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Ann Thorac Surg 1995;60:1683-1685
© 1995 The Society of Thoracic Surgeons

Original Articles: Cardiovascular

Suppressor Cells and Intrathymic Inoculation of Donor Alloantigens in Cardiac Transplantation

Zhenya Shen, MD, Muhammad Mohiuddin, MD, Verdi J. DiSesa, MD

Department of Surgery, Medical College of Pennsylvania, Philadelphia, Pennsylvania

Accepted for publication July 15, 1995.


   Abstract
Top
 Footnotes
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 References
 
Background. Donor-specific tolerance to a rat heterotopic cardiac allograft has been achieved by the pretransplantation intrathymic injection of donor splenocytes and a single intraperitoneal injection of antilymphocyte serum (ALS). Permanent tolerance is achieved without subsequent immunosuppression therapy. This study investigated the mechanisms responsible for maintenance of the tolerant state.

Methods. Tolerance was produced in Lewis rats by the administration of 1 mL of ALS intraperitoneally and 5 x 107 Lewis Brown Norway (LBN) splenocytes intrathymically 21 days before heterotopic transplantation using an LBN donor.

Results. In tolerant Lewis rats bearing LBN allografts for more than 100 days, rejection could not be produced by the intravenous injection of naive Lewis spleen cells (5 x 107 cells x 1 day, n = 5; 5 x 107 cells x 3 days, n = 5) or cells from Lewis rats sensitized to LBN tissues (5 x 107 cells x 3 days, n = 5). Naive Lewis recipients were pretreated with ALS and 6 days later with intravenous spleen cells (25 x 107, n = 5) or lymphoid cells (10 to 15 x 107, n = 5) from a tolerant animal bearing a viable LBN graft. When transplantation with an LBN donor was done the next day, significant prolongation of LBN allograft survival (mean survival time 32.8 days, p < 0.01; and 22.2 days, p < 0.01; respectively) was seen. Wistar-Furth allograft survival was not prolonged by treatment with ALS and intravenous spleen (n = 5) or lymph node (n = 5) cells from rats tolerant to LBN tissues (mean survival time 8.6 and 9.2 days, control 9 days; p = not significant). The administration of ALS alone (n = 5) did not prolong LBN graft survival (mean survival time 11.8 days).

Conclusion. These data suggest that transferable suppressor cells specific for the donor strain are at least in part responsible for the maintenance of long-term allograft survival after intrathymic pretreatment with allogeneic cells.


   Introduction
Top
 Footnotes
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 References
 
Despite improvements in medical therapy, cardiac transplantation continues to be the only effective treatment for patients with end-stage heart disease. The immunologic reaction (rejection) remains an important barrier to the success of this treatment [1]. Recent pharmacologic advances in nonspecific immunosuppression protocols have improved results, but the long-term use of systemic immunosuppression therapy continues to be a major cause of morbidity and mortality [2, 3]. These complications could be avoided if it were possible to achieve a state of immunologic unresponsiveness without subsequent immunosuppression therapy. Indefinite donor-specific tolerance to a fully major histocompatibility complex-disparate rat heterotopic cardiac allograft has been achieved in our laboratory and others [4–8] by the pretransplantation intrathymic injection of donor splenocytes and a single intraperitoneal injection of rabbit anti-rat lymphocyte serum (ALS) without subsequent immunosuppression therapy. The allograft immune response and the processes that produce tolerance are complex phenomena that involve multiple cellular and soluble mediators. The present study was designed to investigate the mechanism of maintenance of the tolerance to a cardiac allograft induced by intrathymic inoculation of donor-specific splenocytes. Specifically, we sought to demonstrate whether transferable suppressor cells played a role.


   Material and Methods
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 Footnotes
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 References
 
Animals
Male Lewis, Lewis Brown Norway (LBN), and Wistar Furth rats were obtained virus free from Harlan Sprague-Dawley Company (Indianapolis, IN). The animals were housed in pathogen-free facilities at the Medical College of Pennsylvania. All animals were handled humanely according to the guidelines published by the National Institutes of Health (NIH publication 85-23, revised 1985).

Preparation of Spleen Cells
Whole spleen cells were harvested and disrupted by use of a tissue homogenizer. Cells were washed with RPMI 1640 medium. Red cells were lysed with NH4Cl. Viability of the cells was evaluated by trypan blue dye exclusion.

Injection of Cellular Alloantigen into Recipient Thymus
On the day before intrathymic treatment, Lewis recipients received 1 mL ALS intraperitoneally. The next day, after metofane inhalation anesthesia was induced, the thymus of the recipient rat was exposed by a partial sternotomy and 2.5 x 107 viable LBN donor splenocytes in a volume of 0.05 mL were injected into both lobes under direct vision.

Heart Transplantation
Three weeks after pretreatment, Lewis rats underwent transplantation under metofane anesthesia with hearts from the LBN donors that were used to obtain splenocytes for intrathymic injection. Hearts were transplanted heterotopically in the manner described by Ono and Lindsey [9]. One intramuscular dose of gentamicin (40 mg/kg) was given to each rat before the operation. Transplant function was determined by daily abdominal palpation and, if palpation was indeterminate, by direct visualization. Rejection was marked by the complete absence of ventricular contractions and was confirmed histologically.

Challenge of the Unresponsive State
After 100 days with a beating LBN heart, tolerant Lewis recipients were challenged by the intravenous injection of splenocytes from a naive or sensitized Lewis rat. Lewis rats were sensitized by transplantation of an LBN cardiac allograft. Sensitized Lewis splenocytes were harvested after the grafts were rejected at 6 to 8 days. Three cell dosage protocols were used in this experiment, with 5 rats in each group: (1) 5 x 107 naive syngeneic Lewis spleen cells once, (2) 5 x 107 naive syngeneic Lewis splenocytes daily for 3 days, and (3) 5 x 107 sensitized syngeneic Lewis splenocytes for 3 days.

Adoptive Transfer of Cells
Naive Lewis recipients were pretreated with 1 mL ALS intraperitoneally 6 days before the intravenous injection of 25 x 107 spleen cells (n = 10) or 10 to 15 x 107 lymph node cells (n = 10) from a tolerant Lewis rat bearing a viable LBN cardiac graft. Seven days after ALS administration, heterotopic heart transplantation was performed. Donors of cardiac allografts in each group of pretreated animals were either LBN (n = 5) or third-party Wistar Furth (n = 5) rats. As a control, Lewis rats were pretreated with ALS only 7 days before receiving LBN cardiac allografts (n = 5). Graft function was assessed in the manner described.

Histopathologic Assessment
Heart allografts were fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Slides were examined by light microscopy.

Statistical Analysis
Groups were compared by the Mann-Whitney U nonparametric test.


   Results
Top
 Footnotes
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 References
 
Donor-specific tolerance to LBN rat heterotopic cardiac allografts was achieved in 35 Lewis rats by the pretransplantation intrathymic injection of donor splenocytes and a single intraperitoneal injection of ALS. No further immunosuppression therapy was given.

Rejection could not be induced in tolerant Lewis rats bearing LBN cardiac allografts by the intravenous injection of either naive syngeneic Lewis splenocytes for 1 or 3 days or by spleen cells from Lewis rats sensitized to LBN antigen for 3 days (5 x 107 cells; no rejection of five grafts). All grafts maintained viability for more than 100 days.

Results of the effect of the transfer of tolerant cells are summarized in Table 1Go. Statistically significant prolongation of LBN cardiac allograft survival was achieved by passive transfer of either splenocytes or lymph node cells from modified Lewis rats bearing beating LBN hearts. In one case, essentially permanent graft survival was achieved. No significant extension of graft survival was seen in recipients treated with ALS alone (mean survival time 11.8 days). Third-party Wistar Furth cardiac allografts were rejected normally in Lewis recipients pretreated with either LBN tolerant splenocytes or lymph node cells and ALS (mean survival time 8.6 and 9.2 days, respectively).


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  		Table 1. . Survival of Heart Allografts in Lewis Recipients After Transfer of Syngeneic Cells from Lewis Brown Norway Tolerant Lewis Rats
 

   Comment
Top
 Footnotes
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
References
 
Apparent tolerance to a cardiac allograft has been obtained in a rat after a single dose of ALS and intrathymic inoculation of donor splenocytes. No subsequent immunosuppression therapy is required for indefinite graft survival. This result has been demonstrated in our laboratory and in others [4–8, 10]. We and other investigators have done a number of experiments to try to characterize this phenomenon, which may have critical importance for clinical transplantation science. Proposed mechanisms of physiologic tolerance include clonal deletion, clonal anergy, and the development of active suppression. Clonal deletion is thought to be the most important mechanism for the induction of self tolerance during immune ontogeny [11]. In this process, self-reactive clones of lymphocytes are eliminated from the immune cell repertoire. In clonal anergy, the potentially reactive cells remain but are rendered inert by processes yet to be defined. Active suppression requires the development of a distinct population of cells whose role is to suppress the effector cells that produce the immune response. The mechanism of the tolerance produced by intrathymic inoculation remains unclear [12]. The present set of experiments was designed to test the hypothesis that transferable suppressor cells were playing a role in maintenance of the tolerant state.

Data from these experiments suggest that suppressor cells may in fact be induced by intrathymic pretreatment in the rat. Failure of either naive or sensitized recipient strain cells to produce rejection when transferred to graft-bearing syngeneic recipients suggests the presence of a suppressor cell population. Presumably, naive and, almost certainly, sensitized Lewis rats have in their immune-cell repertoire the requisite effectors of rejection of an LBN graft. Failure of these cells to induce rejection when transferred into a Lewis rat rendered tolerant to LBN tissue by intrathymic pretreatment suggests the presence of suppressor cells in the tolerant rat. Of course, another possibility is that an adequate number of viable effector cells was not transferred using our regimen. Further work using other transfer protocols may be necessary to strengthen this conclusion.

However, the presumption of suppressor cells is strengthened by the results of our experiments, in which cells from tolerant animals were transferred into naive animals who subsequently underwent transplantation. In all cases, significant prolongation of graft survival was achieved by transfer of cells from tolerant animals. Rather than using a radiation-conditioning regimen, we used ALS in naive recipients before transfer of tolerant cells. Our control animals given ALS alone proved that prolongation in graft survival was not due to the effects of ALS and must have been due to elements in the transferred cells. It is unlikely that a serum factor was responsible for graft prolongation because the transferred cells were washed multiple times before injection.

Although our results suggest that suppressor cells play a role in maintenance of the tolerant state, they do not exclude other mechanisms for the induction or maintenance of tolerance. In experiments in a murine model, we have obtained evidence that a deletion mechanism may also play a role [13]. At present, we do not have any data about the possibility that clonal anergy is also a component of the tolerant state. It is possible that various mechanisms are responsible for the induction and maintenance of the tolerance produced by intrathymic pretreatment. A different mechanism may predominate at different times after cardiac grafting.

These experiments are part of a growing body of knowledge about the mechanisms whereby the intrathymic introduction of alloantigen can lead to subsequent allograft tolerance in rodent models. All investigators acknowledge the increased complexity of the human immune system. Although it is unlikely that these protocols will be transferable directly to human clinical transplantation, deepening of our insights regarding the mechanisms of these experimental phenomena may lead to new and effective clinical treatment protocols.


   Footnotes
Top
 Footnotes
Abstract
 Introduction
 Material and Methods
 Results
 Comment
 References
 
Address reprint requests to Dr DiSesa, Cardiothoracic Surgery, The Medical College of Pennsylvania, 3300 Henry Ave, Philadelphia, PA 19129.


   References
Top
 Footnotes
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 References
 

  1. Medawar PB. The behavior and fat of skin allografts and skin homografts in rabbits. J Anat 1994;78:176–99.
  2. Kahan BD. Cyclosporin. N Engl J Med 1989;321:1725–9.
  3. Walter RG, d'Apice AJF. Azathioprine and steroids. In: Morris PJ, ed. Kidney transplantation: principles and practice. Philadelphia: WB Saunders, 1988:319–41.
  4. Goss JA, Nakafusa Y, Flye MW. Donor-specific cardiac allograft tolerance without immunosuppression after intrathymic injection of donor alloantigen. Transplant Proc 1992;24:2879–80.[Medline]
  5. Kline GM, Shen Z, Mohiuddin M, et al. Successful experimental heart transplantation without immunosuppressive drugs. J Heart Lung Transplant 1993;12:388–93.[Medline]
  6. Shen Z, Kline GM, Mohiuddin M, DiSesa VJ. Histocompatibility differences and cardiac transplant tolerance produced by intrathymic pretreatment. J Thorac Cardiovasc Surg 1994;107:1472–5.[Abstract/Free Full Text]
  7. Posselt AM, Barker CF, Tomaszewski JE, et al. Induction of donor-specific unresponsiveness by intrathymic islet transplantation. Science 1990;249:1293–5.[Abstract/Free Full Text]
  8. Goss JA, Nakafusa Y, Flye MW. Intrathymic injection of donor alloantigens induces donor-specific vascularized allograft tolerance without immunosuppression. Ann Surg 1992;216:409–16.[Medline]
  9. Ono K, Lindsey ES. Improved technique of heart transplantation in rats. J Cardiovasc Surg 1969;57:225–9.
  10. Odorico JS, Posselt AM, Naji A, et al. Promotion of rat cardiac allograft survival by intrathymic inoculation of donor splenocytes. Transplantation 1993;55:1104–7.[Medline]
  11. Kappler JW, Roehm N, Marrack P. T cell tolerance by clonal elimination in the thymus. Cell 1987;49:273–80.[Medline]
  12. Posselt AM, Campos L, Mayo GL, et al. Selective modulation of T-cell immunity by intrathymic cellular transplantation. Transplant Rev 1993;7:200–13.
  13. Mohiuddin M, Shen Z, DiSesa VJ. Molecular biology of cardiac allograft rejection and tolerance: changes in the T cell repertoire induced by intrathymic pretreatment with allogeneic cells. Surg Forum 1994;45:415–6.



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