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Total lymphoid irradiation
Immunology Today, vol. 8, No. 3, 1987
Total lymphoid irradiation The delivery of ionizing radiation to lymph nodes, thymus and spleen while non-lymphoid organs are shielded has profoundly immunosuppressiveeffects that may have value in transplantation and the treatment of autoimmune disorders. Here Shimon Slavin reviews what is known of the cellular consequences of total lymphoid irradiation in animals and discusses the results of its use in various clinical immunosuppressive treatment regimens.
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BM cells to induce chimerism permanently accepted heterotopic donor-type heart allografts, and also pancreatic islet allografts with reversal of all the signs of diabetes induced by streptozotocin 7. The beneficial role of BM cells in generation of the unresponsive state remains an enigma. Permanent survival of a skin allograft was never observed unless BM cells were infused and The unique sensitivity of lymphocytes to ionizing irradichimerism documented 8. In contrast, tolerance to peration is the basis of several radiotherapeutic procedures fused organ allografts has been established in rodents for the prevention of allograft rejection. However, the and primates even without BM cells8.9. Similarly, tolerradiation dose required to prevent rejection of an organ ance to perfused organ allografts was established in 20 allograft was soon found unacceptable due to irrevers- rats and 10 beagles treated with low-dose BM under ible bone marrow and lung radiotoxicity. Patients treated conditions where no residual donor cells could be idenfor malignant lymphomas with fractionated, high dose tified in the recipients. The latter findings suggest that radiotherapy had strikingly impaired cell-mediated re- the state of unresponsiveness that develops following TLI sponses, an observation which revived an old interest in is not an 'all or none' phenomenon, but rather a complex use of ionizing radiation to suppress immune responses. dynamic condition in which immunohemopoietic cells To explore the use of fractionated total lymphoid irradibearing donor's alloantigens seem to play an important ation (TLI) for immunosuppression, protocols were derole. Likewise, in contrast to their resistance to BMsigned in experimental animals which took into account induced GVHD, TLI-treated mice developed acute GVHD factors related to the radiobiology of lymphocytes, the when immunocompetent spleen cells were mixed with anatomy of the immune system and the possible adverse the BM inoculum, which suggests that the development effects of ionizing irradiation. Radiation was adminisof unresponsiveness towards the host by the graft is tered by selective exposure of all major lymph nodes, totally dependent on the proportion of mature T-cells thymus and spleen while non-lymphoid organs were infused. On the other hand, durable chimerism was shielded with lead2.3. High and effective total cumulative difficult to accomplish after infusion of T-cell-depleted doses of irradiation were achieved with few severe marrow allografts into TLI-treated recipients, indicating side-effects by administration of multiple daily fractions that the bilateral state of unresponsiveness in TLIof 100-200 tad per day3. induced BM chimeras represents a delicate dynamic Fractionated TLI resulted in profound and long-lasting balance of interactions between the immunocompetent reduction of the pool of lymphocytes in the circulation cells in the host and those in the donor These considera...... "'~. . .w. ' ;'v' a. ,'y'"l~"u~u ~,,4 .,,' +~, ~ , ^ - ~'~' , . . . . L_-.~ organs, accompanie d by tions should be kept in mind in applying these model impaired humoral and, especially, cell-mediated immune systems to larger experimental animals and man because functions, including strikingly delayed capacity to reject of the high proportion of T iymphocytes present in their an allograft 3. It was originally assumed that the effect of marrow aspirates. TLI was merely due to dose-dependent elimination of the The effects of TLI on the recovery of various cell radiosensitive immunocompetent cells. Indeed, the desubsets in rodents and man have been described in gree of prolongation of skin graft survival across major detail1,2.8,11.12. The most striking effect of TLI seems to histocompatibility complex (MHC) barriers in mice was be the generation of suppressor cells capable of abrogatclearly dose depenBent 3. At an optimal TLI dose of 3400 ing both cellular and humoral responses, including inrad, the mean survival of skin allografts in mice was vitro suppression of T-cell-mediated proliferative reprolonged 4--5-fold 3 and the mean survival of perfused sponses induced by allogenic cells, phytohemagglutinin heterotopic cardiac allografts in rats was prolonged (PHA), concanavalin A (Con A) 8.11.12, suppression of 9-fold, with occasional permanent survival4. plaque formation of normal B cells to T-independent Interestingly, studies of adult mice given TLI of 3400 antigens13, in vivo suppression of adoptive antibody rad have shown that injection of heterologous protein response to dinitrophenyl (DNP)-hapten coupled to antigens after radiotherapy results in a state of permaheteru',o~;.~s proteins such as bovine serum albumin nent antigen-specific tolerance rather than immunity ~. (BSA)s, and suppression of GVHD by allogeneic BM cells Rodents inoculated with histoincompatib!e bone marrow in lethally irradiated recipients 8.14. TLI-induced suppres(BM) cells showed durable chimerism and tolerance to sor cells of the mixed lymphocyte reaction (MLR) are donor-type alloantigens without clinical signs of graftantigen nonspecific 8.11.14. They appear to be large, versus-host disease (GVHD), indicating that host was immature, mononuclear cells, cyclophosphamidetolerant of graft as well as vice versa3. This possible insensitive and extremely radioresistant (I>3000 tolerogenic effect of BM cells in TLI-treated hosts may be rad)8,11.12.14. Unlike other workers 14 we have found that similar to that observed in animals treated with antithese cells bear no cell surface markers of the matlJre lymphocyte serum6. Thus, rats given TLI and allogeneic T-cell lineages. Phenotypically, MLR suppressor cells are Thy-1 -, Lyt 1-, Lyt 2-, TL-, Ig-, asialo GM1- and largely Department of Bone Marrow Transplantationand Immunobiology nonadherent to plastic, 'Sephadex G-IO' and nylon wool Research,HadassahUniversityHospital,91120Jerusalem,Israel surfaces ~2. Suppressor cells have receptors to soybean (~) 1987, ElsevierPubh/ahr)ns, C,Imbrldge 0167 4919/87t$02 O0
Immunology Today, voL 8, No. 3, 1987
agglutinin and, to a lesser extent, peanut agglutinin, similar to immature celis present in the normal marrow 12. Indeed, TLI-induced spleen cells are capable of colony formation in vitro 1s. TLI may therefore be regarded as a procedure that results in marked depletion of mature T cells by exposure of the lymphoid system to fractionated radiation while shielding the non-overlapping bones, which contain active marrow, that serve as the source of uncommitted immunohemopoietic progenitor cells capable of acquiring tolerance to new protein antigens or neoalloantigens. In this respect, TLI induces a state that mimics the immature, tolerance-susceptible, fetal or neonatal immune system. Indeed, cells with characteristics similar to those of the cells induced by TLI can be documented in fetal liver, neonatal spleen, and the spleen of nude mice, as well as the marrow of normal mice 1i. The biological role of the naturally occurring suppressor cells that are enriched after TLI is unknown. Antigennonspecific suppressor cells may block the proliferation of antigen-responding cells, leading to antigen-specific clonal reduction. In the protein antigen tolerance system, antigen-specific suppressor cells have a role s so that although suppressor cells may induce the unresponsive state after TLI, unresponsiveness is probably maintained by clonal reduction or functional clonal deletion 16 Vast cumulative e×perimental data suggest that the overall effects of TLI on the immune system result from a combination of at least four interrelated mechanisms (Table 1). The success rate of tolerance induction after TLI is multifactorial; many of the essential parameters were derived by trial-and-error experiments and are not yet fully understood. The size of the radiation field seems important: in rodents, exposure of a wide field including the whole abdomen seems to be an important prerequisite4. Likewise, whereas permanent transplantation tolerance to perfused organ allografts cannot be established in dogs, pigs, primates and humans exposed to low (600 rad) or high total cumulative doses of TLI (up to 4000 rad) using the classical 'mantle' and 'inverted Y' ports of radiation 8, permanent survival of renal and hepatic allografts were reported following low dose TLI (600-800 rad) using a wide port of radiation including the whole torso, excluding the skull, limbs and lungs9. Modifications of dose fractionation patterns are unquestionably important too, but were never investigated in a systematic way. An optimal dose per fraction seems to be 100-200 rad, but the precise dose-response relationships for large outbred animals in relationship with the field size and fractionation interval have not yet been fully assessed.
Combination of TLI with other immunosuppressiveagents The feasibility of clinical application of low-dose TLI in combination with other immunosuppressive modalities for transplantation of perfused organ allografts was investigated using small and large outbred animal models. A combination of low-dose TLI (200 rad x 5) and low dose cyclosporine (CS-A) (1.25 mg/kg/day) resulted in a marked synergy best documented by extremely prolonged heart allograft survival in rats across MHC barrierslL Prolonged heterotopic cardiac allograft survival was also reported in dogs treated with TLI, antithymocyte globulin (ATG), azathioprine (AZA) or
methotrexate 18, as well as in primates treated with a combination of low dose TLI and ATG (Ref. 19), AZA or CS-A (Ref. 20). Preoperative TLI may pose problems in sensitized patients, who may have to wait for extended periods after completion of TLI until a negatively crossmatched cadaveric kidney can be found. The feasibility of posttransplant TLI was therefore investigated using the heart allograft model in rats21. Although effective, its immunosuppressive effects were clearly less impressive than those of pre-transplant TLI. It proved much more potent in combination with short-term adjuvant immunosuppressive treatment 21. Although there is no disagreement on the potent immunosuppressive effects of TLI, several investigators failed to establish chimerism and/or Ionglasting transplantation tolerance following conditioning with TLI with or without infusion of donor BM in large outbred animals22-24. It appears that the combination of the classical 'mantle' and 'inverted Y' radiation field used for treatment of malignant lymphomas, at a clinically acceptable cumulative dose of irradiation is insufficient to ensure BM or organ engraftment across the MHC in large outbred animals. Because many unknown parameters influence the results of TLI0all interpretations and comparisons between studies should be made cautiously before any firm conclusions of clinical relevance can be made. More data will have to be generated in order to determine the importance of the optimal ports of radiation, dose/fraction, frequency of pulsed irradiation, dr:~e rate, total cumulative radiation dose, timing of trar~splantation and optimal combinations with other immunosuppressive agents, as well as the role of donor BM.
TU in autoimmunedisorders Fractionated irradiation has been proposed as a rational form of therapy for several autoimmune disorders, as documented in several murine models of spontaneous systemic lupus erythematosus (SLE)2s.26. TLI was administered to several groups of patients with intractable rheumatoid arthritis (RA)27'28, drug-resistant SLE (Ref. 29), and multiple sclerosis (C. Devereux, unpublished), with some encouraging pilot clinical results. Studies of peripheral blood lymphocytes after TLI in RA and SLE revealed marked reduction in the absolute number of T cells (Leu-4), to about one third of the pretreatment number. This decrease was associated with more than 4-fold reduction in the number of helper T cells (Leu-3) with only a slight reduction in the number of suppressor/ cytotoxic T cells (Leu-2) ~7.2B. As expected from murine studies, the laboratory manifestations of autoimmunity persisted, due to the resistance of memory cells to TLI (Ref. 8). The beneficial effects of TLI on polyarthritis paralleled the nonspecific immunosuppressive effects of TLI, and hence partial recrudescence of arthritis occurred following restitution of lymphocyte number and in-vitro responsiveness to mitogens 26. Therefore, TLI should not yet be recommended for general clinical use in autoimmune disorders and should still be regarded as an experimental procedure. TLI in clinicalorgan transplantation TLI was first applied in combination with conventional rejection-preventing agents, including AZA and predni-
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sone. A group of 22 patients at high risk for rejection of a renal allograft (ages 5-51) was treated with TLI at the University of Minnesota between February 1979 and July 1981 (Ref. 30-32). Of these patients, 18 received cadaveric and 2 received mismatched living donor transplants; 13 received second and 7 third transplants. All patients had rejected a previous renal allograft in less than 12 months (mean graft survival 4.3 months). Two patients received primary renal allografts from two haplotype-mismatched living donors. All patients were in chronic renal failure and on hemodialysis during radiotherapy. Nine of the patients had diabetic nephropathy. All but one patient underwent splenectomy prior to irradiation. Patients received 100-125 rad for up to 5 times per week to both the 'mantle' and 'inverted Y' fields simultaneously. The total cumulative dose ranged from 1050 and 4050 rad delivered over periods ranging from 24 to 124 days. The interval between completion of TLI and transplantation ranged from 1 to 330 days. Five of the patients received low doses of T-cell-depleted BM cells (5 x 10z nucleated cells/kg). Maximum immunosuppression by in-vitro assays was obtained at a dose of 2500 rad. All patients received maintenance AZA at a dose of 1-1.5 mg/kg/day adjusted for white cell count. Standard prednisone dose of 2 mg/kg/day was tapered to 0.5 or 0.3 mg/kg/day by 3 or 2 weeks, respectively, or alternatively used at low dose (0.4 mg/kg/day) immediately following transplantation. Of the 22 patients, one technical failure of a graft that never functioned was excluded from further analysis. The overall 1 year actuarial survival was 79% graft function with 86% patient survival. At 2 years, there was a 74% graft function and a 78% patient survival. Three grafts were lost to chronic rejection at 7, 16 and 17 months. There were 5 deaths that accounted for the other graft losses. Four years after the study was initiated, 13 of the original 21 patients treated with TLI (62%) and 11 of the original 19 patients that had Dreviouslv rejP~d ~ r~n~l ~llngr~ft I ~ O / _ ~ have functioning grafts at 16--42 months post-transplant with a mean serum creatinine of 1.6+0.5 mg%. The 2 diabet!c recipients of primary grafts from 2 HLA haplotype-mismatched donors have functioning grafts 24 and 36 months post-transplantation, in this particular setting, marrow transplantation did not seem to improve the butcorne, but evidence of chimerism was not observed. It should be noted that the BM was treated with anti-T-cell globulin, which was not done in studies carried out in experimental animals. Compared with a similar group of patients treated with conventional immunosuppression at 1, 2 and 3 years after retransplantation, patient survival was slightly better after TLI. Graft survival was significantly superior in the TLI-treated group, compared with 46% at 1 year, 36% at 2 years and 28% at 3 years in patients treated with conventional immunosuppression. The most significant contributing factors in prediction of rejection episodes were the length of time between completion of TLI and transplantation, and the post-operative immunosuppressive protocol utilized. Of the 14 patients who received standard prednisone taper, 5 experienced acute rejection episodes within the first 2 months post transplant. These 5 were the only patients in this group who had a significant delay between completion of TLI and transplantation. These patients had recovered T-cell number and MLR reactivity by the time a crossmatchnegative cadaver donor became available. A second •
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Table 1. Effectormechanismsof TLI
• Interphasedeath of the pool of unprimedradiosensitivelymphocytesby the directlymphocytotoxiceffeds of ionizingirradiation • Theemergenceof large,immaturestem-cell-derivednon-Tsuppressor cells capableof potent suppressionof in-vitro and possibly in-vivo cell-mediatedimmuneresponsesduringrecoveryfrom TLI • Alteredprocessingof immunocompetentcellsin the microenvironment of peripherallymphoid organs (thymus, lymph nodes and spleen)followingradiotherapy • Formationof a transientphasein which a stablestate of specific unresponsivenessmay be induced,simila:to the neonatalperiod, through an unknownmechanism,most likelyfaci!itated by donor BM cells important variable was related to the post-transplant treatment protocol. All 3 patients given low dose prednisone (0.4 mg/kg/day) from the day of transplant experienced acute rejection episodes within the first month. They were successfully treated and normal graft function is currently maintained. The conclusions from the pilot trial indicated that optimal results were obtained with TLI 2000-2500 tad delivered in 100 tad/fraction followed by transplantation within 2 weeks, with prednisone starting at 2 mg/kg with tapering schedule and maintenance AZA adjusted for white cell count. Therefore, candidates should ideally have no or a low percentage of cytotoxic antibodies to a panel or else a crossmatch-negative, living donor available in order to minimize the time interval between completion of TLI and transplantation. The use of a combination of TLI and CS-A in clinical renal transplantation was pioneered in Israel in 1981 (Shapira, Weshler and Slavin, unpublished). The protocol ~,,o, ~.u,;,u, ;~u pwuudnsplan~TLi ~1200-i600 rad), CS-A (12 mg/kg) and prednisone with rapid taper was very well tolerated and only 1 of 6 patients lost his graft to rejection. The combination of TLI and CS-A is currently being investigated by R. Cortesini and colleagues (unpublished). Renal allograft survival was studied in 11 recipients of TLI (~>2000 rad) and CS-A (12 mg/kg) for one month with rapid tapering, and 10 recipients of standard immunosuppressive protocols, all patients being in a high risk categc,ry. Nine out of 11 in each of the TLI groups has a functioning graft, compared with 1 out of 10 of the control group. Sampson etaL 33 reported the use of TLI in combination with ATG and prednisone without additional immunosuppressive agents, in a follow-up of the original study, Strober, Collins and colleagues studied the effect of combining TLI with various anti-lymphocyte antibodies: 3 out of 34 graft losses to rejection were reported using rabbit ATG; 3 out of 10 using horse ATG (Upjohn) and 3 out of 6 using monoclonal OKT3. No rejection has yet occurred in 5 recipients of CS-A following TLI (unpublished). The use of TLI (2400 + 400 rad) with prednisone (30 mg with tapering to 10 mg from day 120) without additional Immunosuppression was also investigated by M. Waer, Y. Vanrenterghem and colleagues (unpublished). Seven patients out of 22 evaluabte had no rejection episodes at all. Five patients had 1 and 10 patients more than one rejection episodes and the graft survival at 2 years is approximately 75%.
Immunology Today, voL 8, No. 3, 7987
After documenting successful induction of specific transplantation tolerance to renal allografts in baboons using wide ports of TLI, similar to those originally described in rodents 2.4, J. Myburgh went on to apply a similar regimen in 33 patients undergoing kidney transplantation (unpublished). Six patients received TLI 2200 rad and 26 were treated twice weekly with 60-100 rad/fraction to a total of 760-2200 rad. Of 24 evaluable patients, 3 received prednisone with or without AZA and 21 received CS-A and prednisone. One patient died and another lost his graft. With an observation period of 1 month to 5 years, graft survival is 92% and patient survival 96%. One patient is already off therapy and a few additional patients are off CS-A, currently maintained on very low doses of prednisone (approximately 5 rag/day). According to Myburgh, in addition to superior survival of renal allografts following TLI, the creatinine levels were lower in TLI-treated recipients as compared with patients treated by the conventional approach. The data suggest that true transplantation tolerance may be induced in recipients of renal allografts treated by TLI. TLI in clinical bone marrow transplantation (BMT) In clinical BMT, TLI was used either as a single fraction •to reduce the morbidity associated with total body irradiation, or as small multiple daily fractions, as originally described in experimental animals8. Single dose TLI is now routinely used for successful prevention of rejection in multiply transfused patients with severe aplastic anemia in Minnesota 34. Although rejection is a major problem in up to 60% of similar patients conditioned by cytoxam (CTX) (200 mg/kg) alone, 39 out of 40 multiply transfused patients with severe aplastic anemia treated by one dose TLI 750 rad showed sustained engraftment. The use of fractionated TLI in clinical BMT was introduced in 1979 at Hadassah University Hospital, Jerusalem3s,36. Since then, 10 multiply transfused patients with severe aplastic anemia were conditioned by TLI 950-2450 rad (1200 rad in most cases) followed by CTX (200 mg/kg). All patients engrafted successfully with no major radiation toxicity. Acute GVHD was noted in 3 out of 10 patients and chronic GVHD in 2 out of the surviving 7. This pilot study suggested that TLI may be an effective and safe way to prevent graft rejection in multiply transfused recipients, although GVHD is unlikely to be completely eliminated by TLI, since the human marrow contains high proportions of T lymphocytes. The use of TLI in clinical BM transplantation for prevention of rejection of T-lymphocyte depleted BM allografts is also being currently investigated in Jerusalem37-4°. It has now been confirmed by several centers that in-vitro T-lymphocyte depletion increases 9,~-~-~ject the chance of ..... ~ .~ i •on, even in patients undergoing BMT for malignant hematological disorders, a complication that almost never occurs without T-cell depletion. Therefore, we use a monoclonal rat antihuman lymphocyte antibody (CAMPATH-1) that binds human complement for in-vitro purging of mature T lymphocytes in order to prevent GVHD; this is combined with low-dose TLI to overcome host resistance to T-celldepleted allografts 37-a°. It has already been documented that this regimen accomplishes durable engraftment without GVHD can be accomplished without any postgrafting anti-GVHD prophylaxis in patients with leukemia (TLI 150 rad x 4; CTX 120 mg/kg; total body irradiation
1200 rad), beta thalassemia major (TLI 200 rad x 4; busulfan 16 mg/kg; and CTX 200 mg/kg) and severe aplastic anemia (TLI 150 rad twice daily x 6; and CTX 200 mg/kg). The available data suggests that TLI may be a relatively safe approach as part of the conditioning regimen prior to allogeneic BM transplantation for prevention of late graft rejection, whenever T-cell-depleted marrow allografts are to be considered, in both malignant and non-malignant diseases. Summary A great deal of experimental data in animals and man suggest that TLI may be used clinically in combination with other immunosuppressive modalities in order to reduce the likelihood of rejection in recipients of BM transplants and organ allografts. Tolerance of the graft by the host is now r,..inely established in several BM transplantation units through depletion of mature and committed T lymphocytes, suggesting that an immune system developing de novo out of hemopoietic precursors is capable of acquiring an unresponsive state to foreign alloantigens across minor and even major histocompatibility barriers. It is very likely that tolerance of host against perfused organ allografts could be equally well established by using TLI with additional antilymphocyte agents in order to deplete all the mature and committed lymphocyte reservoir in the host, allowing the newly developing immune system to acquire unresponsiveness to the alloantigens of the grafted organ. Clearly, recent data in primates and man raise hope that such a goal may turn into reality in the foreseeable future.
The author gratefully acknowledges the assistance of the Israel Cancer Association, the Michael Robert Koenigsberg Memorial Fellowshipof the Israel Cancer Research Fund, the Basic ResearchFoundation of the Israel Academy of Sciences and Humanities,and the Chief Scientist'sOffice, Israel Ministry of Health. References 1 Fuks,Z., Strober, S., Brobrove,A. M. etal. (1976)./. C/in. Invest. 58-803 2 Slavin,S., Strober, S., Fuks,Z. and Kaplan,H. S. (1976) Science 193, 1252-1254 3 Slavin,S., Strober, S., Fuks,Z. and :,:~n:3n,H. S. (1977) Transplant. Proc. 9, 1001-1004 4 Slavin,S., Reitz,B., Bieber,C. P. etal. (1978)J. Exp. Med. 147, 700-707 5 Zan-Bar,I., Slavin,S. and Strober, S. (1978)J. Immunol. 121, 1400-1404 6 Monaco, A. P. and Wood, M. L (1984)in Tolerance in Bone Marrowand Organ Transplantation (Slavin,S., ed.), pp. 17-74, Elsevier,Amsterdam 7 3r!,tt, L. D., Scharp,D. W., Lacy,P. E. and Slavin,S. (1982) Diabetes 31 (Suppl.4), 63--68 8 Slavin,S., Weiss, I., Morecki, S. etal. (1984)in Tolerance in Bone Marrow and Organ Transplantation (Slavin,S., ed.), pp. 105-153, Elsevier,Amsterdam 9 Myburgh, J. A., Stair, J. A. and Browde, S. (1984)in Tolerance in Bone Marrow and Organ Transplantation (Slavin,S., ed.), pp. 153-166, Elsevier,Amsterdam t0 Howard, R. J., Sutherland,D. E. R., Lure,C. T. etal. (1981) Ann. Surg. 193, 196-200 11 Weigensberg,M., Morecki, S., Weiss, L. etal. (1984) J. Immunol. 132, 971-978 12 Morecki, S., Weigensberg, M. and Slavin,S. (1985) Eur. J. Immunol. 15, 138-148 13 May, R. D.: Slavin,S. and Vitetta, E. S. (1983)J. Irnmunol.
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131, 1108-1114 14 Strober, S., Gottlieb, M., Zan-Bar, I. et al. (1979)ImmunoL Rev. 46, 87 15 Slavin, S. and Seidel, H. J. (1982)Exp. HematoL 10, 206-216 16 Morecki, S., Leshem, B., Weigensberg, M. etaL (1985) Transplantation 4 J, 201--2 i 0 17 Rynasiewicz, ,. J., Sutherland, D. E. R., Kawahara, K. and Najarian, J. S. (!981)J. Surg. Res 30, 365 18 Koretz, S. H., Gottlieb, M. S., Strober, S. etal. (1981) Transplant. Proc 13, 443 19 Bieber, C. F, Jamieson, S., Raney, A. etal. (1979) Transplantatic;: 28, 347 20 Pennock, J. L., Reitz, 3. A., Biebar, C. P. etaL 1981 Transplantatiora 32, 467 21 Bentley, F. R., S,Jtherland, D. E. R. and Najarian, J. S. (1982) J. Surg. Res. 32, 3bO 22 Howa:d, R. J., Sutherland, D. E. R., Lure, C. T. etal. (1981) Ann. Surg. 193, 196 23 Myburgh, J. A., Smit, J. A., Hill, R. R. H. and Browde, S. (1980) Transplantation 29, 409 24 Chu, F., Chaganti, R. S. K., Shank, B. etal. (1981) Transplant. Proc. 13, 429 25 Slavin, S. (1979)Proc. NatlAcad. Sci. USA 76, 5274-5276 26 Kotzin, B. L. and Strober, S. (1979)J. Exp. Med. 150-371 27 Kotzin, B. L., Strober, S., Engelman, E. G. eta/. (1981) N. Engl. J. Med. 305, 969 28 Trentham, D. E., Belli, J. A., Anderson, R. I. etal. (1981)
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N. Engl. J. Med. 305, 976 29 Ben-Chetrit, E., Gross, D. J., Braverman, A. etal. Ann. Int.
Med. (in press) 30 Najarian, J. S., Sutherland, D. E. R., Ferguson, R. M. etal. (1981) Transplant. Proc. 13, 417 31 Najarian, J. S., Ferguson, R. M., Sutherland, D. E. R., etal.
Ann. Surg. (in press) 32 Sutherland, D. E. R., Ferguson, R. M., Rynasiewicz, J. J. etal. Transplant. Proc. (in press) 33 Sampson, D., Levin, B. S., Hoppe, R. T. etal. (1985) [ransplant. Proc 17, 1299-1303 34 Ramsay, N. K. C. and Kersey, J. H. (1984)in Tolerance in Bone Marrow and Organ Transplantation (Slavin, S., ed.), pp. 167-174, Elsevier, Amsterdam 35 Slavin, S., Naparstek, E., Weshler, Z. etaL (1983) Transplant. Proc. 15, 668-670 36 Slavin, S., Naparstek, E., Weshler, Z. and Fuks, Z. (198.3) Recent Advances in Bone Marrow Transplantation (UCLA Symposia on Molecular and Cellular Biology Vol. 7) (Gale, R. P., ed.), pp. 21-27, Alan R. Liss, New York 37 Slavin, S., Waldmann, H., Or, R. etal. (1985) Transplant. , Proc. 17, 465-467 38 Waldmann, H., Polliack, A., Hale, G., Or, R. etal. (1984) Lancet ii, 483-485 39 Slavin, S., Or, R., Weshler, Z. etal. (1985)5urv. Immunol. Res. 4, 238-252 40 Slavin, S., Or, R., Cividalli, G. etal. IsraelJ. Med. Sci. (in press)
Total lymphoid irradiation The delivery of ionizing radiation to lymph nodes, thymus and spleen while non-lymphoid organs are shielded has profoundly immunosuppressiveeffects that may have value in transplantation and the treatment of autoimmune disorders. Here Shimon Slavin reviews what is known of the cellular consequences of total lymphoid irradiation in animals and discusses the results of its use in various clinical immunosuppressive treatment regimens.
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ShimonSlavin
BM cells to induce chimerism permanently accepted heterotopic donor-type heart allografts, and also pancreatic islet allografts with reversal of all the signs of diabetes induced by streptozotocin 7. The beneficial role of BM cells in generation of the unresponsive state remains an enigma. Permanent survival of a skin allograft was never observed unless BM cells were infused and The unique sensitivity of lymphocytes to ionizing irradichimerism documented 8. In contrast, tolerance to peration is the basis of several radiotherapeutic procedures fused organ allografts has been established in rodents for the prevention of allograft rejection. However, the and primates even without BM cells8.9. Similarly, tolerradiation dose required to prevent rejection of an organ ance to perfused organ allografts was established in 20 allograft was soon found unacceptable due to irrevers- rats and 10 beagles treated with low-dose BM under ible bone marrow and lung radiotoxicity. Patients treated conditions where no residual donor cells could be idenfor malignant lymphomas with fractionated, high dose tified in the recipients. The latter findings suggest that radiotherapy had strikingly impaired cell-mediated re- the state of unresponsiveness that develops following TLI sponses, an observation which revived an old interest in is not an 'all or none' phenomenon, but rather a complex use of ionizing radiation to suppress immune responses. dynamic condition in which immunohemopoietic cells To explore the use of fractionated total lymphoid irradibearing donor's alloantigens seem to play an important ation (TLI) for immunosuppression, protocols were derole. Likewise, in contrast to their resistance to BMsigned in experimental animals which took into account induced GVHD, TLI-treated mice developed acute GVHD factors related to the radiobiology of lymphocytes, the when immunocompetent spleen cells were mixed with anatomy of the immune system and the possible adverse the BM inoculum, which suggests that the development effects of ionizing irradiation. Radiation was adminisof unresponsiveness towards the host by the graft is tered by selective exposure of all major lymph nodes, totally dependent on the proportion of mature T-cells thymus and spleen while non-lymphoid organs were infused. On the other hand, durable chimerism was shielded with lead2.3. High and effective total cumulative difficult to accomplish after infusion of T-cell-depleted doses of irradiation were achieved with few severe marrow allografts into TLI-treated recipients, indicating side-effects by administration of multiple daily fractions that the bilateral state of unresponsiveness in TLIof 100-200 tad per day3. induced BM chimeras represents a delicate dynamic Fractionated TLI resulted in profound and long-lasting balance of interactions between the immunocompetent reduction of the pool of lymphocytes in the circulation cells in the host and those in the donor These considera...... "'~. . .w. ' ;'v' a. ,'y'"l~"u~u ~,,4 .,,' +~, ~ , ^ - ~'~' , . . . . L_-.~ organs, accompanie d by tions should be kept in mind in applying these model impaired humoral and, especially, cell-mediated immune systems to larger experimental animals and man because functions, including strikingly delayed capacity to reject of the high proportion of T iymphocytes present in their an allograft 3. It was originally assumed that the effect of marrow aspirates. TLI was merely due to dose-dependent elimination of the The effects of TLI on the recovery of various cell radiosensitive immunocompetent cells. Indeed, the desubsets in rodents and man have been described in gree of prolongation of skin graft survival across major detail1,2.8,11.12. The most striking effect of TLI seems to histocompatibility complex (MHC) barriers in mice was be the generation of suppressor cells capable of abrogatclearly dose depenBent 3. At an optimal TLI dose of 3400 ing both cellular and humoral responses, including inrad, the mean survival of skin allografts in mice was vitro suppression of T-cell-mediated proliferative reprolonged 4--5-fold 3 and the mean survival of perfused sponses induced by allogenic cells, phytohemagglutinin heterotopic cardiac allografts in rats was prolonged (PHA), concanavalin A (Con A) 8.11.12, suppression of 9-fold, with occasional permanent survival4. plaque formation of normal B cells to T-independent Interestingly, studies of adult mice given TLI of 3400 antigens13, in vivo suppression of adoptive antibody rad have shown that injection of heterologous protein response to dinitrophenyl (DNP)-hapten coupled to antigens after radiotherapy results in a state of permaheteru',o~;.~s proteins such as bovine serum albumin nent antigen-specific tolerance rather than immunity ~. (BSA)s, and suppression of GVHD by allogeneic BM cells Rodents inoculated with histoincompatib!e bone marrow in lethally irradiated recipients 8.14. TLI-induced suppres(BM) cells showed durable chimerism and tolerance to sor cells of the mixed lymphocyte reaction (MLR) are donor-type alloantigens without clinical signs of graftantigen nonspecific 8.11.14. They appear to be large, versus-host disease (GVHD), indicating that host was immature, mononuclear cells, cyclophosphamidetolerant of graft as well as vice versa3. This possible insensitive and extremely radioresistant (I>3000 tolerogenic effect of BM cells in TLI-treated hosts may be rad)8,11.12.14. Unlike other workers 14 we have found that similar to that observed in animals treated with antithese cells bear no cell surface markers of the matlJre lymphocyte serum6. Thus, rats given TLI and allogeneic T-cell lineages. Phenotypically, MLR suppressor cells are Thy-1 -, Lyt 1-, Lyt 2-, TL-, Ig-, asialo GM1- and largely Department of Bone Marrow Transplantationand Immunobiology nonadherent to plastic, 'Sephadex G-IO' and nylon wool Research,HadassahUniversityHospital,91120Jerusalem,Israel surfaces ~2. Suppressor cells have receptors to soybean (~) 1987, ElsevierPubh/ahr)ns, C,Imbrldge 0167 4919/87t$02 O0
Immunology Today, voL 8, No. 3, 1987
agglutinin and, to a lesser extent, peanut agglutinin, similar to immature celis present in the normal marrow 12. Indeed, TLI-induced spleen cells are capable of colony formation in vitro 1s. TLI may therefore be regarded as a procedure that results in marked depletion of mature T cells by exposure of the lymphoid system to fractionated radiation while shielding the non-overlapping bones, which contain active marrow, that serve as the source of uncommitted immunohemopoietic progenitor cells capable of acquiring tolerance to new protein antigens or neoalloantigens. In this respect, TLI induces a state that mimics the immature, tolerance-susceptible, fetal or neonatal immune system. Indeed, cells with characteristics similar to those of the cells induced by TLI can be documented in fetal liver, neonatal spleen, and the spleen of nude mice, as well as the marrow of normal mice 1i. The biological role of the naturally occurring suppressor cells that are enriched after TLI is unknown. Antigennonspecific suppressor cells may block the proliferation of antigen-responding cells, leading to antigen-specific clonal reduction. In the protein antigen tolerance system, antigen-specific suppressor cells have a role s so that although suppressor cells may induce the unresponsive state after TLI, unresponsiveness is probably maintained by clonal reduction or functional clonal deletion 16 Vast cumulative e×perimental data suggest that the overall effects of TLI on the immune system result from a combination of at least four interrelated mechanisms (Table 1). The success rate of tolerance induction after TLI is multifactorial; many of the essential parameters were derived by trial-and-error experiments and are not yet fully understood. The size of the radiation field seems important: in rodents, exposure of a wide field including the whole abdomen seems to be an important prerequisite4. Likewise, whereas permanent transplantation tolerance to perfused organ allografts cannot be established in dogs, pigs, primates and humans exposed to low (600 rad) or high total cumulative doses of TLI (up to 4000 rad) using the classical 'mantle' and 'inverted Y' ports of radiation 8, permanent survival of renal and hepatic allografts were reported following low dose TLI (600-800 rad) using a wide port of radiation including the whole torso, excluding the skull, limbs and lungs9. Modifications of dose fractionation patterns are unquestionably important too, but were never investigated in a systematic way. An optimal dose per fraction seems to be 100-200 rad, but the precise dose-response relationships for large outbred animals in relationship with the field size and fractionation interval have not yet been fully assessed.
Combination of TLI with other immunosuppressiveagents The feasibility of clinical application of low-dose TLI in combination with other immunosuppressive modalities for transplantation of perfused organ allografts was investigated using small and large outbred animal models. A combination of low-dose TLI (200 rad x 5) and low dose cyclosporine (CS-A) (1.25 mg/kg/day) resulted in a marked synergy best documented by extremely prolonged heart allograft survival in rats across MHC barrierslL Prolonged heterotopic cardiac allograft survival was also reported in dogs treated with TLI, antithymocyte globulin (ATG), azathioprine (AZA) or
methotrexate 18, as well as in primates treated with a combination of low dose TLI and ATG (Ref. 19), AZA or CS-A (Ref. 20). Preoperative TLI may pose problems in sensitized patients, who may have to wait for extended periods after completion of TLI until a negatively crossmatched cadaveric kidney can be found. The feasibility of posttransplant TLI was therefore investigated using the heart allograft model in rats21. Although effective, its immunosuppressive effects were clearly less impressive than those of pre-transplant TLI. It proved much more potent in combination with short-term adjuvant immunosuppressive treatment 21. Although there is no disagreement on the potent immunosuppressive effects of TLI, several investigators failed to establish chimerism and/or Ionglasting transplantation tolerance following conditioning with TLI with or without infusion of donor BM in large outbred animals22-24. It appears that the combination of the classical 'mantle' and 'inverted Y' radiation field used for treatment of malignant lymphomas, at a clinically acceptable cumulative dose of irradiation is insufficient to ensure BM or organ engraftment across the MHC in large outbred animals. Because many unknown parameters influence the results of TLI0all interpretations and comparisons between studies should be made cautiously before any firm conclusions of clinical relevance can be made. More data will have to be generated in order to determine the importance of the optimal ports of radiation, dose/fraction, frequency of pulsed irradiation, dr:~e rate, total cumulative radiation dose, timing of trar~splantation and optimal combinations with other immunosuppressive agents, as well as the role of donor BM.
TU in autoimmunedisorders Fractionated irradiation has been proposed as a rational form of therapy for several autoimmune disorders, as documented in several murine models of spontaneous systemic lupus erythematosus (SLE)2s.26. TLI was administered to several groups of patients with intractable rheumatoid arthritis (RA)27'28, drug-resistant SLE (Ref. 29), and multiple sclerosis (C. Devereux, unpublished), with some encouraging pilot clinical results. Studies of peripheral blood lymphocytes after TLI in RA and SLE revealed marked reduction in the absolute number of T cells (Leu-4), to about one third of the pretreatment number. This decrease was associated with more than 4-fold reduction in the number of helper T cells (Leu-3) with only a slight reduction in the number of suppressor/ cytotoxic T cells (Leu-2) ~7.2B. As expected from murine studies, the laboratory manifestations of autoimmunity persisted, due to the resistance of memory cells to TLI (Ref. 8). The beneficial effects of TLI on polyarthritis paralleled the nonspecific immunosuppressive effects of TLI, and hence partial recrudescence of arthritis occurred following restitution of lymphocyte number and in-vitro responsiveness to mitogens 26. Therefore, TLI should not yet be recommended for general clinical use in autoimmune disorders and should still be regarded as an experimental procedure. TLI in clinicalorgan transplantation TLI was first applied in combination with conventional rejection-preventing agents, including AZA and predni-
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Immunology Today, vol. 8, No. 3, 1987
6~
sone. A group of 22 patients at high risk for rejection of a renal allograft (ages 5-51) was treated with TLI at the University of Minnesota between February 1979 and July 1981 (Ref. 30-32). Of these patients, 18 received cadaveric and 2 received mismatched living donor transplants; 13 received second and 7 third transplants. All patients had rejected a previous renal allograft in less than 12 months (mean graft survival 4.3 months). Two patients received primary renal allografts from two haplotype-mismatched living donors. All patients were in chronic renal failure and on hemodialysis during radiotherapy. Nine of the patients had diabetic nephropathy. All but one patient underwent splenectomy prior to irradiation. Patients received 100-125 rad for up to 5 times per week to both the 'mantle' and 'inverted Y' fields simultaneously. The total cumulative dose ranged from 1050 and 4050 rad delivered over periods ranging from 24 to 124 days. The interval between completion of TLI and transplantation ranged from 1 to 330 days. Five of the patients received low doses of T-cell-depleted BM cells (5 x 10z nucleated cells/kg). Maximum immunosuppression by in-vitro assays was obtained at a dose of 2500 rad. All patients received maintenance AZA at a dose of 1-1.5 mg/kg/day adjusted for white cell count. Standard prednisone dose of 2 mg/kg/day was tapered to 0.5 or 0.3 mg/kg/day by 3 or 2 weeks, respectively, or alternatively used at low dose (0.4 mg/kg/day) immediately following transplantation. Of the 22 patients, one technical failure of a graft that never functioned was excluded from further analysis. The overall 1 year actuarial survival was 79% graft function with 86% patient survival. At 2 years, there was a 74% graft function and a 78% patient survival. Three grafts were lost to chronic rejection at 7, 16 and 17 months. There were 5 deaths that accounted for the other graft losses. Four years after the study was initiated, 13 of the original 21 patients treated with TLI (62%) and 11 of the original 19 patients that had Dreviouslv rejP~d ~ r~n~l ~llngr~ft I ~ O / _ ~ have functioning grafts at 16--42 months post-transplant with a mean serum creatinine of 1.6+0.5 mg%. The 2 diabet!c recipients of primary grafts from 2 HLA haplotype-mismatched donors have functioning grafts 24 and 36 months post-transplantation, in this particular setting, marrow transplantation did not seem to improve the butcorne, but evidence of chimerism was not observed. It should be noted that the BM was treated with anti-T-cell globulin, which was not done in studies carried out in experimental animals. Compared with a similar group of patients treated with conventional immunosuppression at 1, 2 and 3 years after retransplantation, patient survival was slightly better after TLI. Graft survival was significantly superior in the TLI-treated group, compared with 46% at 1 year, 36% at 2 years and 28% at 3 years in patients treated with conventional immunosuppression. The most significant contributing factors in prediction of rejection episodes were the length of time between completion of TLI and transplantation, and the post-operative immunosuppressive protocol utilized. Of the 14 patients who received standard prednisone taper, 5 experienced acute rejection episodes within the first 2 months post transplant. These 5 were the only patients in this group who had a significant delay between completion of TLI and transplantation. These patients had recovered T-cell number and MLR reactivity by the time a crossmatchnegative cadaver donor became available. A second •
90
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Table 1. Effectormechanismsof TLI
• Interphasedeath of the pool of unprimedradiosensitivelymphocytesby the directlymphocytotoxiceffeds of ionizingirradiation • Theemergenceof large,immaturestem-cell-derivednon-Tsuppressor cells capableof potent suppressionof in-vitro and possibly in-vivo cell-mediatedimmuneresponsesduringrecoveryfrom TLI • Alteredprocessingof immunocompetentcellsin the microenvironment of peripherallymphoid organs (thymus, lymph nodes and spleen)followingradiotherapy • Formationof a transientphasein which a stablestate of specific unresponsivenessmay be induced,simila:to the neonatalperiod, through an unknownmechanism,most likelyfaci!itated by donor BM cells important variable was related to the post-transplant treatment protocol. All 3 patients given low dose prednisone (0.4 mg/kg/day) from the day of transplant experienced acute rejection episodes within the first month. They were successfully treated and normal graft function is currently maintained. The conclusions from the pilot trial indicated that optimal results were obtained with TLI 2000-2500 tad delivered in 100 tad/fraction followed by transplantation within 2 weeks, with prednisone starting at 2 mg/kg with tapering schedule and maintenance AZA adjusted for white cell count. Therefore, candidates should ideally have no or a low percentage of cytotoxic antibodies to a panel or else a crossmatch-negative, living donor available in order to minimize the time interval between completion of TLI and transplantation. The use of a combination of TLI and CS-A in clinical renal transplantation was pioneered in Israel in 1981 (Shapira, Weshler and Slavin, unpublished). The protocol ~,,o, ~.u,;,u, ;~u pwuudnsplan~TLi ~1200-i600 rad), CS-A (12 mg/kg) and prednisone with rapid taper was very well tolerated and only 1 of 6 patients lost his graft to rejection. The combination of TLI and CS-A is currently being investigated by R. Cortesini and colleagues (unpublished). Renal allograft survival was studied in 11 recipients of TLI (~>2000 rad) and CS-A (12 mg/kg) for one month with rapid tapering, and 10 recipients of standard immunosuppressive protocols, all patients being in a high risk categc,ry. Nine out of 11 in each of the TLI groups has a functioning graft, compared with 1 out of 10 of the control group. Sampson etaL 33 reported the use of TLI in combination with ATG and prednisone without additional immunosuppressive agents, in a follow-up of the original study, Strober, Collins and colleagues studied the effect of combining TLI with various anti-lymphocyte antibodies: 3 out of 34 graft losses to rejection were reported using rabbit ATG; 3 out of 10 using horse ATG (Upjohn) and 3 out of 6 using monoclonal OKT3. No rejection has yet occurred in 5 recipients of CS-A following TLI (unpublished). The use of TLI (2400 + 400 rad) with prednisone (30 mg with tapering to 10 mg from day 120) without additional Immunosuppression was also investigated by M. Waer, Y. Vanrenterghem and colleagues (unpublished). Seven patients out of 22 evaluabte had no rejection episodes at all. Five patients had 1 and 10 patients more than one rejection episodes and the graft survival at 2 years is approximately 75%.
Immunology Today, voL 8, No. 3, 7987
After documenting successful induction of specific transplantation tolerance to renal allografts in baboons using wide ports of TLI, similar to those originally described in rodents 2.4, J. Myburgh went on to apply a similar regimen in 33 patients undergoing kidney transplantation (unpublished). Six patients received TLI 2200 rad and 26 were treated twice weekly with 60-100 rad/fraction to a total of 760-2200 rad. Of 24 evaluable patients, 3 received prednisone with or without AZA and 21 received CS-A and prednisone. One patient died and another lost his graft. With an observation period of 1 month to 5 years, graft survival is 92% and patient survival 96%. One patient is already off therapy and a few additional patients are off CS-A, currently maintained on very low doses of prednisone (approximately 5 rag/day). According to Myburgh, in addition to superior survival of renal allografts following TLI, the creatinine levels were lower in TLI-treated recipients as compared with patients treated by the conventional approach. The data suggest that true transplantation tolerance may be induced in recipients of renal allografts treated by TLI. TLI in clinical bone marrow transplantation (BMT) In clinical BMT, TLI was used either as a single fraction •to reduce the morbidity associated with total body irradiation, or as small multiple daily fractions, as originally described in experimental animals8. Single dose TLI is now routinely used for successful prevention of rejection in multiply transfused patients with severe aplastic anemia in Minnesota 34. Although rejection is a major problem in up to 60% of similar patients conditioned by cytoxam (CTX) (200 mg/kg) alone, 39 out of 40 multiply transfused patients with severe aplastic anemia treated by one dose TLI 750 rad showed sustained engraftment. The use of fractionated TLI in clinical BMT was introduced in 1979 at Hadassah University Hospital, Jerusalem3s,36. Since then, 10 multiply transfused patients with severe aplastic anemia were conditioned by TLI 950-2450 rad (1200 rad in most cases) followed by CTX (200 mg/kg). All patients engrafted successfully with no major radiation toxicity. Acute GVHD was noted in 3 out of 10 patients and chronic GVHD in 2 out of the surviving 7. This pilot study suggested that TLI may be an effective and safe way to prevent graft rejection in multiply transfused recipients, although GVHD is unlikely to be completely eliminated by TLI, since the human marrow contains high proportions of T lymphocytes. The use of TLI in clinical BM transplantation for prevention of rejection of T-lymphocyte depleted BM allografts is also being currently investigated in Jerusalem37-4°. It has now been confirmed by several centers that in-vitro T-lymphocyte depletion increases 9,~-~-~ject the chance of ..... ~ .~ i •on, even in patients undergoing BMT for malignant hematological disorders, a complication that almost never occurs without T-cell depletion. Therefore, we use a monoclonal rat antihuman lymphocyte antibody (CAMPATH-1) that binds human complement for in-vitro purging of mature T lymphocytes in order to prevent GVHD; this is combined with low-dose TLI to overcome host resistance to T-celldepleted allografts 37-a°. It has already been documented that this regimen accomplishes durable engraftment without GVHD can be accomplished without any postgrafting anti-GVHD prophylaxis in patients with leukemia (TLI 150 rad x 4; CTX 120 mg/kg; total body irradiation
1200 rad), beta thalassemia major (TLI 200 rad x 4; busulfan 16 mg/kg; and CTX 200 mg/kg) and severe aplastic anemia (TLI 150 rad twice daily x 6; and CTX 200 mg/kg). The available data suggests that TLI may be a relatively safe approach as part of the conditioning regimen prior to allogeneic BM transplantation for prevention of late graft rejection, whenever T-cell-depleted marrow allografts are to be considered, in both malignant and non-malignant diseases. Summary A great deal of experimental data in animals and man suggest that TLI may be used clinically in combination with other immunosuppressive modalities in order to reduce the likelihood of rejection in recipients of BM transplants and organ allografts. Tolerance of the graft by the host is now r,..inely established in several BM transplantation units through depletion of mature and committed T lymphocytes, suggesting that an immune system developing de novo out of hemopoietic precursors is capable of acquiring an unresponsive state to foreign alloantigens across minor and even major histocompatibility barriers. It is very likely that tolerance of host against perfused organ allografts could be equally well established by using TLI with additional antilymphocyte agents in order to deplete all the mature and committed lymphocyte reservoir in the host, allowing the newly developing immune system to acquire unresponsiveness to the alloantigens of the grafted organ. Clearly, recent data in primates and man raise hope that such a goal may turn into reality in the foreseeable future.
The author gratefully acknowledges the assistance of the Israel Cancer Association, the Michael Robert Koenigsberg Memorial Fellowshipof the Israel Cancer Research Fund, the Basic ResearchFoundation of the Israel Academy of Sciences and Humanities,and the Chief Scientist'sOffice, Israel Ministry of Health. References 1 Fuks,Z., Strober, S., Brobrove,A. M. etal. (1976)./. C/in. Invest. 58-803 2 Slavin,S., Strober, S., Fuks,Z. and Kaplan,H. S. (1976) Science 193, 1252-1254 3 Slavin,S., Strober, S., Fuks,Z. and :,:~n:3n,H. S. (1977) Transplant. Proc. 9, 1001-1004 4 Slavin,S., Reitz,B., Bieber,C. P. etal. (1978)J. Exp. Med. 147, 700-707 5 Zan-Bar,I., Slavin,S. and Strober, S. (1978)J. Immunol. 121, 1400-1404 6 Monaco, A. P. and Wood, M. L (1984)in Tolerance in Bone Marrowand Organ Transplantation (Slavin,S., ed.), pp. 17-74, Elsevier,Amsterdam 7 3r!,tt, L. D., Scharp,D. W., Lacy,P. E. and Slavin,S. (1982) Diabetes 31 (Suppl.4), 63--68 8 Slavin,S., Weiss, I., Morecki, S. etal. (1984)in Tolerance in Bone Marrow and Organ Transplantation (Slavin,S., ed.), pp. 105-153, Elsevier,Amsterdam 9 Myburgh, J. A., Stair, J. A. and Browde, S. (1984)in Tolerance in Bone Marrow and Organ Transplantation (Slavin,S., ed.), pp. 153-166, Elsevier,Amsterdam t0 Howard, R. J., Sutherland,D. E. R., Lure,C. T. etal. (1981) Ann. Surg. 193, 196-200 11 Weigensberg,M., Morecki, S., Weiss, L. etal. (1984) J. Immunol. 132, 971-978 12 Morecki, S., Weigensberg, M. and Slavin,S. (1985) Eur. J. Immunol. 15, 138-148 13 May, R. D.: Slavin,S. and Vitetta, E. S. (1983)J. Irnmunol.
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131, 1108-1114 14 Strober, S., Gottlieb, M., Zan-Bar, I. et al. (1979)ImmunoL Rev. 46, 87 15 Slavin, S. and Seidel, H. J. (1982)Exp. HematoL 10, 206-216 16 Morecki, S., Leshem, B., Weigensberg, M. etaL (1985) Transplantation 4 J, 201--2 i 0 17 Rynasiewicz, ,. J., Sutherland, D. E. R., Kawahara, K. and Najarian, J. S. (!981)J. Surg. Res 30, 365 18 Koretz, S. H., Gottlieb, M. S., Strober, S. etal. (1981) Transplant. Proc 13, 443 19 Bieber, C. F, Jamieson, S., Raney, A. etal. (1979) Transplantatic;: 28, 347 20 Pennock, J. L., Reitz, 3. A., Biebar, C. P. etaL 1981 Transplantatiora 32, 467 21 Bentley, F. R., S,Jtherland, D. E. R. and Najarian, J. S. (1982) J. Surg. Res. 32, 3bO 22 Howa:d, R. J., Sutherland, D. E. R., Lure, C. T. etal. (1981) Ann. Surg. 193, 196 23 Myburgh, J. A., Smit, J. A., Hill, R. R. H. and Browde, S. (1980) Transplantation 29, 409 24 Chu, F., Chaganti, R. S. K., Shank, B. etal. (1981) Transplant. Proc. 13, 429 25 Slavin, S. (1979)Proc. NatlAcad. Sci. USA 76, 5274-5276 26 Kotzin, B. L. and Strober, S. (1979)J. Exp. Med. 150-371 27 Kotzin, B. L., Strober, S., Engelman, E. G. eta/. (1981) N. Engl. J. Med. 305, 969 28 Trentham, D. E., Belli, J. A., Anderson, R. I. etal. (1981)
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N. Engl. J. Med. 305, 976 29 Ben-Chetrit, E., Gross, D. J., Braverman, A. etal. Ann. Int.
Med. (in press) 30 Najarian, J. S., Sutherland, D. E. R., Ferguson, R. M. etal. (1981) Transplant. Proc. 13, 417 31 Najarian, J. S., Ferguson, R. M., Sutherland, D. E. R., etal.
Ann. Surg. (in press) 32 Sutherland, D. E. R., Ferguson, R. M., Rynasiewicz, J. J. etal. Transplant. Proc. (in press) 33 Sampson, D., Levin, B. S., Hoppe, R. T. etal. (1985) [ransplant. Proc 17, 1299-1303 34 Ramsay, N. K. C. and Kersey, J. H. (1984)in Tolerance in Bone Marrow and Organ Transplantation (Slavin, S., ed.), pp. 167-174, Elsevier, Amsterdam 35 Slavin, S., Naparstek, E., Weshler, Z. etaL (1983) Transplant. Proc. 15, 668-670 36 Slavin, S., Naparstek, E., Weshler, Z. and Fuks, Z. (198.3) Recent Advances in Bone Marrow Transplantation (UCLA Symposia on Molecular and Cellular Biology Vol. 7) (Gale, R. P., ed.), pp. 21-27, Alan R. Liss, New York 37 Slavin, S., Waldmann, H., Or, R. etal. (1985) Transplant. , Proc. 17, 465-467 38 Waldmann, H., Polliack, A., Hale, G., Or, R. etal. (1984) Lancet ii, 483-485 39 Slavin, S., Or, R., Weshler, Z. etal. (1985)5urv. Immunol. Res. 4, 238-252 40 Slavin, S., Or, R., Cividalli, G. etal. IsraelJ. Med. Sci. (in press)