advances in transplantation immunology

Advances in Transplantation Immunology

Peter J. Mom~IS

ABSTRACT: Although there have been dramatic advances in clinical organ transplantation over the past 20 years, rejection, both acute and chronic, and the complications of immunosuppression remain major prob- lems. Nevertheless as our understanding of the immune response to a vascularized organ allograft develops, so too will our ability to develop more specific immunosuppression. In any strategy for more specific immunosuppression compatibility for the major histocompatibility complex of ant igens (HLA in man) is likely to be important. Monoclonal antibodies to T cell subpopulations, or even to T cells specifically activated by the graft, provide methods of suppressing the immune response at a more specific level. The recognition that stable grafts are maintained, at least in experimental rodent models, by T suppressor cells may allow development of precise methods of inducing the generation of such cells in clinical practice. The induction of tolerance in the adult animal can be achieved in a number of ways, the most promising of which for clinical application, is antigen pretreatment. I f tolerance could be achieved in clinical practice within the not too distant future, then this would represent the attainment of the ultimate goal of transplantation.

KEY WORDS: organ transplantation, histocompatibility antigen, cell mediated immune response, specific immunosuppression, induction of tolerance

INTRODUCTION

kJ rgan transplantation has made enormous advances in the last 20 years. In this time we have seen renal transplantation become the

NuJfield Department of Surgery, University of Oxford John Radcliffe Hospital

Reference requests to: Peter J. Morris, MD, NuJfield Department of Surgery, University of Oxford John Radcliffe Hospital, Oxford OX3 9DU, UK

Members of the department who contributed to the work described in this lecture were M. Dallman, S. Fug- gle, J. Madsen, G. TeUides, A. Ting and I~ Wood

This paper is based on a lecture given at the 87th Annual Congress of the Japanese Surgical Society, Tokyo, Japan, 1987

treatment of choice for most patients with end-stage renal failure, cardiac and liver transplantation become an acceptable treatment for selected patients with end-stage organ failure, and pancreatic transplantation performed in increasing numbers for diabet- ics with renal failure. One might feel that the advances in organ transplantation have been so great, that there is little remaining to be achieved. This is far from the truth for rejection of the grafted organ remains a problem, especially chronic rejection, and the complications o f the immunosuppressive drug therapy that is needed for the duration of the graft remain considerable.

Thus in the future we will be seeking better and more specific immunosuppression,

JnPAtqESE Jou~Ae OF SURGERY, VOL. 17, No. 5 pp. 323-333, 1987

324

such that graft survival is improved, especially in the long-term, with few side effects o f the therapy. Advances in this area must depend on a thorough unders tanding of the immune response to a tissue allograft, for on that basis it will be possible to develop more specific approaches to immunosuppressive therapy. And indeed considerable advances in t r ansp lan ta t ion i m m u n o l o g y have oc- curred in recent years, which bring closer the day when tolerance to a foreign tissue can be induced in the recipient.

IMMUNE R E S P O N S E TO A G R A F T

Histocompatibility antigens Histocompatibility antigens in all species

are divided into major histocompatibi l i ty complex (MHC) antigens and minor histo-

Morris s J l , n. J. Surg. tember 1987

compatibility antigens. It is incompatibility for the MHC antigens between the donor and recipient which predominant ly is responsible for the immune response to a graft and its rejection, unless modified by immunosuppression.

The MHC in man is known as HLA, and is an extremely complex genetic system. That it is the MHC in man is clearly demonstrated by the superior survival o f renal transplants between HLA identical siblings. However minor histocompatibility antigens are clearly important, for immunosuppress ion is still necessary in HLA identical sibling renal transplants to prevent rejection. Indeed the importance of minor histocompatibility antigen differences in transplantation has been clearly demonstra ted in a mouse heart allograft model in my own department, where, in

; moom 6 / \

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/ / Class II

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Dl~ DQwl DR1 DR2

6 16

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Fig. 1. HLA, the major histocompatibility complex in man, is situated on the short arm of chromosome 6. The HLA antigens can be divided into several series, the members of each series behaving as if determined by allelic genes. The number of allelic products within each series is given below the appropriate locus, e.g., there are 23 alleles at the HLA-A locus. Furthermore, the HLA antigens are separated into 2 classes based on tissue distribution and function. The class I antigens (HLA-A, B, C antigens) are widely dis- tributed on all tissues, whereas class II antigens (HI,,A-DR, DQ, DP) have a distribution restricted to macrophages, B lymphocytes and endothelium.

Volume 17 Number 5 Transplantation immunology 325

certain strain combinations, multiple minor differences may cause an early and irreversi- ble rejection even in the presence of identity for the MHC. 1

Of course the genetic complexity o f the HLA system makes matching in unrelated cadaver transplantation extremely difficult if compatibility for the whole system is sought. However simpler approaches to matching appear possible. The HLA system may be divided into several series o f allelic antigens- HLA, A, B, C, DR, DQ and DP-- there being from 3 to 30 or so alleles in each series (Fig. 1). The ABC antigens are ubiquitous in that they are expressed on virtually all cells and are described as class I antigens. The DR, DQ and DP antigens have a restricted tissue distribution be ing expressed on macrophages, endothelium, and B lymphocytes, and are known as class II antigens. Some years ago we were able to show that matching for just one of these series of antigens, HLA-DR, resulted in improved cadaver graft survival, 2,~ and these results have been confirmed widely since then. However it has been said that with the better immunosuppression achieved by cyclosporin, matching

100E

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Fig. 2. The influence of matching for HLA-DR alone in 70 consecutive first cadaver grafts all of whom were immunosuppressed with triple therapy (cyclosporin, azathioprine, and ste- roids).

1 3 6 12 ]st Graft 0 DR mm (19) 100 95 95 95

--0-1st Graft 1 DR mm (35) 89 89 89 84 - l - l s tGraf t2DRmm(16) 75 61 61 61

no longer influences graft survival. Intui- tively one would feel that this is unlikely, and indeed we still find a significant influence of matching for HLA-DR alone on cadaver graft survival in patients treated with triple therapy, i.e., azathioprine, prednisolone and cyclosporin (Fig. 2). This important influence of matching for HLA-DR may be explained by its key role in antigen presentation to the recipient as I will discuss in the next section.

Antigen presentation to the recipient The classical concept of antigen presenta-

tion would suggest that MHC antigens from

�9 ~ -- Cells / ~

Antibody Cytotoxic T eelI Act ivated M e

Fig. 3. A simplified version of the immune response to an antigen. Antigen is presented to the helper T cell (TH) by the antigen presenting cells (APC) of the host, either macrophages (M) or dendritic cells (DC). The APC of the host must share the same Class II antigens with the TH for the antigen to be recognized (MHC restriction). Once triggered the TH produces a number of lymphokines, such as factors (THF) which makes B cells produce antibody, IL2, which is necessary for the matura- tion of the cytotoxic T cell precursor (TCP) to the mature cytotoxic T cell (Tc) and MAF which arms macrophages (M). Furthermore suppressor T cells (Ts). may be activated which can damp down this response at several levels. (reproduced with permission from Kid- ney Transplantation: Principles and Practice 2nd edit. Ed. P.J. Morris, Grune & Stratton, New York).

326 Morris sJPn. J. Surg. tember 1987

the donor organ are processed by antigen presenting cells (APC) in the recipient and are p re sen ted by these special ized cells (probably dendritic cells) to T helper (TH) lymphocytes to initiate the immune response (Fig. 3). The T H cell recognises the foreign antigen in association with recipient class II antigen on the presenting cell (known as MHC restriction). This is the way in which all foreign antigens are recognized. However, in the case of a vascularized graft, these specialized presenting cells or dendritic cells will be found in the grafted organ, but are o f course of donor origin. It seems possible that recipient T H lymphocytes randomly entering the graft can recognize incompatible class II antigen as presented by these specialized dendritic ceils of the donor directly, without the need for processing by host presenting cells. It is also possible that these donor dendritic cells can leave the graft and interact with recipient TH ceils at a more central site such as the spleen. Be that as it may, it is apparent that t h e above concept does provide a significant explanation for the influence of matching for the class II antigens of HLA-DR in kidney transplantation in man.

The dendritic cell, which was formerly known as a passenger leucocyte, has a wide- spread tissue distribution. 4 The Langerhans cells in the skin as well as the interdigitating cells o f the lymph nodes are members of this same family, and these cells are distinct from tile macrophage and have characterist ic markers which allow them to be identified as a separate cell type.

The T cell The T lymphocyte population may be

divided into two on the basis of function and phenotype, namely the T helper (TH) and T cy to tox ic / suppressor (Tc/s) populat ions. The latter population may not only be cytotoxic but may also actively suppress the immune response. Whether these two functional populations are composed of one and the same cell is uncertain at present.

As can be seen from Fig. 3, the T H cell is a pivotal cell in the whole immune response,

for after recognizing antigen it produces a whole series of lymphokines which lead to proliferation of B lymphocytes, which produce antibody against the foreign graft, and Tc cells which can kill directly cells of the graft, while also activating macrophages which also can kill cells of the graft. Thus the induction of the immune response occurs at the level of the T H cell, which provides an amplification mechanism for the response.

When the T H cell recognizes antigen one of the lymphokines it produces is inter- leukin-2 (IL2). In addition the TH cell produces receptors for IL2 (IL2R) so that the IL2 reacts with these receptors to continue to drive the T H cells to proliferate. In addition IL2R appear on the Tc and the I12 produced by the TH drives these cells to proliferate in the presence of the foreign histocompatibility antigen. Thus the IL2R which appears only on T ceils after activation is one of a group of lymphocyte cell surface molecules known as activation antigens. The definition of activation antigens such as the IL2R provides impor t an t possible approaches to immunosuppression.

The Tc cell has for a long time been con- sidered the key cell involved in graft rejection, but considerable doubt has been cast on that concept in recent years, s One school of thought believes that graft rejection is a delayed type hypersensitivity (DTH) reaction which is mediated by the T H cell, other cells, such as the macrophage, being involved nonspecifically; while the other believes that the Tc is the specific mediator of graft des- truction. Experiments in rodents have shown that T H cells adoptively transferred to animals depleted o f T cells can reject skin or heart allografts whereas transfer of Tc does not result in graft rejection, suggesting that the Tc is not necessary for rejection to occur. However doubt can be cast on these results for it is known now that T cell precursors exist in rats allegedly depleted o f T cells, and that these cells can become Tc. Furthermore TH cells can be cytotoxic themselves to targets expressing incompatible class II

Volume 17 Transplantation immunology 327 Number 5

~ ~ +_ Cyclosporin lOmg/kg • lODays.

LEW DA

lOOdays

l o o ~ o o o o o o

Spleen Cells

LEW 2oo~ ~Q

DA DA DA Day-2 Day-1 Day-0

Fig. 4. Adoptive transfer assay. Light irradiation (200R) of the syngeneic recipient into which the putative suppressor cells are transferred is necessary to allow a clear demonstration of suppression on specific challenge with an allogeneic kidney.

antigen. Further confusion has been added to this

picture by experiments from Dallman and Wood in my own department showing that not only can donor-specific Tc be removed in large numbers from rejecting renal allografts in the rat, but also from grafts that are not being rejected and have stable function as a result of prior blood transfusions? Thus although it is obvious that the TH cell is the key cell involved in rejection of a tissue allograft, the interaction of the different subpopulations is quite unclear.

Another population of T cells which is of the same phenotype as the Tc cell may play an important role in graft acceptance, and that is the T suppressor (Ts) cell. These cells have been demonstrated in functional adoptive transfer assays in vivo, such as illustrated in Fig. 4, or in vitro by their ability to suppress proliferation in the mixed lymphocyte reaction. Spleen cells from rats that have ac- cepted a renal allograft permanently, for example after treatment with cyclosporin or

Table 1. Suppressor Cells in DA Rats Bearing LEW Kidneys for more than 100 Days. Third Party WAG Kidneys Do not Show Prolonged Survival, nor Does the Adoptive Transfer of Normal Spleen Cells Prolong Survival

Survival Cells Transferred Kidney No.

(Days)

Normal Spleen Cells LEW 8 10-16 Normal Spleen Cells WAG 6 11-14

LTS* Spleen Cells LEW 8 >100 (X8) LTS Spleen Cells WAG 6 16-30

* LTS, long term surviving

after prior blood transfusions, can be adoptively transferred into a lightly irradiated animal of the same strain as the donor of the spleen cells, and then on transplanting a kidney of the same strain as the long surviving kidney, rejection is prevented and the kidney survives indefinitely (Table 1). The cell subpopulation responsible for this transfer are T cells and are T cells of the Tc / s phenotype. However it is rather more com- plicated than that for it appears that there is a hierarchy of T cells that can produce suppression. The first cell which is involved is o f the T H phenotype, which in turn produces a factor (antigen specific) which triggers a cell of the Tc / s phenotype, cyclophosphamide resistant, which in turn triggers a third cell again o f the Tc / s phenotype which is cyclophosphamide sensitive. 7 The evidence for this hierarchy of T suppressor cells was f i r s t proposed by BenaceratFs group in a model of delayed type hypersensitivity 8 but we have been able to essentially confirm the presence of a similar system in a rat renal allograft model in our own laboratories.

Thus the immune response to a graft is a very complex set of cellular interactions which in the unmodified host generally lead to the graft rejection, but which, with immunosuppression, may lead to predominance of an active suppressor mechanism.

The target The target o f the effector arm of the

328 Morris Jpn. J. Surg. September 1987

i mm une r e sponse are the incompat ib le MHC antigens on the graft, and one would imagine that endothel ium would be a prime target within a vascularized organ graft. Re- cent work in a number of laboratories, including our own, has shown that as rejection of a graft occurs then class II antigen expression can be induced in cells within a graft that do not normally express class II antigen 9,1~ and also that increased expression of class I antigen occursY All of the above observations, as well as others, suggested that this induction of MHC expression was related to the cellular infiltration that occurs as part of the rejection reaction and is a response to the release of inter- feron-gamma by the T H cell. This increased expression of class II antigen might augment

t h e recognition phase of the immune response, while the increased exlSression of class I and II antigen would provide a markedly increased antigen density as a target for the effector arm of the immune response. And indeed weight was leant to this hypothesis by the demonstration, both in man and rat, that renal allografis in recipients treated with cyclosporin showed far less induction of MHC antigen, which appeared to correlate with graft survival? ,t~

However more recently in our own laboratories, using a different renal allograft model to that used by Milton and Fabre we have shown that induction of MHC class I and class 1I antigens may occur as heavily in

. grafts that are not rejecting as in those that are rejecting, n Thus the role of the induction of MHC antigens in an allograft, although a striking phenomenon, remains unclear.

STRATEGIES FOR MORE SPECIFIC

IMMUNOSUPPRESSION

Immunomodulation of the graft There is no doubt that the passenger leuc-

ocyte in an organ graft, now known to be the tissue dendritic cell, can play a key role in the induction of an immune response against the graft, at least in the rodent. For example

removal of the passenger leucocytes by pre- treating the donor animal in such a way as to remove all leukocytes from a renal allograft will lead to prolonged survival of the graft after transplantation in certain strain combinations. Furthermore retransplantation of a long surviving renal allograft in the rat, which will now carry passenger leucocytes of recipient type, into a fresh naive rat of the same strain as the original recipient will lead to prolonged survival of the graft. However bo th the above results are restricted to selected strain combinations in the rodent. And so the dendritic cell, powerful and necessary as it is as an accessory cell in the in vitro response to histocompatibility antigens, may not be essential for the induction of the immune response in vivo. Nevertheless it is an attractive concept for immunomodulation especially in the case of a tissue allograft such as isolated pancreatic islets where only a few cells of dendritic morphology exist in each islet. Indeed treatment of mouse islets in culture with an anti-dendritic cell monoclonal antibody and complement has led to prolonged survival of these islets after transplantation. TM However this has not been a readily reproduced phenomenon. 13

Despite the obvious relevance of the dendritic cell to immunogenicity in the rodent, it may have less relevance in man. This is because in man, in contrast to the rodent, endothel ium expresses class II antigen, and if therefore endothelium can be recognized as foreign by way of this class II expression, for which there is evidence, then removal of the dendritic cell from an organ before transplantation would be of little relevance to the behaviour of that graft after transplantation. Nevertheless for the time being the dendritic cell remains a cell of very considerable interest, with its final role in the immune response to a graft to be determined, and hence the relevance of its deletion from a graft before transplantation.

Monodonal antibodies to T cells Hetero logous antilymphocyte globulin

(ALG) has been used for years to treat or


prevent rejection with considerable success. TM

But such heterologous sera (usually made in horses or rabbits) are very broad in their reactivity not only to leucocytes but often to other targets, such as platelets. Monoclonal antibodies provide the means to target the immuno-suppression much more specifically at different components of the immune response. The first monoclonal antibody to be used therapeutically was OKT3, a pan T monoclonal antibody; if you like a second generation ALG. It removes T lymphocytes from the circulation within minutes of intravenous adminis t ra t ion , and a course of OKT3 will reverse most acute rejection epi- sodes. 14 Thus it is more effective than treatment with intravenous boluses of methyl- prednisolone and this was reflected by a better cadaver renal graft survival in a con- trolled trial comparing OKT3 with methyl- prednisolone for the treatment of the first acute rejection episode. 15 However there is no evidence that OKT3 is necessarily any more effective than an immunosuppressive heterologous ALG. However its advantage is that it would always be a very standard product in contrast to ALG, which is produced by immunising either horses or rabbits with human lymphocytes, where standardisation of the end product in terms of immunosuppressive potency is extremely difficult. Thus it would appear that where ALG is indicated for the treatment o f rejection, then a pan T monoclonal antibody, such as OKT3, would be more consistently effective. However one drawback to the use of OKT3 (and perhaps to most monoclonal antibodies) has been the development of antibodies against mouse globulin which are mostly anti-idiotypic in nature, and which preclude its continuing use once they appear? 4 Other pan T monoclonal antibodies have been used, but in general they have been far less effective than OKT3. This is presumably because the OKT3 antibody is directed against the CD3 molecule which is intimately associated with the T cell receptor. This is not the case with monoclonal ant ibodies d i rec ted against

Table 2. A Rat Anti-L3T4 Monoclonal Antibody (GK1.5, IgG2b) Was Given iv to Recipient Mice in 1 ml Doses from 2 Days before to 10 Days after Heart Transplantation

MST -4- SD Strain Treatment

(Days)

C57BL/10 to DBA2 Nil 29.2-t-31.1 Anti-L3T4 79.2--+25.2*

C57BL/10 to C3H/He Nil 9.2-t- 0.8 Anti-L3T4 56.7-t-34.4"

*, p=0.01

other pan T cell markers. However an antibody directed against all

T cells represents only a marginal improve- ment in specificity, and more specific approaches to the cellular components of the immune response are theoretically possible with monoclonal antibodies. For example the T H cell is a central cell in the immune response and an antibody directed at that cell might be expected to have a p rofound effect on the immune response. Cobbold and colleagues 1~ have shown that a rat monoclonal antibody directed against mouse T H cells (anti-L3T4) will prolong survival o f skin allografts, and we have shown that an identical antibody will markedly prolong survival of cardiac allografts, also in the mousC ~ (Table 2). However in contrast a mouse monoclonal antibody directed against rat T H cells (W3/25) is ineffective in preventing rejection in a rat renal allograft model, despite being a potent inhibitor of proliferation in the mixed lymphocyte reaction (Tellides, Dallman and Morris-unpublished observations). The reasons for this are unclear at present but it obviously is important to find what parameters are important in determin- ing the effectiveness o f a monoclonal antibody against TH cells in preventing rejection before selecting such antibodies for clinical trials.

Returning to specificity, monoclonal antibodies against T H Cells are more specific than pan T antibodies, but still leave much to

330

be desired in this regard. If one could target treatment at T cells which were recognizing and reacting to the particular histocompatibility antigens of the grafted organ, this would be a large step forward in terms of specificity. As ment ioned earlier, a number of activation antigens appear on lymphocytes during the induction of the immune response, and one well defined activation antigen is the IL2R which appears on activated T cells.

Thus m o n o c l o n a l ant ibodies d i rec ted against the IL2R molecule might delete or inactivate the very cells that are reacting to the graft MHC antigens. Indeed it has been shown that a mouse monoclonal antibody against the IL2R (ART18) will prolong survival of cardiac allografts in the +rat and deplete the graft of T lymphocytes expressing the IL2R. TM We have confirmed this in our own laboratories in a different strain combina- tion, but have also shown that the same antibody (ART18) which prolongs survival of cardiac allografts will not prolong survival of renal allografts. This is a surprising finding for which there is no ready explanation as yet, unless it be that there are more IL2R+ T cells in the rejecting renal allograft com- pared to the cardiac allograft, and hence a higher dose of ART18 would be needed to prevent rejection. However another important finding is that another monoclonal antibody against the IL2R (MRC OX39) which appears identical to ART18 in nearly

Table 3. The Survival of LEW Hetero- topic Heart Allografts in DA Recipients Treated with Two Monoclonal Anti- bodies Against the IL2R, ART18 and OX39, Given for the First 10 Days after Transplantation

Treatment Dosage Survival

Nil - - 7,7,7,8,9, 10,10,10,11, 11

ART18 300/xg/kg/day IV X 10 days 21,22,23, 31,~50

OX39 900/lg/kg/day IV • 10 days 6,6,7,7,13

Morris Jpn. J. Surg. September 1987

every respect (e.g. both IgG1, both precipitate 55KD protein, and both block binding of IL2 to IL2R) does not prevent rejection of cardiac allografts (Table 3). Obviously the MRC OX 39 antibody detects a different epitope on the IL2R molecule to ART18, and it is the latter epitope which must be related to function, for which we now have evidence (Tel- lides, Dallman and Morris-unpublished observations). We are at present examining several monoclonal antibodies against the rat IL2R both in vitro and in vivo in order to confirm that the efficacy of an anti-IL2R antibody is dependent on the binding of the antibody to a functional epitope on the molecule.

The potential for the use of monoclonal antibodies in transplantation is enormous, but it is now apparent that considerable investigation is required tO establish the criteria for the effectiveness of monoclonal antibodies as immunosuppressive agents in organ transplantation before embarking on clinical trials. The rodent model should be useful, as suggested in the two examples described above, in defining the properties and the targets of monoclonal antibodies which will determine its effectiveness in clinical trials.

T SUPPRESSOR CELLS AND FACTORS

As ment ioned in an earlier section suppressor cells of the T cell lineage can be shown to play a role in maintaining long- term surviving grafts in the rodent in adoptive transfer assays. Whether there is a separate T cell populat ion which is the Ts population is unknown, and it is possible that a T cell may be suppressive or cytotoxic depend- ing on the immunogenic stimulus and the environment in which it finds itself.

Obviously if we were clear as to how the suppressor cell circuit was induced, and could produce such cells or their factors, there would be considerable clinical potential for their use in organ transplantation. However this is proving to be extremely


difficult. In our own laboratories we have been able to fuse spleen lymphocytes, some of which have been induced as suppressor cells to the hapten TNP, with a rat thymoma cell line to produce a suppressor cell hybri- doma. This hyb r idoma produces factors which will suppress the response of primed lymphocytes to TNP in vitro and will also prevent rejection of renal allografts in rats pretreated with TNP haptenated to donor alloantigen.

The whole suppressor cell area remains rather confused and it has proved extremely difficult to go much further than observe the phenomenon in transplant models. Never- theless it remains an important area in which to try to resolve whether this is truly a mechanism of suppression or an artefact induced by the methods used for assaying suppressor cells.

INDUCTION OF TOLERANCE

Tolerance remains the ultimate goal of all transplantation biologists, for this would mean that the recipient of a transplant would not be able to respond to the transplant, but would be quite normal in their immunological reactivity to other foreign antigenic chal- lenges such as viruses or bacteria. Tolerance to skin allografts was achieved in the mouse by Medawar and his colleagues some 30 years ago by injecting the neonatal animal with foreign lymphoid tissue at a time when the animal is immunologically unreactive? 9 Thus the mouse does not recognize the injected tissue as foreign, but indeed as self, as predicted would be the case by Burnett and Fenner. 2~ Such animals when chal- lenged with a skin graft from the same donor later on, do not reject the graft. This phenomenon is known as neonatal tolerance, but is not applicable to transplantation in the immunologically competent adult. The induction of tolerance in the adult animal is much more difficult, but can be achieved in the rat either by treatment of the recipient animal with donor-specific antibody at the time of a

r ena l t r ansp lan t (known as passive en- hancement) or by pretreatment with donor antigen, e.g., in the form of blood, b e f o r e transplantation (known as active enhance- ment). For a number of reasons passive enhancemen t is not likely to be successful in man, so that investigations in recent years have concentrated on antigen pretreatment. This is given considerable impetus by the observed better renal allograft survival in patients given blood transfusions either from random donors before cadaver transplantation or from the proposed donors before l iving-related t ransplantat ion. 21 Tha t the transfusion effect, as it is known, is produced by r a n d o m b l o o d d o n o r s would a rgue against against immunological specificity, but work in our laboratories in a rat renal allograft model would suggest that the effect can be produced by sharing only part of the MHC and even in some instances minor antigens between the blood donor and kidney donor, thus providing an explanation for the apparent lack of specificity of the transfusion effect in cadaver renal transplantation. 22

However before any rational approach to the induction of even partial tolerance in clinical practice can be attempted we need to know a lot more about this p h e n o m e n o n in the experimental animal. For example such questions as the form of antigen presentation required, the route and timing of antigen pretreatment, and the mechanism by which any effect is induced and then maintained, are all pertinent. In our laboratories we have stuck to the whole blood transfusion model which Fabre and I had shown many years ago could produce permanent survival of renal allografts in the rat3 3 Whole blood, of course, contains very many cellular components all of which express MHG antigens in one form or another, but this seemed an appropriate base from which to start again. We have used two models of organ transplantation: cardiac allografts in the mouse and renal allografts in the rat. Our efforts have been concentrated on class I MHC

332 Morris sJPn. J. Surg. tember 1987

Table 4. DA Rats Were Given 0.5 ml Whole Blood or 8 X 109 Highly Purified Erythrocytes IV from LEW, DA, BN or WAG Donors 7 Days before Transplan- tation of a LEW Kidney. There Was a Specific Prolongation of Survival in Rats Given LEW Whole Blood or Erythro- cytes, the Latter Expressing Class I MHC Antigens only in the Rodent

Mean Survival Treatment (IV, Day-7) No.

(Days)

Nil 15 11 0.5 ml LEW Blood 5 >100 0.5 ml DA Blood 5 10 0.5 ml BN Blood 5 10

8 X 109 LEW r.b.c.* 7 >100 " BN r.b.c. 7 11 " WAG r.b.c. 4 10 " DA r.b.c. 4 9

* Contamination--1 leucocyte/5 X 106 r.b.c.

antigen, for cells such as erythrocytes and hepatocytes express class I antigens only in the absence of class II MHC antigens. In the absence o f class II antigen, sensitisation by pretreatment is less likely to occur (an important consideration in clinical practice) and indeed mechanisms of responsiveness may be induced which selectively lead to tolerance. Thus in the rat pretreatment with pure erythrocytes (Table 4) or hepatocytes or even hepatocyte membrane fractions, all expressing class I MHC antigen only, produce prolonged survival of renal allografts. To even further clarify this effect we have been able to transfect a fibroblast cell line (L cells) derived from a mouse of the same genetic makeup (C3H) as the recipient of the heart graft, with genes for different class I MHC antigen, the gene product being expressed on the cell surface (Fig. 5). Pretreatment of C3H animals with these L cells, the only MHC difference being in the class I product of the transfected gene, followed by transplantation of an incompatible heart from a mouse strain (C57BL) which included the same class I MHC antigen as expressed by the transfected class I gene in L cells used for

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L-K ~ Frmlsfectant

@ --- KK

L CelI

C57BI

H-2 b /

Day-0 r

C3H

H-2 K

Fig. 5. Prolonged survival of allogeneic cardiac allografts in the mouse after pretreatment with the products of a single class I MHC gene. L cells, a fibroblast line derived from the C3H mouse, are transfected with a K

�9 b regton gene of the H-2 haplotype. The b �9 �9 K gene is incorporated into the genome

and its product is expressed on the cell surface. Thus these L ceils which are syngeneic with the C3H mouse (H-2 k) now express the K b gene product. Pre- treatment of the C3H mouse with these transfected L cells leads to prolonged survival of heterotopic cardiac allografts from a C57BL donor (H-2b).

pretreatment, leads to prolonged survival of a fully allogeneic heart (Superina and Mad- sen, Wood and Morris-submitted for publication).

Thus we now have several examples of partial or complete tolerance induction to a subsequent organ allograft in the rodent after pretreatment with class I MHC antigen. Now that we have defined a specific situation in terms of antigen presentation in which tolerance is induced we can begin to exam- ine the mechanisms involved�9 I remain op- timistic that strategies which will allow at least partial tolerance induction in clinical organ transplantation can be developed.

CONCLUSIONS

There have been dramatic advances in


i m m u n o l o g y a n d i n tha t b r a n c h o f i m m u - nology r e l a t ed to o r g a n t r a n s p l a n t a t i o n in recent years. We u n d e r s t a n d m u c h m o r e about t he m e c h a n i s m s o f graft r e j ec t i on t h a n hitherto, b u t at th is m o m e n t i n t ime t h e r e is cons iderab le c o n f u s i o n as to t h e ce l lu l a r mechan i sms o f re jec t ion . H o w e v e r as o u r u n d e r s t a n d i n g o f t h e i m m u n e r e s p o n s e to ant igen a n d a n a l lograf t deve lops so too we have b e e n a b l e to e x p l o r e s t ra tegies fo r bet - ter a n d m o r e specif ic i m m u n o s u p p r e s s i o n i n exper imenta l m o d e l s o f t r a n s p l a n t a t i o n . A n u m b e r o f t he se s t ra tegies s h o u l d p rove o f value in c l in ica l t r a n s p l a n t a t i o n , a n d i n d e e d we may b e ab l e to i n d u c e t o l e r a n c e to a n organ a l lograf t i n c l in ica l prac t ice o n e day, the u l t i m a t e g o a l o f a l l t r a n s p l a n t a t i o n

biologiests.

(Received for p u b l i c a t i o n o n Apr. 1, 1987)

R e f e r e n c e s

1. Peugh WN, Superina RA, Wood KJ, Morris PJ. The role of H-2 and non-H-2 antigens and genes in the rejection of murine cardiac allografts. Immunoge- netics 1986; 23: 30-37.

2. Ting A, Morris PJ. Matching for B-cell antigens of the HLA-DR (D-related) series in cadaver renal transplantation. Lancer 1978; i: 575-577.

3. Ting A, Morris PJ. A powerful effect of HLA-DR matching on survival of cadaveric renal allografis~ Lancet 1980; 2: 282-285.

4. Hart D, FabreJ. MHC antigens in rat kidney, ureter, and bladder: localization with monoclonal antibodies and demonstration of Ia positive dendritic ceils. Transplantation 1981; 31: 318-325.

5. Mason D, Morris PJ. Effector mechanisms in allograft rejection. Ann Rev Immunol 1986; 4: 119-145.

6. Dallman MJ, Wood KJ, Morris PJ. Specific cytotoxic cells are found in the non-rejected kidneys of blood transfused rats. J Exp Med 1987; 165: 566-571.

7. Hutchinson IV. Suppressor T cells in allogeneic models. Transplantation 1986; 41: 547-555.

8. Doff ME, Benaceraff B. Suppressor cells and im- munoregulation. Ann Rev Immunol 1984; 2: 127-000.

9. Fuggle sV, McWhinnie DL, Chapman JR, Taylor HM, Morris PJ. Segmental analysis of HLA-class II antigen expression in human renal allografts: induction of tubular class II antigens and correla- tion with clinical parameters. Transplantation 1986; 42: 144-149.

10. Milton AD, FabreJW. Massive induction of donor- type class I and class II major histocompatibility complex antigens in rejecting cardiac allografts. J Exp Med 1986; 161: 98-112.

11. Wood KJ, Hopley A, Dallman MJ, Morris PJ. Induc- tion of donor class I and class II major histocompatibility complex antigens does not always correlate with graft rejection. 1987; (submitted for publication).

12. Faustman DL, Steinman RM, Gebel HM, Hayfield V, Davie JM, Laccy PE. Prevention of rejection of routine islet allografts by pretreatment with anti- dendritic cell antibody. Proc Natl Acad Sci 1984; 81: 3864-3868.

13. Reece-Smith H, McSbane P, Morris PJ. Pretreatment of isolated adult islets with antibody effect on survival in allogeneic hosts. Transplantation 1983; 36: 228-230.

14. Jaffers GJ, CosimiAB. Antilymphocyte globulin and monoclonal antibodies. In: Morris PJ, ed. Kidney Transplantation: tMnciples and Practice. 2nd edit. New York, London: Grune & Stratton 1984; 281-299.

15. Ortho Multicenter Transplant Study Group. A ran- domized clinical trial of OKT3 monoclonal antibody for acute rejection of cadaveric renal transplants. New EngJ Med 1985; 313: 337-000.

16. Cobbold SP, Waldmann H. Skin allograft rejection by L3T4 and Lyt-2 + T cell subsets. Transplantation 1986; 41: 634-639.

17. Madsen J, Peugh WN, Wood KJ, Morris PJ. The effect of anti-L3T4 monoclonal antibody treatment on first-set rejection of mouse cardiac allografts. Transplantation (in press).

18. Diamenstein T, Osawa H, Kirkman RL, Shapiro ME, Strom TB, Tilney NL, Kupiec-Weglinski JW. Inter- leukin 2 receptor--a target for immunosuppressive therapy. In: Morris PJ and Tilney NL, eds. Trans- plantation Reviews. New York: Grune & Stratton, 1987 (in press).

19. Billingham RE, Brent L, Medawar PB. Quantitative studies on Tissue Transplantation Immunity III. Actively acquired tolerance. Roy Soc (London), Philos Trans B 1956; 239: 375-414.

20. Burnett FM, Fenner F. The production of antibodies. 2nd edit. (Macmillan, Melb.). 1949.

21. Opelz G. Blood transfusions in renal transplantation. In: Morris PJ, ed. Kidney Transplantation: Principles and Practice. 2nd edit. New York, Lon- don: Grune & Stratton, 1984; 323-334.

22. Hutchinson IV, Morris PJ. The role of major and minor histocompatibility antigens in active en- hancement of rat kidney allograft survival by blood transfusion. Transplantation 1986; 41: 166-170.

23. FabreJ, Morris PJ. The effect of donor-strain blood pretreatment on renal allograft rejection in rats. Transplantation 1972; 14: 608-617.

24. Superina RA, Wood KJ, Morris PJ. The effect of pretreatment with a single cloned donor class I gene product on cardiac allograft survival in mice. Transplantation 1987 (in press).

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