Immune modulation by ionized irradiation

Immune

modulation

by ionized

Z. Tochner

irradiation

and S. Slavin*

Departments of Radiation Therapy and Clinical Oncology and ‘Bone Marrow Transplantation and Cancer lmmunobiologicy Research Laboratory, Hadassah University Hospital, Jerusalem, Israel Current

Opinion

in Immunology

Introduction In 1908, shortly after the discovery of Roentgen rays, it was observed that whole body irradiation (WBI), prior to antigen administration, suppressed antibody production. A few years later it was noted that administration of WBI prior to inoculation of aUogeneic tumors aIlowed prolonged tumor growth. Both Iindings were correlated with a rapid faII in the lymphocyte count post-WBI. Despite these early observations, it was not until the 1960s that the entire scope of the relationship between irradiation and the immune response was seriously investigated. Categorization of the effects of radiation on the Immune response according to the type of irradiation is generaIIy preferred. The laboratory and clinical effects of WI31 are different from those obsened following exposure to localized irradiation, and both are different from fractionated total Iyrnphoid irradiation (TLI). The main characteristics of aII three modes of radiation therapy wiII be described in this review. The tymphocytes - the cells that dominate the immune system - are among the most radiosensitive cells, depending on the subclasses of Iymphocytes invohred and obeying the general rule that the more differentiated the cell, the more radioresistant it is (Markoe and SaIuk, AppI Radio1 1977, 6:22; Stewart and Perez, Radiology 1976, 118:201). The lymphocytes have two types of radiation response when morphologic criteria am used to determine cell survival. About 80% of the lymphocytes are extremely radiosensitive and die a prompt interphase death, whereas other lymphocytes die a mitotic death. When assayed on the basis of reproductive capacity, and considering accepted radIobiologIcal methods for determining survival cutves and defining radiosensitivity, their radiation survivaI curves appear similar to those of other hematopoietic cells, with small differences between different subsets of Iymphocytes (Anderson and Warner, Adv Immunol 1976, 24:251). Radiation survivaI curves represent the reproductive capacity of cells in response to mitogens after radiation or by functional end points dependent on ceIIuIar proliferation. It can be best de&red by two major parameters: (1) the slope of the straight portion of the curve, expressed as Do, which represents

1988, 1:261-268

the radiation dose required for reducing the number of surviving cells to 37% of the originaI population. For the majority of lymphocytes, the Do is very low (about 7GlOO cGy) compared with other cell types, and (2) the extrapolation number (n) that measures the initial shoulder of the curve and primariIy represents the ability of the cells to repair sublethal damage. The n for radiation survival curves of lymphocytes ranges between 1 and 1.5, which reflects minimaI ability to repair sublethal radiation damage. Thymocytes, representing the pool of T cell precursor cells, are considered most radiosensitive, and the suppressor T cell precursors represent a uniqueIy mdiosensitive ceII subset. Bone-marrow stem cells, B cells and helper T cells are considered moderateIy radiosensitive, while the mature effector cells, such as plasma cells, macrophages and antigen-committed memory T-cells, are relativeIy radioresistant (Anderson and Warner, 1976; Anderson et al, J Immunoll977,118:1191; Anderson et al, Eur J Immunoll974, 4:199, Cadson and L&et, Radkat Res 1976, 65111) [1,2]. The extreme radiosensitivity of lymphocytes, plus the post-WI31 Iymphocytopenia which can be observed within hours, may serve as a unique clinical tool; during the Chernobyl nuclear power plan accident in 1986, the Russian scientists used the severity and rapidity of the drop of victims’ Iymphocyte counts as a ‘biological dosimeter’. Radiation-induced IymphocytopenIa proved to be a sensitive indicator which cone&d well with other symptoms of radiation sickness observed several days later. Due to the unique properties of radiation on Iyrnphocytes, the effector cells of aII Immune responses, radiation was used experimentaUy and cIinicaIIy for induction of immunosuppression [4] (Markoe and SaIuk, 1977; Stewart and Perez, 1976; Anderson and Warner 1976; Anderson et al, 1977;Anderson et al, 1974; Cadson and Lubet, 1976).

Whole body irradiation response

and the immune

Whole body irradiation is often used In the clinic as part of the preparation for bone-marrow tC.UlSplatlta-

Abbreviations

.

AU-acute lymphoblastic leukemia; AM-acute myeloid leukemia; BMT-bone-marrow transplantation; BSA-bovine serum albumin; ConA-concavalin A; CML- chronic myeloid leukemia; DTH-delayed-type hypersensitivity; GVHD-graft versus host disease; Il-interleukin; AU-mixed lymphocyte reaction; NHL-non-Hodgkin+3 lymphoma; PHA-phytohemagglutinin; Tll-total lymphoid irradiation; WBt-whole body irradiation.

@ Current

Science Ud ISSN 0952-7915

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tion (BMT). In reaiity, however, BMT represents the only commonly used indication for such an aggressive cytoreductive therapy. Due to limited ciinicai experience as to the elkcts of WBI on the normal immune response, most information is based on extensive investigations in experimental animals. As expected from the data presented above, the immunosuppressive effects of acceptable doses of WI31 are effective in preventing immune responses to primary antigens, but have a iimited capacity to modify the response to secondary responses (Taiiaferro et aL, Radiation and Immune Mechanisms. Academic Press, 1964). Given prior to antigen administration, WBI results in an increase in the time interval between immunization and detectable antibody responses through a slow increase of antibody titer, with a reduced peak. The immunosuppressive effects of WI31 are extremeIy potens even if radiation is administered several weeks before the immunization. Administration of radiation therapy foiiowing immunization with a primary antibody may result in enhanced antibody responses (Dixon and McConahey, J&p Med 1%3,117:833; Stratton et aL, J Clin Invest 1975, 56:88, Taliaferro and Taliaferro, A m J Patboll979, 97:456). An accepted assumption is that suppressor ceiis capable of regulating immune response may be more radiosensitive than other effector cells, and hence, radiation may eliminate the negative feedback of immunoreguiatory circuits, thus increasing antibody titers (Anderson and Warner, 1976; Anderson and Iefkovits, Am J Path01 1979,97:456). As a rule, secondary (anamnestic) antibody responses are more radioresistant than primary ones (Stoner et al, In The Effects of Ionizing Radiations on Immune Processes edited by Leone. Gordon and Breach, 1962, ~1983). This is in agreement with the general belief that differentiated cells are more radioresistant than less differentiated ones. Most studies on the effect of WBI on cellular immune responses have focused on the use of delayed-type hypersensitivity (DTH) as an indicator of cell-mediated immunity. Radiation-induced alterations of DTH involve damage to either T Iymphocytes or macrophage precursors. Whole body irradiation given before antigen administration suppresses the acquisition and expression of DTH (Lennox et aL, Br J I&#JPath011952, 33:380;Shin and Nishio, JExp Med 1972,135:985; Qhan, J Immunoll965, 9422; Uhr and SCM, J w Med 1960, 112:65; Visakorpi, Acta Path01 Microbial Scund 1972,80:788; Visakorpi and Kosunen, CeU Immuncl1972, 5:369; Voikman and Collins, J ImmunoZl968,101:846; Voikman and Coiiins, CeU Immunoll971, 2~552; Wara et al, A m J Roentgenol 1975,123:482). In comparison with the primary antibody responses, cell-mediated DTH immune functions are relatively resistant to irradiation. Thus, WBI is much less effective in prolonging the survivai of second-than first-set skin allografts, although temporary suppression of DTH reactions do occur in sensitized animals (Lennox et al, 1952; Viiorpi, 1972). The exact assessment of the effect of WBI on T cell mediated immune functions may be complicated by complex interactions between different Iymphocyte subsets, monocytes and tissue macrophages (VoIkman and CoIIins, 1971). Analysis of several studies

on the role of radiation therapy on cell-mediated immunity suggest that humorai responses, DTH and allogeneic interactions may be suppressed by irradiation due to direct effects on either the afferent or efferent loops of the immune response, though T lymphocytes, B lymphocytes or radiosensitive monocyte precursors play a role in antigen presentation and activation of cellular interactions by a cascade of cytokines and direct cell-to-cell interactions

[31. Radiation-induced augmentation of immune responses may also result in enhanced cell-mediated cytotoxicity against allogeneic tumors (Sabbadini, J Eq Med 1974, 140:470). Enhancement of tumorogenicity following WI31 might, in principle, also result from elimination of suppressor ceils, leading to removal of negative regulatory feedback and thereby enhancing anti-tumor effector mechanisms negatively affected by suppressor cells under normal circumstances (Sabbadini, 1974; Heiistrom ef al, J Exp Med 1978, 148:799). Under such experimentai conditions, the radiation-induced inhibition of tumor growth may be ablated by reconstitution of the mice with normal, radiosensitive T lymphocytes (Hellstrom et al, 1978). In fact, sub-lethal WI31 can be tumor-promotive as well as tumor-ablative, depending on the stage of development of the underlying anti-tumor immunity, irradiation may thus lead to regression of well-established tumors, whereas the same dose of irradiation may not et&uinate the tumor in animals treated by the same amount of tumor ceils treated shortly following tumor inoculation 161. Similarly T cell-deficient mice (i.e. following thymectomy and irradiation) may faii to show tumor regression after administration of otherwise curative doses of sublethal WBI. This suggests that anti-tumor effector cells or destruction of radiation-sensitive supressor cells during immunologic induction of anti-tumor reactivity may play a major role in determining radiation sensitivity of certain immunogenic tumors [6]. Antigen or mitogen-stimulated lymphocytes may also gain increased radioresistance compared with naive cells, a phenomenon observed at dose response ranges of 5-25 cGy (Ilbery et al, Br J Radio11971,44:834; Rickinson and Ilbery, Cell TissueKinet 1971, 4:549). The transition from resting to proliferating stage following exposure to mitogens is associated with activation of a mechanism for repair of a radiation-induced single strand DNA break in lymphocytes that have little or no ability to repair such damage (Drewinko and Humphrey, Int J Radiat Biol 1971, 20:169; Prempree and Merz, Mutut Res 1%9,7:441). The unique immunosuppressive and penetrating anti-tumor effects of WI31 place this mode of radiation herapy as an essential therapeutic modality and a crucial part of the conditioning in patients undergoing aIiogenic and even autologous BMT for a variety of maiignant hematological disorders and certain solid tumors (O’ReiiIy, Blood 1983, 62:941-346; Gorin. In Proceedings of the First International Symposium, University of Texas Press, 1985, pp 17-22) [7-141. In recent years, the effects of various dose rates and different fractionated and hyperfractionated regimen on the normal tissues were compared with the hematopoi-

Immune

etic bone-marrow compartment, suggestingthat protection of non-hematopoietic tissuesmay be accomplished with fractionation and low-dose rate without protecting hematopoietic stem cells. This principle is currently applied by most bone-marrow transplantationcenters [ 151. Although radiation therapy given as WBI is intended to be primarily against tumor and host immune cells, late complications due to damage to normal tissues cannot be prevented, including bone growth and development and late effects on the endocrine tissues(Shalet,Arc.5Dis cbildbood 1987, 62:461-464) [ 161. Radiation-inducedsecondarymalignanciesalso represent one of the recognizedrisks following the use of localized or extended WI31radiation therapy [ 191. localized

irradiation

and the immune response

Localizedradiation, commonly used in radiation therapy for tumors, affects the immune response by decreasing the number of circulating lymphocytes, presumably by destroying them as they pass through the irradiated volume. The consequencesof the irradiation are volume-related and can be easily measured as chronic lymphopenia, which affects both T and B cells (Alexander, Int J I&diol Oncol Biol Pbys 1976,1:369;Stjenswardet aL, Luncet 1972, i:1352). Interestingly, even very small amounts of localized irradiation may cause measurablesystemic effects. The animal data on the effects of localized irradiation is lim ited, therefore most of the data on the influence of localized irradiation on the immune responseswas obtained from patients with neoplastic diseases,which may also be associatedwith independent immune dysfunctions. In patients undergoing radiation therapy, the in vitro proliferative responsesof lymphocytes to T cell-dependent m itogens such a sphytohemagglutininare markedly depressed, regardlessof whether treatment is directed to the head and neck, chest, pelvis, or abdomen (Braeman, Luncet 1973, i&33; Slater et al, Am J iZoentgeno1 1976, 126:313). Maximal suppression occurs during, or shortly after, termination of treatment. Proliferative responsesto B cell-dependentm itogens such as pokeweed results in marked suppression of lymphocyte reactivity. This suggeststhat B lymphocyte function may be more radiosensitivethan T lymphocyte function in man as well as in experimental animals. The clinical signiiicance of the lymphocytopenia and the suppressed responsesof lymphocytes in response to m itogens is far from being fully understood. Interestingly,patients are capableof developing secondaryimmune reactions to recall antigens, even when they show marked suppression of primary immune responses.As a result of the latter (excluding those suffering from lymphoreticular or hematopoietic neoplasms),they are not susceptibleto the complications characteristicof immunocompromizedpatients,with the exception of Herpes zoster (Kaplan, Hodgkin’s Disease. Harvard University Press, 1972). There’have been reports (Stjemward, Luncet 1974, ii:1285-1286; Levitt and McHugh, Luncet 1975, iiJ2561259; Cancer Research

modulation

by ionized

irradiation

Tochner and Slavin

Campaign, Luncet 1980, ii:55-60) of an increased incidence of early mortality among breast carcinoma patients undergoing postoperative radiotherapy caused by immune suppression,but this does not appear to be the case in either the original or most subsequentstudies. lmmunosuppression and induction of tolerance to allo-antigens using total lymphoid irradiation In order to further explore the unique effects of ionizing irradiation on cells of the immune system without subjecting the non-lymphoid tissues to the harmful effects of radiation, a new model was introduced in 1975, the TLI. The use of total nodal irradiation was originally introduced for treating Hodgkin’s and nonHodgkin’s lymphomas primarily affecting the lymphatic tissues.It was to provide maximal tolerable doses of radiation therapy to the lymphatic tissueswhile shielding nonlymphoid organs (Kaplan, 1972). Interestingly, subsequent analysisof the immune status of patients apparently cured of Hodgkin’s disease- known to be associated with increasingimmunosuppression- revealeda substantialdegree of long-term immunosuppressionof T lymphocyte-mediatedimmune responsesmanifested by suppression of proliferative responses to phytohemagglutinin (PI-IA), con& A (ConA) and m ixed lymphocyte reaction (MLR; Slavin et al, J Ea$ Med 1977, 146:34-48X In order to investigate the immunosuppressiveeffects of TU, an animal model was constructed from irradiated (BALB/c) m ice exposed to daily ionizing radiation of 2OOcGywith an increasing number of fractions for a maximal tolerated dose of 34OOcGy(Slavin et al, 19n, Slavin et al, Science 1976, 193:1252-1254).The two major principles of the new TLI regimen designed for immunomodulation included exposure of the major lyrnphoid tissues(including peripheral lymph nodes, thymus and spleen) to irradiation while shielding non-lymphoid tissues and bone-marrow-containingbone for effective rescue from the irreversible effects of radiation therapy on the normal hematopoieticcells falling within the wide radiation field in m ice. In addition, radiation was administered in multiple daily fractions. The subsequentnumerous investigationsin m ice, rats, dogs, primates and, subsequentlyman, indicated that the procedure was relatively well tolerated while causing potent immunosuppressiwz effects in both T’and B cell-mediatedresponses(Slavin et al, 1976). Total lyrnphoid irradiation (Prernpree and Merz, 1969) causeda dose-relatedreduction of circulating and tissueT and B lymphocytes,reduced proliferative responsesto PI-IA,ConA and MLRand prolonged survival of histoincompatibleskin grafts (five-fold increaseof skin survival time across major histoincompatibility barriers (Slavin et al, 1977).Within severalweeks following completion of radiation therapy, absolute B lyrnphocytosis occurred despite persistent T lymphocytopeniatith an increaseof the proportion of circulating large mononuclear cells bearing no B or T cell surface markers. Latge mononuclearcells were also apparent (up to 70%) in the

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spleen and these ceils were proven to be positive for soybean agglutinin and partiaky for peanut aggIutinin receptors (We&en&erg et al, J Immunoll984, 132:97X)78; More&i et al, Eur J Immunoll985,15:138-148). There appears to be a marked increase in hematopoietic cells in the irradiated spleen, with documented Increase in the proportion of colony formation of committed (GM-CFU) and pluripotent (CFU-S) progenitor ceks (SIavin et al, Transpht PTWC 1981, 13:439-442). In p&a&I, it was shown that spleen cells obtained from TLI treated mice were capable of inducing potent in vitro suppression of T cell-mediated prokferative res~nses to predominantly MLE in an aIIo-antigen non-specific patient (Weigensberg et aL, 1984; More&i et aA, 1985). Suppressive capacity could be ‘got rid of almost entirely by eIimination of SBA positive cells. Suppressor cells showed no evidence of Thy-l, Lfl-1, LyT-2 or I3T4 cell surface determinants (Weigensberg et al, 1984). It is now agreed that, despite some evidence to the contrary in the previousIy published literature, most of the non-specific suppressor ceU activity induced by TLI is mediated by non-T cell origin (Weigensberg et al, J Immunoll984,132:971-978). Based on the avaiIable information, the potent immunosuppressive effects of TLI seem to stem from a combination of the foIlowing: (1) elimination of radiosensitive Iymphocytes during all cell cycles, including interphase, through the direct effect of ionized radiation; (2) impairment of the function of T lymphocytes on a cell-per-cell basis; (3) abnormal maturation patterns of recovering Iymphocytes following radiation therapy, i.e. reduced T ceII number and function and increased suppressor ‘T ceII phenotype, and (4) increased number and function of non-T suppressor cells. Whereas the in vim capacity of TLI-induced suppressor cells to suppress a variety of proliferative responses to allo-antigens and mitogens was clearly shown, their biological role in ho remains unclear. As will be suggested later, it is tempting to suggest that suppressor cells may play a role in some of the more subtle immunomodulatoty effects of TLI; however, conclusive evidence for such a role is still Iacking [24]. Natural suppressor cells are believed by the Stanford group [25,26] to play a major role in the induction of unresponsiveness following TLI. Our own data suggest that functional clonal reduction rather than aUo-antigen-specific suppression is the final outcome following induction of unresponsiveness to bone-marrow dografts following TLI [ 211. Similar mechanisms have been recentIy suggested in our model of tolerance to ceII protein [ 22,231. The most novel effect of TI.I on the immunologicaI system is UnquestionabIy on induction of unresponsive states to heterologous protein antigens and ako-antigens. As previously shown TLI-treated animals could be readily and consist.entIy made to tolerate aIIo-antigens foUowing inocukrtion of donor-type bone-marrow cells across major histocompatibihty barriers. BALB/c, C57BL/6, C3H (HeJ) and (BAIB/c x C57BL/6) Fl recipients accepted

permanently C57BI./6, BALB/c and 4/J bone-marrow allografts, respectively, whiIe showing no clinical evidence of graft versus host disease (GVI-ID) [ 241. Bone-marrow recipients showed permanent survivaI of donor-type skin aU0graft.s without immunosuppressive treatment despite major histoincompatibility between hosts and donors. The clonogenic effects of TLI to histoincompatible antigens was subsequently documented in rats, who permanently accepted heart and skin aIIografts, as well as in primates (Myburgh et al, 1980; J Immunoll984; Myburgh and Emit, Tran.pkant Proc 1985,171. Dogs seemed more resistant to histoincompatible bone-marrow aIIografts in the majority of experiments reported (Pennock et al, Traqixkznt Proc 1981,32:467). In our own hands, induction of tolerance to bone-marrow aUograits was inconsistent and mostly negative among outbred dogs [24]. Interest@&, a much higher success rate in induction of tolerance to aIIo-antigens was accomplished by Myburgh et al. (Tran.sphtution 1980, 29:401404; J Immunol 1984, 132; Transpkant Proc 1985, 17) in baboons utilizing wide-field TLI with radiation ports similar to those used originaUy in rodents. These investigators found that splitting the time intervals between radiation doses to 2-3 days, or even 1 week, stik accomplished very satisfactory, if not superior, results. In addition, vulnerability to induction of unresponsive state could be further extended by increasing the time interval between termination of radiation therapy and organ transplantation by several months, provided that one additional dose fraction of TI.I was given prior to organ transpIantation. Interestingly, specikc unresponsiveness to kidney or liver alIografts was successfuIIy accomplished in 30-50% of animals utilizing 800-1200 cGy without using donor’s marrow prior to organ transplantation. This indicated that induction of tolerance to perfused organ aIiografts may not require coinfusion of donor-type marrow cells [25]. In baboons, larger cumulative doses of TLI post-TLI or addition of cytotoxic agents did not help obtain better success rates for organ aIIografting (Myburgh, personal communication). In contrast, several studies in dogs and pilot clinical trials in renal akografts in man suggest a marked synergistic effect between TLI and cyclosporine 4 as weII as additional immunosuppressive agents such as methotrexate and ALG (Pennock et al, 1981; Strober et al, J Immunol 1974: 32). More recently, it has been shown that TLI treated beagles may accept permanentIy ( > 296 days) kidney allografts from DIA-compatible donors depleted of dendritic cells in bone-marrow transplantation from the kidney recipients before kidney transplantation [ 271. The reason for discrepancy between tolerance induction in baboons by Myburgh et al (1980) and not in dogs and pigs (reviewed in Slavin et al, In Tolerance in Bone Marrow and Organ Transplantation, Elsevier, 1984, pp105-153), in contrast to GottIieb et al (Transpkzntation 1980,29:487) may stem from the fact that most investigators have used narrow field irradiation in dogs similar to fields used for TLI in man. The originaI investigations in rodents as well as their reproducible counterparts in baboons were, however, performed using wide parts of

Immune

radiation, including the whole abdomen. We have previously documented [45] that m inor variations in the radiation field may make a major difference in susceptibility to induction of unresponsivenessfollowing TLI; m inimal extension of the cervical,as well as the lower abdominal, fields in ratswas sufficient for induction of chimerism and tolerance to heart allografts in 100%of the recipients, in contrast to none of those treatedwith a slightly smaller radiation field (Slavin et al, J Exp Med 1978,147:700-707). Nevertheless,it should be noted that complete unresponsivenessto skin allografts was not achieved in rodents without infusion of bone-marrow cells of donor origin. In contrast, tolerance to perfused heart allografts was consistently induced in rats even without such transplantation and also in recipients of a relativelysmall number of bone-marrow cells not proven to be chimeric (Slavin et al, 1978) 1281. It has been recently shown that engrafment of allogenic bone-marrow cells and aquisition of donor-type unresponsivenessmay be accomplishedfollowing conditioning of the recipients with low-dose TLI followed by sublethal WI31(Shin et al, 1984) [28]. Induction of antigen-specific unresponsiveness to protein antigens following TLI Pretreatment with TLI gave BAIB/c m ice - normally resistant to induction of tolerance to soluble protein antigens- antigen-specihcunresponsivenessto bovine serum albumin (BSA; &n-Bar et al, J Immunoll978, 121:1400-1404). BALB/c m ice receiving intraperitoneal injections of BSA became permanently unresponsive to BSA, even if given the complete Freund adjuvant. They did, however, recover normal responsivenessto unrelated bovine gammaglobulin. Tolerance to BSA could be rendered in other species as well (C3HiHe J and CVB1/6), with the exception of (NZB x NZW)/Fl, in which resistanceto induction of toleranceto soluble protein antigensmay be associatedwith the pathogenic@of SIP-like autoimmunedisease@an-Baret al, CliniculEzp Med 1983, 51558-564) Specihcunresponsivenessto BSAcould be transferred by lim iting the number of spleen T lymphocytes obtained from tolerant m ice transferred into sublethally irradiated syngeneichosts. This suggeststhat the mechanismfor induction of tolerance to soluble protein antigenswas mediated by Thy-l.2 positive suppressor lymphocytes@anBar et al, 1978). The use of TLI in clinical trials of allogeneic bone-marrow transplantation The unique immunosuppressiveand tolerogenic effects of TLI in experimental animals prompted us to investigate its use in the treatment of patients with severeaplastic anemia receiving allogeneic bone-marrow transplantation. Our protocols consisted of a combination of TLI: 6-i-12daily fractions of 200cGy, followed by four daily intravenousdoses of cyclophosphamlde50 mg/kg [24,25].

modulation

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irradiation

Tochner and Slavin

I-&4-matched bone-marrow grafts were obtained from siblings in 10 consecutive transplants of multiple-transfused patients with severe aplastic anemia None of the patients rejected their graft, however, in contrast to experimental animals in which it could be shown that TU had a marked effect on both host versus graft and graft versushost reactivi~, acute,as well as chronic, GVHD was observed in severalpatients conditioned with TLI and cyclophosphamide. Marked anti-rejection effects of TLI in patients with severeaplasticanemiawere also confirmed by other centers, using one single higher dose of radiation of 750 cGy (Ramseyet al, Blood 1980, 55:344) (41. The absence of anti-GVHD effects in man is not surprising. We have previously shown that TLI did not convey anti-GVHD resistancefollowing inoculation of a large number of immunocompetent T lymphocytes derlved from the spleen in BAD/c m ice. It did, however, convey protection againstGVI-IDInduced by bone-marrow cells, which in the murine system include up to 5-7% T lymphocytes, in contrast to man, where bone-marrow cells obtained as a m ixture of peripheral blood cells contain relatively higher levels of immunocompetent T lymphocytes. In a subsequentstudy, TLI was combined with T lymphocyte depletion pre-transplantationin order to establish a method in which two new principles were combined; the use of TLI for immunosuppression of graft rejection and T lymphocytedepletion for prevention of GVHD [ 241.T lymphocyte depletion was carded out using monoclonal rat anti-humanlymphocyteantibodies (CAMPTH1, kindly supplied by Drs G. Hale and H. Waldma~, Cambridge University School of Medicine, UK) using autologous donor’s serum obtained on the morning of transplant as a source of complement [24]. The risk of rejection of marrow allograftsin previouslytransfusedpatients with severe aplastic anemia is great, even without lymphocyte depletion. Most investigatorsattempting T lymphocyte depletion, which is known to further augment the incidence of graft rejection follawing allogeneicBMT, have failed to accomplishsustainedengraftmentin severe aplasticanemia Our current seriesconsistsof 19 patients with severeaplastic anemiawho receiveda combination of TLI 18OOcGygiven as two daily fractions of 15OcGy with cyclophosphamide200mg/kg over 4 days.With the exception of one patient who was particularly sensitized with three transfusions from family members, none of the patients rejected his graft, although most were multiply transfused(personal communication,1988).Our data in severeaplastic anemiasuggestthat TLI may help overcome rejection of allogenelcmarrow allografts,even following T lymphocyte depletion. Successfulbone-marrow transplantationwith T lymphocyte-depletedmarrow allograftsfor complete prwention of GVHD using no post-transplant anti-GVHD prophylaxis was also accomplishedin patientswith severep thalassemiamajor. To date, 12 patients haveundergoneB m with a protocol that consisted of TLI gOo-1OOOcGy (4-5 daily fractions of 200cGy) followed by busulfan 16 mg&g over 4 days and cyclophosphamlde 2OOn@‘kgover 4 days prior to graft.@. Patients received no post-trans-

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plant anti-GVHD immunosuppressive prophylaxis. Only one patient (receiving TLI 800 cGy, thereby increasing the total dose to 1000cGy) rejected the graft, while the others showed durable engraftment and no evidence of GCHD. Overall, our pilot clinical trials suggest that TLI may represent a useful component of the conditioning regimen prior to BMT for accomplishing consistent engraftment, men in situations susceptible to graft rejection such as in recipients of T-depleted skin allografts. The use of TLJ for overcoming rejection of histoincompatible marrow allografts has not yet been challenged. The use of TLI for prevention renal allografk

of rejection

of

In view of the potent immunosuppressive effects of TIJ in large anlmal models using perfused organ allografts, its use in the prevention of rejection of renal allografts in patients at high risk was pioneered in 1979 (Najarian et al, Ann Sung 1982, 196442452). Twenty-two such patients have been treated with TLI prior to transplantation of primary, secondary or tertiary renal allografts. All patients undergoing retransplantation rapidly rejected previous grafts. At 24 months follow-up, 72% of grafts were functioning in the TLI group as compared with 38% in the historical control group of recipients receiving secondary or tertiary grafts and treated with conventional immunosuppression. Eight of the patients treated with TLJ had diabetes mellitus in addition to endstage renal function. Interestingly, a comparison (Kim et al, Longterm clinical and immunological studies of high risk renal transplant recipients given total lymphoid irradiation. XII Int Cong of the Transplant Sot, Sydney,Australia, p 319) between patients at high risk of rejecting renal allografts and patients treated with TJJ and low-dose imuran and prednisone rather than cyclosporine A indicates with a minimal observation of 5 years, a signiticantly greater patient and graft survival in the latter group. As with the initial reports by the Minnesota group, successful application of TLI for renal transplantation was reported by Saper et aL, [29] Myburgh et al (World J Surg 1986, 10:369-380) Cortesini et al (Tran.@ant P?wc 1985, 17~1291) and Waer et al (I Immunoll984, 132:1041). Pilot observations in patients undergoing renal allografts by Strober’s group at Stanford [29] suggest that adjunct immunosuppressive therapy may be gradually downgmied, in which case specific alloresponsiveness to donor allo-antigens could be documented in 0irr0 by showing responsivenessto donor’s lymphocytes with concomitant normal responsivenesstowards thirdparty lymphocytes. This would mean, for the first time, induction of true transplantation tolerance to perfused organ allogmfts in man. This was recently induced in our own institution (unpublished observations) utikzing the combination of TLI and monoclonal antibodies with lowdose cyclosporine A that was tapered off within a few weeks. The above clinical trials are extremely encouraging since they suggest that TLI could be used for potent&zing im-

munosuppression, thus reducing the incidence of rejection even in patients at high risk. More interestingly, TLI can most likely be utilized for induction of true transplantation tolerance in man. Intensive clinical trials for induction of true transplantation tolerance to organ allografts without the need for long-term administration of post-transplant immunosuppression are currently under investigation. Conclusion Ionizing irradiation has a profound effect on circulating and tissue lymphocytes - the major component of the immune system. However, manipulation of the immune system for organ allografting does not require exposure of the whole body to irradiation. TLI is a form of radiation therapy that can be applied clinically for the induction of potent immunosuppression and the facilitation of induction of antigen-specific unresponsiveness.The unique immunomodulatory effects of TLI on the immune system make this procedure a clinically applicable approach to the prevention of rejection in bone-marrow and organ transplantation. Encouraging results of experiments in bone-marrow and organ transplantation. Encouraging results of experiments in large outbred animals, particularly those of Myburgh et al (1986) showing permanent and allo-antigen specific unresponsivenessto donor allo-antigens in baboons carrying renal and liver transplants for up to 6 years,with no rejection occurring in animals that maintained an allograft at one year post-transplant, plus pilot results in renal allografts documented by Myburgh, Strober and our own group suggest that true transplantation tolerance might be accomplished in man without any need for continuous non-specific immunosuppression following transplantation. Although induction of permanent transplantation tolerance is certainly feasible with TLI, additional experience with large outbred animals, particularly in man, is mandatory for the design of an optimal protocol. This is to ensure the projectible results with minimal possible risk in using TLI, such as optimal dose/fraction, time intervals between doses, and total cumulative doses, as well as the necessityof adjunct agents concomitantly with TLI for reduction of the amount of radiation required for optimal induction of transplantation tolerance. The combination of TH and potent in viva monoclonal antibodies seemsparticularly encouraging, but further studies are required to assessthe full potential of such combinations. Annotated reading l l e

1.

references and recommended

Of interest Of out.standing interest

KBHIHAM K, YOSHINAKA Y, hW~~uwa G, TOMOOKAS, NCIMOTO K: ‘RadioresMant’ intrathymic T-cell percursors express Tcell receptor C-gamma4-specific and Cdekaspeci6c gene messages. Eur J fmmunol1988, 18:83%!40. This paper describes resistence of radioresistent intmthymic T-cell procursors of CD3+, cD4-, CM- T lymphocyte precursors which exl

Immune

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Tochner and Slavin

press T-ceil receptor y and 6 chain messagesafter exposing mice to the radiation of 6OOcGy,detected 7 days after irradiation.

cur in mice failing to generate anti-tumor immunity after thymectomy and irradiation.

2.

7. a

FERRARA JIM, hf.ICHAEISON J, BURAKOFF SJ, tvfMJCH P: Engraftment tbllowing T cell-depleted bone marrow transplantation: In. Diimtial &cts of in creased total-body irradiation on semiallogeneic and allogeneic recipients. Truns pkantatzbn 1988, 45948-952. This work documents differential effects of a brief exposure of recipients of bone-marrow cells to total body irradiation in recipients of bone-marrow aRograft.sacross semi-allogeneicand allogeneic barriers in mice. Recipients of ailogeneic grafts showed improved survival and complete donor eqraftment as the dose of irradiation was increased from 1000 to 1500 cGy. By contrast survival of semi-allogeneicrecipients was independent of the dose of WBI. Mixed chimeras were more frequent in those given lower doses of radiation. l

3. l

i+ASEGAWA Y, NAKSHIMA I, ANDO K, MIZOCXJCHI K, tt4GASE F, ISOBE K, KAw~stttht~K, S~~~MOKATA K, YOSIUDA T, IWAMOTOT:

Dynamics of cytotoxic T lymphocytes percuraors in vivo assessedby change in the radiation sensitivity. Evidence for development of radiation-sensitive memory cells without clonal expansion. Scund J Immurwll988, 28:4353. This paper presents evidence for development of radiation-sensitive memory cells without clonal expansion. Although whole body irradiation of 400 cGy abolished the capacityof spleen cells of mice sensitized or non-sensitizedwith allogeneic spleen cells to proliferate and generate cytotoxic T tymphocytes (CTL), impaired generation of CD. in primary MLR was restored by R-2, whereas impaired activity in secondary MIR was not restored by E-2, suggestingthat helping cells whose activity was replaceablewith IL2 were functionaRy more radiation-sensitive than precursors of CTl in unprimed mice. Suprisii memory ptecurson3of CTL in mice primed by allogeneic cells were sensitive to radiation. KIM TH, SHANKBM, VNLERADA Ta~vt.5El, SONGcw: Immunosuppreasive techniques using radiation. Am J Clin Oncd 1988, 11362367. A review of immunosuppressiveeffects of various radiation procedures, including WBI and total lymphoid irradiation, and a discussionof potential applications of radiation therapy in bone-marrow and organ transplantation, respectively.

4. l

J,BoNuuM~O’REULYRJ, WELIE K: Effects of in vfvo administration of human recombinant IL-2 qnomolgus monkeys after autologous bone marrow transplantation. Trurz.@nl Pnx 1987, 19(suppl 7):157-161. The effects of administration of L-2 to monkeys undergoing autologous bone-marrow transplantations were studied during the early pe tiod after marrow infusion, attempting to facilitate the recovery of T lymphocytes and NK cell numbers and functions. In one animal, treatment wirh E-2 increased the number of lymphocytes following conventional BMT without a sign&ant increase in the in r&-o prolifetatlve responses,compared with the normal control, whereas the effects of IL-2 therapy in another animal treated after transpIantation of stem cell-enriched, T-lymphocytedepleted bone-marrow autograft was less remarkable. There was an insigni6cant incmase of the number in the latter lymphocytesanimal, which began during the 3rd week of therapy. Lymphocytecounts fell to the control level 1 week after the cessationof IL-2 treatment ,%sfn~tiwas obsetved after IL-2 treatment in animals nxeivhg cornentional or T-cell depleted bone-marrow grafts. 5.

~!XVER~GIlLlo~,hS'lCK

l

AwwAp M, Notrrtt RJ: Sublethal, whole-body ionizing irradia00 tion can be tumor promotive to tumor destructive depending on the stage of development of underlying antitumor immunity. Gzner Immunol Immunotber 1988, 26~5560. This paper indicates ditTerentialeffects of sublethal WBI on tumor development as a function of time of administration of radiation relative to tumor inoculation. h-t mice with an establishedimmunogenic tumor, sublethal radiation of 500&y resulted in complete tumor ablation and long-term sun&al. Mice exposed to the same dose of radiation several hours after inoculation of the tumor e&axed growth of the primary tumors and earlier deaths due to systemicdiseasewere observed. The authors suggestthat low-dose irradiation may act through elimination of radiosensitivesuppressor cells. This phenomenon was obseryed only after administmtion of immunogenic tumors, against which T effector cells may respond, since radiationinduced tumor mgressiondid not oc6.

267

CWmU R Bone marrow transplantation for acute leukemia A prelimimuy report from the International Bone Mamnv Transplant Registry. Trunsphrt PRX 1987, 1926262631. This paper represents p&minary data from the International BoneMarrow Transplantation Registtyon the outcome following bone-marrow transplantation for acute leukemia, in which WBI represents a major component during the conditioning. 8.

Foaht~~sJ, KRANCE RA, O'DONNELLMR, NADE~MEEAF', SNYDER DS, FAHEYJl, SCHMW GM, ZNA J& Lrt’strrr JA F~‘DLEYDD, SNIECINSKI IJ, ME-tTERGE, HUL lR, NA’~+X’ANI MB, BLLJMEKG: Bone marrow transplantation for acute non lymphoblastic leukemia during first complete remission. An analysis of prognostic factors. Tranqhntation 1987, 43:650-653. This paper summa&es n3dts of bone-marrow transplantation for acute non-lymphoblastic leukemia in patients undergoing the procedure during iirst complete remission. The role of radiation therapy in elimination of leukemia is a part of the conditioning regimen in bonemarrow transplantation. l

SNYDERDS, F~NDLEYDo, FORMAN SJ, N&XMANEE AP, O’DONNELL. MR, SCHMIDTGM, B~ERMAN PJ, FAH!ZYJI, KRANCE RA, SNIECWKIIJ, DOELKENG, Lrpsnr JA, LUK KH, NATHwANl MB, HIU LR, BWME KG: Fractionated total body irradiation and high dose cyclophosphamidez a preparative regimen for bone marrow transplantation for patients with hematologie malignancies in iirst complete remission. Blut 1988, 57:7-13. This paper reviews the outcome of patients undergoing allogeneic bone-marrow tmnsplantation following fractionated total body irradiation and a single dose cyclophosphamide (lOOm@g) in All, AML and high-grade NHL, undergoing transplantation during tirst complete remission, as well as in patients with chronic phase CML

9. l

Urrt-os GW, SARALR, Butots WH, BRNNEHG, SENSENBRENNER LI, W~NGARD JR, YEAGERAM, JOHN R, AhmtNDEttRF, R0wt.Y SD, MAY S, VOGELSANG GB: Allogeneic. syngetteic and autologous marrow transplantation in the acute le&umias and lymphomas - Baltimore experiences. Actu Huemud 1987, 78 (suppl 1):175-180. This Is a report of the Baltimore experience in bone marrow ttansplantation for acute leukemias and lymphomas in patients undergoing aUogeneic,syngeneicand autokqous BMT. Patientswith ALL were conditioned with WBI cyclophosphamide,whereas patients with AML were conditioned with busulfan and cyclophosphamide,with a high dose of cyclophosphamide of 2OOm&g in both cases.Recipients of autologous marrow grafts received bone-marrow lxlls purged with 4Hc. 10.

l e

KRANCERA, Fotth~~ SJ,BWME KG Total-body hmdiation and higbdose teniposide: A pilot study in bone marrow tcansplantation for patients with relapsed acute lymphoblastlc leukemia Carrcer Treat Rep 1987,71&l-647. Another paper describing the use of WBI hmdiation as part of the conditioning prior to aRogeneicbone-marrow transplanration in patients with AU foRowing telapse. It investigatesa pilot trial utiRzing vhf-26 (teniposide) in combination with WBI. 11. l

IATINIF, CHECRAGUNI F, MMAMANO E, Atust~ C, PANQZABM, GOBBI G, R~MONDI C, Avtstei~F, MARlXUJMF:HypChCtionated total body irradiation for Tdepleted HLA identical bone marrow transplants. Radio oncd 1988, 11:113118. This study hmstigates intensitication of immunosupptessiwsconditioning prior to aRoger& BMT for mal&nant hematologic disorders in pa tie% treated w& a combination of hyperfractionaied WBI in combination with cvcloohosohamide.Dose of TBA (120 &y/3 hactior&ayJ was escalatei frdm 1440 cGy to 1560 cGyper 4 days: 12.

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13. l e

PHILIPT, ARM~TAGE JQ, Sprtzatt G, CBA~MNF, G~cxtq~~ S, CAHNJ-Y, COKXBAT IP, GO~NE AH, GOtttn NC, FtESH M, IAPORTE J-P, WCtit D, Ptco J, BOSLYA, ANDERsON C, saion R, BYRONP, CAEUNUUF,~K:Highdosetherapy and autologous bone marrow transpkll.ltation after 6illue of conventionai chanottlerapy in adults with iIt--

grade or high-grade non-Hoddgkin’s lymphoma N En@ J Med 1987, 31614951498. This is a review of the outcome foilowing autologous BMT in pa tients with intermediate or high-grade NHL who bave failed conven tional frontline therapy. The authors suggestthat patients with chemosensidverelapsehad a signiiicantly higher disease-freesurvivaifoiiowing BMT, compared with those with resistant relapse. 14.

WOLPFSN, MCCURLEY TI+ GIANNONEL High-dose chemoradiothefapy with syngeneic bone marrow transplantation for -multiple myeloma: a ewe report and literature review. Am J Hematoll987, 26:191-198. Thisisareponofacaseandreviewof19reportedcasesofbonemamnv tmnspiantation foiiowing conditioning with chemotherapy and WBI in patients with pteviousiy treated multiple myeioma, suggesting potential benefits of such an aggressiveapproach for patients with reslsmnt disease. l

TARBEU.NJ, WTCI DA, DOUJNJD, MAUCHP, HEM S: Fractionation and dose rate e&x.% in mice: a model for bone manow transpkintation in man. Int J Rad Oncol Biol 1987, 13:106>1869. This study represents a murine modei for designing a better conditioning prior to BMT in man, with data suggestingprotection of nonhematopoietic tissues following fractionated irradiation and low-dose tates without protection of hematopoietic stem ceils by these factors. 15. l

16.

Sm SM, GIBSONG, SWINDEUR, PEA~OND: Effect of spinal imdiation. on gnnvth. AI& LXs Cbik&xd 1987, 62:%1-464. l?tkcts of spinal irradiation on growth was investigatedand indicated thatgrowth exposue to higher doses of radiation therapy may represent a long-term complication in patients undergoing high-dose radiochemotherapy. 17.

LEPERAD, STANHOPE R hu T, GM DB, BLACKILXKH, l C~EUS JM, PICKMANPN: The elect of total body irradiation and bone marrow transplantation during childhood and adolescence on growth and endocrine function. Br J H-1987, 67~419-426. The long-tern efkts of BMT foiiowing conditioning with cyclophosphamide, and single&se WBI in chiidren and adolescentswere investigated, focusing on gmwth and endocrine functions. Growth Miiure was obserd in the majority of chUdren with evidence of growth hormone deficiency. Rduced growth hormone response to insui.in-inducedhy pog@xmh was rlwumented in over 50% of the children. In adults, ovarian failure was common in ail girls, requiring sex steroid repiacemerit In males, gonadal failure may be compensated unless additional irradiation to the testicleswas used. Unlike growth hormone deiiciency, gonadal and thyroid dysfunctions showed no correlation with previous ctanial radiotherapy. 18.

SAND= JE, BUCKNERCD, AMOSD, brv~ W, APPELBAUM FR, mNEY K, %ORB R, SUulvAN I&& Wi’tHERsPoONRP, THOMAS ED Ovarian function !bEowing marrow transplantation for aphsdc anemia or leukemia J CIin On& 1988, 6:813-818. This summary represents the largest followup of ovatian function following allogeneic BMT for malignant and non-maUgnant(aspiastic anemia) diseaes in Seattle.The majority of younger women with apiastic anemiaretmmedcn5rian function with 9 of 43 having 12 pregnancies, resulting in 8 live births. In contrast, most women transplanted for leukemhneverrec~redovalian functions, which resulted in 3 pregnancies in 2 women, with spontsneous abortion in 2 and elective abor. tion in 1. WBI was the only factor that signilicsntly intluenced owian MUUte. l

CHANGQ~NG P, SHUQtNGGE, YUANY: Relationships between ionizing radiation bone marrow transplantation and leukemogenesk?.Sckwfa SInica &des B) 1987, 38~161-168. This paper inwstigates relationship between ioniaing radiation and leukogenesisby comparing the effect of radiation of the hosts on the one hand and the recipients on the other. It is suggestedthat indirect ~,~~caused by irnidiation is not the only factor that gives 19.

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20.

GONGM G~~MSKICA BRUCEAR Iron kinetics effects of Cd Bic&sfcs 1988, 1315-27. This paper indicates changes in iron kinetics even foliowing low-dose irmdlation comparing partiai with total body irradiation. l

88 millids.

21.

MORECK~ S, LESHEM B, EID A, Si~vt~ S: AUoantigen persistence in induction and maintenance of transplantation tolerance. J E @ Med 1987, 165:14&1480. This article descibesan experimental system in mice made tolerant with TLI, which documents the importance of the system of host’s aiio-antigens in the maintenanceof tolerance of donor cells to host’s aiio-anti gem.;tolerant C57BV6 spleen ceils with no cytotoxic T-lymphocyte precursors against tolerogenic BALB/c alio-antigenslost unresponsiveness following ‘parking’ in heavily irradiated synergeic recipients, whereas spleen ceiis obtained from tolerant mice retained unresponsiveness when parked in aliogeneic, BALB/c recipients. l e

22.

KAPPLER JW, ROEHMN, M++IUL&CK P: T cell tolerance by clonal eUmination in tbe thymus. Cell 1987, 49:273280. The authors present physical evidence supporting the clonai detection model for the induction of tolerance to self proteins. A Extremely interesting and vety important piece of work - the iirst demonstration of this kind. l

WPiER JW, STAEPZU, WHITEJ, MAIUL4CK PC: Self-tolerance eliminates T ceiis specitic for Mls-mod&led products of tbe major histocompatibiity complex. Nature 1988, 332:35-39. A continuation of V817a-1Estoty reported in Cell [22] but looking at Vj38.1and reactivity to the Mls antigen. An interesting paper conihming previous observations concerning clonai deletion. 23. l

24. Si~vt~ S: Total lymphoid hmdiation (TLI). Immunol Today me 1987, 888-92. This is a review of the basic sNcks in animals and ciinicai results in man using TLI for immunosuppression and for induction of tmnspiantation tolerance,with an overview of the possible mechanismsof TLI-induced transpiantation tolerance.

25.

WU~FFBH, PALA‘IHUMPAT V, SCHUYADRON R, STROBER S: Prevention of graft-versus host disease by natural suppressor cells. Tratzpkmt Ptuc 1987, 10:1087. This paper suggests that MNtd suppressor ceiis may piay a role in prevention of gmft versus host disease.Suppressor ceils may block ailoreactivity by immunocompetent T iymphocytes. l

26. 0

SCHVXDRON RB, STROBER S: Cloned natural suppressor celis derived from the neonatal spleen: in vitro action and iincage. Tran.$&anl Pnx 1987, 19533535. CuiNring of spleen cells obtained from neonatal mice suggestthat naturai suppressor ceils may be cloned. The phenotype of these ceiis has been described. 27. 0

RAPPAPOKT Fl., MEEK A, MiUM S, HAYAsHlR AltNOLDAN, S~OBERS: Synergistic effects of combined immunosuppressive modulation: I. Unresponsiveness to dendritic cell-depleted renai aUografts in dogs exposed to total-iymphoid in-abtion. Tramfxkantation 1988, 45682688. This paper documents synergistic effects between various therapeutic modalities that may lead to induction of transplantation tolerance in dogs, investigating effects of depletion of the dendritic ceiis from the graft in renai aiiograft recipients conditioned by TLJ. 28.

BLAUR BR, SODERUNG CCB, ROBINSON U, VNIERA DA Shortcourse total-lymphoid irmdiadon combined with total-body irradiation to facilitate engraftment of T cell-depleted marrow across a major histocompatlbility barrier in mice. Transpkmkation 1988, 46~324327. This paper suggests that combination of low-dose TD may be combined with WBI to facilitate engraftment of T-cell depleted bone-marrow aiMgrafts across major histocompatibiiity barriers in mice. Some authors have previousiy indicated that after conditioning with the TLI, regimen effective for engrahment of non-T cell-depleted bone-marrow aUogral3.s may not be suificient for consistent engrahment of T ceii-depleted aiiogmfts. l

SAPER V, CHOWD, ENCXEMAN ED, HOPPERT, LRrIN B, COLUNS G, STROBER S: Ciinicai and immunological studies of cadaveric renal transplant recipients given total iymphoid irradiation and maintained on lowdose predniaone. Trun.plunmfon 1988, 45540-546. A report of the resuits of renai transplantation foUowing conditioning with TLI and ATG. Maintenancewith p&&one in 25 patients resulted in effective immunosuppression after conditioning with TLI 29. l