Induction of tolerance to human renal allografts with bone marrow and antilymphocyte globulin

Induction of Tolerance to Human Renal Allografts With Bone Marrow and Antilymphocyte Globulin w. Henry Barber urvival of cadaver and related donor renal allografts remains suboptimal despite recent advances in immunosuppressive therapy. I·. Acute and chronic rejection of transplants continues to be the most important source of allograft loss, and chronic nonspecific immunosuppression leads to significant morbidity and mortality in transplant recipients with long-term allograft function. 5·9 Therefore, the induction of donor antigen-specific unresponsiveness to tissue allografts by administration of donor antigen with reduction or elimination of nonspecific immunosuppression remains an important goal in clinical transplantation. Immunologic modulation induced by random donor blood transfusion was an important step in the improvement of cadaver kidney allograft survival,I 0-15although more recently the importance of this apparently nonspecific enhancement of allograft survival has been questioned. ":" Similarly, in related donor transplantation. donor-specific blood transfusion has been used in an effort to improve functional allograft survival. 19·21 This approach, like the use of random donor transfusions, has limited usefulness because of the risk of recipient sensitization against the donor 22•25 ; indeed, several studies have shown that the apparent benefit of donor-specific transfusions may be negated by more effective immunosuppressive protocols.P'" Medawar's experimental results in 1944 showing that rejection of tissue allografts is an immune phenornenon'" has led to intensive efforts to alter this response both chemically and with histocompatibility antigens. Early evidence demonstrated that transplantation tolerance could be produced in neonatal animals" by the infusion of viable donor

S

From the Department of Surgery. University of Alabama at Birmingham, Birmmgham, AL.

Supported by Grants No. 1 ROIA/27985-01 and 1 K04ARO1818-01 from the us Public Health Sennce. Address reprint requests 10 W. Hen ry Barber, MD, D PhIL, Department of Surgery, Umversity of Alabama at BIrmingham, Btrmingham, AL 35294 . 0 / 990 by W.B. Saunders Company. 0955-470X/90/0401-0003$3.00/0

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lymphoid cells and followed upon the observations that in dizygotic cattle. erythrocytes of the twins were shared, that these cattle were tolerant to skin grafts from their dizygotic sibling.":" The induction of functional allograft tolerance in adult animals has proven somewhat more difficult, and numerous methods have been investigated since the feasibility was first reported." Passiveenhancement with alloan_ tiserurn, although effective in rodents. has numej-; ous logistical problems in clinical transplantation and has proved to be only weakly effective in large animal models. 5'>-58 Similarly, alloantigen-alloanjj, body immune complexes, which have been used effectively under certain conditions to suppress varj; ous types of immune responses, also have been used successfully in experimental organ allograft systems. The same logistic problems of the need for dorioj-; specific alloantisera and limited usefulness in large outbred animals have not been circumvented by the more recent use of haptenated alloantigen-antihajy; ten antibody complexes, the effectiveness of which are highly strain-dependent. even in rodents. 59-. 2 Donor-antigen induced unresponsiveness, Used with or without concomitant immunosuppression. usually requires a period of recipient pretreatment ranging from several days to several weeks, thus als~ limiting its usefulness in the transplantation of hu_ man cadaver organs. Antigen alone may be benefi_ cial in some situations, and a variety of experimental protocols are described wherein soluble antigen. blood, platelets, lymphoid cells, and whole spleen allografts have been used to induce donor unrespon_ siveness . As with antibody and immune complex allograft enhancement, only modest success has been possible in outbred species grafted withOUt conventional immunosuppression."?' A recurrent problem with the use of antigen alone as a tolerogen is the unpredictable variability of graft survival even in inbred species. Most studies show biphasic SUr_ vival curves with some grafts being greatly pro_ longed and others demonstrating accelerated rejec_ tion . The addition of nonspecific immunosuppressive agents to donor antigen treatment has led to irn; proved efficiency in tolerance induction. Several

Transplantation Reviews, Vol 4, No 1 Uuly), 1990: pp 68-78

Donor Bone Marrow in Renal Allografts

combinations of drug and antigen pretreatment have been shown to have potential usefulness. Antilymphocyte sera (ALS) have been used most commonly and have been extensively investigated with a number of antigens, including lymphoid cells, platelets. and soluble tissue extracts. The addition or substitution of other agents, including procarbazine, cyclosporin A, cyclophosphamide , or azathioprine in lieu of ALS has also been shown to be advantageous in some models.t':" Recipient irradiation has been used as an irnmunoablative agent and is capable of tolerance induction. Both total lymphoid irradiation and whole body irradiation with autologous reconsitution have been examined. The clinical applicability of the radiation protocols is hampered by the need for long-term pretreatment of recipients, a common problem with many methods of active tolerance induction. 6~·69

The Use of Donor-Specific Bone Marrow and ALS for the Induction of Allograft Tolerance Monaco et al were the first to show that the nonspecific immune suppression induced by adult thymectom y and ALS could be converted to a state of stable immunologic tolerance by the pretransplant infusion of donor antigen in the form of viable lymphoid cells after the cessation of ALS therapy." Subsequently, the demonstration by Lance and Medawar that extended graft survival could be achieved by donor antigen administration after organ transplantation has stimulated further research into the applicability of tolerance induction in human cadaver allograft recipients." The concept of allograft transplantation with concomitant ALS administration, followed by a critically timed transfusion of donor bone marrow cells (which has been developed by Monaco and Wood), has been found to be a potent and reliable method of obtaining prolonged and sometimes indefinite allograft survival in mice," dogs," and rhesus monkeys" without the use of long-term nonspecific immunosuppressive agents. When used in combination with an induction course of ALS, donor bone marrow cells were found to be the optimal cellular antigen for the induction of immunologic tolerance. compared with lymphocytes obtained from peripheral blood, spleen, lymph nodes or thymus." Details of the experimental aspects of tolerance induction using ALS and donor bone marrow have been reviewed extensively":": therefore, the remaining discussion will be primarily

69

concerned with the clinical applications of this protocol.

Clinical Application of the Use of Donor-Specific Bone Marrow and Antilymphocyte Globulin The initial clinical trial using ALS and donor bone marrow was conducted in a group of one haplotypemismatched living related donor allograft recipients who were highly reactive in mixed lymphocyte culture to their prospective donors." The use of related donors was required in this study because it was done before the demonstration that cryopreserved marrow cells could be equally efficacious to those transfused immediately after procurement, a condition that is necessary for use in cadaveric transplantation." Immunosuppression consisted of prednisone in tapering doses, azathioprine 2 mg/kg! d , and Upjohn (Kalamazoo, MI) antithymocyte globulin (ATG) 10 to 15 mg/kg/d from da ys 0 through 14 posttransplantation. Donor bone marrow was harvested from the kidney donor under general anesthesia on the 21st posttransplant da y and immediately transfused to the allograft recipient. A total of six patients completed the marrow transfusion protocol. A seventh recipient developed severe acute rejection on postoperative day 7 during ATG therapy and underwent a transplant nephrectomy before the time of marrow transfusion. An eighth recipient was withdrawn from the study after a myocardial ischemic event 3 days after transplantation. Of this group, 3 patients achieved long-term stable graft function. A diabetic patient died of a stroke 1 week after the completion of the marrow infusion. Two other grafts were lost from rejection: one in the second postoperative month secondary to noncompliance with immunosuppressive medications , and the other approximately one year posttransplant due to an irreversible late rejection episode. The three long-term survivors have been maintained on low doses of prednisone and Imuran (Burroughs Wellcome, Research Triangle Park, NC). Mixed lymphocyte culture analysis of these three patients has shown that specific anti-donor reactivity has remained depressed, while anti-third party reactivity, which is initiall y nonspecifically depressed after antilymphocyte globulin (ALG) treatment, recovered to pretransplant levels. Monaco et al have also transplanted a single patient with a cadaver kidney in whom cryopreserved donor bone marrow was used after transplantation in a protocol analo-

70

W. Hmry Barber

gous to that described above for their group of related donor recipients." This patient sustained no rejection episodes but died with normal renal function 8 months after transplantation due to sepsis caused by a ruptured diverticulum of the sigmoid colon. The results of renal transplantation with donorspecific bone marrow transfusion in these initial patients were significant in that they showed the feasibility of undertaking a larger prospective study. Importantly, there was evidence of neither sensitization (precipitation of rejection) in these patients nor graft-versus-host disease (GVHD). The implications of specific hyporeactivity in mixed lymphocyte culture are somewhat uncertain; however. the fact that rejection episodes did occur in some instances after the administration of donor bone marrow would indicate that complete allograft tolerance is not uniformly achieved. Therefore, some form of concomitant immunosuppression is necessary in human transplantation until such time as a means of defining functional tolerance (other than the complete cessation of all immunosuppression with the continued survival ofthe allograft) is available.

Clinical Trial With Donor Bone Marrow and ALG at the University of Alabama Hospital The organization of the transplantation services at the University of Alabama at Birmingham has been conducive to the initiation of a large scale clinical trial of tolerance induction in renal transplantation. An independent organ procurement agency. the Alabama Organ Center. provides the institution with cadaver organs for transplantation. We are the sole renal transplant program in our procurement area, and most kidneys that are made available to us are used locally. A total of245 renal allografts were done at our center in 1989. and 79 vascular organ donors were managed and procured by our program. This system has allowed us to obtain donor bone marrow from a relatively high percentage of organ donors, and has facilitated the production of a controlled trial in which the kidneys from a single donor are transplanted into two local recipients on similar immunosuppressive protocols. one being entered into the bone marrow transfusion group and the other into the control group. On August 23. 1987, bone marrow was obtained by aspiration through multiple needle punctures from the iliac crests of a 16-year-old female organ

donor who had died in an automobile accident. The heparinized bone marrow was returned to the labo., ratory, washed. and cryopreserved. On the follOWing day, both kidneys were transplanted. The recipient of the right kidney was a 20-year-old man with end stage renal disease of uncertain etiology who had been on hemodialysis for approximately 12 months. The transplant functioned immediately, and his initial immunosuppression consisted of prednisone 30 mg daily, azathioprine 25 mg daily, and Minne_ sota antilymphoblast globulin (MALG) 20 mg/kg intravenously from the first through the l Oth postojs., erative days. Cyclosporin A was instituted at 10 rng/kg on the fourth postoperative day. The renal function rapidly stabilized with daily serum crear; inines of approximately 1.7 mg/dL. On the 15th postoperative day. 5 days after cessation of MALG, the patient's creatinine rose from 1.7 to 2.0. On this day he received a transfusion of cryopreserved donor bone marrow. which was thawed and immedj; ately transfused by standard blood administration sets without further preparation. Because of the noted rise in the creatinine. a renal biopsy was done that showed mild acute cellular rejection. On the day after marrow transfusion. the patient was begun on high dose intravenous methylprednisolone, to which he responded in a favorable fashion. At the time of his hospital discharge. 4 weeks after transplantation, he had satisfactory renal function with a creatinine of 1.9. Maintenance immunosuppression at that time included cyclosporin A. azathioprine. and pred, nisone. By 3 months after transplantation, his preel, nisone dose had been tapered to 0 and he has since been maintained without evidence of rejection and with stable renal function on no corticosteroids. The patient has returned to full-time employment. Since this initial patient was transplanted. a total of 51 patients have been entered into the protOCol and have received donor-specific bone marrow trans; fusions. The early results showing the feasibility of this immunosuppressive regimen have been reported."

Bone Marrow Procurement After routine skin preparation and surgical draping for cadaver organ procurement. bone marrow is harvested immediately after completion ofthe initial incisions and cephalad retraction of the abdominal viscera. Procurement usually precedes dissection Of other organs. The bone marrow is obtained from the iliac crests of heart-beating cadaver organ donors by

DonorBone Marrowin Renal Allografts

multiple aspirations into heparinized syringes using disposable Jamshidi needles. In most cases, approximately I L of marrow aspirate can be obtained. The heparinized marrow is placed on ice pending completion of other organ and tissue procurement and is then transported to the laboratory for further processing. On those occasions when permission has been obtained for bone procurement, ribs are obtained as an alternative marrow source and can be procured after completion ofthe removal ofvascularized organs.

Cryopreservation of Bone Marrow Under sterile conditions, donor ribs are cut into 2 to 3 cm segments, and the contained marrow is expressed into RPMI 1640 medium containing 10% fetal calf serum. This process is facilitated by the use of a specially made device that resembles the geardriven rollers of a sugarcane mill. The rib marrow and the iliac crest aspirates are filtered through wire screens to remove bone spicules and are washed twice in medium. Typical cell yields from I L of bone marrow aspirate are 2.1 x 10 10 viable nucleated cells, and from six donor ribs, 1.8 X 10 10 viable nucleated cells. The donor rib and iliac crest samples are then placed in a cryoprotectant mixture containing RPMI 1640 with a final concentration of 10% fetal calf serum and 10% dimethylsulfoxide. Aliquots of 200 mL are sealed in freezing bags and frozen in a programable controlled rate unit with temperature reduction at 1°C per minute to -40°C, after which the samples are placed in the vapor phase of liquid nitrogen for storage. Bone marrow samples were processed and cryopreserved within 8 hours of procurement.

Immunosuppression A sequential protocol using an induction course of MALG was combined with cyclosporin, azathioprine, and prednisone." In the bone marrow group, beginning the day after transplantation, a 10- to 14-day course of MALG was given. Cryopreserved bone marrow was transfused approximately 7 days after cessation ofMALG treatment. The first 20 bone marrow recipients received cyclosphosphamide, beginning the day after marrow transfusion for 14 days," after which time azathioprine was again given. Immunosuppression for the control group recipients of the contralateral kidney from the marrow donor was similar, with the exception of those

71

marrow patients who received cyclosphosphamide. We have now eliminated cyclosphosphamide from the protocol. Maintenance doses ofimmunosuppression for both groups included cyclosporin, 3 to 7 mg/kg, azathioprine 1.25 mg/kg/d, and prednisone 0.125 mg/kg/d. Patients enrolled in the bone marrow protocol with stable renal function have prednisone tapered by 5 mg per week, beginning approximately 3 months after transplantation. Prednisone is then usually discontinued 4 to 6 months after transplantation. Episodes of acute rejection are diagnosed by a combination of clinical symptoms, an increase in the baseline serum creatinine of more than 0.5 mg/dL above baseline, diminished renal plasma flow with increased cortical retention on radionuclide scan, or by renal biopsy. Rejection is treated with pulse intravenous methylprednisolone, I g on 3 consecutive days, often with a concomitant increase in oral prednisone to 5 mg/kg. This is tapered over a period of 10 to 20 days to a level of 15 to 20 mg/d. Patients whose renal function does not improve with steroid therapy receive OKT3 as a means of rescue from steroid-resistant rejection, at a dose of 5 mg/d for 10 to 14 days.

Infusion of Donor Bone Marrow Before marrow infusion, recipients are hydrated with lactated Ringer's solution containing 2.5% mannitol. This infusion is continued throughout the course of the transfusion and for several hours thereafter. Aliquots of bone marrow are rapidly thawed in a 37°C waterbath and transferred into blood administration sets. The marrow solution is then transfused intravenously to the recipient within a half-hour of thawing. The initial 10 patients enrolled in the protocol received cryopreserved marrow that contained the entire volume of cryoprotective solution. There was also a considerable amount of hemoglobulin and red blood cell debris present, and renal dysfunction with a histologic pattern of acute tubular necrosis occurred in six of these patients. Subsequently, patients have received bone marrow that has been washed once in RPMI to remove the cryoprotectant and cellular debris. There is approximately a 10% cell loss during the washing process. Nucleated cells remained 85% to 90% viable during the preservation process. The dose of cells given to each recipient is 2 to 3 X 108 viable nucleated cells per kilogram of body weight.

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W. Henry Barber

Long-Term Results

Bone

Thus far, 51 patients have received a cadaver donor kidney allograft and have been transfused with donor specific bone marrow. The median follow-up time posttransplantation is 16 months, with a range of 3 to 27 months. A kidney from three of the marrow donors was exported to a six-antigen matched recipient at another transplant center; therefore, 48 patients have been entered into the control group who received the contralateral kidney and no marrow transfusion. The demographic data of the bone marrow and the control groups of patients listed in Table 1 are representative of our overall population of renal transplant recipients. A total of six allografts have been lost in the bone marrow group. Two losses have been due to cardiovascular deaths in two diabetic patients who had satisfactory allograft function at the time of death. A single patient experienced the rapid recurrence of focal sclerosis in the allograft, which was lost due to recurrent disease. There have been three irreversible acute rejection episodes. One of these graft losses was in a patient who had become noncompliant and had discontinued his immunosuppressive medications. In the control group, sixteen grafts have been lost. There have been three patient deaths: two from cardiovascular disease and one from viral sepsis. Thirteen transplants have had irreversible acute rejection episodes. The allograft survival curves for the bone marrow recipients, contralateral control patients, and our overall renal transplant experience with patients using quadruple immunosuppressive therapy during the same time period is depicted in Fig 1. Allograft survival of the patients receiving donor bone marrow transfusions is significantly improved compared with the other two groups. The crude allograft survival rate for bone marrow recipients at 18 months is 88% (confidence limits, 85 to 94) and for the control group patients is 67% (confidence Table 1. Characteristics of Donor Marrow Transfused Renal Allograft Recipients Versus Control Group Patients

Characteristics Graft losses (all causes) Deaths Black White Diabetes > 1 Transplant

Bone Marrow (N = 51)

Control (N = 48)

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31

32

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Figure 1. Bone, bone marrow transfused renal allograft recipients. Control, recipients of the con; tralateral kidney, no marrow transfusion. Quad concurrent quadruple therapy cadaver kidney recip: ients, Statistical significance determined by Gehan , Wilcoxon analysis. limits 58 to 74). Although allograft survival in the bone marrow recipients is superior. the overall number of rejection episodes is not significantly different between the two groups. This is primarily due to a relatively high incidence of early aCUte rejection before the transfusion of bone marrow either during or shortly after cessation of MALG therapy. The two groups are also compared for differences in serum creatinine. estimated renal plasma Row, glomerular filtration rate, and urinary protein. There is no significant difference in the function of surviving allografts at 12 months. Although no apparent improvement in renal function is noted by the above parameters, perhaps impor_ tautly, five patients in the control group have biopsy_ proven chronic rejection compared with one patient in the bone marrow group.

Immunologic Mechanisms Complete allograft tolerance, as judged by allograft survival without maintenance immunosuppression, is clearly not achieved in all cases after bone marro~ transfusion. One patient in our experience and one in Monaco et al's study" experienced irreversible acute rejection after cessation of immunosuppres_ sive therapy. Both of these individuals preViously had excellent and stable allograft function. We haVe also noted rejection episodes occurring in patients maintained on apparently adequate immunosuppres_ sive protocols. However, we have been able to taper and discontinue prednisone in 17 patients in the bone marrow group. Three of these have had mild rejection episodes. Thus, in the majority of the patients in the bone marrow group in whom prec:l_

73

Donor BoneMarrow in RenalAllografts

nisone is stopped, allograft function is maintained. Since no patients in the control group had prednisone discontinued, the issue of whether the majority of patients on triple immunosuppressive therapy with stable renal function can have steroids discontinued remains unanswered. Clinical trials are underway at several institutions to address this question. There are currently no in vitro assays capable of predicting the existence of functional allograft tolerance." Until such methods are developed, clinical trials of the induction of allograft tolerance will require the use of concomitant maintenance immunosuppression. We have done mixed lymphocyte cultures in a standard one-way system, 6 to 12 months after transplantation in both bone marrow transfused patients and in the contralateral control group. The results are shown in Fig 2 and are expressed as a percentage of the relative response to third party controls. All patients had normal third party responses. The proliferative response was specifically suppressed to a significant (P < .004) degree in the bone marrow recipients compared with the control group. All patients were noted to have some degree of specific suppression. The amount of mixed lymphocyte culture reactivity does not appear to correlate with the number of previous rejection episodes in these patients. The mechanisms of immune regulation responsi-

ble for allograft maintenance in ALG-bone marrow transfused recipients have not been elucidated in humans. Studies in experimental animals have shown that several factors may be involved. The presence of adoptively transferable suppressor T cells has been demonstrated in the spleens of ALS-treated marrow transfused animals," and at least a subpopulation of these cells appear to be of donor origin." In agreement with this observation, Thomas et al" have found persisting nodules ofFc IgG-receptor positive CD8 positive cells in long-term surviving bone marrow-treated primates and have proposed that a veto-type mechanism":" may be active. It was established by Liegeois et al that skin grafted mice treated with ALS and donor bone marrow display a state of microchimerism with long-surviving donor type cells being present on the order of 1% in the spleens ofthese animals." Whether or not the production of local" or systemic chimerism is necessary for long-term allograft unresponsiveness in the ALS-bone marrow model has not been established. However, the establishment of allogenic chimerism per se is not adequate in all cases to render recipients unresponsive to vascularized allografts.":" Animals with stable allografts in the ALS-bone marrow protocol show markedly depressed mixed lymphocyte reactivity and do not generate lympho-

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Figure 2. Mixed lymphocyte reactivity of bone marrow and control recipients. Mixed lymphocyte cultures were done in a standard fashion using cryopreserved responder and stimulator cells. Stimulators were treated with mitomycin-C. The responses are calculated as a percentage of the relative response to donor versus third party. Recipient samples were taken 6 to 12 months posttransplant. All third party responses were within the normal range. The mean relative response ± SEM for bone marrow patients was 20.1 ± 5.7 and for control patients was 52.0 ± 7.8. Bars show the SD. Comparison by a two-tailed paired t test shows a P value of .004.

74

W. Henry Barb"

eyre-mediated cytotoxIcIty to donor antigens. In spite of the abrogation of the early proliferative response in mixed lymphocyte culture, spleen cells from unresponsive mice are capable of differentiating into effector cells demonstrating cytotoxic activity upon resensitization to donor specific alloantigens . The presence ofthis cell population shows that the specific unresponsiveness is not due to clonal deletion." It is likely that a comb ination of immune mechanisms are involved in the development of specific unresponsiveness in ALS-donor bone marrow treated allograft recipients. The relative contributions of alloantigen spec ific suppressor cells, the development of clonal anergy, antigen reactive cell opsonization," or anti-idiotypic cellular and antibody networks remains to be determined.

Bone Marrow Inoculum In our clinical trial of ALS and donor bone marrow treatment, we have used the entire cell population that was present in the cryopreserved marrow preparations. However, the characteristics of the cell in the bone marrow responsible for the induction of unresponsiveness have been studied. In mice, it has been shown that the whole marrow can be fractionated by continuous or discontinuous density gradient centrifugation, and an active fraction can be obtained. The cells in this fraction are small mononuclear cells and are Ia negative, Thy-I negative; however, they are Fc gamma receptor positive .r'''" Interestingly, this cell would appear to correlate with the population ofdonor origin cells shown to be present in nodules in long-term surviving primate allografts treated with ALS and marrow." Importantly, this same active fraction has been found in the peripheral blood and, thus, may expand the availability of appropriate donor antigen in human cadaver organ donors.f: "

Other Immunosuppressive Agents The use of concurrent nonspecific immunosuppression is presently a requirement in human trials of tolerance induction using the ALS-donor bone marrow model. While the commonly used drugs azathioprine, cyclosporin, and prednisone have not been investigated in detail regarding their effect on tolerance induction, it would appear that Cyclosporin A fortuitously has a beneficial effect when given to both mice and primates using this model. 99 , l oo The action of azathioprine is uncertain .!" However, it is clear that other agents, such as cyclophosphamide, may

have both beneficial and detrimental influences on tolerance induction, based on the dosage and timing of administration." The influence of conventional nonspecific immunosuppression is being actively investigated in a canine model in my laboratory. In add ition , newer immunosuppressive drugs, such as FK506, lOl.105 may prove to be useful in tolerance induction protocols. There appears to be general agreement that the most satisfactory current method of tolerance induc_ tion in immunocompetent adults is by the use of transient , profound immunosuppression with subse_ quent exposure to alloantigenic stimulation, either via the transplanted organ or by the administration of other forms of donor-specific transplantation antigens. In our clinical study, we have used a polyclonal anti-human lymphoblast preparation for immunologic modulation before antigen adrninisn-g; tion in the form of donor bone marrow. Unfor nj., natel y, there have been numerous early rejection episodes, even with the use of concomitant chemical immunosuppression. Polyc1onalreagents seem to be somewhat variable in their immunosuppressive effects; therefore, a monoclonal preparation such as OKT3 may in the future be found to have clinical usefulness in tolerance induction. Currently, the most promising antibodies would appear to be those of the anti-CD4 group, which have proved to be useful both alone and in combination with donor antigen in organ allograft prolongation.!" Total lymphoid irradiation (fLI) has been shOwn to be a potent immunosuppressant modality. When used preoperatively, TLI is capable of inducing functional allograft tolerance in human cadaveric renal allograft recipients, albeit in a minority ofthose patients thus treated.!" The combined use of pre_ transplant TLI and donor bone marrow adminisn-g., tion has been found to be highly effective in the ind uction of tolerance to allografts in rodents. 106. 10' On the other hand, similar protocols have been far less successful in producing indefinite allograft SUr_ vival in large outbred species. 9 1.9'l . IOM , 109 As mentioned previously, the pretransplaru use of TLI greatly limits its usefulness for large scale application in human cadaver organ transplantation. The recent demonstration that treatment posttransplantatio n with both TLI and anti -L3T4 antibody produces a synergistic prolongation of allograft survival is an important observation and may extend the clinical usefulness of TLl . " O We have experiments in progress to determine the usefulness of ALS, TLI. and donor bone marrow administration posttrans_ plantation in a canine model.

Donor BoneMarrow in Renal Allografts

Conclusions Patients with end stage organ failure require either transplantation or organ replacement therapy for the remainder of their lives. Recent advances in immunosuppressive treatment have led to greatly improved early patient and allograft survival; however, the long-term results remain decidedly suboptimal. With current treatment modalities, we can not reasonably expect that a child or young adult can maintain satisfactory allograft function free from the side effects of bone disease, infections, and neoplasia that occur with the indefinite use of nonspecific immunosuppression, as is now necessary for continued allograft function. While the development of improved and more specific chemical agents may lead to beneficial survival results in the short-term, it is likely that any drug capable of promoting a high rate of allograft acceptance will be plagued by the development of unacceptable complications with long-term use. Therefore, we have undertaken a clinical trial to examine the usefulness of ALS and donor bone marrow, as developed by Monaco and Wood", for the induction of tolerance or partial tolerance in cadaveric renal allograft recipients. The goals of this treatment have been not only to improve allograft survival, but to decrease the amount of immunosuppressive medications needed for chronic therapy. We have shown that renal allograft recipients who receive donor bone marrow transfusions have improved allograft survival compared with patients treated with similar immunosuppressive protocols with no marrow transfusion. Most bone marrow transfused patients can have steroid treatment withdrawn within the first year posttransplantation. The marrow transfused group also demonstrates significantly less specific anti-donor reactivity in mixed lymphocyte cultures. While these results are encouraging, there are several problems and questions that remain to be answered. Many of our patients experienced early rejection episodes, either during or shortly after cessation of antilymphocyte therapy. Therefore, it is clear that a more effective form of inductive treatment would be beneficial. Also, the transfusion of donor-specific bone marrow has not uniformly led to allograft tolerance in that rejection episodes have been seen to occur, albeit infrequently, after the infusion of marrow. Although we have been able to withdraw prednisone in a number of these patients, we have no reliable method of testing recipients for the presence or absence of functional allograft tolerance. The relationship of

75

other immunosuppressive agents to the induction of tolerance in the ALS-donor bone marrow protocol needs further clarification, as does the optimal dosage and timing of administration of the marrow antigen. The use ofother immunomodulatory agents, such as monoclonal antibodies or TLI with or without polyclonal ALS, are also possible avenues that may lead to improvements in the success oftolerance induction. These questions form the basis for experimental approaches in our unit and will hopefully lead to future improvements in clinical transplantation.

References 1. Stratta Rj, Oh CS, Sollinger HW, et al: Kidney

retransplantation in the cyclosporine era. Transplantation 1988,45:40 2. Gordon RD, Starzl TE. Hakala Rj, et al: Long-term results of cyclosporine-steroid therapy in 131 nonmatched cadaveric renal transplants. Clin Transplant 1987, 1:44 3. Simmons RL, Canafax OM, Fryd OS, et al: New immunosuppressive drug combinations for mismatched related and cadaveric renal transplantation. Transplant Proc 1986, 18:76 4. Najarian jS, Strand M, Fryd OS, et al: A single institution randomized prospective trial of cyclosporine versus azathioprine-antilymphocyte globulin for immunosuppression in renal allograft recipients.Ann Surgery 1985,201:142 5. Clunie GjA: Skin cancer in caucasian renal allograft recipients living in a subtropical climate. Surgery 1980,87:117 6. MahonyJF, Sheil AGR, Etheredge SB: Delayedcomplications of renal transplantation and their prevention. Medj Australia 1982,2:426 7. Weir M, Kirkman RL, Strom TB, et al: Liver disease in recipients of long functioning renal allografts. Kidney Int 1985,28:839 8. Kelly GE, Mahony jF, Sheil AGR, et al: Risk factors for skin carcinogenesis in immunosuppressed kidney transplant recipients. Clin Transplant 1987, 1:271 9. Rao KV,Anderson RC: Long-term resultsand complications in renal transplant recipients. Transplantation 1988,45:45 10. Opelz G, Terasaki PI: Improvement of kidney-graft survival with increased numbers of blood transfusions. N Engl] Med 1978, 12:799 11. Opelz G, Terasaki PI: Dominant effectoftransfusions on kidney graft survival. Transplantation 1980, 29: 153 12. Ting A, Morris PJ: The influence of HLA-A, Band OR matching and pregraft blood transfusions on graft and patient survival after renal transplantation in a singlecenter. Tissue Antigens 1984, 24:256 13. Terasaki PI: The beneficial transfusion effect on kidney graft survival attributed to clonal deletion. Transplantation 1984, 37:119 14. Opelz G: Current relevance of the transfusion effect

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in renal transplantation. Transplant Proc 1985. 17: 1015 15. Sanfilippo F. Vaughn WK, LeFor WM, et al: Multivariate analysis of risk factors in cadaver donor kidney transplantation . Transplantation 1986.42:28 16. Kerman RH. VanBuren CT . Lewis R~f. et al: Successful rransplantation of 100 untransfused cyclosporinetreated primary recipients of cadaveric renal allografts. Transplantation 1988.45:37 17. Opelz G: Improved kidney graft survival in nontransfused recipients. Transplant Proc 1987. 19:149 18. Lundgren G, Albrechtsen D. Brynger H. et al: Role of blood transfusions and HLA matching in cyclosporine treated renal transplant recipients. Transplant Proc 1987. 19:1248 19. Salvatierra 0, Vincinti F, Amend W. et al: Deliberate donor specific blood transfusions prior to living related renal transplantation. Ann Surg 1980. 192:543 20. Anderson CB. Sicard GA, Etheredge EE: Pretreatment of renal allograft recipients with azathioprine and donor specific blood products. Surgery 1982. 92:315 21. Whelchel JD. Shaw JF. Curtis JJ. et al: Effect of pretransplant stored donor-specific blood transfusion on early renal allograft survival in one-haplotype living related transplants. Transplantation 1982. 34: 326 22. Salvatierra 0, Iwaki Y, Vincinti F, et al: Incidence. characteristics and outcome of recipients sensitized after donor-specific blood transfusions. Transplantation 1981.32:528 23. Ettenger RB, Jordan SC. Arnett J . et al: Specific anti-donor lymphocyrotoxic antibodies following blood transfusion from non-related donors. Transplant Proc 1982, 14:347 24. Glass NR, Miller DT, Sollinger HW, et al: Comparative analysis of the DST and Immuran-plus-DST protocols for live donor renal transplantation. Transplantation 1983.36:636 25. Ettenger R. Kerman R. Arnett J. et al: Sensitization following donor-specific transfusions for living related renal transplantation. Transplant Proc 1983. 15:943 26. Leivestad T. Flatrnark A. Thorsby E: Transplants of kidneys from one-haplotype mismatched living related donors. Experience with donor-specific transfusions and cydosporine. Transplant Proc 1985. 17: 2679 27. Sommer BG, Ferfuson RM: Mismatched living related donor renal transplantation: A prospective randomized study. Surgery 1985.98:267 28. Sommer BG. Bowers VD. Henry ML. et al: Mismatched living related donor transplantation: Donor specific transfusions vs. cyclosporine, Clin Transplant 1988.2:36 29. Medawar PB: The behaviour and fate of skin autografts and skin homografts in rabbits . J Anat 1944. 78:176 30. Billingham RE. Brent L, Medawar PB: Actively acquired tolerance of foreign cells. Nature 1953. 172: 603 31. Owen RD: Dizygotic cattle bear erythrocytes from the twin. Science 1945. 102:460

32 . Owen RD. Davis HB. Morgan RF: Quintuplet calves and erythrocyte mosaicism. J Hered 1946.37:291 33. Anderson D. Billingham RE. Lampkin GH. et al: Grafts between dizygotic cattle twins accepted. Hered_ ity 1951. 5:379 34. Shapiro F. Martinez C. Smith J~f, et al: Tolerance of skin homografts induced in adult mice by multiple injections of homologous spleen cells. Proc Soc Exp Bioi Med 1961. 106:472 35. French ME. Batchelor JR: Immunological enhance_ mem of rat kidney allografts. Lancer 1969. 2: 1103 36 . Stuart FP. Saitoh T . Fitch FW: Rejection of renal allografts:Specific Immunologic suppression. Science 1968,160:146:3 37. Marquet RL. van Es AA. Heystek GA. et al: Attempts to induce immunological enhancement for kidney allografts in rhesus monkeys . Transplantation 1978, 25:188 38. Jeekel J. Obertop H. Vriesendorp H. et al: Effect of anti-donor serum (ADS) and donor cells on renal allograft survival in DL-A tissue-typed littermate bea; gles. Transplant Proc 1975, 7:435 39. Hutchinson IV. Brent L: Immunological enhance_ ment of tumour allografts following treatment or mice with TNP-conjugated alloantigen and anti-TNp antibody. Nature 1981, 292:353 40. Hutchinson IV. Barber WHo Brent L: Enhancement of tumor skin and kidney allografts using TNp_ conjugated alloantigens and anti-TNP antibody. Transplant Proc 1983. 15:819 41. Barber WHo Hutchinson IV. Morris PJ: Enhance_ ment of rat kidney allografts using haptenated alloan_ tigens and anti-hapten antibody. Transplantation 1983.36:475 42. Hutchinson IV. Barber WHo Morris PJ: Specific suppression of allograft rejection by trinitrophenYI (TNP)-induced suppressor cells in recipients treated with TNP.haptenated donor alloantigens. J Exp Med 1985.162:1409 43. Medawar PB: The use of antigenic tissue extracts to weaken the immunological reaction against skin ho, mografts in mice. Transplantation 1963,1:21 44. Kim J P, Shaipanich T. Sells RA. et al: Active enhance_ ment of rat renal allografts with soluble splenic antigen. Transplantation 1972. 13:322 45. Brent L. Hansen JA. Kilshaw PJ. et al: Specific unresponsiveness to skin allografts in mice. I. Proper_ ties of tissue extracts and their synergistic effect With antilymphocyte serum. Transplantation 1973. 15: 160 46. Foster S, Cranston D, Wood KJ, et al: Production or indefinite renal allograft survival in the rat by pretreat_ ment with viable and non-viable hepatocytes or liver membrane extracts. Transplantation 1988.45:228 47. Stark RB, Dwyer E: The enhancement of homografts of skin in the adult rabbit using elements of homologous whole blood. Surgery 1959.46:277 48. Marquet RL. Heysteck GA. Tinbergen WJ: Specific inhibition of organ allograft rejection hy donor blOOd. Transplant Proc 1971. 3:708 49. FabreJW. Morris PJ: The effect of donor strain blOOd pretreatment on renal allograft rejection in rats Transplantation 1972. 14:608 . 50. Hibberd AD, Scott LJ: Allogeneic platelets increase

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the survival of rat renal allografts. Transplantation 1983, 35:622 51. Bitter-Suerman H, Shevach EM: Induction of transplantation tolerance in guinea pigs by spleen allografts. III. Transfer of tolerance to normal hosts. Transplantation 1983, 36: 161 52. Halasz NA, OrioffMJ, Hirose F: Increased survival of renal homografts in dogs after injection of graft donor blood. Transplantation 1964,2:453 53. Zimmerman CE, Busch GJ, Stuart FP, et al: Canine renal homografts after pretreatment with subcellular splenic antigens. Surgery 1968,63:437 54. Holl-Allen RTJ, Scharli AF, Maggs PR, et al: Active enhancement of canine renal allografts using solubilized lymphocyte antigen. Transplantation 1970, 10: 472 55. Rowinski W, Ryffa T, Ruke M, et al: Platelet-induced donor-specific unresponsiveness to kidney allografts in mongrel dogs treated with ALG. Transplant Proc 1981,13:586 56. Kilshaw PJ, Brent L, Thomas AL: Specific unresponsiveness to skin allografts in mice. II. The mechanism of unresponsiveness induced by tissue extracts and antilymphocytic serum. Transplantation 1974, 17:57 57. Floershiem GL: A study of combined treatment with chemical immunosuppressants and antilymphocyte serum to prolong skin allograft survival. Transplantation 1969,8:392 58. Floershiem GL: Induction of unresponsiveness to skin and heart allografts in mice by a synergistic treatment with procarbazine, antilymphocyte serum and donor-type cells. Transplantation 1973, 15:195 59. Brent L, Opara SC: Specific unresponsiveness to skin allografts in mice: V. Synergy between donor tissue extract, procarbazine hydrochloride and antilymphocyte serum in creating a longlasting unresponsiveness mediated by suppressor cells. Transplantation 1979, 27:120 60. Al-Mahdi N, Hutchinson IV, Brent L: Specific unresponsiveness to fully allogeneic kidney allografts in rats induced by procarbazine hydrochloride and antilymphocyte serum. Transplantation 1983,36:480 61. Homan WP, Williams KA,Millard PR, et al: Prolongation of renal allograft survival in the rat by pretreatment with donor antigen and cyclosporin A. Transplantation 1981,31 :423 62. Yasamura T, Kahan BD: Prolongation of rat kidney allografts by pretransplant administration of donor antigen extract or whole blood transfusion combined with a short course of cyclosporine. Transplantion 1983,36:603 63. Strober S, Slavin S, Gottlieb M, et al: Allograft tolerance after total lymphoid irradiation (fLI). Immunol Rev 1979, 46:87 64. Bieber CP, Jamieson S, Raney A, et al: Cardiac allograft survival in rhesus primates treated with combined total lymphoid irradiation and rabbit antilymphocyte globulin. Transplantation 1979,28:347 65. Sampson D, Levin BS, Hoppe RT, et al: Preliminary observations on the use oftotallymphoid irradiation, rabbit anti-thymocyte globulin, and low dose prednisone in human cadaver renal transplantation. Transplant Proc 1985,17:1299 66. MyburghJA, Meyers AM, BothaJR, et al: Wide field

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low-dose total lymphoid irradiation in clinical kidney transplantation. Transplant Proc 1987, 19:1974 67. Rapaport FT, Watanabe K, Cannon FD, et al: Histocompatibility studies in a closely bred colony of dogs. J Exp Med 1972, 136:1080 68. Blumenstock DA, Cannon FD: Prolonged survival of lung allografts in nonidentical beagles by treatment with lethal total-body irradiation, autologous bone marrow transplantation, and methotrexate. Transplantation 1978, 26:203 69. Norin AJ, Goodell EM, Kamholz SL, et al: Immunologic, morphologic, and functional evaluation oflongterm-surviving beagle lung allograft recipients treated with lethal total-body irradiation, autologous bone marrow, and methotrexate. Transplantation 1987, 44:179 70. Monaco AP, Wood ML, Russell PS: Studies on heterologous anti-lymphocyte serum in mice. III. Immunologic tolerance and chimerism produced across the H-2 locus with adult thymectomy and anti-lymphocyte serum. Ann NY Acad Sci 1966, 129: 190 71. Lance EM, Medawar PB: Quantitative studies on tissue transplantation immunity. IX. Induction of tolerance with antilymphocyte serum. Prog R Soc Lond [Bioi) 1969, 173:447 72. Monaco AP, Wood ML: Studies on heterologous antilymphocyte serum in mice. VII. Optimal cellular antigen for induction of immunologic tolerance with ALS. Transplant Proc 1970,2:489 73. Caridis DT, Liegeois A, Barrett I, et al: Enhanced survival ofcanine renal allografts ofALS-treated dogs given bone marrow. Transplant Proc 1973,5:671 74. Thomas FT, Carver FM, Foil MB, et al: Long-term incompatible kidney survival in outbred higher primates without chronic immunosuppression. Ann Surg 1983, 198:370 75. Monaco AP, Wood ML: The potential for induction of specific unresponsiveness to organ allografts in clinical transplantation. Heart Transplantation 1982, I: 257 76. Monaco AP, Wood ML, Maki T, et al: The use of donor-specific antigen for the induction of immunologic unresponsiveness to experimental and clinical allografts. Transplant Proc 1988, 20: 122 (sup pi 2) 77. Monaco AP, Wood ML, Maki T, et al: Future strategies in immunosuppression: Problems and potential for the induction of specific unresponsiveness to organ allografts in clinical transplantation. Transplant Proc 1989,21:3939 78. Monaco AP, Wood ML, Maki T, et al: Attempt to induce unresponsiveness to human renal allografts with antilymphocyte globulin and donor-specific bone marrow. Transplant Proc 1985, 17:1312 79. Hartner WC, DeFazio SR, Maki T, et al: Prolongation of renal allograft survival in antilymphocyte-serumtreated dogs by postoperative injection of densitygradient-fractionated donor bone marrow. Transplantation 1986,42:593 80. Monaco AP, Clark AW, Wood ML, et al: Possible active enhancement of a human cadaver renal allograft with antilymphocyte serum (ALS) and donor bone marrow: Case report of an initial attempt. Surgery 1976, 79:384 81. Barber WH, Diethelm AG, Laskow DA, et al: Use of

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cryopreserved donor bone marrow in cadaver kidney allograft recipients. Transplantation 1989.47:66 Deierhoi MH, Sollinger HW , Kalayoglu M, et al: Quadruple immunosuppression in 305 consecutive cadaver renal allografts. Clin Transplant 1987, 1:71 Wood ML, Monaco AP: The effect of cyclophosphamide on the specific unresponsiveness to skin allografts induced in ALS-treated mice infused with donor bone marrow.J Immunol 1977, 118: 1456 Streilein JW, Strome P, Wood PJ: Failure of in vitro assays to predict accurately the existence of neonatally induced H-2 tolerance. Transplantation 1989. 48:630 Wood ML, Monaco AP: Adoptive transfer of specific unresponsiveness to skin allografts by spleen cells from ALS-treated, marrow-injected mice. Transplant Proc 1979, 11: 1023 Maki T, Gottschalk R, Wood M, et al: Specific unresponsiveness to skin allografts in anti-lymphocyte serum-treated, marrow injected mice: Participation of donor marrow derived suppressor T cells. J Immuno11981,127 :1433 Thomas J , Carver M, Cunningham P, et al : Promotion of incompatible allog raft acceptance in rhesus monkeys given posttransplant antithymocyte globulin and bone marrow. Transplantation 1987. 43:332 Rammensee H, Fink P. Bevan M : The veto concept: An economic system for maintaining self tolerance of cytotoxic T lymphocytes. Transplant Proc 1985, 17: 689 Muraoka S, Miller R: Cells in bone marrow and in T cell colonies grown from bone marrow can suppress generation of cytotoxic T lymphocytes directed against their self antigens. J Exp Med 1980, 152:54 Liegeois A, Charriere J , Brennan JL: Allograft enhancement induced by bone marrow cells. Surg Forum 1974,25:297 Koretz SH, Gottlieb S, Strober S, et al: Organ transplantation in mongrel dogs using total lymphoid irradiation (TLl). Transplant Proc 1981, 13:443 Strober S, Modry DL, Hoppe RT, et al: Induction of specific unresponsiveness to heart allografts in mongrel dogs treated with total lymphoid irradiation and anti-thymocyte globulin. J Immunol 1984, 132: 1013 Hutchinson IV. Zola H: Antigen-reactive cell opsonization (ARCO). Transplantation 1977.23:464 Gozzo J, Crowley M, Maki T , et al: Functional characteristics of a ficoll-separated mou se bone marrow cell population involved in skin allograft prolongation. J Immuno11982,129:1584 De Fazio S, Hartner W. Monaco A, et al: Mouse skin graft prolongation with donor strain bone marrow and antilymphocyte serum: Surface markers of the active bone marrow cells. J Immunol 1985, 135:3035 De Fazio SR, Kowolenko M, Monaco AP, et al: Isolation by continuous density centrifugation and characterization of bone marrow cells active in prolonging allograft survival in antilymphocyte serum (ALS)-treated mice. Transplant Proc 1987. 19:547

97 . Gozzo JJ . Hartner WC, Monaco AP. et al: Effect of posuransplant injection of peripheral blood lymphocytes on skin graft prolongation in mice treated with antilymphocyte serum (ALS)or ALS plus donor bone marrow. Transplant Proc 1987. 19: 1409 98 . De Fazio SR. Monaco AP, Gozzo JJ: Prolongation of skrn allograft survival in antilymphocyte serurn_ treated mice by posuransplam administration of peripheral blood lymphocytes. Transplantation 1989. 48 :163 99 . Wood ML. Oouschalk R, Monaco AP: The effect of cyclosporine on the induction of unresponsiveness in ALS-treated marrow-injected mice. Transplantation 1988.46:449 100. Thomas J. Carver M. Sash C. et al: Beneficial effect of cyclosporine in posuransplantation induction ofunre_ sponsiveness of renal allografts in rhesus monkeys. Transplant Proc 1988,20: 134 (suppl !) 101. Yosimura N, Matsui S, Hamashima T , et al: A new Immunosuppressive agent, FK506, inhibits the expres_ sion of alloantigen-activated suppressor cells as well as the induction of alloreactivity. Transplant Proc 1989.21 :1045 102. Sanghvi A. Warty VS. Diven WF. et al: Increased cyclosporine uptake by cells pretreated with FK506 and evidence for binding of both drugs to a common int racellular protein . Transplant Proc 1989, 21: I 050 103. StaTZ! TE, Todo S. Fung J, et al : FK506 for liver. kidney, and pancreas transplantation. Lancet 1989. 2:100 104. Madsen JC. Wood KJ, Morris PJ: Induction of spe., cific unresponsiveness to heart grafts by treatment with donor MHC antigen and monoclonal antibody to L3T4 . (manuscript subm itted) 105. Strober S. Dhillon M, Schubert M. et al: Acquired immune tolerance to cadaveric renal allografts-a stud y of three patients treated with total lymphoid irradiation. N EnglJ Med 1989.321 :28 106. Slavin S, Strober S, Fuks Z, et al : Induction of specific tissue transplantation tolerance using fractionated total lymphoid irradiation in adult mice: Long-terrn survival of allogeneic bone marrow and skin grafts. J Exp Med 1977. 146:34 107. Slavin S, Reitz B, Bieber C, et al : Transplantation tolerance in adult rats using total lymphoid irradia_ tion : Permanent survival of skin. heart and marrow allografts.J Exp Med 1978. 147:700 108. Howard RJ, Sutherland DER. Lum CT. et al: Kidney allograft survival in dogs treated with total lymphoid irradiation. Ann Surg 1981. )93 :196 109. Myb urgh JA. Smit JA, Browde S, et al: Current status of total lymphoid irradiation . Transplant Proc 1983 15:659 • 110. Trager OK. Banks BA, Rosenbaum GE. et al : Cardiac allograft prolongauon in mice treated with combined post -transplantation total-lymphoid irradiation and anti-L3T 4 antibody therapy. Transplantation 1989 47 :587 •