BAND30-HEFT1 JANUAR 1975

Blut VEREINIGT ZEITSCH

MIT FOLIA HAEMATOLOGICA, NEUE RIFT FOR DIE GESAMTE BLUTFORSCH

FOLGE UNG

Organ der Deutschen Gesellschaft f6r H~matologie Organ der Deutschen Gesellschaft for Bluttransfusion und Imrnunh~matologie

ZUSAMMEN

FASSENDER

BERICHT

Abteilung Immunologie, Institut ftir H~matologie der Gesellschaft ftir Strahlen- und Umwehforschung mbH, Mtinchen

Experimental Bone Marrow Transplantation Stefan Thierfelder Attempts to transfer bone marrow met with little success before the 1940's. It had still to be realized, that bone marrow differentiated from transplantable stem ceils, which engraft only in recipients needing stem cells. Also, the importance of histocompatibility between donor and recipient was not recognized. One thing that was puzzling, due to its simplicity rather than its complexity, was the method of administering bone marrow: Earlier applications of bone marrow per os, by intramedullary or intraperitoneal injections were finally replaced by the intravenous route (for historical review see [1], [10]). In the 1950's, when the fear of nuclear warfare had mobilized biologists in the United States to study systematically the effects of whole body irradiation, Loren z et al. [2] reported in 1951 that lethally irradiated mice could be saved by the intravenous injection of bone marrow from other mice, and not only by shielding the spleen of the irradiated mice as Jacobsen et al. [5] had demonstrated shortly before. Experimental data by Barnes et al. from Harwell in England [4] supported the "cellular hypothesis" arising from Loren z' observation of a cell-born repopulation of the bone marrow cavity. In contrast the "humoral hypothesis" postulated factors in cell-free homogenates of spleen or bone-inducing hemopoietic regeneration after irradiation [3], [5]. The final confirmation of successful bone marrow transplantation was the proof in 1956 of a persistence in the marrow recipient of mouse [6] or rat [7,8] donor cells, which could be distinguished by cytogenetic, histochemical or serological techniques. When highly inbred strains of mice became available it was recognized that bone marrow could be easily grafted only within the same strain. Experiments with irradiated mice which had been given marrow from foreign, histoincompatible strains [9,11,13] remained less successful. While the laws of transplantation were explored on the basis of skin- or tumor graft rejection by the host, researchers attempting bone marrow transplantations realized that they were dealing additionally with the reverse situation: an immunocompetent graft "rejecting" its host. Eingegangen am 20.9. 1974.

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S. Thierfelder

This gra•versus-host reaction [! 1,9,12,14], common to homologous and secondary disease, was usually fatal. It damaged the epithelia of gut and skin, the liver, and also affected hemopoiesis and immune defense. It was observed after transplantation of incompatible marrow in mice (for review see [ 1]) and confirmed in rabbits [ 133 ], rats [132], monkeys [16], dogs [17,73], chicken embryos [15], and man [18]. Bone marrow transplantation, having overcome the "primary" disease characterized by radiation-induced aplasia, was thus complicated by the immune reactions of secondary disease (sec. dis.). Other problems encountered in bone marrow transplantation were by no means negligible, such as the preoperative conditioning and the postoperative care of the patient; however sec. dis. as the "stumbling block" (Matbd) discouraged as well as challenged many investigators during the past decade. The high mortality in the first series of marrow grafted patients [19] made it clear that progress in clinical bone marrow transplantation depended on further investigation with experimental models, in particular with a view to the handling of the immunological complications. Various approaches in many animals models have contributed to the modification of this dangerous immune disease. Some advantages of experimenting with mice became drawbacks later on: It was observed that unrelated bone marrow of man, monkeys and dogs induced a secondary disease fatal within 2 to 3 weeks, while murine bone marrow did not kill histoincompatible recipients before day 20 post transplantation. This raised the critical question as to whether the mouse could at all be used as a model for human bone marrow transplantation. However, murine spleen cells injected into irradiated H~incompatible recipients caused an acute mortality comparable to that observed after transplantation of marrow cells in man. The discovery of the thymus dependent (T)-cell, its causal role in sec.dis., its occurrence in the spleen and its scarcity in the murine bone marrow [121,134,122,123,20], finally explained why, in mice, spleen rather than bone marrow produced a sec.dis, similar to that in man. When spleen marrow or bone marrow mixed with lymph node cells of homozygous parental donors is transferred to incompatible heterozygous, semiallogeneic F1 hybrids [21], acute mortality from sec. dis. can be suppressed quite easily. This was another reason why the mouse model appeared inadequate for studying the suppression of sec. dis. as it occurs in larger animals. One should bear in mind, however, that individuals of outbred species are usually heterozygous at the loci of the polymorphic histocompatibility systems. It was probably the choice of this very special immunogenetic combination of inbred strains with semiallogeneic recipients lacking host-versus-parental-graft reactivity, which gave the impression that murine sec.dis, is deceptively easy to suppress. Sec.dis. in other strongly histoincompatible donor-recipient pairs remained as difficult to overcome as in larger animals (see below). The immunogenetic approach to secondary disease A major advance in circumventing sec.dis, was achieved by the selection of histocompatible donors. In 1968 Epstein et al. [124] demonstrated in dogs that bone marrow could be grafted successfully from littermates matched to the recipient in the reaction pattern of allogeneic, lymphocytotoxic antisera. The clinical of in vitro

Experimental bone marrow transplantation

3

tests of histocompatibility could be evaluated only in an outbred species such as the dog. Evidence of one multiallelic locus coding for strong histocompatibility antigens had been obtained in mice [28] where grafts between strains with the same strong histocompatibility antigens caused only weak immune reactions [27]. Human geneticists made great efforts in defining the human locus for strong histocompatibility antigens, viz. the HL-A system (Human Leukocyte antigen A) [22]. They pointed out that-according to the Mendelian laws-1 out of 4 siblings differing at the HL-A locus should be HL-A identical to a given sibling if the strong histocompatibility antigens belonged to a single locus [29]. The ratio of 1 in 4 was experimentally confirmed in humans when immunocompetent ceils of the donor and the recipient were put together in the mixed lymphocyte culture (MLC): The predicted proportion of siblings did not stimulate each other in the MLC [30]. Chances of finding unrelated, HL-A identical donors are considerably lower, since the polymorphism of the HL-A system includes over 15 400 genotypes [41 ]. Furthermore, recombinations within the major histocompatibility complex revealed several linked loci of potential importance for sec.dis. In selecting the donor one has therefore not only to consider the HL-A but also the MLC system for which polymorphism has been demonstrated in man [42] and dogs [143]. A further locus Ir (Immune response) was reported recently to affect graft-versus-host reactivity in mice [32]. These additional polymorphismus minimize the chances of finding unrelated compatible donors. There are now data available in patients correlating sec.dis, with in vitro histocompatibility tests: The majority of patients grafted with bone marrow from HL-A identical MLC non-stimulatory sibling donors do well with appropriate postgrafting therapy, while most of the patients grafted from histoincompatible donors have died [146]. However, even in the group of patients receiving bone marrow from HL-A identical siblings about 20% still died from untreatable severe sec.dis. [88]. This mortality was attributed to minor histocompatibility antigens undetected by currently employed in vitro techniques. It is often lower in dogs than in man, which may be explained by a comparatively lower degree of random mating. The existence of weak histocompatibility antigens causing all levels of graft-versus-host reactions was demonstrated in inbred mice by in vivo procedures [31]. Manipulating cells responsible for secondary disease The mortality of patients and dogs encountered in sec.dis, despite the selection of histocompatible sibling donors, indicates that attempts to modify sec.dis, are still required. These attempts can be classified according to the 3 phases of bone marrow transplantation: pre-, intra- and postoperative. 1-. Before transplantation Advances in basic cellular immunology have delineated the causal role of the thymusdependent (T)-cell in graft-versus-host reactions [121,122,134,135]. The number of T-cells may be reduced by treating the marrow donor in various ways. T-cells are radiosensitive. Attempts to affect sec.dis, by irradiation [33-35] of the donor have not gained general interest because of the radiation sensitivity of hemopoietic stem

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S. Thierfelder

cells. Reduced graft-versus-host reactions were reported with donor cells from spleens undergoing erythromyeloid metaplasia after phenylhydrazine or decreased oxygen tension [37]. Xenoantigens such as foreign serum proteins or sheep red cells also reduced sec.dis, probably by competing with the recipient antigens for the immune reactivity of the marrow of the donor [40,46]. The injection of antigens of the recipient strain into the donor partially suppressed graft-versus-host reactions as a consequence of low zone tolerance [60] or active enhancement [43,44,119]. Antithymocyte or antilymphocyte serum (ALS) affects T-cells [45]. When given to the marrow donor, ALS delayed sec.dis, in unrelated strongly H o-incompatible mice for about 2 weeks [47,48] and had no effect on sec.dis, in dogs [50] and monkeys [49]. Almost complete suppression of graft-versus-host reactions was observed only in Fl-recipients of spleen cells from parental donor mice treated with ALS [36]. It has not been determined whether the good results in this particular mouse model are due to the inhibition of T-cells, the erythromyeloid metaplasia of ALS-treated spleens with only a relative reduction of lymphocytes [46], the lack of host-versusgraft reactivity, the single semiallogeneic dose of incompatibility, or a combination of these and other factors. A delay of sec.dis, in dogs [141], monkeys [49,77] and man [100] was reported when ALS was administered to the recipient rather than to the donor shortly before grafting. Whether this was due to a decrease of host leukocytes as the targets of graft-versus-host disease or caused by residual ALS suppressing donor T-cells is not clear. Higher doses of ALS in this type of treatment, of course, always bear the risk of interfering with the engraftment [141] because of stem cell toxic antibodies (see below). Since the murine thymus starts producing T-cells around birth, fetal marrow was used to avoid sec.dis. A significant number of long-term survivals were obtained with H2-incompatible unrelated recipients of marrow containing fetal liver [52,167].

2. During transplantation There is of course little clinical relevance in treating healthy marrow donors with agents causing dangerous side effects. The collection of large numbers of fetal marrow cells poses logistic problems. Elimination of T-cells without harmfull effects to the donor and the host may best be achieved by treating the marrow inoculum. Even a simple incubation of marrow at 37 ~ C or 4 ~ C appeared to affect the graft-versus-host reactive cells more than the cells restoring hemopoiesis [21,25]. Dicke et al. [39] tried to separate T-cells from stem cells by physical means. They obtained stem cells from fractions of albumin gradients showing enriched colony formation (stem cell activity) and low stimulation to mitogens (T-cell activity). They reported suppression of sec.dis, in the murine parent-to-F~ model and a delay of :several weeks in a series of monkeys [53]. Research on graft-versus-host reactivity of fractionated mouse or dog marrow from unrelated donors has not been reported. An immunological inhibition of T-cells in the marrow suspension was first tried with ALS but failed because of stem cell toxicity of ALS [54,168] which prevents hemopoietic engraftment. Rodt et al. [57,58] succeeded in purifying rabbit-antimouse-brain sera [ 144] by eliminating antibodies which crossreacted with stem cells. The residual antibodies reacted only with the theta-antigen present on both brain

Experimental bonemarrow transplan*ation

5

and T-cells [59]. When admixed to the donor cell inoculum, they suppressed sec.dis. completely in donor-recipient combinations incompatible at the H3 locus and in the murine parent-to-F1 combination [57,58]. In the latter model Trentin andJudd [56] reported similar success with anti-thymocyte globulin absorbed with spleen cells. Also, spleen cell absorbed anti-thymocyte sera did not diminish colony-forming stem cells while suppressing immunological splenomegaly in unirradiated, H2different F1 hybrids [155]. Reduced graft-versus-host reactivity was also observed following an incubation with rabbit antisera against rat lymphocytes absorbed with erythrocytes, peritoneal exudate cells and fetal liver cells [65], or against chicken thymocytes absorbed with bursal cells [67]. Golub [145] reported on brain-associated theta-antigen bearing stem cells, which would, of course, prevent engraftment of marrow preincubated with anti-theta antibodies. These observations are as yet difficult to reconcile with the above data. Fab fragments of an unabsorbed horse-anti-mouse lymphocyte globulin were reported to spare stem cells and to decrease mortality from sec.dis, during the first 15 days post transplantation [55]. No such effect on graft-versus2host reactions was observed using Fab or F(ab)~ fragments of rabbit antisera against lymphocytes, brain or IgG of mice [ 136] or thymocytes of chicken [ 137]. Enhancing or blocking antisera directed against the recipient's antigens and given together with the donor cells were also effective, though less so than the above mentioned anti-T-cell globulin preparations [43,44,119]. The same applies to the incubation of the mitogen Concanavalin B [63], thymic extracts (chalones?) [62], host red cells [63] or host-anti-donor recognition structure serum [66] with donor marrow. Whether the retention of donor T-cells on host fibroblasts is a practical way to reduce sec.dis, has still to be determined [64]. Another open question is whether the selective killing of replicating T-cells by tritiated thymidine ("thymidine suicide") can be used against sec.dis. [68].

3. After transplantation Attempts at modifying sec.dis, after transplantation have been made with antiproliferative drugs, ALS and antiinfectious measures. Antiproliferative drugs may exhibit a selective action on T-cells during the burst of killer cells which occurs when donor T-cells are stimulated by the incompatible antigens of the recipient. Timing and dosage in this type of treatment, which has to spare hemopoietic ceils and suppress immunocompetent lymphocytes responsible for graft-versus-host reactivity, is of great importance [69,138]. Amethopterin (Methotrexate) and Cyclophosphamide are two drugs most widely tested in sec.dis. Amethopterin delayed sec.dis, in unrelated dogs [71,72] and completely suppressed sec. dis. in DL-A compatible dog littermates [73] and in H,incompatible Fl-mice which had been grafted with parental bone marrow instead of the more aggressive spleen cells [69]. Cyclophosphamide considerably reduced the mortality of sec.dis, following transplantation of strongly incompatible mouse or rat marrow [23,74,75]. It prolonged survival in monkeys [23] but not in dogs [71]. Combinations of immunosuppressive drugs including Amethopterin, 6-Mercaptopurine, Cyclophosphamide and Prednisone delayed the onset of sec.dis, in dogs [139], but hemopoietic toxicity with decreased defense against infections limited their use.

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Treating the marrow recipients with ALS delayed sec.dis, in mice [140], dogs [49] and monkeys [49,77]. Administration of ALS soon after transplantation complicated the postgrafting course by serious infections in monkeys [49] and jeopardized hemopoietic engraftment in dogs [142]. ALS was used with greater success in dogs after the graft had been established and sec.dis, had become clinically apparent [76]. Judging from the experience in man [141], an even better effect of ALS on sec.dis, should be expected in recipients of marrow compatible at the strong histocompatibility locus. However, complicating virus infections still remain a serious problem in these patients. An immunodeficient state is part of chronic sec.dis, in mice [78] in short-term canine chimaeras [149] and man [156]. Mortality in chronic sec.dis, could therefore be reduced with antibiotics [ 147] and gnotobiotic measures [ 116,148], whereas acute sec.dis, was not clearly influenced by anti-infectious treatment. A key question concerning sec.dis, and immune defense in chimaeras has still to be answered experimentally, namely whether transplanted donor stem cells produce T-cells that gain immunocompetence without attacking the host. Rodt et al. [ 109] have recently shown in the murine parent-to-F1 hybrid system that T-cells in fact develop from donor precursor cells and regain immunocompetence under the influence of the recipient's thymus even if the latter differs at the H2-1ocus. On the other hand, blocking antibodies or antigen-antibody complexes, produced by the grafted antibody forming cells against the recipient's antigens and protecting them against T-cells of the donor, would reconcile the existence of intolerant donor T-cells with suppressed sec.dis. Such antibodies have been reported in long-term mouse and dog chimaeras [79,80]. How regularly they occur and whether they really mediate chimaeric tolerance has still to be determined. Conditioning of the recipient Engraftment of incompatible bone marrow became possible after suppression of host-versus-graft reactivity by total-body irradiation. Larger animals needed supportive care with platelet transfusions and antibiotics during the aplastic phase following irradiation. In rodents [81], dogs [72] and monkeys [150] between 750-1000 rads of x- or y-rays were needed for foreign marrow to take. Fast neutron irradiation conditions poorly because of its gastrointestinal toxicity [125]. An increase of exposure rates to the range of 40 R/rain [82] or a combination of ALS with total or partial body irradiation [83,107] enhanced marrow engraftment in mice. Higher doses of radiation increase the risk of irreversible injury of the gut, but lower than optimal doses have also often been found to cause mortality in several strains of mice, a "midlethal dose effect" attributed to an acute rejection of the graft [84] and subsequent death from marrow aplasia. The introduction of cyclophosphamide by Santos et al. [24,26], as a conditioning alternative to irradiation in mice and rats had important clinical implications. Less well supported in dogs [85] and better in monkeys [86,87], high doses of cyclophosphamide became the conditioning treatment of choice in aplastic patients [88] in spite of occasional cardiotoxicity accompanied by fluid retention. An advantage, at least concerning non-leukemic patients, lies in a lower stem cell toxocity of cyco-

Experimental bone marrow lransplantation

7

phosphamide as compared to irradiation. In contrast to cyclophosphamide, conditioning irradiation accompanied by a generalized stem cell death depends entirely on successful marrow engraftment and is therefore particularly dangerous. Radiation also affects the growth centers of bones in young patients [151]. Another advantage of cyclophosphamide is obvious: It does not depend on radiation facilities which are very expensive for larger animals. Candidates for bone marrow transplantation are frequently presensitized by multiple transfusions. Presensitization was systematically studied by Storb et al. [89,106] in dogs. An important finding was that even a single transfusion of DL-A matched donor blood can prevent engraftment while several transfusions of DL-A incompatible donors did not regularly interfere with the engraftment of DL-A identical marrow. Blood transfusions from the prospective marrow donor or his relatives can therefore jeopardize transplantation even though the donor is compatible at the major histoc0mpatibility locus; this is further evidence of the importance of minor histocompatibility antigens in bone marrow transplantation. Total body irradiation alone cannot abrogate this presensitization, but ATS combined with procarbazine [90] was found to prolong skin graft survival in mice sensitized with lymphocytes of the donor [105] and recently also in dogs presensitized by blood transfusions against marrow engraftment [90]. Using the murine parent-to-F1 model, Cudkowicz and Bennett [92] defined a resistance of recipients against marrow of certain donor strains. The significance of this ALS-sensitive, but radiation-insensitive 'hybrid resistance' [115], 'poor growth phenomenon' [91] and 'CFU repression' [93], which-though genetically determined-does not obey the classical laws of immunology, is not clear. It interferes with engraftment in certain recipient strains, but can often be overridden by increasing the cellular inoculum. Growth resistance, immunologic and non-immunologic, plays an important role in bone marrow transplantation between different species [95]. Xenogeneic bone marrow grafts were reported in the rat-to-mouse [114] and hamster-to-mouse [94] system and recently, after combined conditioning treatment with ALS and irradiation, following transfer of mouse marrow to rats [83]. About ten times as many donor marrow cells are needed to override the growth resistance in a mouse receiving rat marrow [1]. No successful hemopoietic xenografts have so far been reported in larger mammals.

Conditioning of the donor The collection of marrow with little risk for the healthy donor has been technically solved [154]. That circulating blood also contains stem cells which can be used to repopulate marrow cavities, was discovered in mice [112] and confirmed in dogs [II0,111] and baboons [170]. Stem cell activity and graft-versus-host reactivity of a given cell suspension can be determined by cell colony forming assays and labelling techniques which measure the concentration of T-cells [152]. These tests can now help select the better site for collecting marrow in the donor. They would, for instance, favor bone marrow

Thymus

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Bone Marrow

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tissue typing ALS, ATS recipient antigens (active enhancement, low zone tolerance) fetal marrow xeno antigens (immuno competition) hyperplasia (Phenylhydrazine, low oxygen tension)

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marro w suspe#sfoR

gradient centrifugation anti-T-cell globulin Fab fragments of ALS blocking antibodies (passive enhancement) Concanavalin B thymic chalones? host red cells (maternal modification) anti-recognition site serum Thymidine suicide T-cell retention (on fibroblast layers)

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Methotrexate Cyclophosphamide ALS gnotobiotic conditions

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stem cells

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Fig. 1: Approaches to suppress graft-versus-host reactions.

Experimental bone marrow transplantation

9

transplantation rather than stem cell transplantation from peripheral blood with its low number of stem cells and its high T-cell concentration. Conditioning marrow donors with ALS or other means of immune manipulation has been mentioned above.

Bone marrow transplantation and microenvironment Animal models for human aplastic anemia are difficult to obtain, since its etiology is unknown for the most part. However, congenital anemia has been found in mice. Mice carrying the WW v genotype suffer from macrocytic anemia and can be cured by transplantation of healthy bone marrow [153,167]. Mice with the genotype S1S1d also possess a reduced number of hemopoietic stem cells, but cannot be cured by transplantation of bone marrow [101]. When marrow of S1S1d mice was transferred into irradiated, previously healthy mice, normal functioning hemopoiesis was restored. These findings indicate that there is a stem cell defect in the former strain of mice, whereas in the latter strain environmental conditions are defective. Another microenvironmental defect, which prevents the homing of transfused but not the proliferation of endogenous stem cells, has been described in NZC mice [102]. Bone marrow transplantation is thus a valuable tool in differentiating stem cell defects from environmental defects. Cyclic neutropenia, for instance, was cured by marrow of healthy dogs, indicating an inherent stem cell defect which could be transplanted into healthy littermates [ 104,130]. In contrast, canine hemophilia could not be reversed by transplantation of nonhemophilic marrow which indicates that this tissue does not take part in factor VIII production [103]. High partial-body irradiation caused a localized aplasia 2 to 3 months later. This late aplasia was attributed to a defect of the microenvironment and permanent damage of sinusoidal cells [ 120] which prevents normal hemopoietic stem cells from taking and multiplying. Experimental bone marrow transplantation also revealed osteogenic precursor cells present in bone marrow inocula [126,127]. Successful clinical bone marrow transplantation is the best indication of a stem cell defect in human aplastic anemia [88]. It does not, of course, exclude the existence of aplasia caused by microenvironmental changes which either can be reversed by the conditioning treatment of the marrow recipient or which still disappear in the group of unexplained engraftment failures.

Chimerism and transplantation tolerance An important approach to transplantation problems was Billingham, Brent and Medawar's [96] definition of immunological tolerance as a weakening or suppression of allograft responsiveness through exposure of the neonatal host to donor antigens before the development of immunological competence. Ever since a young housewife laboratory assistant [97] discovered in 1955 that permanent immune tolerance of the donor's tissue occurred also in adult mice preirradiated and repopulated with donor :narrow, bone marrow transplantation has intrigued researchers of organ transplantation. Correspondingly, dog chimaeras tolerate skin and kidney of the marrow donor [128].

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Under this aspect xenogeneic marrow transplantation may be justified by the promise that it mediates tolerance of unpair organs for which no healthy human donor can be found. Surprisingly, mortality from sec;dis, is not higher in rat-tomouse chimaeras than in strongly incompatible allogeneic donor-recipient pairs. Mice surviving sec.dis, after transplantation of rat marrow have tolerated rat tissue

[981. Exceptions to the complete tolerance of donor tissue were found in chimaeras rejecting skin grafts of certain donor strains, which was explained by the loss of the grafted marrow's tolerance of skin antigens present in the donor strain but lacking in the recipient [117]. Split chimaerism and little sec.dis, were reported in rabbits conditioned with ALS [99]. In this model transient takes of hemopoietic cells and prolonged survival of B-cells were observed, while T-cells remained of the recipient type. These data are of particular interest in the light of split chimaerism observed in humans [100]. Bone marrow transplantation and leukemia

Barnes et al. [ 118] demonstrated that spleens of leukemic mice dying of severe sec.dis. were often free of leukemic cells. Cells causing graft-versus-host reactions bad not only damaged the host's hemopoiesis but had also killed its leukemia. Boranic studied the time pattern of the antileukemic effect in sec.dis. [131] and tried to use transient graft-versus-host reactions for the treatment of leukemia [ 13 I, 157]. Further studies evaluated cell dose and antigenic disparity requirements in the destruction of leukemic or non-malignant hemopoietic tissue by graft-versus-host reactions [159,158]. Many murine leukemias are preserved by passage in the strain of origin. They quickly become very malignant, killing their host within 2 weeks after inoculation. Since sec.dis, is no less vigorous, the problem of timing treatment against leukemia and sec.dis, is very difficult. Eradication of spontaneous leukemias in AKR mice irradiated and grafted with allogeneic marrow has been reported [ 171]. Chronic sec.dis, became subclinical in this system because the mice were kept under germ free conditions. Additional measures which increase the immune response against leukemias with proven antigenicity were investigated. Chemoimmunotherapy combining antileukemic drug therapy with the transfer of weakly incompatible spleen cells prolonged the survival in mice carrying a moloney lymphoma [164] when the donor cells were immunized against the antigenically related routine sarcoma virus. In man, successful treatment of leukemias with whole-body irradiation followed by allogeneic and even isogeneic marrow grafts have been reported [161,160]. An eradication of leukemia with anti-leukemic drugs and isogeneic bone marrow transplantation is almost impossible in mice carrying the highly malignant leukemias which result from continuous passage in the strain of origin. Better results with the syngeneic approach renouncing on the antileukemic effect of graft-versus-host reactions may be expected in early spontaneous leukemias of AKR mice in view of the above mentioned experience with identical twins [162]. So far, the absolute necessity of graft-versus-host reactions to eradicate leukemias has not been proved in patients treated with allogeneic, H2-identical bone marrow grafts.

Experimental bone marrow transplantation

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An unexpected complication in clinical bone marrow transplantation was the recurrence of leukemia in the grafted donor cells [ 162,163]. The interesting question of leukemic transformation of engrafted marrow cells from healthy persons may stimulate the experimental work in animal models. An activation of congeneic viruses after total body irradiation [172] in mice has been reported also during chronic graft-versus-host reactions [165], though-in the latter model-lymphomas were only found in graft-versus-host reactions due to strong histoincompatibility [166]. Conclusions Almost all the technical and biological facts of clinical bone marrow transplantation had to be worked out in animals. The many variables and the complicated interrelationship of histocompatibility, pre- and postoperative conditioning, suppression of graft-versus-host and host-versus-graft reactions, cooperation between donor stem cells and recipient thymus, microenvironment etc., could only be studied in animal models. The choice of the best animal model for a particular investigation was of great importance. The conditioning effect, for instance, of cyclophosphamide was discovered in rodents. The dog would not have been the best choice because cyclophosphamide is, as mentioned, less well tolerated in dogs than in rodents, monkeys and man. In contrast, the significance of selecting histocompatible sibling donors to avoid or decrease sec.dis, was discovered in random bred dogs. Inbred strains of mice were not suitable for these studies and siblings in the outbred monkey model were rare and expensive. The lack of randomly distributed histocompatibility antigens in mice makes it often difficult to decide how relevant experimental data from this model are. Though one may expect fundamental biological laws, formulated from experiments on mice, to hold true for other mammals, the competition for "positive" results often leads to "easy systems" in mice which are rare in outbred species or to high dosage schedules which are difficult to apply in larger animals. With these reservations the rodent model is appropriate for research on many unsettled questions in bone marrow transplantation. Manipulation of sec.dis., less harmful ways to condition marrow recipients, tests to predict presensitization and the severity of sec.dis, for a given donor-recipient pair and the eradication of malignant cell clones, will continue to preoccupy investigations in this field. The answer to the question whether T-cell deprived donor marrow can become tolerant towards the antigens of the recipient but immunocompetent against other antigens by way of the recipient's thymus, just as it was found in the parent-to-F1 model [I09], would have logistic consequences. There is little doubt that bone marrow transplantation between non-siblings deserves further experimentation, and it is to be hoped that it will profit from the remarkable evolution of cellular immunology.

Acknowledgement: I thank Drs H. J. Kolb, G. Santos and R. Storb for their advice and criticism.

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