Specific Immunosuppression in Cardiac Allografting Using Antithymocyte Sera and Soluble Transplantation Antigen

Transcription

1 Specific Immunosuppression in Cardiac Allografting Using Antithymocyte Sera and Soluble Transplantation Antigen Francis T. Thomas, M.D., J. S. Wolf, M.D., Judith M. Thomas, Ph.D., Richard R. Lower, M.D., Buddy Hudson, and Stephen Burke, B.S. ABSTRACT Two techniques of specific immunosuppression, using antithymocyte globulin (ATG) alone and in combination with soluble transplantation antigen (STA) prior to treatment, were studied in heterotopic outbred canine cardiac alloprafts. Both techniques resulted in prolonged survival of dogs having cardiac allografts with a low incidence of infections and drug toxicity complications as compared to animals treated with Prednisone and Imuran. This study demonstrates the ability of short-course ATG treatment to produce long-term (greater than 2 to 3 months) cardiac allograft survival without chronic immunosuppression. Combined ATG and STA pretreatment gave the longest allograft survival (mean, 93 days) in a small group of animals. This improved allograft survival, associated with a reduction in infections and drug toxicity complications, suggests that these techniques of specific immunosuppression may have the potential for improving survival and reducing the complications of immunosuppressive therapy following cardiac transplantation. C linical transplantation of cadaver organs from nonrelated donors was introduced approximately 10 years ago. During the interim period, considerable progress has been made in the technical aspects of transplantation of kidneys, livers, hearts, and lungs. In particular, kidney transplantation has now become an established therapeutic technique which offers the recipient the potential of full rehabilitation from end-stage organ failure. Cardiac transplantation has met with limited success, and the clinical results of transplantation done by the Stanford University group have indicated that this procedure does offer some degree of palliation to patients who otherwise have no potential for rehabilitation [5]. The experience with clinical transplantation of the human kidney now encompasses more than 13,000 procedures, and valuable lessons can be learned from this experience which may be applicable to transplantation of From the Medical College of Virginia, Virginia Commonwealth University, Richmond, Va. Presented at the Tenth Anniversary Meeting of The Society of Thoracic Surgeons, Los Angeles, Calif., Jan , Address reprint requests to Dr. F. T. Thomas, Medical College of Virginia, Virginia Commonwealth University, MCV Station, Richmnd, Va

2 THOMAS ET AL. other organs. The standard immunosuppressive therapy in clinical transplantation today is Imuran and Prednisone given on a long-term basis. The use of this nonspecific immunosuppressive therapy and inadequate measures to prevent rejection are responsible for a high incidence of infectious complications and loss of graft function in present-day transplantation. In general, current therapies lack the ability to specifically inhibit the recipient response to donor alloantigens while allowing maintenance of host immunological responses against other antigens encountered in the environment. Thus there is an urgent need in transplantation for clinically feasible regimens of specific immunosuppression. Numerous attempts to induce specific unresponsiveness to allotransplants in laboratory animals have included the technique of treating the adult recipient with viable donor cells or cell extracts. With a view toward the possible future clinical application of such treatment protocols, the use of cellular extracts has a major advantage over viable cells in avoiding serious graft-versus-host complications in the treated recipient. Early efforts using donor lymphoid cell homogenates in mice resulted in prolonged skin allograft survival across weak histocompatibility differences [9]. Subsequently, Medawar [ 101 showed that semisoluble extracts prepared by ultrasonic disruption and administered intravenously could increase survival of murine skin allografts across major histocompatibility barriers. The addition of immunosuppressive drugs to the treatment regimen could facilitate the effect. Other studies using a combination of extracted donor antigen and conventional immunosuppression methods have yielded promising results, not only in murine skin allografts [4, 6, 151 but in canine [3, 181, rabbit [ 141, and porcine [3] renal allografts. Results in canine renal allografts demonstrated that long-term maintenance with Imuran and Prednisone after transplantation was required for significant prolongation of graft survival in animals receiving soluble antigen pretreatment [18]. The advantage of using a more selective agent for adjuvant immunosuppressive treatment was suggested by the studies of Lance and Medawar [8], which showed that a limited course of potent antilymphocyte serum (ALS) administered only during the first week following transplantation could facilitate the induction of long-lasting, specific tolerance to H-2 incompatible skin grafts in adult mice treated with donor-strain lymphoid cells. More recently other workers have demonstrated that a strictly limited treatment regimen of ALS and soluble antigen can produce permanent skin allograft survival in a high proportion of H-2 incompatible mice [2] and significantly prolong skin allograft survival across a strong histocompatibility barrier in rats [17]. The use of a combination of antithymocyte globulin (ATG) and soluble transplantation antigen (STA) to initiate specific immunosuppression in canine recipients is being studied in our laboratories at the present time. Results so far indicate that heterotopic cardiac allograft survival can be 242 THE ANNALS OF THORACIC SURGERY

3 Iminunosuppwssion in Cardiac Allogrufting prolonged by this technique for up to 4 to 5 months and that the treatment does appear to produce some degree of specific immunosuppression since the rate of infectious complications and drug toxicity are low in these animals. To our knowledge, this is the first application of this technique to canine cardiac allografting. Materials and Methods Mongrel dogs weighing 10 to 15 kg. were used as cardiac allograft recipients in various treatment groups. The operative model used was a heterotopic cervical cardiac allograft obtained from unrelated 6- to 8-weekold mongrel puppies. As a large animal organ allograft model it has the advantages of simplicity and a high technical success rate. The heart is placed subcutaneously in the cervical region and is easily accessible to palpation. Palpation is a sensitive indicator of early rejection in that an increase in size and prominent stiffening of the organ are apparent at the onset of rejection prior to detection of ECG voltage changes. The allografts become increasingly stiff until the time of end-stage rejection, when the cardiac beat and ECG voltage cease. Figure 1 illustrates the fall in cervical voltage seen in the heterotopic cardiac allograft during rejection. It should be noted that the rates in these allografts fluctuate from day to day, but normal sinus rhythm is maintained throughout the period of allograft survival. Histological sections were taken to confirm rejection in all cases. Antithymocyte serum was prepared in both horses and rabbits using a two-pulse immunization schedule of puppy thymocytes. The primary injections were given in complete Freund s adjuvant followed by an intravenous booster injection 2 weeks later. All dogs treated with ATS were given a total dose of 150 mg. of gamma globulin per kilogram of body weight administered intramuscularly. ATS was injected in five divided doses on days 0 to 4 (cardiac allografting was done on day 0). Semisoluble canine transplantation antigen extracts were prepared from washed spleen cells suspended in phosphate-buffered saline at ph 7.2. The cells were disrupted in an SlOSA sonicator for 4 minutes at 10 kilohertz with maximal energy applied. A water-cooling system was used to ensure maintenance of a constant temperature of 4 C. during sonication. The soluble extract used for animal pretreatment was centrifuged at 106,000 g for 2 hours. Protein content was determined by the Buiret test, and the average protein concentration in soluble antigen preparations used for injection was 40 mg. per milliliter. Characteristics of the canine solubilized antigen preparations used in these studies are listed below: Protein concentration = 31 T, Lipid concentration = 64% Carbohydrate concentration = 3y0 Soluble at 106,000 g ultracentrifugation

4 THOMAS ET AL. FIG. 1. Fall in cervical ECG age with rejection of heterotopic cardiac transplant. Preparations of STA were made from specific individual donor spleens and also from pools of random donor (more than 5) spleens. Antigen activity in specific donor preparations was demonstrated in vitro by the ability to inhibit cytotoxic activity of specific alloantisera. The antisera used were antidog lymphocyte antigen typing sera (obtained from Dr. Joseph Ferrebee s laboratory in Cooperstown, New York). Specific inhibition of lymphocytotoxic activity was demonstrated by preincubation of STA with cytotoxic antiserum for 30 minutes at room temperature prior to addition of donor lymphocytes and guinea pig complement. Appropriate controls are included for anticomplementary activity. The experimental treatment groups were divided, by treatment protocol, as follows: Group 1-untreated controls (n = 6); Group 2-Imuran, 3 mg. per kilogram of body weight, and Prednisone, 2 to 3 mg. per kilogram of body weight per day (n = 12); Group 3-limited ATS, total dose of 150 mg. per kilogram of body weight administered between days THE ANNALS OF THORACIC SURGERY

5 in Cardiac Allografting and 4 (relative to cardiac allografting at day 0) (n = 11); Group 4-limited ATG (150 mg. per kilogram of body weight) plus injections of donor bone marrow at the time of transplantation (the ATG injections were given at varying times prior to and following transplantation, but always within minus 5 days or plus 5 days relative to the transplant); Group 5-5 to 10 mg. per kilogram of body weight of pooled soluble antigen prepared from at least 5 unrelated mongrel donors injected intravenously over a 2-week period prior to transplantation, followed by 3 injections of antithymocyte globulin after transplantation (n = 5). R esii 1 ts A total of 52 heterotopic cervical cardiac allografts were studied. As shown in Figure 2, the survival of control dogs with cardiac allografts was 6.8 f 3.5 days. Animals given 3 mg. per kilogram of body weight of Imuran and 2 to 3 mg. per kilogram of body weight of Prednisone (Group 2) had an increase in mean graft survival of 16.8 & 6.8 days. The groups receiving only a limited course of ATS, that is, 9 to 5 injections, and no subsequent immunosuppressive treatment (Group 3) had a mean graft survival of 63 f 11.8 days. Animals given limited ATS injections plus bone marrow (Group 4) had a mean cardiac allograft survival of 23 f 8.9 days, which was less than the survival time of the ALS group (p < 0.05), suggesting that some degree of sensitization from the bone marrow injections must have occurred. Furthermore, experimentation with several time-dosage schedules for the bone marrow injections did not result in any apparent satisfactory improvement in the allograft survival rate. In the group receiving soluble antigen pretreatment and limited ATS injections, the mean graft survival was 93 f 18.6 days. As seen in Figures 3 and 4, respectively, the incidence of infectious complications and drug toxicity complications in this group of prolonged cardiac allograft survivals was significantly lower than the rate ' P k SURVIVAL IN DAYS FIG. 2. Results of cardiac transplantation by various immunosuppression treatments. VOL. 18, NO. 3, SEPTEMBER,

6 THOMAS ET AL. c CONTROL IMURAN RABBIT HORSE 8 ATG ATG PREDNISONE.w.:m ATG 8 SOLUBLE ANTIGEN FIG. 3. Comparative rate of infectious complications in the groups using different immunosuppression regimens. seen in animals treated by Prednisone and Imuran. The dogs on the Prednisone-Imuran regimen suffered a variety of infections and drug toxicity complications, the most common being gram-negative infections, distemper, leukopenia, and gastrointestinal toxicity. The differences in both infections and drug toxicity complications between the group treated by Prednisone and Imuran and the other groups was statistically significant at the p < 0.05 level. Comment This study using the heterotopic cervical cardiac allograft has demonstrated that a limited course of 3 to 5 injections of potent antithymocyte globulin could prolong graft survival significantly beyond that seen in animals given standard immunosuppressive drugs. Furthermore, the low rate of infectious and drug toxicity complications in these animals leads to the conclusion thjat some degree of selective immunosuppression has been produced. i loo l5 t c 2 50 FIG. 4. Comparative rate of drug toxicity complications in the different trentmen / group.?. 25 n " IMURAN a RABBIT ATG HORSE ATG ATG 8 SOLUBLE PREDNISONE ANTIGEN 246 THE ANNALS OF THORACIC SURGERY

7 Immunosupfiression in Cardiac Allograjtzng From early studies on the action of potent heterologous antilymphocyte serum in mice, it was apparent that the short-course administration of this material about the time of transplantation had a powerful immunosuppressive effect in prolonging skin allograft survival while leaving host humoral antibody defense mechanisms intact [7, Although numerous theories have been advanced to explain the mechanism of action of ALS, it remains to be proved precisely how a brief treatment with this agent can produce permanent renal allograft survival in dogs [16] and facilitate the induction of permanent allograft survival in rodents receiving histoincompatible donor lymphoid cells [8, 121 or cell extracts [2, 171. Recent evidence has demonstrated that the mechanism by which ALS causes indefinite renal allograft survival in rats may relate to the selective suppression of effector cell populations and cellular immunity, with concomitant generation of serum blocking factors [l]. There is relatively little information on the mechanism of specific unresponsiveness generated by combined antilymphocyte serum and soluble alloantigen treatment, although the recent reports of Brent and associates [2] and Sumerska and his colleagues [17] would suggest that blocking antibodies or complexes may be involved. In vitro studies of cellular immunity and serum blocking factors have been initiated in our laboratory to attempt to determine the mechanism of prolonged cardiac allograft survival in dogs treated with ALS and soluble antigen. It is hoped that they may shed some light on the relevant mechanisms involved, since this information would be of critical importance in understanding and improving the technology and thus its potential application in human transplantation. The results of this study tend to confirm the earlier work of Wilson and co-workers [ 181 using canine kidney allografts. Myburgh and Smit [ 131 have also demonstrated the ability of soluble antigen extracts to prolong liver graft survival in baboons. Thus, at least three studies are available to date which suggest that soluble antigen pretreatment can be valuable in prolonging allograft survival in large outbred animals. These studies would seem to offer at least a partial answer to the critiques leveled at many previous antigen pretreatment studies-namely, that they were performed in related lines of small animals whose immunological responses are apparently somewhat unlike those seen in the larger animals and man. This study would also suggest that a state of specific immunosuppression can be utilized in cardiac as well as kidney and liver allografting; and it does not support the contention that specific unresponsiveness in allografting is more easily induced in kidney and liver allografting [3]. The results of studies by Wilson and his co-workers [18] are encouraging in their demonstration of marked kidney allograft prolongation with soluble antigen pretreatment. However, the requirement of adjuvant chronic Prednisone-Imuran therapy to achieve prolongation detracts

8 THOMAS ET AL. somewhat from the potential attractiveness of their system. In most instances neither the canine nor the human can be maintained in a completely healthy state during long-term Prednisone-Imuran therapy. Therefore, the cardiac allograft prolongation demonstrated in this study (roughly in the range of Wilson and colleagues results with kidneys), which was achieved without long-term Prednisone-Imuran therapy, appears promising as a potential technique for inducing specific immunosuppression in cardiac allografting. References 1. Biesecker, J. L., Fitch, F. W., Rowley, D. A., and Stuart, F. P. Cellular and humoral immunity after allogeneic transplantation in the rat. Transplantation 16:441, Brent, L., Hansen, J., Kilshaw, P., and Thomas, A. Specific unresponsiveness to skin allografts in mice. Transplantation 15: 160, Calne, R., Davis, D., and Hadjiyannakis, E. Immunosuppressive effects of soluble cell membrane fractions, donor blood and serum on renal allograft survival. Nature 227:903, Chunta, J. The mechanism of immunological enhancement of H-2- incompatible skin grafts in mice. Transplantation 12:28, Griepp, R. B., Dong, E., Jr., Stinson, E. B., and Shumway, N. E. Advances in human heart transplantation. Transplant. Proc. 5: 835, Halle-Pannenko, O., Martyre, M., and Jolles, F. Conditioning of allogeneic mice with crude and purified H-2 extracts, alone and combined with cyclophosphamide, for skin graft prolongation. Transplant. Proc. 3:257, Lance, E., and Batchelor, J. Selective suppression of cellular immunity by antilymphocyte serum. Transplantation 6:490, Lance, E., and Medawar, P. Quantitative studies on tissue transplantation immunity: IX. Induction of tolerance with antilymphocyte serum. Proc. R. SOC. Lond. [Biol.] 173:447, Martinez, C., Smith, J., Blaese, M., and Good, R. Production of immunological tolerance in mice after repeated injections of disrupted spleen cells. J. Exp. Med. 118:743, Medawar, P. The use of antigenic tissue extracts to weaken the immunological reaction against skin homografts in mice. Transplantation 1:21, Medawar, P. Biological Properties of Heterologous Antilymphocyte Antisera. In F. Rapaport and J. Dausset (Eds.), Human Transplantation. New York: Grune & Stratton, Monaco, A., and Wood, M. Studies on heterologous antilymphocyte serum in mice: VII. Optimal cellular antigen for induction of immunological tolerance with antilymphocyte serum. Transplant. Proc. 2:489, Myburgh, 1. A., and Smit, 1. A. Enhancement and antigen suicide in the outbred primate. Transplant. Proc. 5:273, Owen, E., Slome, D., and Waterston, D. Prolongation of Rabbit Allograft Survival by Desensitization. In 1. Dausset and G. Math6 (Eds.), Advances in Transplantation. Copenhagen: Munksgaard, P Rosenberg, E., Mann, D., Hill, I., and Fahey, 1. Prolonged skin allograft survival in mice pretreated with soluble transplant antigens. Transplantation 12:402, Shanfield, I., Wren, S. F. G., and Blennerhassett, J. B. Permanent survival of dog renal allografts after a limited course of antilymphocyte serum treatment. Surg. Forum 20:304, Sumerska, T., Betel, I., Balner, H., and Warren, H. Prolongation of skin allograft survival with antilymphocyte serum and soluble histocompatibility antigens. Transplantation 17: THE ANNALS OF THORACIC SURGERY

9 Immzinosuppl-ession in Cardiac Allografting 18. Wilson, R., Rippin, A., Dagher, R., Kinneart, P., and Busch, G. Prolonged canine renal allograft survival after pretreatment with solubilized antigen. Transfilantation. 7:360, Discussion DR. RANDALL B. GRIEPP (Stanford, Calif.): I should like to congratulate Dr. Thomas and his associates on a very nice piece of work. We certainly agree with the Richmond group that specific immunosuppression must be worked out if heart transplantation is to develop its true therapeutic potential. Work in our laboratory with the orthotopic heart graft would tend to confirm their work with the heterograft heart, at least with respect to the use of antithymocyte globulin as a potent immunosuppressive agent. Our work with solubilized antigen pretreatment, however, has not been as encouraging as theirs. In a recent series of experiments in our laboratory with canine orthotopic heart graft recipients, mean survival in an untreated group was 5 days. With ATG, mean survival was 16 days; and with cyclophosphamide and ATG, mean survival was 40 days. However, when donor-specific solubilized antigen pretreatment was combined with ATG, mean survival was 10 days, a figure not significantly different from that of ATG treatment alone. Somewhat more disturbing was the fact that 3 pretreated animals died within 24 hours of transplantation. At postmortem examination their hearts had the typical findings of hyperacute rejection. The myocardium was swollen and purple, and histopathological examination revealed massive interstitial hemorrhage and polymorphonuclear infiltration. Perhaps the explanation for the difference in our results from those of Dr. Thomas involves the amount of antigen administered preoperatively. Our animals received rather large doses of solubilized antigen, averaging 200 mg. per kilogram of body weight. I should like to ask Dr. Thomas if he thinks that donor antigen prepared from a pool could be used for antigen pretreatment, inasmuch as in the clinical situation donor-specific antigen is not available until several hours prior to operation. In conclusion, I should like to strike a note of caution with respect to antigen pretreatment. I think any system proposed for clinical work must first be extensively evaluated in animals to obviate the possibility of presensitization with resultant hyperacute rejection. DR. F. T. THOMAS: We would certainly agree with Dr. Griepp that hyperacute rejection can be produced by soluble antigen, especially if given at high doses. In point of fact, this is one of the in vivo techniques which can be used to assay transplant antigen activity in these preparations. In inappropriate timedosage scheduling, an active preparation ought to be able to produce hyperacute rejection. This is one of the variables that has to be worked out before clinical application of this system is begun. With regard to the question of pool antigen, this is also, of course, of critical importance in the logistics of clinical cardiac transplantation if soluble antigen pretreatment is to be used. We have in fact studied this and believe at the present time that with the appropriate combination of pooled antigen we can get results equal to those using donor-specific antigen. I would add in closing that certainly we are all aware of the tremendous contributions of the Stanford group in both experimental and clinical cardiac transplantation. Their work is a stimulus to all. They have occupied the leading position in this field, with some short-lived interruptions, since 1960, when Drs. Shumway and Lower originally demonstrated the technique which made clinical cardiac transplantation possible.