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Heart and kidney transplantation using total lymphoid irradiation and donor bone marrow in mongrel dogs
J THORAC CARDIOVASC SURG 80:125-128, 1980
Heart and kidney transplantation using total lymphoid irradiation and donor bone marrow in mongrel dogs Heart and kidney allografts showed markedly prolonged survival in unrelated mongrel dogs following total lymphoid irradiation (TLl) and donor bone marrow without any other immunosuppression. In every animal the heart survived longer than the kidney; genetic disparity was ruled out. since the heart and kidney from the same donor were placed into one recipient. Placing the kidney allograft in the abdomen with the bone marrow given intraperitoneally doubled kidney survival over placement in the neck. but heart survival was equally prolonged in the abdomen or neck. Splenectomy before TLI or after TLI, bur just before transplantation, almost completely eliminated the prolonged survival of both heart and kidney allografts. Thus there is suggestive evidence from the work of Slavin and his colleagues and from our study that TLI plus bone marrow from the donor may be valuable for transplantation in man, particularly heart transplantation.
Donald R. Kahn, M.D., John H. Dufek, P.A., Richard Hong, M.D. William L. Caldwell, M.D., Frank J. Thomas, M.D., Donald R. Kolenda, B.S., David K. Swanson, Ph.D., and Robert A. Struble, M.D., Madison, Wis.
In
1953, Billingham, Brent, and Medawar' induced tolerance to transplanted tissue in mice by injecting allogeneic bone marrow cells intravenously into newborn recipients. When allogeneic bone marrow cells are injected into adults, the cells are readily rejected unless the immune system of the adult has been totally annihilated by such measures as total body irradiation; however, graft-versus-host disease then occurs and the new marrow destroys the host. Recently, Slavin and his colleagues-" achieved successful allogeneic bone marrow, skin, and heart transplantation without graftversus-host disease in mice and rats following treatment of the recipients with fractionated total lymphoid From the Division of Thoracic and Cardiovascular Surgery, Department of Surgery, and the Departments of Microbiology, Pediatrics, Human Oncology, and Radiology. University of Wisconsin, Madison, Wis. 53792. Grant support: American Heart Association Grant No. 78-983, Wisconsin Heart Association Grant No. 78-GA-38, Surgical Associates of the University of Wisconsin Grant No. 133-B575-A, and National Institutes of Health Grant No. HD-07778. Received for publication Nov. 9, 1979. Accepted for publication Dec. 18, 1979. Address for reprints: Donald R. Kahn, M.D .• University of Wisconsin, Clinical Science Center, Department of Surgery, 600 Highland Ave., Madison, Wis. 53792.
irradiation (TLI). To achieve this permanent chimerism required TLI consisting of 17 daily fractions of 200 rads directed selectively to the lymphoid organs including the thymus and 30 x 106 donor bone marrow cells per mouse (or roughly I billion cells per kilogram of body weight). Our experiments were designed to test the effect of TLI and donor bone marrow on kidney and heart transplantation in totally unrelated mongrel dogs. We modified the technique of Slavin and associates somewhat so that this approach could be applied to transplantation in man.
Methods Irradiation. Male, mongrel, unrelated dogs received a total of 3,300 rads of TLI divided into 15 treatments of 220 rads daily over 3 weeks. During irradiation the dogs were supine with forelimbs and hind limbs outstretched on a specially constructed treatment board. The irradiation protocol consisted of separate upper (supradiaphragmatic) and lower (abdominal) mantle fields ranging from the angle of the mandible to the level of the obturators. Cerroband (Lipowitz metal) blocking of the nonirradiated areas allowed selective treatment to the cervical, axillary, and mediastinal
0022-5223/80/070125+04$00.40/0 © 1980 The C. V. Mosby Co.
125
The Journal of Thoracic and Cardiovascular
1 26 Kahn et al.
Surgery
Table I. Pretreatment of each recipient with total lymphatic irradiation and bone marrow Heart Dog No. I 2
3 4 5
Position Abdomen Neck Abdomen Neck Abdomen
I
Kidney Survival (days)
30 41 19 62
91
Position Neck Abdomen Neck Abdomen Neck
I
Survival (days)
17 38 11 36
18
creatinine clearance dropped precipitously to zero over 2 to 3 days. Animals were evaluated for chimerism by looking for female chromosomes in the male recipients by karyotypic analysis. Stimulation of lymphocytes and mixed leukocyte cultures were performed essentially as described by Shifrine ," Dutta,? and their associates. In three animals the spleen was removed after TLI at the time of the transplantations, and in two animals a splenectomy was done prior to TLI.
Results lymph nodes and thymus in the upper field and to the para-aortic, iliac, and inguinal lymph nodes and part of the spleen in the lower field. Surgical technique. Seven to 15 days after irradiation was completed, each dog received a heterotopic heart and kidney transplant and a bone marrow transplant from the same female donor. The heart was placed either in the pelvis (anastomosing the aorta to the iliac artery and the pulmonary artery to the iliac vein) or in the neck (anastomosing the subclavian artery to the carotid artery and the pulmonary artery to the jugular vein). The kidney was placed either in the pelvis (anastomosing the renal artery to the iliac artery and the renal vein to the iliac vein, and bringing the ureter out in the groin as a cutaneous ureterostomy) or in the neck (anastomosing the renal artery to the carotid artery, the renal vein to the jugular vein, and the ureter to the skin in the neck as a cutaneous ureterostomy). Bone marrow treatment. Bone marrow was obtained by flushing the long bones with heparinized saline. The cell suspension was washed in RPMl 1640 three times and viable mononuclear cells were counted. Viability was always greater than 95%. From I to 6 billion cells per dog were injected intraperitoneally immediately after the transplantations. Examination of the fatty marrow layer revealed abundant mononuclear cells. These cells were also injected, although an accurate count of the cellular content could not be made. It is estimated that at least an equal number of cells was given in this way. No untoward reactions were noted following cellular infusions. All animals received cephalexin monohydrate (Keflex) and intravenous 5% glucose in normal saline for the first few postoperative days, but no blood and no immunosuppressive drugs were given. Rejection of hearts was assessed by palpation. When rejection occurred they became enlarged from edema and then ceased beating in 2 to 3 days. Kidney rejection was evaluated by measuring creatinine clearances on the transplanted kidney. Once rejection started, the
In five control dogs not receiving irradiation or bone marrow, the hearts and kidneys were rejected after the same interval-7 days. When only bone marrow from the donor was given (three dogs), the heart and kidney again were rejected after the same time interval- 7 days-similar to the control dogs. With TLI to the host without bone marrow therapy (three dogs), the grafts remained viable slightly longer-8Y2 days-with no difference between the heart and kidney. Five animals were treated with TLI and bone marrow, and survival of both the heart and kidney was prolonged significantly (Table I). Regardless of whether the heart was placed in the abdomen or neck, it always survived longer than the kidney from the same donor. This difference was much greater when the heart was in the abdomen and the kidney in the neck, mainly because the kidney survived 5 times as long as the control kidney when placed in the abdomen and 2V2 times as long as the control kidney when placed in the neck. Three animals had a splenectomy after TLI just before the transplantations. The hearts survived only II days and the kidneys 10 days, only slightly longer than control organs, almost completely abolishing the prolonged acceptance achieved when the partially irradiated spleen was left in place. Two animals had a splenectomy prior to TLI. In both of these animals the heart and kidney were rejected in 6 days, similar to the control organs. One animal was treated with azathioprine, 2 mg/ kg/ day, and prednisone, 2 mg/kg/day, after TLI and bone marrow treatment. The heart survived 97 days in the abdomen. The kidney became infected and was removed at 9 days with normal function. Stimulation of lymphocytes by phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM) was tested in five animals, all of which had been treated with TLI and bone marrow. All but one showed no response to these mitogens in the early post-transplant period. This was true even though rejection occurred soon after the absence of reactivity had
Volume 80 Number 1 July, 1980
been demonstrated. In one animal which had a splenectomy just before transplantation, a modest reaction to PHA, Con A, and PWM was seen (l1.4x, 8.3 x, and 6.0x compared to normal values of 61.6x, 9.41 x , and 17.7x, respectively). These values gradually decreased over a 2 week period, during which time the kidney and heart were rejected (10 and 14 days, respectively). We could not demonstrate whether or not partial chimerism was achieved, because the poor cellular proliferation prevented karyotypic analysis. In no animals, including the longest term survivors, did we see any suggestion of graft-versus-host disease.
Discussion The methods used in these experiments differed from those used by Slavin and associates in several respects: (1) Totally unrelated mongrel dogs were used; (2) transplantation was performed I to 2 weeks after TLI rather than immediately afterward; (3) the amount of bone marrow administered was much less (we took all we could get from the one donor dog) and was given intraperitoneally rather than intravenously, so that all the cells trapped in the fatty layer could be given, too; and (4) it is difficult to irradiate the spleen completely in the dog because of its position in the abdomen. Despite these differences, survival of heart and kidney transplants was prolonged markedly, without any other immunosuppression. Both the TLI and bone marrow were necessary to obtain these results, as there was little prolongation of graft survival with TLI alone and none with bone marrow alone. In every animal, the heart survived longer than the kidney. In several animals there was a twofold to threefold difference that cannot be accounted for by different means of determining the end points of rejection. When rejection did occur in both the kidney and heart, the reaction took place quickly like a typical acute rejection, not a chronic rejection. The kidneys lasted longer in the abdomen than in the neck. Whether placing the bone marrow intraperitoneally produced some additional local lymph node effect is still speculation. The intraperitoneal route was chosen for transplantation of bone marrow inasmuch as this route has been used repeatedly with success in immunodeficient patients. We believe that minimal handling of the marrow allows engraftment with relatively low cell doses. Although we were unable to demonstrate chimerism, it is clear that the intraperitoneal installation of marrow cells at a dose approximately 20 times less than that employed by Slavin and associates still had a marked
Heart and kidney transplantation
I27
effect upon graft prolongation. This factor may be of great importance in translating this experience to efforts in human transplantation, where it would not be feasible to obtain such large doses of marrow. One of the most fascinating parts of this experiment was that the marked beneficial effect of TLI and bone marrow on graft survival was blocked almost completely by performance of a splenectomy just before the transplantations or before TLI. Slavin and colleagues" showed a permanent and specific acceptance of C 57BLlKa* skin allografts that could be adoptively transferred to normal BalBI c* mice by use of spleen cells from C.';7BLlKa-BALBI c bone marrow chimeras; thus it appears that specific suppressor cells play a major role in the tolerance to tissue transplantation antigens following TLI. Our first thought was that removing the spleen eliminated a large source of suppressor cells and allowed graft rejection to proceed more easily. The failure of two dogs to show any increased lymphocyte response to PHA, Con A, and PWM after splenectomy, despite a vigorous rejection of both heart and kidney grafts, and the decreasing reaction to these mitogens in another splenectomized dog during the period of rejection suggest that alternative mechanisms are involved. Enomoto and Lucas" showed that splenectomy in rats abolished both active and passive immunologic enhancement of renal allografts when done 7 days before or up to 6 days after grafting. A similar mechanism may be present in our studies, as we needed donor bone marrow to achieve the prolonged graft acceptance that TLI alone did not accomplish. Mitogen response does not predict adequately the capability for organ rejection. Despite essentially no response to stimulation by three different mitogens, animals still possessed normal vigorous rejection capability. This profound degree of in vitro nonresponsiveness was due to the irradiation and was present prior to transplantation. The poor in vitro responses persisted for approximately I month with very gradual return thereafter. The numbers of dogs in these experiments are too small for statistical significance at a highly reliable level, but the trend is definite and the results are of such interest that reporting seems warranted. *Particular mice strains. REFERENCES Billingham RE, Brent L, Medawar PB: "Actively acquired tolerance" of foreign cells. Nature 172:603-606, 1953 2 Slavin S, Reitz B, Beiber CP, Kaplan HS, Strober S: Transplantation tolerance in adult rats using total lymphoid
128
3
4
5
6
Kahn et al.
irradiation. Permanent survival of skin, heart, and marrow allografts. J Exp Med 147:700-707, 1978 Slavin S, Strober S, Fuks Z, Kaplan HS: Use of total lymphoid irradiation in tissue transplantation in mice. Transplant Proc 9: 1001-1004, 1977 Slavin S, Strober S, Fuks Z, Kaplan HS: Induction of specific tissue transplantation tolerance using fractionated total lymphoid irradiation in adult mice. Long-term survival of allogeneic bone marrow and skin grafts. J Exp Med 146:34-48, 1977 Slavin S, Fuks Z, Kaplan HS, Strober S: Transplantation of allogeneic bone marrow without graft-versus-host disease using total lymphoid irradiation. J Exp Med 147:963-972, 1978 Shifrine M, Taylor NJ, Rosenblatt LS, Wilson FD: Com-
The Journal of Thoracic and Cardiovascular Surgery
parison of whole blood and purified canine lymphocytes in a lymphocyte-stimulation microassay. Am J Vet Res 39:687-690, 1978 7 Dutta SK, Novilla MN, Bumgardner MK, Ingling A: Lymphocyte responsiveness to mitogens and quantitation of T and B lymphocytes in canine malignant lymphoma. Am .I- Vet Res 39:455-458, 1978 8 Slavin S, Reitz BA, Beiber CP, Hoppe R, Fuks Z, Gottlieb M, Grumet C, Kaplan HS, Strober S: Transplantation tolerance after total lymphoid irradiation. Transplant Proc 11: 1032-1038, 1979 9 Enomoto K, Lucas ZJ: Immunological enhancement of renal allografts in the rat. III. Role of the spleen. Transplantation 15:8-16, 1973
Heart and kidney transplantation using total lymphoid irradiation and donor bone marrow in mongrel dogs Heart and kidney allografts showed markedly prolonged survival in unrelated mongrel dogs following total lymphoid irradiation (TLl) and donor bone marrow without any other immunosuppression. In every animal the heart survived longer than the kidney; genetic disparity was ruled out. since the heart and kidney from the same donor were placed into one recipient. Placing the kidney allograft in the abdomen with the bone marrow given intraperitoneally doubled kidney survival over placement in the neck. but heart survival was equally prolonged in the abdomen or neck. Splenectomy before TLI or after TLI, bur just before transplantation, almost completely eliminated the prolonged survival of both heart and kidney allografts. Thus there is suggestive evidence from the work of Slavin and his colleagues and from our study that TLI plus bone marrow from the donor may be valuable for transplantation in man, particularly heart transplantation.
Donald R. Kahn, M.D., John H. Dufek, P.A., Richard Hong, M.D. William L. Caldwell, M.D., Frank J. Thomas, M.D., Donald R. Kolenda, B.S., David K. Swanson, Ph.D., and Robert A. Struble, M.D., Madison, Wis.
In
1953, Billingham, Brent, and Medawar' induced tolerance to transplanted tissue in mice by injecting allogeneic bone marrow cells intravenously into newborn recipients. When allogeneic bone marrow cells are injected into adults, the cells are readily rejected unless the immune system of the adult has been totally annihilated by such measures as total body irradiation; however, graft-versus-host disease then occurs and the new marrow destroys the host. Recently, Slavin and his colleagues-" achieved successful allogeneic bone marrow, skin, and heart transplantation without graftversus-host disease in mice and rats following treatment of the recipients with fractionated total lymphoid From the Division of Thoracic and Cardiovascular Surgery, Department of Surgery, and the Departments of Microbiology, Pediatrics, Human Oncology, and Radiology. University of Wisconsin, Madison, Wis. 53792. Grant support: American Heart Association Grant No. 78-983, Wisconsin Heart Association Grant No. 78-GA-38, Surgical Associates of the University of Wisconsin Grant No. 133-B575-A, and National Institutes of Health Grant No. HD-07778. Received for publication Nov. 9, 1979. Accepted for publication Dec. 18, 1979. Address for reprints: Donald R. Kahn, M.D .• University of Wisconsin, Clinical Science Center, Department of Surgery, 600 Highland Ave., Madison, Wis. 53792.
irradiation (TLI). To achieve this permanent chimerism required TLI consisting of 17 daily fractions of 200 rads directed selectively to the lymphoid organs including the thymus and 30 x 106 donor bone marrow cells per mouse (or roughly I billion cells per kilogram of body weight). Our experiments were designed to test the effect of TLI and donor bone marrow on kidney and heart transplantation in totally unrelated mongrel dogs. We modified the technique of Slavin and associates somewhat so that this approach could be applied to transplantation in man.
Methods Irradiation. Male, mongrel, unrelated dogs received a total of 3,300 rads of TLI divided into 15 treatments of 220 rads daily over 3 weeks. During irradiation the dogs were supine with forelimbs and hind limbs outstretched on a specially constructed treatment board. The irradiation protocol consisted of separate upper (supradiaphragmatic) and lower (abdominal) mantle fields ranging from the angle of the mandible to the level of the obturators. Cerroband (Lipowitz metal) blocking of the nonirradiated areas allowed selective treatment to the cervical, axillary, and mediastinal
0022-5223/80/070125+04$00.40/0 © 1980 The C. V. Mosby Co.
125
The Journal of Thoracic and Cardiovascular
1 26 Kahn et al.
Surgery
Table I. Pretreatment of each recipient with total lymphatic irradiation and bone marrow Heart Dog No. I 2
3 4 5
Position Abdomen Neck Abdomen Neck Abdomen
I
Kidney Survival (days)
30 41 19 62
91
Position Neck Abdomen Neck Abdomen Neck
I
Survival (days)
17 38 11 36
18
creatinine clearance dropped precipitously to zero over 2 to 3 days. Animals were evaluated for chimerism by looking for female chromosomes in the male recipients by karyotypic analysis. Stimulation of lymphocytes and mixed leukocyte cultures were performed essentially as described by Shifrine ," Dutta,? and their associates. In three animals the spleen was removed after TLI at the time of the transplantations, and in two animals a splenectomy was done prior to TLI.
Results lymph nodes and thymus in the upper field and to the para-aortic, iliac, and inguinal lymph nodes and part of the spleen in the lower field. Surgical technique. Seven to 15 days after irradiation was completed, each dog received a heterotopic heart and kidney transplant and a bone marrow transplant from the same female donor. The heart was placed either in the pelvis (anastomosing the aorta to the iliac artery and the pulmonary artery to the iliac vein) or in the neck (anastomosing the subclavian artery to the carotid artery and the pulmonary artery to the jugular vein). The kidney was placed either in the pelvis (anastomosing the renal artery to the iliac artery and the renal vein to the iliac vein, and bringing the ureter out in the groin as a cutaneous ureterostomy) or in the neck (anastomosing the renal artery to the carotid artery, the renal vein to the jugular vein, and the ureter to the skin in the neck as a cutaneous ureterostomy). Bone marrow treatment. Bone marrow was obtained by flushing the long bones with heparinized saline. The cell suspension was washed in RPMl 1640 three times and viable mononuclear cells were counted. Viability was always greater than 95%. From I to 6 billion cells per dog were injected intraperitoneally immediately after the transplantations. Examination of the fatty marrow layer revealed abundant mononuclear cells. These cells were also injected, although an accurate count of the cellular content could not be made. It is estimated that at least an equal number of cells was given in this way. No untoward reactions were noted following cellular infusions. All animals received cephalexin monohydrate (Keflex) and intravenous 5% glucose in normal saline for the first few postoperative days, but no blood and no immunosuppressive drugs were given. Rejection of hearts was assessed by palpation. When rejection occurred they became enlarged from edema and then ceased beating in 2 to 3 days. Kidney rejection was evaluated by measuring creatinine clearances on the transplanted kidney. Once rejection started, the
In five control dogs not receiving irradiation or bone marrow, the hearts and kidneys were rejected after the same interval-7 days. When only bone marrow from the donor was given (three dogs), the heart and kidney again were rejected after the same time interval- 7 days-similar to the control dogs. With TLI to the host without bone marrow therapy (three dogs), the grafts remained viable slightly longer-8Y2 days-with no difference between the heart and kidney. Five animals were treated with TLI and bone marrow, and survival of both the heart and kidney was prolonged significantly (Table I). Regardless of whether the heart was placed in the abdomen or neck, it always survived longer than the kidney from the same donor. This difference was much greater when the heart was in the abdomen and the kidney in the neck, mainly because the kidney survived 5 times as long as the control kidney when placed in the abdomen and 2V2 times as long as the control kidney when placed in the neck. Three animals had a splenectomy after TLI just before the transplantations. The hearts survived only II days and the kidneys 10 days, only slightly longer than control organs, almost completely abolishing the prolonged acceptance achieved when the partially irradiated spleen was left in place. Two animals had a splenectomy prior to TLI. In both of these animals the heart and kidney were rejected in 6 days, similar to the control organs. One animal was treated with azathioprine, 2 mg/ kg/ day, and prednisone, 2 mg/kg/day, after TLI and bone marrow treatment. The heart survived 97 days in the abdomen. The kidney became infected and was removed at 9 days with normal function. Stimulation of lymphocytes by phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM) was tested in five animals, all of which had been treated with TLI and bone marrow. All but one showed no response to these mitogens in the early post-transplant period. This was true even though rejection occurred soon after the absence of reactivity had
Volume 80 Number 1 July, 1980
been demonstrated. In one animal which had a splenectomy just before transplantation, a modest reaction to PHA, Con A, and PWM was seen (l1.4x, 8.3 x, and 6.0x compared to normal values of 61.6x, 9.41 x , and 17.7x, respectively). These values gradually decreased over a 2 week period, during which time the kidney and heart were rejected (10 and 14 days, respectively). We could not demonstrate whether or not partial chimerism was achieved, because the poor cellular proliferation prevented karyotypic analysis. In no animals, including the longest term survivors, did we see any suggestion of graft-versus-host disease.
Discussion The methods used in these experiments differed from those used by Slavin and associates in several respects: (1) Totally unrelated mongrel dogs were used; (2) transplantation was performed I to 2 weeks after TLI rather than immediately afterward; (3) the amount of bone marrow administered was much less (we took all we could get from the one donor dog) and was given intraperitoneally rather than intravenously, so that all the cells trapped in the fatty layer could be given, too; and (4) it is difficult to irradiate the spleen completely in the dog because of its position in the abdomen. Despite these differences, survival of heart and kidney transplants was prolonged markedly, without any other immunosuppression. Both the TLI and bone marrow were necessary to obtain these results, as there was little prolongation of graft survival with TLI alone and none with bone marrow alone. In every animal, the heart survived longer than the kidney. In several animals there was a twofold to threefold difference that cannot be accounted for by different means of determining the end points of rejection. When rejection did occur in both the kidney and heart, the reaction took place quickly like a typical acute rejection, not a chronic rejection. The kidneys lasted longer in the abdomen than in the neck. Whether placing the bone marrow intraperitoneally produced some additional local lymph node effect is still speculation. The intraperitoneal route was chosen for transplantation of bone marrow inasmuch as this route has been used repeatedly with success in immunodeficient patients. We believe that minimal handling of the marrow allows engraftment with relatively low cell doses. Although we were unable to demonstrate chimerism, it is clear that the intraperitoneal installation of marrow cells at a dose approximately 20 times less than that employed by Slavin and associates still had a marked
Heart and kidney transplantation
I27
effect upon graft prolongation. This factor may be of great importance in translating this experience to efforts in human transplantation, where it would not be feasible to obtain such large doses of marrow. One of the most fascinating parts of this experiment was that the marked beneficial effect of TLI and bone marrow on graft survival was blocked almost completely by performance of a splenectomy just before the transplantations or before TLI. Slavin and colleagues" showed a permanent and specific acceptance of C 57BLlKa* skin allografts that could be adoptively transferred to normal BalBI c* mice by use of spleen cells from C.';7BLlKa-BALBI c bone marrow chimeras; thus it appears that specific suppressor cells play a major role in the tolerance to tissue transplantation antigens following TLI. Our first thought was that removing the spleen eliminated a large source of suppressor cells and allowed graft rejection to proceed more easily. The failure of two dogs to show any increased lymphocyte response to PHA, Con A, and PWM after splenectomy, despite a vigorous rejection of both heart and kidney grafts, and the decreasing reaction to these mitogens in another splenectomized dog during the period of rejection suggest that alternative mechanisms are involved. Enomoto and Lucas" showed that splenectomy in rats abolished both active and passive immunologic enhancement of renal allografts when done 7 days before or up to 6 days after grafting. A similar mechanism may be present in our studies, as we needed donor bone marrow to achieve the prolonged graft acceptance that TLI alone did not accomplish. Mitogen response does not predict adequately the capability for organ rejection. Despite essentially no response to stimulation by three different mitogens, animals still possessed normal vigorous rejection capability. This profound degree of in vitro nonresponsiveness was due to the irradiation and was present prior to transplantation. The poor in vitro responses persisted for approximately I month with very gradual return thereafter. The numbers of dogs in these experiments are too small for statistical significance at a highly reliable level, but the trend is definite and the results are of such interest that reporting seems warranted. *Particular mice strains. REFERENCES Billingham RE, Brent L, Medawar PB: "Actively acquired tolerance" of foreign cells. Nature 172:603-606, 1953 2 Slavin S, Reitz B, Beiber CP, Kaplan HS, Strober S: Transplantation tolerance in adult rats using total lymphoid
128
3
4
5
6
Kahn et al.
irradiation. Permanent survival of skin, heart, and marrow allografts. J Exp Med 147:700-707, 1978 Slavin S, Strober S, Fuks Z, Kaplan HS: Use of total lymphoid irradiation in tissue transplantation in mice. Transplant Proc 9: 1001-1004, 1977 Slavin S, Strober S, Fuks Z, Kaplan HS: Induction of specific tissue transplantation tolerance using fractionated total lymphoid irradiation in adult mice. Long-term survival of allogeneic bone marrow and skin grafts. J Exp Med 146:34-48, 1977 Slavin S, Fuks Z, Kaplan HS, Strober S: Transplantation of allogeneic bone marrow without graft-versus-host disease using total lymphoid irradiation. J Exp Med 147:963-972, 1978 Shifrine M, Taylor NJ, Rosenblatt LS, Wilson FD: Com-
The Journal of Thoracic and Cardiovascular Surgery
parison of whole blood and purified canine lymphocytes in a lymphocyte-stimulation microassay. Am J Vet Res 39:687-690, 1978 7 Dutta SK, Novilla MN, Bumgardner MK, Ingling A: Lymphocyte responsiveness to mitogens and quantitation of T and B lymphocytes in canine malignant lymphoma. Am .I- Vet Res 39:455-458, 1978 8 Slavin S, Reitz BA, Beiber CP, Hoppe R, Fuks Z, Gottlieb M, Grumet C, Kaplan HS, Strober S: Transplantation tolerance after total lymphoid irradiation. Transplant Proc 11: 1032-1038, 1979 9 Enomoto K, Lucas ZJ: Immunological enhancement of renal allografts in the rat. III. Role of the spleen. Transplantation 15:8-16, 1973