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J Thorac Cardiovasc Surg 1994;107:1472-1475
© 1994 Mosby, Inc.

CARDIAC AND PULMONARY REPLACEMENT

Histocompatibility differences and cardiac transplant tolerance produced by intrathymic pretreatment

Philadelphia, Pa.

From the Division of Cardiothoracic Surgery, Medical College of Pennsylvania, Philadelphia, Pa.

Received for publication July 30, 1993. Accepted for publication Nov. 8, 1993. Address for reprints: Verdi J. DiSesa, MD, Division of Cardiothoracic Surgery, Medical College of Pennsylvania, 3300 Henry Ave., Philadelphia, PA 19129.

Abstract

Control of cardiac transplant rejection without toxic immunosuppressive drugs remains an unreached goal. Our laboratory and others have shown that intrathymic inoculation of donor-specific allogeneic spleen cells can produce tolerance to a subsequent cardiac allograft. The present experiments were designed to investigate whether the degree of donor-recipient histoincompatibility influenced the efficacy of this technique. Four congeneic strains of rats with different degrees of histoincompatibility were studied. Heterotopic cardiac transplantation was done with the following congeneic strain combinations: DA donor into PVG recipient (full major histocompatibility complex and nonmajor histocompatibility complex incompatibility); PVG.RT1a donor into PVG recipient (full major histocompatibility complex incompatibility); PVG.RT1a donor into PVG.R1 recipient (partial major histocompatibility complex incompatibility). Prospective graft recipients underwent intraperitoneal injection of 1 ml antilymphocyte serum and intrathymic injection of 5 x 10 ⁷ prospective donor spleen cells. Three weeks later, heterotopic cardiac transplantation was done with a heart from a donor of the same strain as that used to obtain splenocytes for intrathymic injection. Prolongation of graft survival was observed in pretreated recipients in all strain combinations but was greatest in recipients that differed from donors at fewer histocompatibility loci. Complete graft tolerance was not seen in strain combinations that included nonmajor histocompatibility complex incompatibilities. DA heart survival in PVG recipients was 50.6 days ( p < 0.04 versus controls); PVG.RT1a graft survival in PVG hosts was 165.8 days ( p < 0.02 versus control) and in PVG.R1 recipients 163.8 days ( p < 0.02 versus controls) with four of five grafts in each group surviving indefinitely (more than 200 days). (J THORAC CARDIOVASC SURG 1994;107:1472-5)

The obvious limitations of current immunosuppressive therapy for heart transplantation have stimulated efforts to develop improved methods to control the immune response to grafted organs. In previous experiments we and others have shown that intrathymic injection of donor allogeneic spleen cells can produce tolerance to a subsequent cardiac allograft. ^1-4 The tolerance produced by this pretreatment is major-histocompatibility-complex donor strain specific. Long-term systemic immunosuppression is not required to maintain it. However, a report by Nakafusa and colleagues ⁵ suggested that this method fails to induce tolerance to renal and skin allografts. They supposed that tissue-specific nonmajor histocompatibility complex (non-MHC) antigens might play a role in the rejection of kidney and skin allografts and, therefore, that intrathymic pretreatment was ineffective in producing tolerance to these grafts. These observations underscore the fact that the mechanism of inducing allograft tolerance by intrathymic inoculation of donor allogeneic splenocytes is still not unraveled. In transplant models with congeneic rats that differ only at major histocompatibility complex (MHC) or minor histocompatibility (non-MHC) antigenic loci, it has been shown that various genetic subregions differ in their ability to cause allograft rejection. ^6,7 The goal of the present study was, therefore, to use congeneic lines of rats to investigate whether the degree of donor-recipient histoincompatibility influences the extent to which intrathymic pretreatment induces donor-specific transplant tolerance.

MATERIALS AND METHODS

Animals
The rat MHC (RT1) has been subdivided into five loci (A, B, C, D, and E). ⁸ In brief, the rat RT1A and RT1C regions encode, respectively, class I (major) and class I (medial) transplantation antigens. The RT1B/D regions encode class II transplantation antigens. PVG (AcBcDcCc), PVG.RT1a (AaBaDaCa), PVG.R1 (AaBcDcCc), and DA (AaBaDaCa + non-MHC) rats were used in these experiments. The DA and PVG strains have both full MHC and non-MHC incompatibilities. PVG.RT1a and PVG are MHC incompatible alone. PVG.RT1a and PVG.R1 have partial MHC incompatibility (class I [medial] and class II). All rats studied were male adults (200 to 250 gm) and obtained virus-free from Harlan Olac Ltd. (Bicester, Oxon) in England. Animals were stored in pathogen-free facilities and were handled humanely according to guidelines published by the National Institutes of Health (NIH Publication No. 86-23, revised 1985).

Preparation of donor spleen cells
Whole spleens were harvested and disrupted by use of a tissue homogenizer. Cells were washed with RPMI 1640 medium. Lymphoid cells were isolated with the use of a Ficoll density gradient. Viability of cells was evaluated by trypan blue dye exclusion.

Injection of cellular alloantigen into recipient thymus
On the day before intrathymic treatment, animals received 1 ml antilymphocyte serum (ALS) intraperitoneally. The next day, after metofane inhalation anesthesia was induced, the thymus of the recipient rat was exposed by a partial medial sternotomy, 2.5 x 10⁷ donor splenocytes in a volume of 0.05 ml were injected into both lobes of the recipient thymus under direct vision.

Heart transplantation
Three weeks after pretreatment the animals underwent transplantation under metofane anesthesia with hearts from donors of the same strains as those 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 confirmed histologically.

Experimental design
Heart grafts were done with the following strain combinations and across the following histoincompatibilities: full MHC and non-MHC, DA into PVG; full MHC, PVG.RT1a into PVG; and partial MHC, PVG.RT1a into PVG.R1. Recipient animals were divided into three subgroups: A, no pretreatment; B, intraperitoneal injection of ALS only; and C, ALS and intrathymic pretreatment with splenocytes from prospective donors.

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

Statistical analysis
Comparison between groups was done by an unpaired two-tailed t test.

RESULTS

Results of heart graft survival are summarized in Table I. Heart grafts transplanted across the three levels of tissue mismatch were all rejected (mean graft survival: 6.3 days, 8.3 days, and 10.6 days, respectively) by untreated recipients. Graft survival in untreated animals was longest in strain combinations that differed at fewer loci. The survival of grafts in strains with partial MHC mismatch was significantly longer than that across full MHC and non-MHC mismatch (p < 0.05) but not markedly longer than graft survival in strains with full MHC mismatch alone (p = not significant). Interestingly, we observed a similar phenomenon in grafts of recipients receiving intraperitoneal injection of ALS alone. After administration of ALS, cardiac graft survival was prolonged most in strain combinations with fewer histocompatibility differences.

Histologic evaluation of rejected hearts showed active cellular rejection and widespread myocyte destruction. Hearts removed from tolerant animals 200 days after transplantation had minimal mononuclear cellular infiltration without myocyte necrosis or evidence of active rejection.

DISCUSSION

This study reiterates the observation that cardiac allograft survival in naive recipients is dependent on the extent of donor-recipient histoincompatibility. These findings are consistent with those of previous reports that correlate the vigor of the rejection response with the degree of donor-recipient tissue incompatibility. ^10,11 This study also shows that pretreatment with ALS alone or in combination with intrathymic inoculation of donor spleen cells produces prolongation of graft survival or in some cases complete graft tolerance. Graft survival appears to be longer when there are fewer histocompatibility differences between donor and recipient.

As seen again in this study graft survival is longer in untreated animals when donor-recipient mismatch is less. This observation is probably also the basis for the results seen when ALS only was given 3 weeks before grafting. It is not difficult to imagine that ALS alone prolonged graft survival in less incompatible strains on the basis of a generalized immunosuppressive effect. In previous studies we have shown that one dose of ALS produces transient reduction in all classes of circulating lymphocytes. ² The time course of this reduction is such that the recipient animal has not completely regained normal circulating levels of immune competent cells by 3 weeks, the time of engraftment. Presumably this nonspecific immunosuppressive effect leads to slight prolongation of graft survival especially in less incompatible strain combinations.

It is potentially instructive to speculate about the mechanism responsible for the response to ALS plus intrathymic inoculation observed in the various strain combinations. The most striking conclusion to be drawn from our data is that the presence of non-MHC incompatibilities almost completely abrogates the ability of this pretreatment regimen to produce permanent graft acceptance. When DA donor hearts were placed in PVG recipients graft survival ranged from 7 to 21 days except in one animal that accepted its graft for more than 200 days. In contrast, in the two other groups that differed at the MHC locus alone intrathymic pretreatment plus ALS produced complete graft acceptance in all but one animal in each group. This striking difference suggests that the presence of non-MHC incompatibilities significantly reduces the efficacy of intrathymic pretreatment in producing graft tolerance.

It may be useful to consider this observation in the context of one theory about the mechanism by which intrathymic pretreatment produces prolongation of graft survival. As noted, ALS eliminates most mature circulating T lymphocytes. Subsequent introduction of allogeneic cells into the thymus alters the immunologic milieu in which new T cells mature. When immature lymphoid cells from the bone marrow circulate through the thymus their development takes place in the presence of alloantigens that are now presented as "self." In normal immune ontogeny, self-reactive cells are eliminated in the thymus. This process leads to the development of a repertoire of T cells that do not recognize the allogeneic cells in the thymus as foreign. When a subsequent graft of the same strain is introduced into this altered immune system the newly reconstituted T cells recognize the graft as self and therefore do not reject it. This scenario is speculative. ^2,3 However, in combination with the results of this study, it suggests a possible mechanism whereby non-MHC incompatibilities produce failure of graft prolongation.

Proper presentation of the foreign antigens in the thymus must be required for tolerance induction by intrathymic pretreatment. In normal immune development, the MHC antigens play a critical role in this process. It is plausible that allogeneic non-MHC transplant antigens are somehow inaccessible to lymphocytes that circulate through the thymus. If these antigens are not detected by T cells they cannot take part in the recognition events of the thymus that produce tolerance to tissue antigens. Under these circumstances it would not be surprising that in strain combinations that include non-MHC disparities intrathymic pretreatment does not lead to subsequent allograft acceptance. This hypothesis requires further investigation but may provide important clues regarding the mechanism whereby intrathymic pretreatment produces allograft tolerance. Our findings are also consistent with those of Goss, Nakafusa, and Flye, ¹² who showed that graft tolerance is dependent on the expression of donor class II but not class I MHC antigens in the intrathymic inoculation. Specifically, in our experiments the survival of PVG.RT1a donor hearts, in pretreated PVG.R1 recipients (partial, predominantly MHC class II, incompatibility) was equivalent to the survival of these grafts in PVG animals (full, class I and class II, incompatibility). Our studies did not include MHC class I only incompatible strains.

This study has shown that the efficacy of intrathymic pretreatment with allogeneic cells in prolonging cardiac allograft survival is related to the degree of donor-recipient histoincompatibility. We have also shown that the presence of a non-MHC histoincompatibility essentially eliminates the ability of intrathymic pretreatment to produce permanent graft acceptance. In addition, the presence of class II MHC incompatibilities appears to be an important requisite for tolerance induction by intrathymic pretreatment. These observations provide potential insight about the mechanism whereby intrathymic pretreatment produces significant prolongation of allograft survival. Understanding how this pretreatment works may lead to the development of more effective strategies for the prevention of allograft rejection and improvement in the care of patients who require cardiac transplantation.

References

1. 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]
   
2. Kline GM, Shen Z, Mohiuddin MM, et al. Successful experimental heart transplantation without immunosuppressive drugs. J Heart Lung Transplant 1993;12:388-93. [Medline]
   
3. 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]
   
4. 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]
   
5. Nakafusa Y, Goss JA, Mohanakumaa T, Flye NW. Induction of donor-specific tolerance to cardiac but not skin or renal allografts by intrathymic injection of splenocyte alloantigen. Transplantation 1993;55:877-82. [Medline]
   
6. Lim SML, White DJG, Calne RY. Unresponsiveness to class I antigens is not equal to tolerance to class I antigens induced by cyclosporine A. Transplant Proc 1988;20(Suppl):1031-3. [Medline]
   
7. Rozing J, Bonthuis P, Joling L, et al. The influence of RT1 subregion difference on cardiac allograft survival. Transplant Proc 1983;15:1647-8.
   
8. Butcher GW, Howard JC. Handbook of experimental immunology. 3rd ed. London: Blackwell Scientific, 1985:101.1-101.18.
   
9. Ono K, Lindsey ES. Improved technique of heart transplantation in rats. J THORAC CARDIOVASC SURG 1969;57:225-9. [Medline]
   
10. Katami M, Lim SML, Kamada N, et al. A pure class II MHC disparity does not induce rejection of cornea or heart grafts in the rat. Transplant Proc 1990;22:2200-1. [Medline]
    
11. Paul WE. Fundamental immunology. 2nd ed. New York: Raven Press, 1989:891-4.
    
12. Goss JA, Nakafusa Y, Flye M. MHC class II presenting cells are necessary for the induction of intrathymic tolerance. Ann Surg 1993;217:492-501.[Medline]
    

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This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Zhenya Shen Gary Kline Verdi J. DiSesa Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Shen, Z. Articles by DiSesa, V. J. Search for Related Content PubMed PubMed Citation Articles by Shen, Z. Articles by DiSesa, V. J.