Rejection UnansweredQuestions Peter J. Morris
ABBREVIATIONS
APC CML DC GVH
antigen presenting cells cell-mediated lysis dendritic cells graft-versus-host
IFN MHC
interferon major histocompatibility complex
INTRODUCTION Although our understanding of the immune response to exogenous antigens, and thereby to a tissue allograft, has grown rapidly in recent years, we still remain relatively ignorant about many aspects of the response to an allograft which in the nontreated recipient will result in an aggressive reaction leading to rejection of the graft or, on the other hand, usually with some manipulation of the recipient, a suppressive reaction preventing rejection. Indeed, as our knowledge increases, so, too, do the unanswered questions, some of which I will touch upon in this paper, remembering, of course, that others may not agree with what I perceive as the more important unanswered questions. It would seem most appropriate to deal with these areas of uncertainty within the concept of an afferent, or induction, phase and an efferent, or effector, phase of the immune response to an allograft.
HISTOCOMPATIBILITY
Major histocompatibility complex (MHC) antigens and minor histocompatibility antigens: their relative roles. Incompatibility for the M H C antigens is the major determinant of graft rejection, as confirmed in humans by the superior survival of kidneys transplanted between HLA-identical siblings. Nevertheless even in this situation immunosuppression is required to prevent rejection and occasionally kidneys will be lost from irreversible rejection. This can only be due to incompatibility for multiple minor antigens, presumably especially so if the recipient is sensitized against minor antigens. However, we know little about minor antigen systems in humans, for their definition in the mouse (where over 40 have been defined) is based on rejection of skin allografts in vivo or cell-mediated lysis (CML) in vitro assays because it is not possible to detect them serologically as can be clone with most M H C antigens. Indeed, in the mouse incompatibility for multiple minor systems in the presence of M H C compatibility can result in
From the Nuffield Department ofSurgery, University of Oxford,John Radcliffe Hospital, Oxford, England. Address reprint requests to PeterJ. Morris, Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, England. Received December 1, 1989," acceptedJanuary 3, 1990.
104 0198-8859/90/$3.50
Human Immunology 28, 104-111 (1990) © American Society for Histocompatibility and Imunogenetics, 1990
Rejection--Unanswered Questions TABLE 1
105
Survival of vascularized cardiac allografts and skin allografts across H-2 and multiple minor histocompatibility antigen differences, or either of these alone a Survival (days)
Histoincompatibility H-2 + multiple minor H-2
Multiple minor
Donor
Recipient
Heart
Skin
C57BL/10 (b) Balb/c (d) Balb/c (d) C57BL/10 (b) C57BL/10 (b)
Balb/c (d) C3H/He (k) Balb/B (b) B10.BR (k) B10.D2 (d)
9 11 13 >100 9
ND ND 11 10 14
B10.BR (k) Balb/c (d) Balb/c (d)
C3H/He (k) DBA 2 (d) B10.D2 (d)
13 >100 11
11 13 11
Adapted from [1]
rejection of a cardiac allograft in just as vigorous a manner as occurs with incompatibility for the MHC, even in the absence of sensitization ([1] and Table 1). In bone marrow transplantation incompatibility for minor antigens certainly results in graft-versus-host (GVH) disease after HLA-identical sibling marrow grafts, and the elegant work of Goulmy has allowed a small number of minor systems to be defined on the basis of CML, compatibility for which reduces the incidence of G V H reactions [2]. In organ grafting there is little information available about minor systems other than that some of the minor blood groups, e.g., Lewis, might act as histocompatibility antigens, and evidence has been presented for the importance of endothelial antigens as minor histocompatibility antigens. Thus, it does seem likely that multiple minor differences are likely to influence graft outcome in certain situations, especially in the presence of HLA compatibility or prior sensitization.
Are all MHC antigens equally immunogenic, and what determines immunogenicity? All M H C antigens are not equally immunogenic as determined by the ability to sensitize a naive recipient and allograft survival. In the mouse class I K locus products are more immunogenic than D locus products, and in general incompatible M H C class II antigens also appear to be more immunogenic than class ! antigens. In humans although there are data suggesting increased immunogenicity for certain incompatible antigens, e.g., HLA-A1, over others in kidney transplantation, none of the findings has been widely confirmed. As to what determines immunogenicity, this must depend on a number of factors, presumably based both on the mode of presentation and recognition of the alloantigen in the first instance, which in turn may depend on the type of allograft (e.g., skin vs. kidney vs. liver) as well as on the genetic control of the response, which may be MHCor non-MHC-linked. Does the induction of MHC antigens during rejection augment either the induction or effector phase of the response or merely represent an epiphenomenon? The increased expression of M H C class I antigens and the induction of expression of M H C class II antigens in the cells of a tissue allograft at a very early stage of the immune
106
P.J. Morris response to an allograft has been demonstrated in allografts in rodents, dog, and humans and is due mainly to the production of the cytokine interferon ~/(IFN5/) by the specific T-cell response to the allograft (reviewed in [3]). Obviously in the case of class II MHC antigens the increased expression could augment the recognition phase of the response, while the increased expression of both class I and II antigens could provide an increased level of target antigen for the effector response. Nevertheless the increased expression of MHC antigens, both class I and class II, in a rat renal allograft model in which rejection does not occur, must shed some doubt on the above hypothesis [4].
T H E PASSENGER LEUKOCYTE The passenger leukocyte in organ allografts is the interstitial dendritic cell (DC), and in skin allografts it is the Langerhans cell, also of the DC lineage. Furthermore the DC is a potent immunostimulatory cell of the resting T cell in vitro (reviewed in [5]). But is the DC a major component of the induction phase of the response to an allograft? The role of the passenger leukocyte, now identified as the interstitial DC, has been controversial since the recognition years ago that in selected strain combinations in the rat, pretreatment of a kidney donor before transplantation with a protocol that would be expected to delete the organ of passenger leukocytes led to prolonged survival of the allograft and rejection could be induced with the administration of donor leukocytes enriched for dendritic cells. However, in general these experiments were successful in relatively weak combinations, e.g., F 1 hybrid to parental strain, and clinical trials of donor pretreatment were unconvincing. Nevertheless, in recent years evidence in favor of an important role of the DC has grown. For example, the deletion of DC in pancreatic islets with a specific monoclonal antibody for DC before transplantation resulted in prolonged survival of the islet allografts [6]; the survival of cardiac allografts in the rat correlates with the number of DC in the heart at the time of transplantation following attempts to remove the DC before transplantation [7]; and the recognition that the DC is several orders of magnitude more potent as a stimulatory cell in vitro in the mixed lymphocyte reaction than other lymphoid cells and that very few DC are required for immunostimulation [5], all point to a prominent role for the DC in sensitization of the recipient against the donor allograft. However, the role of the DC has not yet been established as major in all allograft reactions. ALLOANTIGEN RECOGNITION
The conventional recognition of an exogenous antigen involves processing of the antigen and presentation by host antigen presenting cells (APC) to host T-helper or cytotoxic T cells where the processed antigen is recognized as a peptide in association with class II or class I M H C antigens, respectively." But how is alloantigen recognized? Obviously alloantigen from the graft could be processed and presented by recipient APC in the conventional way. But, as well, it seems likely that MHC aUoantigen in an organ graft might be recognized directly, perhaps as presented by the interstitial DC. This would be a different mechanism of recognition, for obviously this recognition could not be based on MHC restriction as conventional recognition of an exogenous antigen requires. One can only speculate at this time, but it is possible that the alloreactive T cells in the recipient recognize a relatively public determinant of the alloantigen of the allograft, perhaps in association with a spectrum of exogenous peptides, assuming that peptides of either exogenous or endogenous origin are always present in association with MHC antigens. Such a
Rejection--Unanswered Questions TABLE 2
107
Balb/c hearts grafted into naive or sensitized CBA recipients treated with an ~-CD4 (YTS 191.1) or an ~-CD8 (YTS 169.4) antibody for 2 days before and 10 days after transplantation Survival in days (range)
Treatment Nil a-CD4 q-CD8
Naive recipient
Sensitized recipient
10 (9-11) >100 (21->100) 13 (10-14)
4 (3-6) 10 (4-15) 20 (12-58)
hypothesis is required also to explain the relatively high numbers of alloreactive cells for any given M H C antigen that will be found in the normal individual. Does induction of the allograft response occur peripherally or centrally ? Conventional wisdom suggests that in the case of a vascularized organ allograft, such as a kidney or heart, sensitization occurs peripherally within the organ, while in the case of a nonvascularized skin allograft, it occurs centrally. However, recent experiments by Larsen and Austyn in my own laboratories showing that DC from cardiac allografts in mice migrate quite quickly to the spleen provide a basis for a mechanism of central sensitization for vascularized allografts as well [8]. EFFECTOR MECHANISMS The effector area of the immune response comprises a specific component, both cellular and humoral, as well as a nonspecific component which is predominantly cellular, but the relative contributions of these various components to the aggressor reaction which results in graft rejection has not been defined, probably because there are a spectra of reactions depending on the type of graft, the site of implantation, and the state of priming of the recipient. Are C D 4 + or C D 8 + T cells the primary cells involved in rejection? The importance of the T lymphocyte in rejection is beyond dispute, as illustrated, for example, by the indefinite survival of skin allografts on athymic nude mice or B-cellreconstituted rats. However, controversy has raged for some years on whether the CD4 + T-helper cell is the primary cell involved in the specific effector arm of the rejection reaction (with other leukocytes being attracted secondarily in a nonspecific manner as occurs in a delayed-type hypersensitivity reaction) or the destruction of the graft is mediated specifically by CD8 + cytotoxic T cells. Conflicting data have been presented in rodent models in which T-cell-depleted animals were reconstituted with CD4 + or with CD8 + T cells; on the whole they support a more central role for the CD4 + cell, with or without participation of CD8 + cells (reviewed in [9]). On the other hand, a cyclosporine protocol that leads to indefinite renal allograft survival in the rat also results in loss of donorspecific cytotoxicity in leukocytes harvested from the graft itself, implying thereby a prominent role for CD8 + cells [10]. Both of these types of experiments are open to other interpretations, but perhaps experiments in which either CD4 + or CD8 + T cells are deleted or inactivated in vivo with monoclonal antibodies are more persuasive, and as seen in Table 2, mouse heart allografts in naive recipients are prolonged markedly with anti-CD4 antibody treatment while anti-CD8 antibodies were ineffective. On the other hand in the sensitized recipient treatment
108
P.J. Morris with an anti-CD8 antibody was far more effective than treatment with the antiCD4 antibody [11]. More recently we have been able to extract cells from grafts that were not rejecting and show that these cells also exhibited donor-specific cytotoxicity in vitro, suggesting that CD8 ÷ cells themselves are not sufficient for rejection [4]. To some extent the argument revolves around semantics, for it is recognized now that CD4 + cells may be cytotoxic and that CD8 + cells may provide help. Nevertheless, for the moment the balance of evidence favors a major role for the CD4 + T cell in the naive recipient of an allograft.
Are natural killer (NK) cells involved in graft rejection? Although an increasingly prominent role for these cells is being established in tumour surveillance models, there is little evidence suggesting an important role in graft rejection. Indeed in several aUograft models in the rat in our laboratories we have noted prolonged survival of renal allografts in the presence of high levels of N K activity in leukocytes harvested from the graft (e.g., [10]).
Donor-specific lymphocytotoxic HLA antibodies in humans produce hyperacute rejection of a renal allograft, but can hyperacute rejection be mediated by cells? Hyperacute rejection is mostly mediated by antibodies directed at donor HLA class I and possibly class II antigens. Nevertheless on relatively uncommon occasions renal grafts have failed to function in the presence of a current and historical negative serologic crossmatch with the donor. As this usually only occurs in patients receiving a regraft, it is strongly suggestive of an immunologic reaction, and biopsies done before infarction will confirm the presence of severe rejection. It seems possible that this is mediated by cells in the absence of preexisting antibody, but one must postulate the presence of donor-specific cellular immunity in the recipient against either M H C or non-MHC antigens. This type of allograft rejection has been demonstrated against minor histocompatibility antigens in a miniature swine model [12], and it seems very likely to me that failure to function in patients with regrafts is sometimes due to immediate cellular destruction of the graft in a sensitized recipient in the absence of any evidence of humoral sensitization.
Are antibodies involved in acute and chronic rejection? Although antibody is undoubtedly responsible for most cases of hyperacute rejection, its role in acute and chronic rejection is unclear. Antibody of donor specificity can certainly be demonstrated in rejecting grafts and in most patients after removal of a rejected graft, and the histologic picture of acute and chronic rejection in many instances may be predominantly one of vascular damage, which has always been attributed to antibody. On the other hand, donor-specific cytotoxic antibodies have been demonstrated in patients with functioning renal transplants. Antibody may also play a role in the antibody-dependent cellular cytotoxicity (ADCC) reaction, the final mediator being K cells reacting with the FC portion of the donor-specific antibody. Whether this reaction plays a part in rejection in vivo is unknown. S U P P R E S S O R CELL M E C H A N I S M S Since the recognition that the response to sheep red blood cells in mice could be suppressed by a population of spleen cells from tolerized mice (first termed infectious tolerance) by Gershon and Kondo [13], suppressor cells have been demonstrated in a variety of immune response models in vitro and in vivo by
Rejection--Unanswered Questions
109
adoptive transfer. In general the suppressor cells are T cells, and in general they are specific for the antigen concerned, although nonspecific suppressor cells have been also described within the macrophage lineage as well as in a bone-marr o w - d e r i v e d non-T-cell population. However, the suppressor cells described in allograft models are T cells [14]. Both suppressor cells of CD4 + and CD8 + phenotype have been shown to adoptively transfer suppression in different experimental models, although in general the suppressor cells demonstrated at an early stage of the response have been described as CD4+and during the maintenance phase of a long surviving graft as CD8 + [ 15 ]. Do suppressor cells represent a true phenomenon or do they represent an artifact of the systems used for their demonstration? The failure to clone suppressor T cells or to identify a separate population of suppressor T cells together with the somewhat contrived experiments needed to demonstrate their existence in vitro or in vivo (e.g., adoptive transfer to lightly irradiated syngeneic recipients) has led to a good deal of scepticism about the real existence of suppressor T cells. Nevertheless the p h e n o m e n o n has been so widely demonstrated, not only in many experimental models but in a variety of immune responses, that it seems unreasonable to suggest that this is not a true phenomenon that may in vivo regulate the immune response to an allograft. Is there a separate population ofT-suppressor cells? Certainly no phenotypic marker has been identified for such a population and although it is possible that within a CE4 ~ or CD8 + T-cell population there might be a subpopulation of suppressor cells, it is probable that these same cells can behave as suppressor cells given the appropriate stimulation or environment. I f there is not a separate suppressor T-cell population, what induces the T cell to act as a suppressor cell rather than an aggressor cell? The answer to this question becomes even more speculative, for although there are a variety of ways of inducing suppressor cells in allograft models in the rodent, e.g., antigen pretreatment with blood, passive enhancement, or cyclosporine, there would appear to be little in common with these models other than perhaps an inhibition of the interleukin 2 (IL-2) pathway in the induction phase of the response. In the blood transfusion model in the rat, a single transfusion in a selected strain combination produces indefinite survival of renal allografts with complete suppression of rejection. Suppressor T cells can be demonstrated in vitro and in vivo in this model in the mixed lymphocyte reaction and by adoptive transfer, respectively, both early in the response to the transfusion and during the course of the graft [16]. Early in the course of the graft, T cells can be separated from the graft which exhibit donor-specific cytotoxicity in vitro but do not produce interleukin 2 (IL-2) or respond to IL-2 in vitro, nor can these cells break the transfusion effect on adoptive transfer to transfused rats. However if IL-2 is administered to the rats from the time of transplantation, then rejection occurs normally ([17] and manuscript in preparation). At the moment it is impossible to reconcile these findings, but it would appear that pretreatment with blood inhibits the IL-2 pathway in the T-ceU response of the recipient to the graft such that the T cells are inactive in vivo. It is just possible that these inactivated cells have the functional characteristics of suppressor cells, or, on the other hand, that a separate population of suppressor cells is responsible for the inhibition of the IL-2 pathway. The maintenance phase of tolerance to a renal or cardiac allograft in the rodent is extremely hard to break, which would suggest that the active suppression, putatively mediated by T-suppressor cells,
110
P.J. Morris must be very strong. Why, then, does one need to manipulate a syngeneic recipient, by irradiation for example, to demonstrate the adoptive transfer of suppression by cells?
CYTOKINES
What is the role of the ever-growing family of cytokines in regulating the immune response to an allograft? This would appear to me to be one of the major areas of investigation required over the next few years, for it would seem that many of the questions that I have already posed above will be answered as we understand more of the complex interactions of the cytokines and cells in both the induction phase and the effector phase of the allograft response. THE TARGET
Is the major targetfor the effector response to a vascularized allograft the endothelium? It is assumed, probably correctly, that the endothelium is the prime target of the response, both cellular and humoral, against the allograft. But this has never been clearly established; more importantly, are there special features which make endothelium the prime target, other than that the circulating blood is in direct contact with it as it passes through the allograft? We know that the endothelium o f the grafted organ, at least in the case of a kidney, remains of donor phenotype (e.g., [17]). Furthermore, not only does endothelium express class I and class II M H C antigens (the latter in the human), but it can be induced to express both in abundance. Furthermore, endothelium expresses a variety of specialized receptors which enhance adhesion of lymphocytes in the presence of an inflammatory reaction. In addition endothelial cells are functionally specialized and extremely active metabolic cells, and, for example, via the arachidonic acid pathway are capable o f significantly influencing the damage produced by the allograft reaction. CONCLUSIONS There are no doubt many questions which I have not discussed which others would see as major problems, but I hope to have touched on sufficient areas of uncertainty to indicate that there is still a vast area of ignorance in our understanding of the immune response to an allograft, some of which may be dispelled during this meeting. If we are ever to manipulate the response in a truly specific manner in clinical practice, it is essential that we continue to strive to answer these questions. REFERENCES 1. Peugh WN, Supervisor RA, Wood KJ, Morris PJ: Immunogenetics 23:30, 1986. 2. Goulmy E: In Morris PJ, Tilney NL (eds): Transplant Rev 2:29, 1988. 3. Fuggle SV: In Morris PJ, Tilney NL (eds): Transplant Rev 3:81, 1989. 4. Dallman MJ, Wood KJ, Morris PJ: J Exp Med 165:566, 1987. 5. AustynJM, Steinman RM: In Morris PJ, Tilney NL (eds): Transplant Rev 2:139, 1988. 6. Faustman D, Steinman RM, Gebel H, Hauptfield V, Davis J, Lacy P: Proc Natl Acad Sci USA 81:3864, 1984. 7. McKenzie JL, Beard ME, Hart DN: Transplantation 38:371, 1984. 8. Larsen CP, Morris PJ, Austyn JM: J Exp Med 171:307, 1989.
Rejection--Unanswered Questions
111
9. Dallman MJ, Morris PJ: In Morris PJ (ed): Kidney Transplantation: Principles and Practice, 3d ed. Philadelphia, Saunders, 1988. 10. Bradley JA, Mason DW, Morris PJ: Transplantation 39:169, 1985. 11. Madsen JC, Wood KJ, Morris PJ: Transplant Proc 21:1022, 1989. 12. Kirkman RL, Colvin RB, Flye MW, Williams GM, Sachs DH: Transplantation 28:24, 1979. 13. Gershon RK, Kondo K: Immunology 18:723, 1970. 14. Hutchinson IV: Transplantation 41:547, 1986. 15. Rodriquez MA, Hutchinson IV, Morris PJ: Transplantation 47:847, 1989. 16. Quigley RL, Wood KJ, Morris PJ: J Immunol 142:463, 1989. 17. Wood KJ, Dallman MJ, Morris PJ: Transplant Proc 21:338, 1989.
ABBREVIATIONS
APC CML DC GVH
antigen presenting cells cell-mediated lysis dendritic cells graft-versus-host
IFN MHC
interferon major histocompatibility complex
INTRODUCTION Although our understanding of the immune response to exogenous antigens, and thereby to a tissue allograft, has grown rapidly in recent years, we still remain relatively ignorant about many aspects of the response to an allograft which in the nontreated recipient will result in an aggressive reaction leading to rejection of the graft or, on the other hand, usually with some manipulation of the recipient, a suppressive reaction preventing rejection. Indeed, as our knowledge increases, so, too, do the unanswered questions, some of which I will touch upon in this paper, remembering, of course, that others may not agree with what I perceive as the more important unanswered questions. It would seem most appropriate to deal with these areas of uncertainty within the concept of an afferent, or induction, phase and an efferent, or effector, phase of the immune response to an allograft.
HISTOCOMPATIBILITY
Major histocompatibility complex (MHC) antigens and minor histocompatibility antigens: their relative roles. Incompatibility for the M H C antigens is the major determinant of graft rejection, as confirmed in humans by the superior survival of kidneys transplanted between HLA-identical siblings. Nevertheless even in this situation immunosuppression is required to prevent rejection and occasionally kidneys will be lost from irreversible rejection. This can only be due to incompatibility for multiple minor antigens, presumably especially so if the recipient is sensitized against minor antigens. However, we know little about minor antigen systems in humans, for their definition in the mouse (where over 40 have been defined) is based on rejection of skin allografts in vivo or cell-mediated lysis (CML) in vitro assays because it is not possible to detect them serologically as can be clone with most M H C antigens. Indeed, in the mouse incompatibility for multiple minor systems in the presence of M H C compatibility can result in
From the Nuffield Department ofSurgery, University of Oxford,John Radcliffe Hospital, Oxford, England. Address reprint requests to PeterJ. Morris, Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, England. Received December 1, 1989," acceptedJanuary 3, 1990.
104 0198-8859/90/$3.50
Human Immunology 28, 104-111 (1990) © American Society for Histocompatibility and Imunogenetics, 1990
Rejection--Unanswered Questions TABLE 1
105
Survival of vascularized cardiac allografts and skin allografts across H-2 and multiple minor histocompatibility antigen differences, or either of these alone a Survival (days)
Histoincompatibility H-2 + multiple minor H-2
Multiple minor
Donor
Recipient
Heart
Skin
C57BL/10 (b) Balb/c (d) Balb/c (d) C57BL/10 (b) C57BL/10 (b)
Balb/c (d) C3H/He (k) Balb/B (b) B10.BR (k) B10.D2 (d)
9 11 13 >100 9
ND ND 11 10 14
B10.BR (k) Balb/c (d) Balb/c (d)
C3H/He (k) DBA 2 (d) B10.D2 (d)
13 >100 11
11 13 11
Adapted from [1]
rejection of a cardiac allograft in just as vigorous a manner as occurs with incompatibility for the MHC, even in the absence of sensitization ([1] and Table 1). In bone marrow transplantation incompatibility for minor antigens certainly results in graft-versus-host (GVH) disease after HLA-identical sibling marrow grafts, and the elegant work of Goulmy has allowed a small number of minor systems to be defined on the basis of CML, compatibility for which reduces the incidence of G V H reactions [2]. In organ grafting there is little information available about minor systems other than that some of the minor blood groups, e.g., Lewis, might act as histocompatibility antigens, and evidence has been presented for the importance of endothelial antigens as minor histocompatibility antigens. Thus, it does seem likely that multiple minor differences are likely to influence graft outcome in certain situations, especially in the presence of HLA compatibility or prior sensitization.
Are all MHC antigens equally immunogenic, and what determines immunogenicity? All M H C antigens are not equally immunogenic as determined by the ability to sensitize a naive recipient and allograft survival. In the mouse class I K locus products are more immunogenic than D locus products, and in general incompatible M H C class II antigens also appear to be more immunogenic than class ! antigens. In humans although there are data suggesting increased immunogenicity for certain incompatible antigens, e.g., HLA-A1, over others in kidney transplantation, none of the findings has been widely confirmed. As to what determines immunogenicity, this must depend on a number of factors, presumably based both on the mode of presentation and recognition of the alloantigen in the first instance, which in turn may depend on the type of allograft (e.g., skin vs. kidney vs. liver) as well as on the genetic control of the response, which may be MHCor non-MHC-linked. Does the induction of MHC antigens during rejection augment either the induction or effector phase of the response or merely represent an epiphenomenon? The increased expression of M H C class I antigens and the induction of expression of M H C class II antigens in the cells of a tissue allograft at a very early stage of the immune
106
P.J. Morris response to an allograft has been demonstrated in allografts in rodents, dog, and humans and is due mainly to the production of the cytokine interferon ~/(IFN5/) by the specific T-cell response to the allograft (reviewed in [3]). Obviously in the case of class II MHC antigens the increased expression could augment the recognition phase of the response, while the increased expression of both class I and II antigens could provide an increased level of target antigen for the effector response. Nevertheless the increased expression of MHC antigens, both class I and class II, in a rat renal allograft model in which rejection does not occur, must shed some doubt on the above hypothesis [4].
T H E PASSENGER LEUKOCYTE The passenger leukocyte in organ allografts is the interstitial dendritic cell (DC), and in skin allografts it is the Langerhans cell, also of the DC lineage. Furthermore the DC is a potent immunostimulatory cell of the resting T cell in vitro (reviewed in [5]). But is the DC a major component of the induction phase of the response to an allograft? The role of the passenger leukocyte, now identified as the interstitial DC, has been controversial since the recognition years ago that in selected strain combinations in the rat, pretreatment of a kidney donor before transplantation with a protocol that would be expected to delete the organ of passenger leukocytes led to prolonged survival of the allograft and rejection could be induced with the administration of donor leukocytes enriched for dendritic cells. However, in general these experiments were successful in relatively weak combinations, e.g., F 1 hybrid to parental strain, and clinical trials of donor pretreatment were unconvincing. Nevertheless, in recent years evidence in favor of an important role of the DC has grown. For example, the deletion of DC in pancreatic islets with a specific monoclonal antibody for DC before transplantation resulted in prolonged survival of the islet allografts [6]; the survival of cardiac allografts in the rat correlates with the number of DC in the heart at the time of transplantation following attempts to remove the DC before transplantation [7]; and the recognition that the DC is several orders of magnitude more potent as a stimulatory cell in vitro in the mixed lymphocyte reaction than other lymphoid cells and that very few DC are required for immunostimulation [5], all point to a prominent role for the DC in sensitization of the recipient against the donor allograft. However, the role of the DC has not yet been established as major in all allograft reactions. ALLOANTIGEN RECOGNITION
The conventional recognition of an exogenous antigen involves processing of the antigen and presentation by host antigen presenting cells (APC) to host T-helper or cytotoxic T cells where the processed antigen is recognized as a peptide in association with class II or class I M H C antigens, respectively." But how is alloantigen recognized? Obviously alloantigen from the graft could be processed and presented by recipient APC in the conventional way. But, as well, it seems likely that MHC aUoantigen in an organ graft might be recognized directly, perhaps as presented by the interstitial DC. This would be a different mechanism of recognition, for obviously this recognition could not be based on MHC restriction as conventional recognition of an exogenous antigen requires. One can only speculate at this time, but it is possible that the alloreactive T cells in the recipient recognize a relatively public determinant of the alloantigen of the allograft, perhaps in association with a spectrum of exogenous peptides, assuming that peptides of either exogenous or endogenous origin are always present in association with MHC antigens. Such a
Rejection--Unanswered Questions TABLE 2
107
Balb/c hearts grafted into naive or sensitized CBA recipients treated with an ~-CD4 (YTS 191.1) or an ~-CD8 (YTS 169.4) antibody for 2 days before and 10 days after transplantation Survival in days (range)
Treatment Nil a-CD4 q-CD8
Naive recipient
Sensitized recipient
10 (9-11) >100 (21->100) 13 (10-14)
4 (3-6) 10 (4-15) 20 (12-58)
hypothesis is required also to explain the relatively high numbers of alloreactive cells for any given M H C antigen that will be found in the normal individual. Does induction of the allograft response occur peripherally or centrally ? Conventional wisdom suggests that in the case of a vascularized organ allograft, such as a kidney or heart, sensitization occurs peripherally within the organ, while in the case of a nonvascularized skin allograft, it occurs centrally. However, recent experiments by Larsen and Austyn in my own laboratories showing that DC from cardiac allografts in mice migrate quite quickly to the spleen provide a basis for a mechanism of central sensitization for vascularized allografts as well [8]. EFFECTOR MECHANISMS The effector area of the immune response comprises a specific component, both cellular and humoral, as well as a nonspecific component which is predominantly cellular, but the relative contributions of these various components to the aggressor reaction which results in graft rejection has not been defined, probably because there are a spectra of reactions depending on the type of graft, the site of implantation, and the state of priming of the recipient. Are C D 4 + or C D 8 + T cells the primary cells involved in rejection? The importance of the T lymphocyte in rejection is beyond dispute, as illustrated, for example, by the indefinite survival of skin allografts on athymic nude mice or B-cellreconstituted rats. However, controversy has raged for some years on whether the CD4 + T-helper cell is the primary cell involved in the specific effector arm of the rejection reaction (with other leukocytes being attracted secondarily in a nonspecific manner as occurs in a delayed-type hypersensitivity reaction) or the destruction of the graft is mediated specifically by CD8 + cytotoxic T cells. Conflicting data have been presented in rodent models in which T-cell-depleted animals were reconstituted with CD4 + or with CD8 + T cells; on the whole they support a more central role for the CD4 + cell, with or without participation of CD8 + cells (reviewed in [9]). On the other hand, a cyclosporine protocol that leads to indefinite renal allograft survival in the rat also results in loss of donorspecific cytotoxicity in leukocytes harvested from the graft itself, implying thereby a prominent role for CD8 + cells [10]. Both of these types of experiments are open to other interpretations, but perhaps experiments in which either CD4 + or CD8 + T cells are deleted or inactivated in vivo with monoclonal antibodies are more persuasive, and as seen in Table 2, mouse heart allografts in naive recipients are prolonged markedly with anti-CD4 antibody treatment while anti-CD8 antibodies were ineffective. On the other hand in the sensitized recipient treatment
108
P.J. Morris with an anti-CD8 antibody was far more effective than treatment with the antiCD4 antibody [11]. More recently we have been able to extract cells from grafts that were not rejecting and show that these cells also exhibited donor-specific cytotoxicity in vitro, suggesting that CD8 ÷ cells themselves are not sufficient for rejection [4]. To some extent the argument revolves around semantics, for it is recognized now that CD4 + cells may be cytotoxic and that CD8 + cells may provide help. Nevertheless, for the moment the balance of evidence favors a major role for the CD4 + T cell in the naive recipient of an allograft.
Are natural killer (NK) cells involved in graft rejection? Although an increasingly prominent role for these cells is being established in tumour surveillance models, there is little evidence suggesting an important role in graft rejection. Indeed in several aUograft models in the rat in our laboratories we have noted prolonged survival of renal allografts in the presence of high levels of N K activity in leukocytes harvested from the graft (e.g., [10]).
Donor-specific lymphocytotoxic HLA antibodies in humans produce hyperacute rejection of a renal allograft, but can hyperacute rejection be mediated by cells? Hyperacute rejection is mostly mediated by antibodies directed at donor HLA class I and possibly class II antigens. Nevertheless on relatively uncommon occasions renal grafts have failed to function in the presence of a current and historical negative serologic crossmatch with the donor. As this usually only occurs in patients receiving a regraft, it is strongly suggestive of an immunologic reaction, and biopsies done before infarction will confirm the presence of severe rejection. It seems possible that this is mediated by cells in the absence of preexisting antibody, but one must postulate the presence of donor-specific cellular immunity in the recipient against either M H C or non-MHC antigens. This type of allograft rejection has been demonstrated against minor histocompatibility antigens in a miniature swine model [12], and it seems very likely to me that failure to function in patients with regrafts is sometimes due to immediate cellular destruction of the graft in a sensitized recipient in the absence of any evidence of humoral sensitization.
Are antibodies involved in acute and chronic rejection? Although antibody is undoubtedly responsible for most cases of hyperacute rejection, its role in acute and chronic rejection is unclear. Antibody of donor specificity can certainly be demonstrated in rejecting grafts and in most patients after removal of a rejected graft, and the histologic picture of acute and chronic rejection in many instances may be predominantly one of vascular damage, which has always been attributed to antibody. On the other hand, donor-specific cytotoxic antibodies have been demonstrated in patients with functioning renal transplants. Antibody may also play a role in the antibody-dependent cellular cytotoxicity (ADCC) reaction, the final mediator being K cells reacting with the FC portion of the donor-specific antibody. Whether this reaction plays a part in rejection in vivo is unknown. S U P P R E S S O R CELL M E C H A N I S M S Since the recognition that the response to sheep red blood cells in mice could be suppressed by a population of spleen cells from tolerized mice (first termed infectious tolerance) by Gershon and Kondo [13], suppressor cells have been demonstrated in a variety of immune response models in vitro and in vivo by
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adoptive transfer. In general the suppressor cells are T cells, and in general they are specific for the antigen concerned, although nonspecific suppressor cells have been also described within the macrophage lineage as well as in a bone-marr o w - d e r i v e d non-T-cell population. However, the suppressor cells described in allograft models are T cells [14]. Both suppressor cells of CD4 + and CD8 + phenotype have been shown to adoptively transfer suppression in different experimental models, although in general the suppressor cells demonstrated at an early stage of the response have been described as CD4+and during the maintenance phase of a long surviving graft as CD8 + [ 15 ]. Do suppressor cells represent a true phenomenon or do they represent an artifact of the systems used for their demonstration? The failure to clone suppressor T cells or to identify a separate population of suppressor T cells together with the somewhat contrived experiments needed to demonstrate their existence in vitro or in vivo (e.g., adoptive transfer to lightly irradiated syngeneic recipients) has led to a good deal of scepticism about the real existence of suppressor T cells. Nevertheless the p h e n o m e n o n has been so widely demonstrated, not only in many experimental models but in a variety of immune responses, that it seems unreasonable to suggest that this is not a true phenomenon that may in vivo regulate the immune response to an allograft. Is there a separate population ofT-suppressor cells? Certainly no phenotypic marker has been identified for such a population and although it is possible that within a CE4 ~ or CD8 + T-cell population there might be a subpopulation of suppressor cells, it is probable that these same cells can behave as suppressor cells given the appropriate stimulation or environment. I f there is not a separate suppressor T-cell population, what induces the T cell to act as a suppressor cell rather than an aggressor cell? The answer to this question becomes even more speculative, for although there are a variety of ways of inducing suppressor cells in allograft models in the rodent, e.g., antigen pretreatment with blood, passive enhancement, or cyclosporine, there would appear to be little in common with these models other than perhaps an inhibition of the interleukin 2 (IL-2) pathway in the induction phase of the response. In the blood transfusion model in the rat, a single transfusion in a selected strain combination produces indefinite survival of renal allografts with complete suppression of rejection. Suppressor T cells can be demonstrated in vitro and in vivo in this model in the mixed lymphocyte reaction and by adoptive transfer, respectively, both early in the response to the transfusion and during the course of the graft [16]. Early in the course of the graft, T cells can be separated from the graft which exhibit donor-specific cytotoxicity in vitro but do not produce interleukin 2 (IL-2) or respond to IL-2 in vitro, nor can these cells break the transfusion effect on adoptive transfer to transfused rats. However if IL-2 is administered to the rats from the time of transplantation, then rejection occurs normally ([17] and manuscript in preparation). At the moment it is impossible to reconcile these findings, but it would appear that pretreatment with blood inhibits the IL-2 pathway in the T-ceU response of the recipient to the graft such that the T cells are inactive in vivo. It is just possible that these inactivated cells have the functional characteristics of suppressor cells, or, on the other hand, that a separate population of suppressor cells is responsible for the inhibition of the IL-2 pathway. The maintenance phase of tolerance to a renal or cardiac allograft in the rodent is extremely hard to break, which would suggest that the active suppression, putatively mediated by T-suppressor cells,
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P.J. Morris must be very strong. Why, then, does one need to manipulate a syngeneic recipient, by irradiation for example, to demonstrate the adoptive transfer of suppression by cells?
CYTOKINES
What is the role of the ever-growing family of cytokines in regulating the immune response to an allograft? This would appear to me to be one of the major areas of investigation required over the next few years, for it would seem that many of the questions that I have already posed above will be answered as we understand more of the complex interactions of the cytokines and cells in both the induction phase and the effector phase of the allograft response. THE TARGET
Is the major targetfor the effector response to a vascularized allograft the endothelium? It is assumed, probably correctly, that the endothelium is the prime target of the response, both cellular and humoral, against the allograft. But this has never been clearly established; more importantly, are there special features which make endothelium the prime target, other than that the circulating blood is in direct contact with it as it passes through the allograft? We know that the endothelium o f the grafted organ, at least in the case of a kidney, remains of donor phenotype (e.g., [17]). Furthermore, not only does endothelium express class I and class II M H C antigens (the latter in the human), but it can be induced to express both in abundance. Furthermore, endothelium expresses a variety of specialized receptors which enhance adhesion of lymphocytes in the presence of an inflammatory reaction. In addition endothelial cells are functionally specialized and extremely active metabolic cells, and, for example, via the arachidonic acid pathway are capable o f significantly influencing the damage produced by the allograft reaction. CONCLUSIONS There are no doubt many questions which I have not discussed which others would see as major problems, but I hope to have touched on sufficient areas of uncertainty to indicate that there is still a vast area of ignorance in our understanding of the immune response to an allograft, some of which may be dispelled during this meeting. If we are ever to manipulate the response in a truly specific manner in clinical practice, it is essential that we continue to strive to answer these questions. REFERENCES 1. Peugh WN, Supervisor RA, Wood KJ, Morris PJ: Immunogenetics 23:30, 1986. 2. Goulmy E: In Morris PJ, Tilney NL (eds): Transplant Rev 2:29, 1988. 3. Fuggle SV: In Morris PJ, Tilney NL (eds): Transplant Rev 3:81, 1989. 4. Dallman MJ, Wood KJ, Morris PJ: J Exp Med 165:566, 1987. 5. AustynJM, Steinman RM: In Morris PJ, Tilney NL (eds): Transplant Rev 2:139, 1988. 6. Faustman D, Steinman RM, Gebel H, Hauptfield V, Davis J, Lacy P: Proc Natl Acad Sci USA 81:3864, 1984. 7. McKenzie JL, Beard ME, Hart DN: Transplantation 38:371, 1984. 8. Larsen CP, Morris PJ, Austyn JM: J Exp Med 171:307, 1989.
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9. Dallman MJ, Morris PJ: In Morris PJ (ed): Kidney Transplantation: Principles and Practice, 3d ed. Philadelphia, Saunders, 1988. 10. Bradley JA, Mason DW, Morris PJ: Transplantation 39:169, 1985. 11. Madsen JC, Wood KJ, Morris PJ: Transplant Proc 21:1022, 1989. 12. Kirkman RL, Colvin RB, Flye MW, Williams GM, Sachs DH: Transplantation 28:24, 1979. 13. Gershon RK, Kondo K: Immunology 18:723, 1970. 14. Hutchinson IV: Transplantation 41:547, 1986. 15. Rodriquez MA, Hutchinson IV, Morris PJ: Transplantation 47:847, 1989. 16. Quigley RL, Wood KJ, Morris PJ: J Immunol 142:463, 1989. 17. Wood KJ, Dallman MJ, Morris PJ: Transplant Proc 21:338, 1989.
