a “null” hypothesis for every experiment and selecting rigorous tests to disprove—or in the terms of his friend Carl Popper, to falsify—the postulated null hypothesis [
]. Medawar's null hypothesis passed the first set of tests but was falsified in subsequent tests in free martins, mixed chimeras after BM transplants, dogs after kidney allografts, and MSC transplants in fetuses and neonates with IO described above. Medawar died before the second set of tests of his null hypothesis.
We speculate that Medawar and van Bekkum would have agreed to drop the acquired immunotolerance hypothesis based on the new information described above.
Three types of GvHD
Three types of GvHD were identified after allogeneic BMT in large-animal models (dog, rhesus monkey) as well as in human patients [
The relative role of the DLA complex in bone marrow transplantation.
in: Zaleski M.B. Abeyounis C.J. Kano K. Immunobiology of the Major Histocompatibility Complex. 7th International Convention on Immunology. Karger,
Basel1981: 214-223
]: (1) acute GvHD, leading to death of the recipient within 30 days of transplant (A); (2) delayed GvHD, causing mortality between posttransplant days 30 and 100 (D); and (3) chronic GvHD, causing immune deficiencies and immune dysfunction after posttransplant day 100 (C). Two GvHD survival studies in dogs are illustrated in Figure 5, Figure 6.
Four variables determine the type of GvHD the recipient dog will experience.
1.
MHC differences between donor and recipient. DLA-matching and the intravenous administration of 4 × 108 dog BM cells/kg of body weight after exposure to 7.5 Gy of moderately homogeneous TBI leads to 95% survival without the need for GvH prophylaxis/treatment with immunosuppressive medications (Fig. 6).
2.
Number of donor lymphocytes per kilogram body weight: Increasing the donor lymphocyte dose stepwise to 1 × 109/kg increases the incidence of acute GvHD in DLA-identical donor–recipient combinations from 0%–5% to 95% in dogs receiving HSCs isolated from peripheral blood, which equals the survival of dogs receiving DLA-mismatched marrow (Fig. 6).
3.
Use of posttransplant chemical immunosuppressive agents: GvHD prevention with methotrexate decreases the incidence of acute GvHD in DLA-mismatched situations from 95% to 60% (Figure 5, Figure 6).
4.
Total or selective decontamination of canine gastrointestinal microflora [
]: Total gastrointestinal decontamination is difficult to maintain in DLA-mismatched combinations. Selective removal of Gram-negative microorganisms decreases the severity of endogenous infections after TBI and lowers the incidence of severe GvHD in dogs receiving 1 × 108 lymphocytes/kg body weight.
Chronic GvHD
Chronic GvHD, developing 100 days after BMT, consists of opportunistic infections and autoimmune disorders. Immunosuppressive therapy is not an option. In humans, the best way to decrease the incidence of chronic GvHD is to reduce the incidence of acute and delayed GvHD by lowering the dose of donor lymphocytes per kilogram. The use of fetal liver cells for transplantation in dogs leads to slow immune recovery and autoimmunity, likely due to chronic GvHD [
in: van Bekkum D.W. Lowenberg B. Bone Marrow Transplantation in Animals and Man: Biological Mechanisms and Clinical Practice. Marcel Dekker,
New York, NY1985: 383-407
Influence of gastrointestinal microorganisms on development of GvHD
When Dick van der Waaij became the microbiologist of the Radiobiological Institute of TNO in Rijswijk, The Netherlands, he, along with van Bekkum and Heidt, started a series of allogeneic BMT studies in gnotobiotic (germ-free and defined flora) mice. The absence of microorganisms prevented death from GvHD after allogeneic (H2-mismatched) BMT (107 bone marrow cells administered intravenously) in 100% of the animals [
]. Between 90% and 100% of the recipients that harbored a strict anaerobic nonpathogenic microflora survived after allogeneic BMT without developing life-threatening GvHD [
]. When mice were selectively decontaminated of potential pathogenic microorganisms, they, too, survived H2-mismatched BMT. The animals could be re-conventionalized as early as day 40 after BMT without adverse effects [
Gastrointestinal decontamination in dogs and rhesus monkeys
After conditioning with TBI (7.5, 8.5, or 9.0 Gy), dogs receive DLA-identical bone marrow cells enriched for BM donor lymphocytes. Dogs treated with daily oral antibiotics to eliminate gram-negative microorganisms only are treated in conventional animal rooms. Dogs receiving total decontamination are treated in laminar-airflow cabinets. The systemic infections occurring in selective decontaminated dogs after high-dose TBI are less severe, possibly because the remaining gram-positive microorganisms lack endotoxins. Total decontamination of dogs is difficult to maintain. The animals in which total decontamination is successful produce odorless stools, just like the first bowel movements of human neonates. The decontaminated dogs are able to survive 9.0 Gy TBI, which often causes a lethal gastrointestinal syndrome in conventional dogs [
Twenty-five rhesus monkeys were completely or selectively decontaminated of gastrointestinal microorganisms, irradiated with a single dose of 8.5 Gy x-rays or two fractions of 7.0 Gy separated by 3 days, and transplanted with stem cell-enriched, lymphocyte-depleted allogeneic BM. The BM cell dose is 5 × 107/kg of body weight [
Influence of gastrointestinal decontamination on graft-versus-host disease in rhesus monkeys after (partially) mismatched allogeneic bone marrow transplantation.
]. The occurrence of GvHD could be determined in 16 of the 25 transplanted animals. Four monkeys rejected the donor BM cells, and five monkeys were not evaluable because of early death caused by severe electrolyte imbalances, possibly as a result of the gastrointestinal toxicity of the high 1 × 8.5 or 2 × 7 = 14 Gy TBI dose. The incidence rates of lethal GvHD in the different donor–recipient combinations are given in Table 1.
Table 1Incidence of lethal GvHD in different gnotobiotic groups of monkeys (different donor/recipient combinations)
Donor/recipient combination
Gnotobiotic state
RhLA
Family relationship
Complete GID
Selective GID
Clean conventional
A/B
D/DR
=
≠
None
0/4a
—
5/9b
≠
=
Sibling
0/2c
0/1d
4/5e
≠
≠
None
6/8f
1/1g
5/5h
RhLA = major histocompatibility complex of the Rhesus Monkey.
χ2 test: (a + c + d) vs. (b + e), p < 0.05; (f + g) vs. h, p > 0.05 (not significant).
Decontamination of the gastrointestinal tract results in the prevention of lethal GvHD in recipients of partially matched, T lymphocyte–depleted allogeneic BM grafts in rhesus monkeys. A possible difference between the two types (complete and selective) of gastrointestinal decontamination could not be evaluated because of the small size of the individual groups in this study.
Gastrointestinal decontamination in pediatric patients
In 1990, Vossen et al. reported on the effect of gnotobiotic measures on moderate to severe GvHD after allogeneic BMT in children treated in the Department of Pediatrics of the Leiden University Medical Center [
Prevention of infection and graft-versus-host disease by suppression of intestinal microflora in children treated with allogeneic bone marrow transplantation.
]. Their analysis includes 65 cases of BMT for either severe BM failure or leukemia, performed in a strict protective environment with either complete (n = 44) or selective (n = 21) gastrointestinal decontamination (GID). All BM grafts were taken from HLA-identical siblings and were not depleted of donor T lymphocytes. Complete GID was successful in 12 of 44 patients; selective GID was successful in 17 of 21 patients. Complete GID was superior to selective GID in lowering the incidence of grade ≥2 GvHD to only 7 of 40 (17.5%) in completely decontaminated children versus 9 of 18 (50%) in selectively decontaminated children (p < 0.01).
]. All 112 patients in this study were completely decontaminated using nonabsorbable antibiotics, and they received non–T-cell–depleted marrow from HLA-matched sibling donors. All patients were nursed in strict, protective isolation (Fig. 8). Total GID was successful in 57 (51%) of the 112 evaluable graft recipients. After discontinuation of the antibiotics for total decontamination, the patients were reconventionalized on posttransplant day 40 by oral administration of strictly anaerobic intestinal enteric flora from an unrelated human donor [
]. The number of patients who developed GvHD was small: 9 of 112 (8%). Eight children developed grade I GvHD (only involvement of the skin) and 1 patient developed grade II GvHD (skin stage 2, gut stage 1, and liver stage 2). No patients died from GvHD.
Figure 8Down-flow isolator for the sterile nursing of pediatric BMT patients (Department of Pediatrics, Leiden University Medical Center).
Other battles between two allogeneic immune systems
Fetal–maternal interactions
Newborns with serious, life-threatening hemolytic were treated with exchange transfusions of a single, healthy, Rhesus-negative blood donor. The babies died from TaGvHD [
]. In a study not likely to be repeated or approved by current internal review boards, a “rhesus” baby having survived an exchange blood transfusion tolerated a skin graft from the blood donor much longer than a skin graft from another unrelated donor [
]. The slower rejection of the donor skin is best explained by the immunosuppression induced by the immunosuppression generated by the blood donor and baby lymphocytes are inactivating each other.
A transplantation surgeon in Scotland, who shall remain anonymous, advised his male residents to donate a pint of blood to their newborn babies, so they could become organ donors if their child ever needed an allograft later in life (Thomas Starzl, personal communication, 2002). One can only hope the Scottish residents did not heed this irresponsible advice from their department head. Currently, all exchange transfusions for rhesus antagonism are radiated with 15–25 Gy prior to administration to prevent TaGvHD. Milder cases of rhesus antagonism do not need to be transfused and will respond to phototherapy or require no treatment. Sensitization of a rhesus-negative mother by a rhesus-positive offspring can be prevented by the intramuscular administration of anti-rhesusD IgG within 72 hours of delivery [
The lymphocytes of a healthy blood donor will attack the histocompatibility antigens of the patient receiving the transfusion. An Internet search for information on TaGvHD invariably produces the following definition: “TaGvHD is a rare complication that develops 4 to 30 days after a blood transfusion (incidence: 0.1% of all transfusions),” a serious underestimate. “Recognition is often delayed because nonspecific symptoms are attributed to the patient's underlying diagnosis. Unlike GvHD associated with bone marrow transplantation, TaGvHD destroys the recipient's bone marrow. Therefore, TaGVHD is almost always fatal.” (Shimoda reported in 1955 that only 6 of his 12 POE = TaGvHD patients died [
].) “After allogeneic BMT the bone marrow cells of the recipient are of donor origin and are therefore exempted from this attack” (UpToDate, December 24, 2014, and most national blood bank websites, e.g., national blood banks in the United Kingdom, New Zealand, Australia, The Netherlands; accessed in 2016).
The U.S. Food and Drug Administration approved the use of blood irradiation for the prevention of lethal, acute TaGvHD as early as 1990 [
]. For years, the Dutch National Blood bank filtered all cellular blood products, not realizing until many years later that filtration removes granulocytes, platelets, and macrophages but not lymphocytes. Filtration does not prevent TaGvHD, as is now acknowledged at www.sanguin.nl.
The decision to limit the diagnosis of TaGvHD/POE to those patients who die from GvHD within 30 days of transfusion is surprising. Why would delayed and chronic GvHD occur only after allogeneic BMT and not after HLA-mismatched blood transfusions? In 1995, two different reports indicated TaGvHD in human patients can be chronic and is not always lethal [
Morbidity and mortality in patients receiving more than one blood transfusion
In 1965, English heart surgeons noticed early postoperative jaundice in 13% of their patients. Eighteen of 31 patients with jaundice died. The incidence and severity of the jaundice decreased when the surgeons used fewer blood transfusions. The biochemistry and scant histologic analysis of autopsies were compatible with GvHD-induced liver lesions [
A group of orthopedic surgeons randomly assigned patients requiring an artificial hip to receive their own blood back during surgery or to receive nonirradiated donor blood. The incidence of postoperative infection was low after autologous blood transfusion (1/34) and significantly higher in the patients receiving allogeneic blood (16/50) [
]. It was concluded that delayed or chronic TaGvHD might have caused immunosuppression and thereby increased postoperative infections in the patients receiving nonirradiated donor blood.
Table 2 lists studies of patients receiving more than one blood donor transfusion [
Blood transfusions in colorectal surgery: Incidence, outcomes and predictive factors: An American College of Surgeons National Surgical Improvement Program Analysis.
]. Multiple blood transfusions cause mortality and morbidity (M&M). M&M are positively correlated to the number of transfusions received. In multivariate analyses, blood transfusions are not correlated to other factors causing M&M such as comorbidities of the patient, lifestyle issues (smoking, obesity, diabetes), and age. Blood transfusions are also correlated to M&M occurring later than 30 days after transfusions. The most frequently heard, but inaccurate, explanation for the delayed transfusion effect has been that poor patients do poorly; they do require blood transfusions but would have died or remained in poor condition regardless of whether they received blood transfusions.
Table 2Delayed morbidity and mortality after multiple blood transfusions
Blood transfusions in colorectal surgery: Incidence, outcomes and predictive factors: An American College of Surgeons National Surgical Improvement Program Analysis.
Orthopedic surgeons decided to start a campaign against perioperative blood transfusions: the battle against blood transfusions. Autologous blood, blood aspirated from the surgical bed and filtered before re-infusion, or the administration of erythropoietin did not improve the fate of the nontransfused orthopedic patients. Patients who received a new prosthesis without incurring serious M&M did better because they were healthy enough to undergo a hip or knee replacement without a blood transfusion. However, the patients considered too anemic to receive a new prosthesis might have become proper surgical candidates with irradiated blood transfusions administered before and/or during surgery.
Irradiation of blood bags
To improve the logistics of irradiating (25 Gy) multiple transfusion bags every day, 25 Gy can be delivered to transfusion bags of O-positive blood donors with a 6-MV linear accelerator. Ten blood bags can be irradiated in 15 to 30 min, delivering a more homogeneous dose than the off-site, radiation safety-intensive, cesium chloride blood irradiators.
Radiation kills cells by inflicting double-strand DNA breaks [
]. Another potential benefit of almost universal blood bag irradiation might be the inactivation of single- or double-stranded DNA and RNA viruses carried in the blood, lymphocytes, and macrophages of the blood donor.
Conclusions
The only difference between BMT GvHD and TaGvHD is that the latter produces BM aplasia. Both types of GvHD can occur as an acutely/lethal, delayed/less lethal, and chronic/nonlethal illness. All forms of GvHD induce immune suppression.
In The Netherlands, where every year between 0.5 and 1.0 million cellular blood products are administered, radiating packed red cell transfusions and platelet concentrates with 25 Gy will eliminate mortality and morbidity caused by lethal as well as delayed TaGvHD and decrease the incidence of chronic GvHD. This review supports the almost universal radiation of cellular blood products with 25 Gy in adults who receive more than one blood transfusion on a given day or a single five-donor platelet concentrate. All single blood transfusions or platelet concentrates for children 10 years of age or younger should be irradiated with 15–25 Gy before administration. The indications for granulocyte transfusions collected from normal donors are unclear, and this procedure is most likely ill-advised for donors and recipients. The use of granulocytes from patients with benign-phase chronic myelogenous leukemia (CML) was stopped after recipients developed CML (personal communication of R. Graw and E. Freireich, 1975 and 1995).
The mortality and morbidity of GvHD after BMT can be diminished by applying the reciprocal interference concept: The donor lymphocyte dose in HLA-identical BMT should not exceed 5 × 107 lymphocytes/kg of body weight of the recipient. This will lower the severity and incidence of GvHD. The reciprocal increase in HvGD can be prevented by a higher TBI dose, fractionated in 3 × 4-Gy or 4 × 3-Gy fractions, one fraction per day, and one fraction should include five half-value layer lung blocks, to prevent radiation-induced pneumonitis. Obviously lung blocks would not be necessary if, instead of TBI, fractionated total lymphoid irradiation is delivered [
T lymphocytes can be removed from BM cell suspensions by density gradients or ex vivo incubations with monoclonal anti–T lymphocyte antibodies and complement [
Elimination of graft versus host disease by the in-vitro depletion of alloreactive lymphocytes with a monoclonal rat anti-human lymphocyte antibody (Campath-1).
The selective elimination of immunologically competent cells from bone marrow and lymphatic cell mixtures: II. Mouse spleen cell fractionation on a discontinuous albumin gradient.
]. Such nonirradiated blood transfusions will induce a chronic low degree of immune suppression before the recipient receives a kidney allograft. Postoperation, the patients should receive regular immune suppression and irradiated blood transfusions as needed.
Mixed chimeras are immunologically handicapped individuals. They are not appropriate candidates for studying allograft survival with little to no chemical immunosuppressive therapy based on the concept of acquired immunotolerance.
Acknowledgments
Dick van Bekkum never relinquished his role as the director and mentor of his team members, including the authors of this article. As we completed the manuscript for articles, his input and guidance provided an excellent excuse for wild laughter, deep despair, impossible deadlines, and new insights, and these are once more gratefully acknowledged. The Radiobiological Institute of TNO, in Rijswijk, The Netherlands, no longer exists. This acknowledgment is almost identical to the acknowledgment written by HMV for Reference [
] in 2003. The repeat acknowledgment does reflect a certain degree of denial. Dick passed away July 2015 at almost 90 years of age. Ada van Bekkum-Kylstra died in April 2016. Any merit this article might have is due to their inspiring personalities.
References
Dicke K.A.
van Noord M.J.
van Bekkum D.W.
Attempts at morphological identification of the hemopoietic stem cell in rodents and primates.
Vriesendorp HM. Interviews with Dick and Ada van Bekkum in Emmalaan 4, Rotterdam, The Netherlands, for a lecture titled “Dick van Bekkum, Past, Present and Future,” presented at the International Panorama Stem Cell Conference in the World Trade Center, Rotterdam, The Netherlands. [Dick van Bekkum is interviewed in his office seated between his percussion set and his computer. Ada van Bekkum is answering questions at the living room dining table while serving tea and cookies.]
The relative role of the DLA complex in bone marrow transplantation.
in: Zaleski M.B. Abeyounis C.J. Kano K. Immunobiology of the Major Histocompatibility Complex. 7th International Convention on Immunology. Karger,
Basel1981: 214-223
in: van Bekkum D.W. Lowenberg B. Bone Marrow Transplantation in Animals and Man: Biological Mechanisms and Clinical Practice. Marcel Dekker,
New York, NY1985: 383-407
Influence of gastrointestinal decontamination on graft-versus-host disease in rhesus monkeys after (partially) mismatched allogeneic bone marrow transplantation.
Prevention of infection and graft-versus-host disease by suppression of intestinal microflora in children treated with allogeneic bone marrow transplantation.
Blood transfusions in colorectal surgery: Incidence, outcomes and predictive factors: An American College of Surgeons National Surgical Improvement Program Analysis.
Elimination of graft versus host disease by the in-vitro depletion of alloreactive lymphocytes with a monoclonal rat anti-human lymphocyte antibody (Campath-1).
The selective elimination of immunologically competent cells from bone marrow and lymphatic cell mixtures: II. Mouse spleen cell fractionation on a discontinuous albumin gradient.
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