Suppression of Rejection of Organ Allografts by Alloantibody

tmmunologicai Rev. (1980). Vol. 49

Published by Munksgaard. Copenhagen. DenmarkNo par! may be reproduced by any process wilhoul written permission from the author(s)

Suppression of Rejection of OrganAUografts by Alloantibody*

PHTER J . MORRIS

I. INTRODUCTION

Passively administered donor specific alloantibody was first shown to enhancethe growth of allogeneic tumours in the mouse by Kaliss and Molomut in 1952(and hence the term passive enhancement). Subsequently the exquisite donorspecificity of this phenomenon was demonstrated by Moller (1%3), again in amouse tumour model. However attempts to prolong the survival of skinallografts by the passive administration of alloantibody were relatively unsuc-cessful, so clinical interest in the phenomenon waned. With the advent ofmicrosurgical techniques in the late sixties, it became possible to performvascularised organ allografts in the rat which allowed both orthotopic renalallografts and heterotopic cardiac altografts to be performed between inbredstrains of rats in which the major histocompatibility system was reasonably welldefined, although not to the degree of sophistication that existed in the mouse.This allowed the phenomenon of passive enhancement to be reexamined in arenal allograft model. Stuart and colleagues (1968) and French & Batchelor(1969) first showed a dramatic suppression of rejection ofrenal allografts by thepassive administration of donor specific antibody.

The term passive enhancement implies suppression of the immune responseby passively administered donor specific alloantibody. in contrast to activeenhancement where suppression of the immune response is achieved by prioradministration of antigen, perhaps mediated by antibody. The terms, passiveand active enhancement, might be replaced with those of antibody-induced andantigen-induced suppression. In this review I shall confine my remarks tosuppression of the immune response to major histocompatibility systems (MHS)by passively administered alloantibody, as this appears the method which mightbe applicable to clinical transplantation in the first instance. I shall use the termpassive enhancement synonomously with antibody-induced suppression.

Nulticld Ucpjirtment of Surgery, University of Oxford, John Radcliffe Hospital, Headington,Oxford. 0X3 9DU.* Supported in part by grants from the Medical Research Council of the U.K.. the WellcomeTrust, and the National Kidney Foundation.

94 MORRIS

IL MODELS OF ANTIBODY-INDUCED SUPPRESSION OF REJECTION OF

TISSUE ALLOGRAFTS r

I. Commonly used models

The orthotopic renal allograft in the rat, in which end-to-end renal artery andvein and ureteric anastomoses are performed using microsurgical techniques, isnow the standard model of a vascularised organ aliograft. the technique havingbeen modified over the years with developing microsurgical skills (Fabre et al.1971, Lee 1967). The heterotopic cardiac allograft. in which the aorta and venacava of the heart are anastomosed end-to-side to the recipient aorta and venacava, is also widely used (Ono & Lindsey 1969) because of its les.s demandingtechnique. Some studies have been performed also using the vascularisedsegmental pancreatic graft in the rat (Poole et al. - unpublished). Skin andtumour allografts in the mouse, especially between congenic strains, have givenvaluable information concerning specificity of alloantibody. And models ofrenal and cardiac transplantation in the mouse have also been used to a limitedextent (Russell et al. 1978).

ENHANCEMENT IN THE (DAXLEWISJFI -*- tEWI5 MODEL

Da

Lewis anti Doenhancing serum

cFigure I. A typical model of enhancement of renal allografts In the rat. Spleen lymphocytes areused to produce a hyperinimune anti-serum in the recipient .strain. This antiseruni is thenpassively administered to the recipient at or about ihe time of renal transplantation. Bilateralnehpreclomy of tlie recipient is performed at the time of transplantation or a eontralatenilnephrectomy is delayed for 7 days.

SUPPRESSION OF REJECTION BY ALLOANTIBODY 95

In all models donor specific antiserum is administered at or shortly after thetime of transplantation (Figure 1). It is most effective when given intravenously;continued administration does not produce any augmentation of the effect andindeed may be harmful (Fabre & Morris 1972a).

2. Production of antisera

Donor specific antisera are produced generally by hyperimmunisation of therecipient with donor lymphoid cells. Freund's complete adjuvant may be usedwith the initial immunisation to ensure a hyperimmune serum with immuno-suppressive activity. Antisera with immunosuppressive activity in the renalallograft model have been produced by skin allografts (Fabre & Morris 1975,Gallico & Mason 1978) and kidney homogenates in the DA to Lewis straincombination only (Gallico & Mason 1978. McKenzie et al. 1979).

Although some correlation between the lymphocytotoxin titer of an anti-scrum and its enhancing activity has been seen this is neither a consistent nor areliable finding. We have also attempted to correlate the enhancing activity ofantiserum with the binding capacity of anti-serum to donor lymphoid targetcells, which did show a rather better correlation with enhancing activity than thelymphocytotoxin titre (McKenzie et al. 1979). However no reliable index of theimmunosuppressive activity of an allo-antiserum has been defined; but it doesappear that the antiserum has to be produced by hyperimmunisation of therecipient with donor tissues.

Xenoantisera, absorbed to leave donor specific activity, have been used byHutchin.son et al. (1976) to produce enhancement of rat renal allografts, anapproach to the problem now probably superseded by the advent of hybridomasproducing monoclonal antibodies. Such monoclonal antibodies will be of greatvalue both for definition of the target antigen of passive enhancement and evenlor therapeutic use. It is of considerable interest in this respect that McKearn etal. (1979) have reported passive enhancement of rat renal allografts with aputative mouse anti-rat la monoclonal antibody.

3. Variation of effect related to strain combination and type of allograft

There is an enormous variation in the immunosuppressive effect produced bypassive enhancement of the same organ in the same species in different straincombinations although all represent transplants across the major histocompat-ibility system. Furthermore there is a striking difference between the behaviourof the organ of a homozygous parental donor and the heterozygous Fl hybriddonor. Examples of this variation in the survival of renal allografts are shown inTable I. It is usually far more difficult to enhance homozygous renal allograftsthan heterozygous renal allografts, and indeed in the DA to Lewis renal

96 MORRIS

TABLE I.The effectiveness of passive enhancement of kidney grafts across different major histocompaii-

hilitv barriers in the rat*

Strain combination

AS2 to DA(DAXLewis)Fl to DALewis to DA(ASXAugust)Fl to ASAugust to AS(DAXLewis)FI to LewisDA

Mean blood urea (mg/lOO ml) at weeksNo.

467

5556

1

429262

6410484210

2

6265

151146167

201617

3

607264

81201730

—

20

45ND41

68--—

Mediansurvival (days)

>300>100>300>300

432214

In all strain combinations untreated recipients undergo a severe acute rejection, fromwhich they generally die, usually In the second week after transplantation. Lewisrecipients receiving DA kidneys die within 12 days.(Adapted from Fabre & Morris 1973a, 1974b, and Fabre & Batchclor 1975).

allograft model complete suppression of rejection by alloantibody alone cannotbe achieved. This difference between the homozygous and heterozygous donorcannot be explained on the basis of a simple gene dosage effect.

In addition to the above variation between different strain combinations,there is also a variation in the immunosuppressive activity of donor specificantisera for different tissue allografts in the same strain combination. Forexample rejection of renal allografts may be suppressed completely withindefinite survival of the recipients, while only a moderate prolongation ofsurvival of cardiac allografts, and a very modest prolongation of skin allografisurvival is achieved (Tilney & Bell 1974, Nash et al. 1977). There may be anexplanation for this strain and tissue variation in response to passive enhance-ment, to which I shall return later in the discussion of mechanisms.

4. Modification of the immune response and histology of an allograft h\aUoanlihody

The immune response to a renal allograft across the MHS in the rat ischaracterised by a humoral complement dependent lymphocytotoxin response,and the appearance of cellular immunity as shown in vitro by direct lymphocytekilling of target lymphoid cells, this latter phenomenon being independent ofantibody and complement (Burgos et al. 1974, Biesecker et al. 1973, Strom et ai.1975). Following the administration of donor specific alloantibody whichsuppresses rejection of a renal allograft, the humoral response is suppressedeither completely or shows an IgM response only, while the cellular response isdelayed by a day or two but shows little or no decrease in the strength of the


response (Figure 2). Both the humoral response, if present, and the cellularresponse, wane after 10 days or so, and are not usually detectable in the animalwith a long surviving kidney. The demonstration of cellular immunity is to someextent dependent on the technique used (Biesecker et al. 1973); and it must beadmitted that the relevance of the in vitro findings to what occurs in vivo is insome question.

Nevertheless support for the relevance of the in vitro finding is provided by thehistological changes seen in the renal allografts, for these are compatible withthe in vitro demonstration of humoral and cellular immunity. In the unmodifiedhost, rejection of a renal allograft in the rat is characterised by glomerular andtubular necrosis, gross oedema, flbrinoid necrosis of arteriolar walls, throm-bosis, and deposition of IgG and complement in arteries, all features whichcould be attributed to antibody-mediated damage, and which are fullydeveloped seven days after transplantation (Abbas et al. 1974a, b). Theenhanced renal allograft, on the other hand, shows no such evidence ofantibody-mediated damage at 7 days but does show quite marked foci ofmononuclear cellular infiltrates (Abbas et al. 1974a, b). Thus all the evidence atpresent suggests that passive enhancement suppresses predominantly thehumoral response to a vascutarised organ allograft, but does not have a strikingeffect on the initial cellular response.

LYMPHOCYTOTQXINS

r\/ \/ \/ \/ \/ \/ \ '

/ •• \

/ '

/ /

/ // /

/ . . - - ' •

1

I

\\\\\

untroaled

enhanced

\\\\\\\\\I1\\\1V\\

\ \

untreoted

enhanced

/ \/ \

/ \/ \

/ \/ \/ \

/ \/ \

/ \/ \

/ \/ \

/ \/ \

/ \/ \

/ \

/ ' ' ' ~ ^ - ^ \

10 15

days

0 5after Iransplontation

10 IS

ligure 2. A characteristic lymphocyloloxin response and a cell mediated immune (CMI)response in an untreated and an enhanced rat receiving a renal ailograft. The CMI assaymeasures the in vitro killing of donor target lymphocytes (thymocytes labelled with Cr") bylecipient lymphocytes.

98 MORRIS

TABLE IIDose re.sponse studies of passive enhancement of renal allografts in the (DA X Lewis)Fl

to Lewis and DA models (all antisera given IV).

Treatment

Nil2 ml day 0.2 ml day 1,3.51 ml day 00.25 ml day 0.05 ml day 0.01 ml day 0

(DAX

No.

6

5

5555

Lewis) to LewisMedian

Survival (days)

11

26

17171510

{DA X LewisjFI to DA

No.

8

5

6556

MedianSurvival (days)

19

>100

>100>I00>100>100*

* These animals suffered an early rejection episode from which they recovered to surviveindefinitely.

5. Dosage and Timing

Dose response studies of passive enhancement of renal allografts in the rat havebeen performed in the rat by Fabre & Morris (1973a), and show thatsuppression of rejection can be achieved by quite small doses of antisera (TableII). In the weaker (DA X Lewis) Fl to DA model an effect is seen with as little as0.01 ml of DA anti-Lewis serum, and even in the strong (DA X Lewis) FI toLewis model 0.05 ml prolongs survival. This has quite important implications inany discussion on the mechanism of passive enhancement, to which I shallreturn later. Continuing administration of antisera does not augment the effectand may be even harmful (Fabre & Morris 1972a). The timing of administrationfor an effect is relatively broad. Antiscrum is usually given at the time oftransplantation but may be given with equal effect from 24 hours beforetransplantation as well as up to 48 hours after transplantation, at least in therenal allograft rat model (Sen, Fabre and Morris - unpublished observations).

6. Class of antibody

Enhancing activity is confined to the IgG class (Mullen et al. 1977), but there isone report of enhancing activity in the IgA class (Voisin et al. 1969). Igm doesnot enhance and will damage a renal atlograft (Mullen & Hildemann 1971).There is some controversy concerning the subclass of IgG which enhances. Thismay depend on the model tested, and very likely the purity of the preparationtested in earlier studies. More recently Jansen et al. (1975) isolated IgGsubclasses from a 7S fraction by affinity chromatography. Pure preparations ofIgGl. IgG2, IgG2a, and IgG2b were thus obtained. The IgGl was non-cytotoxic. IgGl was able to enhance skin allografts in the mouse, but not


TABLE IIIlewis to DA renal aitofjrafts treated with DA anti-Lewis serum. IgG. or Ffah'):. Finally0..S ml Ffab'): was given before IgG. The F(ab'h was non cytotoxic tm had unimpairedbinding to DA targets (modified from Winearls et at. 1979a). In these experiments therecipients had a detayed contrataterat nephrectoy at 7 days.

Treatment

NilDA anti l.vwis serum,025 ml days O.I.05 ml days 0.1i1.4 anti Lewis IgG.05 ml days 0.1(0.38 mg).005 ml days O.I(0.038 mg)DA anti Lewi.s F(at)')2.05 ml days 0.1

0,5 ml (F(ab'):followed by 0.05 mlIgG

No.

5

55

5

4

6

4

Mean Blood urea (mg/lOO mi)10 days

762

150102

120

324

636

96

14 days

—

96114

198

222

180*

108

28 days

—

6666

108

108

72*

66

MedianSurvival-days (range)

I]

>100>100

>I00

>100

11

>100

(10- 11)

(17->IO0)( 5 2 - > 100)

(37- too)

(28-> 100)

( 8->I00)

(all>100)

* 2 animals only

produce hyperacute rejection, while the IgG2 was able to produce bothenhancement and hyperacute rejection. Thus it appears that both subclasses ofIgG, including the nonconiplement fixing IgGl, are able to enhance, althoughonly IgG2 has been shown to enhance rat renal allografts (Mullen cl al. 1977).

Although it has been considered that the F(ab')2 fraction of IgG is able toenhance, even if less effectively than whole IgG, there is considerable doubt nowconcerning the purity of F(ab)2 preparations used in earlier experiments. Asdose response studies (Table 2 and Fabre & Morris 1973a) have shown that verysmall doses of whole antiserum are able to enhance, any contamination ofI7ab')2 preparation with IgG could result in apparent enhancement. Indeedstudies from our laboratories of a preparation of F(ab'): which had less than I %contamination of IgG showed that F(ab')3 was at least 100 fold less effective thanwhole IgG (Winearls et al. 1979a; and Table III). Even 0.05 ml of DA anti LewisIgG was able to significantly prolong survival of Lewis renal atlografts in mostrecipients., albeit after an early rejection episode, whereas animals receiving 100fold the amount of specific antibody in the form of F(ab')> had a severe acuterejection and only 2of6animals went on to survive. Of some clinical relevance isthe failure of a large dose (0.5 ml) of F(ab')2 to block the enhancement producedby a subsequent injection of IgG (Table III), lor as I shall discuss in a later sectionIhis might allow F(ab')2 to be used to block the potential damaging effects of

1(X) MORRIS

whole antiserum to a graft. Thus it appears that the Fc port ion of the IgGmolecule is necessary for passive enhancement , at least of renal aliografts.Similar observations have been made by Sinclair et al. (1970) who showed thaithe suppression of the anti-sheep erythrocyte haemolysin response in the mouseproduced by specific F(ab')2 was 100 to 1000 fold less effective than wholeantibody, and by Capel et al. (1979) who showed that mouse skin allograftscould not be enhanced by F(ab')2, even in enormous doses. These observationstoo are relevant to the subsequent discussions of mechanisms.

7. Specificity of enhancement

Earlier studies of the specificity of passive enhancement of tumour allografts inthe mouse (Moller 1963) and skin allografts in the mouse (Jeekel et al. 1972)suggested a marked degree of specificity of the enhancing antisera for theincompatible H2 specificities. In view of these studies, the later specificitystudies of enhancement in renal allograft models in the rat seemed to indicate asurprising lack of specificity, or to express this more accurately, a considerabledegree of cross reactivity in enhancement between strains (Fabre &. Morris1974b Fabre & Batchelor 1975). However more recent detailed serologicalstudies in our laboratories of cross reactivity between enhancing sera do showthat there is much less cross reactivity than previously supposed. And when itdoes occur, sera that are not donor specific are substantially weaker than donorspecific antisera. As the M H S in the rat becomes better defined it is possible thata similar degree of specificity ofenhancing sera for donor antigens, as seen in themouse, will become evident (McKenzie et al. 1980).

The nature of the target antigen of enhancing al loantibody has been clarifiedto a great extent since Davies & Alkins (1974) first showed that enhancementof heterotopic cardiac allografts in the rat could be achieved by antiseraabsorbed with erythrocytes (expressing SD antigens but not la antigens in therat) , but not by antisera absorbed with lymphoid cells (expressing both S D andla antigens). This led to the concept that enhancement was produced by the ant ila antibody in enhancing antisera. And these findings were substantiated byStaines et ai. (1975) and McKenzie & Henning (1977) using skin allograftsbetween congenic mouse lines, and by Soullilou et al. (1975) in a rat renalallograft model. However this initial concept that enhancement was producedby anti-la antibodies alone is now not so firm, as it has been shown that ant i-SDantibody, without anti-la activity, will enhance skin allografts in the mouse(Staines et al. 1977, Davis 1977), cardiac allografts in the rat (Jeekel et al. 1977),and tumour allografts in the mouse (Due et al. 1978). Fur thermore , recentlyGallico and colleagues (1979) have shown by raising antisera in recombinant ratstrains that antisera to SD antigens only will produce enhancement of rat renalallografts. It seems that antibodies against donor SD or la antigens can induce

1-1.5 ml day 00.5 ml days 1.3,5

0.15 ml dayO0.1 ml days 1,3,5

1-1.5 ml day 00.5 ml days 1.3,5

6/10

2/13

0/6


Table IVThe incidence of hyperacute rejection of DA or (DA X Lewis)Fl kidneys in lewis recipients

treated with large ami small doses of two Lewis anti DA sera.

Hyperacute Rejection

Strain Combination Lewis anti-DA serum Serum 1 Serum 2

DA to Lewis l~ l .5 ml day 0 6/lO 2/5

N.T.

(DAXLewis)Fl to Lewis l- l .5mldayO 0/6 0/6

N.T. = not tested* Modified from Eabre & Morris 1974a.

enhancement, but that the enhancing capacity of antisera resides predominantlyin the anli la traction of an enhancing antiserum. However, at least in the rat,this might be explained by the fact that the bulk of alloantibody produced byhyperimmunisation in most strain combinations appears to be directed against\d specificities iGallico & Mason 1978, McKenzie et al. 1980).

8. Antibody-mediated damage

A major reason, perhaps, that the rat has proved to be such a good tnodel inwhich to produce enhancement of vascularised organ allografts, is its relativeresistance to antibody mediated datnage in the presence of donorspecificcytotoxic antibody. The reasons for this have not been adequately explained, butmay be due to a defective complement pathway, inadequate fixation ofcomplement by antibody in the rat, or inappropriate MHS antigen density onthe target organ. Certainly the administration of heterologous guinea pig seruma.s a source of complement together with enhancing antiserum will result inimmediate destruction of a renal allograft (French 1972; Winearls et al. 1979b).However hyperacute rejection of a renal allograft does occur in the DA to Lewisrat combination in the absence of heterologous complement if large doses ofenhancing antiserum are given at the time of transplantation (Fabre & Morris,1974a). This phenomenon is dose dependent and is only seen if the homozygousdonor is used (Table IV). Furthermore hyperacute rejection of renal allografts isseen in the rabbit after passive administration of alloantibody and is also dosedependent (McDowall et al. 1973). This does underline the need for caution inconsidering the clinical application of passive enhancement, for antibodymediated damage might be expected in this situation, I will discuss approachesto its prevention in a later section.

102 MORRIS

III. MECHANISMS OF PASSIVE ENHANCEMENT

Two phases of the mechanism of passive enhancement of renal allografts need tobe considered. The first is the induction phase, in which the immune re.sponse isdelayed or suppressed, and the second is the maintenance phase, in which thegraft may survive indefinitely in the host in the absence of any further treatmi-nt.Whatever the mechanism by which the maintenance phase is induced in theadult rat, for example by antigen pretreatment or passive administration ofalloantibody, the mechanism by which it is maintained is probably identical ineach situation.

1. Induction phase

The induction phase might operate peripherally or centrally. A peripheralmechanism would involve antigen masking which either would delay or preventrecognition of the alloantigens carried by the graft or protect it from the effectorarm of the established immune response. A central mechanism implies eitherprevention of the induction or active suppression of the immune response afterantigen recognition has occurred. Although masking of antigens peripherallymay play a part in the induction of a state of enhancement if a sufficiently largeamount of antiserum is given to the recipient animal, the dose response studiesof Fabre & Morris (1973a) make a peripheral effect most unlikely (Table II).For the demonstration that as little as a single injection of 0.01 ml of antiserumcould produce enhancement of a renal allograft must argue against a peripheraleffect. The rapid disappearance of passively administered alloantibody alsoargues against any peripheral antigen masking effect of the alloantibody(French & Batchelor 1972).

Thus the evidence favours a central effect of passively administered alloanti-body in the induction of enhancement, but one can only speculate about possiblemechanisms. One such explanation is based on the dominant role for anti-laantibody in this induction phase. For if passenger leucocytes, including the Blymphocyte .subpopulation expressing la antigens, played a major role in theinduction of the immune response against a vascuiarised organ allograft assuggested by Stuart et al. (1971) and Batchelor etal. (1977b), then a direct effecton these cells by the enhancing serum might be responsible for the inductioti ofenhancement. Although the concept of a key role for passenger leukocytes in theinduction of an immune response to a renal allograft is not established in all ralstrain combinations (Fabre & Morris 1973b), the concept of the necessity for asecond signal, perhaps mediated by la, to induce the immune response to a tissueallograft as developed and proposed by Lafferty (Lafferty & Woolnough1977) is attractive. Of considerable relevance to this discussion is the recentdemonstration by Hart & Fabre (1979) in our laboratory that renal


parenchyma also expresses la antigen in very considerable amounts. Indeed onekidney was shown to express as much la antigen as half a spleen in the rat (Figure}]. Similar observations have been made in the mouse (O'Halloran et al. 1980)and in man (Williams et al. 1979). Thus these findings must be taken intoiiccounl in any explanation of the induction phase based on the anti la activity ofihc enhancing antiserum.

How then can we explain the induction of enhancement based on a directeffect of anti-la antibody on the B lymphocyte subpopulation of passengerleukocytes, bearing in mind the expression of large amounts of la on renaltissue? This would be possible if the la on non-lymphoid tissue was irrelevant inthe induction of the immune response against the graft. In other words the majorstimulus for the induction of the immune response are the la-antigens expressedon viable passenger lymphocytes, perhaps in association with SD antigens.Batchelor and his colleagues have produced considerable data in the rat tosupport this hypothesis, in that they have shown that allogeneic platelets,erythrocytes, partially purified lymphocyte, liver membranes and liposomesbearing Ag-B antigens fail to elicit a primary humoral or a cellular response.

PROTEIN PER ASSAY)

ii<{ RAH

[•OUNll

LfcH A5SAV

• » LEWIS

P • LEWIS

• • LEWIS

KIDWEY

KIDHEY

O O DA KIDNEY

• 'OA KU

• * LEMISK 1

»1KV

L,N. LYMTHOCVTUS

ASSORfilNG CELLS PER ASSAV

ligiirc J. Presence of la antigens on Lewis kidney. Exhaustively RBC - absorbed F(ab')3 DAanti-Lewis at 0.5 mg/ml absorbed with the tissues indicated and assayed on Lewis lymph nodelvmphocyte targets. The dashed lines, continuing the absorption curves of Lewis and Flkidney, indicate that the amount of kidney tissue for absorption was increased by additionalabsorplions. using the pellet of the kidney homogcnate after centrifugation. to avoid dilutionof the antibody (sec Hart & Fabre 1979 for further details).

104 MORRIS

despite the presence of la on the lymphocyte membranes and liposomes (Welshet al. 1979, Batchelor et al. 1978). If then la antigen must be presented to therecipient on the surface of viable lymphocytes in the grafted organ for animmune response to be induced, then anti-la antibody could interact directlywith these ceils, perhaps leading to their opsonisation and phagocytosis. Theenhancing effect of very small amounts of antiserum would be compatible withthis hypothesis, if it weren't for the presence of such large amounts of la on thekidney as a whole. For it is hard not to imagine that after the administration of asmall amount of antibody, most would be mopped up by the la antigen on renalparenchyma.

This then leads us to another hypothesis which takes into account the largeamount of la antigen present in the kidney. After the administration ofalloantibody considerable amounts of la antigen-antibody complexes areformed, and it is these complexes which produce the suppression of the immuneresponse. For example these complexes may be presented to antigen specific Thelper cells, resulting in opsonisation and phagocytosis of these cells by thereticulo-endothelial system, a mechanism based on that suggested by Hutchin-son & Zola (1977). The need for the Fc portion of the enhancing antibodywould be compatible with a hypothesis requiring opsonisation and phago-cytosis. It is also possible that such complexes would directly stimulatesuppressor T cells, which in turn would inactivate T helper cells. In bothinstances the humoral response to an allograft would be suppressed, but perhapsnot the cellular response, again compatible with the observations of theseresponses in the enhanced rat renal allograft.

Another possibility is that the passively administered alloantibody inducesantibodies to the antigen combining sites of the antibody itself (anti-idiotypeantibody). This anti-idiotype antibody would also react with receptor sites forthat antigen on T lymphocytes e.g. T helper lymphocytes, again blocking theinduction of the immune response at a central level. Some striking examples ofthe suppression of immune responses to histocompatibility antigens in this wayhave been demonstrated, as for example illustrated by the experiments of Binz etal. (1973) in the rat. where they showed that Lewis anti- (Lewis anti-DA)antibodies in the presence of complement were able to eliminate Lewisresponder T cells to DA antigens in both the MLC and G VH reactions. Binz &Wigzell (1976) have also shown a striking prolongation of (DA X Lewis) Fl skingrafts on Lewis rats stimulated to produce Lewis anti-(Lewis anti-DA)antibodies. Stuart et al. (1976) have shown that the acceptance of LBN kidneysby Lewis recipients after pretreatment with LBN spleen cells and anti-donorantibody was best if transplantation was performed at the peak titre of Lewisanti- (Lewis-anti-BN) antibody. However attempts to prolong renal graftsurvival by the production of anti-idiotype antibodies in the rat have not provedsuccessful (Batchelor & Welsh 1976). Thus attractive as this concept is, there is


no evidence to support its role in the induction phase of enhancement of renalallografts over that of any other.

Any explanation must take into account the difficulty in enhancing organsfrom homozygous donors, as this appears to be more than a gene dosage effect,in that increasing the amount of antiserum still does not permit more than atemporary delay in rejection of renal allografts across strong histocompatibilitybarriers. The enormous variation in the amount of la antigen expressed ondifferent tissues (Hart & Fabre 1979) might also explain the variation in theability of antisera to enhance different tissue allografts in the same straincombination, an observation which is not otherwise readily explained. Howeverlor the present the mechanism by which passively administered alloantibodysuppresses the immune response to an allograft remains subject to speculation,although the final pathway for the effect is likely to be the T helper ceU.

2. Maintenance phase

The induction phase merges into the maintenance phase in those models whererejection is suppressed and the allografts survive indefinitely. Once themaintenance phase is established it appears to be quite stable. In general nodonor specific antibody or cellular immunity as demonstrated by cytotoxicity ina CMI assay (Burgos et al. 1974, Mullen et al. 1973) can be detected.Restimulation of the recipient with donor spleen cells results in a weak IgMresponse only, without any apparent eftect on the graft and a normal cellularresponse in a CMI assay (Burgos et al. 1974), an observation also compatiblewith T helper cell inactivation. Again if one replaces the long surviving kidneywith a fresh syngeneic kidney, then this too survives indefinitely (Fabre &Morris 1972b). More impressively we have shown recently in our laboratory thatif a long surviving (DA X Lewis) F1 kidney in a Lewis recipient is replaced by ahomozygous DA kidney (which cannot be enhanced in the conventional way),then this kidney too shows no evidence of rejection. Similarly Poole et al. (1976)have shown that (AS X Aug) Fl leg allografts in the rat, which cannot beenhanced, wilt show prolonged survival in AS rats bearing long survivingenhanced Fl kidneys. Donor strain skin grafts placed on animals with enhancedrenal allografts, show prolonged survival which is more marked both with thepassage of time after renal transplantation and the strength of the histocom-patibility barrier (Mullen et al. 1973, Fabre & Morris 1975). Thus themaintenance phase represents a very powerful state of persistent immuno-suppression.. whatever the mechanism. This state will also persist for some weeksafter removal of the long surviving kidney, an interesting observation to which Ishall return shortly.

Four po.ssible mechanisms have been proposed to explain the maintenancephase of enhancement. These are (a) the presence of blocking factors (b) clonal

106 MORRIS

25O0-

2OOQ

C.P.M.

(fl)

I5OO.

100Q

5OO-

Effect of Enhancing wrutn on MLfi

(FI(DAXL)— DA)

OS 1 2Stimulating Cells x 10%!

I — Dft + DA anti Lewis serum)

Effect of Sonjm from long Mjrviving ml ^15R) on MLR.

CPM.

2500.

30OO.

I5OO.

5 O O .

Ih)

— DA + LSRsefum

-V.^Ji:'-'-":::;' - controls

O-5 IStimulating Cells

Figure 4. Mixed lymphocyte reaction after 6 days in culture Responder cells—DA lymplinode lymphocytes (IX 10̂ ) Stimulator cells - (DAXLewis)FI spleen lymphocytes irradiatedwith 1000 R (0.5 to 4X 10* lymphocytes Controis= Fl spleen (irradiated) + FI lymph node andDA spleen (irradiated)+DA lymph node.(a) (DAXL)F1+DA in presence of 10% DA serum or DA anti Lewis serum known loenhance renal allografts.(b) (DAXL)F1 + DA in presence of 10% DA serum or serum from long surviving DA rai(LSR) with enhanced (DAXL)Fl kidney.

SUPPRESSION OF REJKC'I ION BY ALLOANTIBODY 107

deletion of antigen reactive cells (c) active suppression of the immune responseby suppressor cells (d) graft adaptation.

(a) Blocking factors

These could exist in the form of antibody or Immune complexes and would blockthe effector arm of the immune response. However the passive transfer of serumfrom animals with long surviving grafts to syngeneic recipients have failed toinduce enhancement, with one notable exception seen in the experiments ofTilney & Bell (1974). In their experiments cytotoxic antibody was present inihe recipients at the time of serum transfer, which is not usually the case.Furthermore although enhancing antiserum will block completely the mixedlymphocyte reaction (MLR) in the rat, serum from long surviving animals hasno effect on this reaction (Figure 4). Thus there is little evidence supporting arole for blocking factors, although it must be admitted that whatever isresponsible for the maintenance phase of an enhanced allograft may not be ablela induce enhancement in a transfer experiment.

{b) Clonal deletion

Deletion of a specific clone of antigen reactive cells during the induction phaseUnds little support as an explanation. Lymphocytes from long surviving animalswith enhanced kidneys produce a normal GVH reaction in a popliteal node

o( long vKviving ( i s ) Dfc rot tymphocyt« m MLR

39OO

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aooc

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coo

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Figure .̂ . As in figure 4, but responder lymphocytes from a normal DA rat are compared withihose from a long surviving DA rat (LSDA) with an enhanced (DAXL)Fl kidney.

108 MORRIS

TABLE V(DA X LewisjFI kidneys were transplanted into passively enhanced DA recipients with one oftheir own kidneys left in situ. 8 weeks after transptantation the enhanced kidney was removed.the animat surviving wiih its own kidney- A fresh (DA X LewisjFl kidncv was implanted

2. 4 or 8 weeks later, and at that time the remaining kidney in the recipient was removed.

Time from removal of Second Kidney Second Kidneyenhanced kidney toimplantation of .second Mean blood urea (mg/lOO ml)kidney (No. of animals) 2 weeks 4 weeks 10 weeks Survival (days)

2 weeks (2)4 weeks (1)8 weeks (2)

554450

514742

554260

>80.>77>77,

>50

>77

assay (French et al. 1971, Mullen et al. 1973), although they do show a ratherdampened response in the MLR reaction (Figure 5). Injection of syngeneiclymphocytes (3X10^) into animals with long surviving grafts produced noevidence of rejection (McKenzie & Morris - unpublished observations). AndBowen and colleagues (1974) were not able to produce rejection of a longsurviving kidney by parabiosis of the recipient with a syngeneic hyperimmunisedanimal for 9 days, nor by injection of lymphocytes from hyperimmunisedanimals. Thus there is no evidence which suggests that clonal deletion isresponsible for the maintenance phase of enhancement.

I

(c) Suppressor cells i

From what has been said already, one is led indirectly to an explanationrequiring active suppression of the immune response to the graft, presumablythrough the mediation of suppressor cells. Althoug suppressor T cells have beendemonstrated in adoptive transfer experiments in some models of unrespon-siveness to tissue allografts (Kilshaw & Brent 1977, Jirsch et al. 1974), theywere not demonstrated in enhanced renal allograft recipients (Batchelor et al.1977a) nor in rats rendered unresponsive by repeated blood transfusions (Fabre& Morris 1972b). However Stuart and colleagues (1976) did prolong survival ofrenal allografts by the adoptive transfer of spleen cells from recipients withenhanced kidneys. Experiments in which enhancement of renal allografts wasproduced in the presence of one of the recipients own kidneys, thus allowing therecipient to survive after removal of the enhanced kidney and replacement with asecond kidney from the same donor strain, showed that this state of immuno-suppression persisted for at least up to 8 weeks (Table V). This experiment inwhich the maintenance phase persists in the absence ofantigen not only wouldseem to exclude blocking factors as a mechanism, but would be compatible witheither clonal deletion (which I have excluded from consideration) or the

(DA X Lewis)FI to LewisLong surviving (DA X L)F1kidney to riiiive LewisDA to Lewi.sLong surviving DA kidney10 naive Lewis

59

106

582499

594571


TABLE VILong surviving DA or (DA X Lewis}FI kidneys have been produced in Lewis recipients bytreatment with ALS andpas.sive enhancement. The.se kidney.s have been retransplantedinto naive

Lewis recipient.s (Hart et at. 1980)-

Mean blood urea (mg/lOO ml)at weeks Median survival

Strain combination No, l'/: 2 3 -days (range)

- - 12 (12-13)283 338 >50 (12-90)

II (10-14)12.5 (10-16)

generation of relatively long lived suppressor cells. Dorsch & Roser (1977)have presented evidence that tolerance to skin grafts, induced in the neonatalrat, is maintained in adult rats by long lived rapidly recirculating suppressor Tcells. Thus although the role of suppressor cells in the maintenance phase ofpassive enhancement remains to be confirmed, for the present it must remain themost attractive explanation. For both blocking factors and clonal deletion canbe excluded with some confidence as possible mechanisms, at least in most of therenal allograft models that I have discussed.

(d) Graft adaptation

Some alteration in an enhanced graft, such that it no longer provides anadequate immunogenic stimulus to the recipient, must be considered in anydiscussion of mechanisms. There is no doubt that some form of adaptationoccurs in a long surviving renal allograft in that removal of the graft andreimplantation of this same graft into a fresh syngeneic recipient does result insome prolongation of survival of the reimplanted graft. This prolongation ofsurvival is much more marked when the long surviving kidney is an Fl hybridthan when it is a homozygous parental kidney (Table VI). again showing thisstriking difference between the survival of homozygous and heterozygouskidneys. Batcheior's group (Batchelor et al. 1979, Welsh et al. 1979) haveretransplanted enhanced (AS X Aug)Fl kidneys in AS recipients into naive ASrecipients, and have shown a markedly prolonged survival and also failure ofthese AS animals to mount an immune response against August targets. But ifihe same kidneys were retransplanted into sensitised recipients, a vigorous IgM,IgG and cytotoxic T cell response was mounted and the graft was rejectedacutely. Batchelor and colleagues attribute this lack of response to theretransplanted kidney to the absence of passenger leukocytes expressing laantigen in the enhanced kidney such that the appropriate amplification

110 MORRIS

TABLE VII(DA X Lewis)FI renal allografts were implanted in Lewis recipients, either naive or previouslysensitised by two (DA X Lewis)FI skin allografts 6 weeks apart, Lewis anti-DA xerum being

administered as shown.

Treatment No.

Mean

I

435

98

389

401

Btoodat

Urea (mg/lOO ml)weeks2 3

-

180

-

—

-

387

-

_

Mediandays

10

Survival(Range)

(9-10)

26 (20->200)

14

11

(7-15)

(9-95)

Naive reeipientsNil 6Lewis anti-DAL5mldayO 60.5 ml days 1.3,5

Sensitised reeipientsNil 5Lewis anti-DA1.5 ml day 0 50.5 ml days 1,3,5

mediated through the helper T cell does not occur. In a sensitised recipient this himediated amplification is unnecessary, and as might be expected thereforepassive enhancement in sensitised recipients is relatively ineffective (Fabre &Morris 1975) (Table VII). Thus graft adaptation is probably best explained onthis basis, although obviously the rejection of retransplanted enhanced homo-zygous kidneys suggests that where strong histocompatibility barriers existbetween donor and recipient, the presentation of the MHC (including la) onrenal parenchyma is an adequate stimulus.

Any alteration in donor antigen in the enhanced kidney is unlikely as donorspecific antibody labelled with i' ' given to recipients with long survivingkidneys is rapidly absorbed by the enhanced kidney and its uptake in theenhanced kidney is similar to that of a normal allogeneic kidney (Fincet al. 197.̂ .French 1973). Furthermore in our laboratories Hart et al. (1980) have shown inradioimmune binding assays that quantitatively there is no difference in theamount of SD or la antigen in enhanced (DA X Lewis)Fl kidneys to that innormal kidneys. Graft adaptation has also been explained on the basis ofreplacement of graft endothelium by host endothelium. However Hart et al.(1980) have shown that a long surviving DA kidney retransplanted into a naiveLewis recipient given Lewis anti-DA scrum plus guinea pig complementundergoes hyperacute rejection. This would be a most unlikely outcome if thegraft vasculature were lined by host endothelium.

Therefore graft adaptation, using this term in its broadest sense, does occur inthe enhanced kidney, and is best attributed to the loss of whatever provides theamplification message for the immune response in a naive host. At present the


most likely candidate is la antigen expressed on passenger leucocytes within theenhanced kidney.

IV INTERACTION OF PASSIVE ENHANCEMENTWITH OTHER FORMS OF IMMUNOSUPPRESSION

Attempts to apply passive enhancement to clinical transplantation will requirethe concurrent use of more conventional immunosuppressive agents at least inthe first instance. Thus the interaction of passive enhancement with other non-specific agents has been an interest in our laboratories for some time. Severaleffects, namely synergistic, additive, and antagonistic, have been sought in thesestudies.

1. Antilymphocyte serum (ALS)

ALS has proved to be markedly synergistic with passive enhancement in the ratrenal allograft model (Batchelor et al. 1972, Fabre & Morris 1974a), and even inthe very strong homozygous DA to Lewis model the combination is extremelypotent (Table VIII). But in the sensitised recipient the combination of ALS andpassive enhancement was much less effective (Fabre & Morris 1975). Thissyncrgism with ALS is compatible with the suggestion that the cytotoxic T cellresponse is suppressed by the ALS, while passive enhancement predominantlysuppresses the humoral response, perhaps by suppressing T helper activity, asdiscussed earlier.

2. Azathioprine and Prednisolone

Extensive dose response studies in the rat renal allograft model failed to showany additive or synergistic effect of azathioprine, prednisolone, or both drugswith passive enhancement. Indeed there was some evidence that azathioprineinhibited the effect of enhancement (Winearls et al. 1978).

3. Cychphosphamide (Table IX)

Dose response studies of this drug produced some interesting results which arealso relevant to any consideration of mechanisms in view of the relativelyselective action that cyclophosphamide is supposed to have on B lymphocytes(Turk et al. 1976), and possibly on suppressor T cells (Ferguson & Simmons1978). Cyclophosphamide in relatively small doses appears to augment theeffectiveness of passive enhancement if given for 28 days; given for a shorterperiod it is less effective. Both cyclophosphamide and passive enhancementvirtually abrogate the humoral lymphocytotoxic response, but higher doses of

112 MORRIS

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TABLE IXCyclophosphamide and passive enhancement in (DA X Lewis)Fl to Lewis renal allografts

(modified from Winearls et al. 1979c).

rreatment

NilCyclo. 5.3 mg/kgX 28 daysLewis anli-DA0.5 ml IV (days0,1, & 3)Cyclo.-1-Lewisanti-DA (as above)

No.

54

6

7

Median bloodurea

10 d.

600532

125

70

14 d.

—

530

680

66

Cytotoxin titre(reciprocal)7 d. 14 d.

512

0 0

1 4

I 1

Median survival -days (Range)

12(12-13)27(19-55)

16(14->100)

>100(35->150)

cyclophosphamide are needed to prevent the vascular changes of rejection in therenal allografts at 7 days and the onset of acute rejection. In fact cyclophospha-mide in lower doses than shown in Table IX still abrogates the antibodyresponse to the renal allograft without producing any prolongation of graftsurvival. The failure of cyclophosphamide to inhibit enhancement of renalallografts despite its suppression of the humoral response, certainly is notcompatible with a role for antibody in the maintenance of the steady state of along surviving graft. This does suggest that graft rejection in the rat is mediatedby more than just simply alioantibody. Again if a suppressor cell mechamism isresponsible for the maintenance phase of a renal allograft, then these cells arepresumably not selectively sensitive to cyclophosphamide in the relativelytherapeutic dose levels used in our studies.

4. Cyclosporin A

As this agent also appears to have a selective action directed at T cells (Borel1977). it seemed possible that synergism similar to that with ALS might be(bund. However in the same rat renal allograft model, dose response studiesrevealed no evidence either of a syergistic or an additive effect (Table X). despitethe marked potency of Cyclosporin A alone in this model (Homan etal. i980a).

However there is a remarkable parallel in the histological and immunologicalchanges produced by Cyclosporin A to those produced by passive enhancement.And indeed we have suggested that Cyclosporin A might have a relativelyselective action at the T helper cell level also (Homan et al. 1980b). which wouldexplain the lack of synergism between these two immunosuppressive agents andthe similar effect of these two agents in the rat model.

114 MORRIS

TABLE XCyclo.fporin A and passive enhancement in the DA to Lewis renal allograft model (modified

from Homan et al. 1979)

Treatment

NilLewis anti DA-0.5 ml IV (days 0.1)Cy A 2 mg/kgX14 daysCy A 5 mg/kgX 14 daysLewis anti-DA(as above)Cy A (2 mg/kg)

No.

95

5

7

5

Cytotoxin titre(reciprocal)

1 week

80!

64

0

1

Median survival

- days (range)

11 (10-11)12 (10-14)

11 (10-11)

100(24->100)

13(10-57)

V. OTHER TISSUES

Passive enhancement has proved to be most effective in suppressing rejection ofvascularised organ allografts in the rat, the renal allograft being the mostsuccessfully enhanced organ. Heterotopic cardiac allografts in the same animalare also enhanced, but do not behave in the same way as renal allografts in thesame strain combination as already mentioned. Enhancement of skin aliografts.results in a modest but definite prolongation of survival in the mouse and rat.

The difficulty in enhancing skin allografts has not been adequately explained.although we have discussed it in some detail previously (Fabre & Morris 1975).Not only are skin allografts difficult to enhance, but skin allografts of donorstrain placed on rats with long surviving renal allografts are rejected also, albeitwith some prolongation of survival, and without any effect on the renal allograft(Mullen et al. 1973). Skin specific transplantation antigens have been heldresponsible for these results, but even if enhancing sera are raised with skingrafts, there is still only a modest prolongation of survival achieved (Table XI).As mentioned in a previous section, there is a wide variation in the amount of laantigens present on different tissues to which differing effects of passiveenhancement might be attributed, but certainly la antigen is present in skin. 11passive enhancement predominantly suppresses the humoral response ratherthan the cellular response to an allograft, and skin allografts are rejectedpredominantly by the latter in contrast to vascularised organ allografts, thiscould explain the difference between the behaviour of the two types of tissueallografts. However in certain strain combinations differing only at minorhistocompatibility loci, a renal allograft will not be rejected while skin allograftswill still be rejected quite briskly (Mullen & Hildemann 1971). Thus the immune


TABLE XIPa.wive enhancement of (DA X Lewis)Fl skin and renal allografts in DA recipients using

antiserum raised in DA rats by (DA X LewisjFl skin allografls.

Treatment

Renal allograft:a) Nilb) DA anti Lewis

(0.5 ml days 1,3)Skin allograft;a) Nilh) DA anti Lewis

Mean blood ureaI 3

433

94 80

— —— —

at week20

—

61

——

Median Survival- days (Range)

19.5 (13->300)

>300

8 ( 8 - 9 )12(12-14)

response to a skin allograft appears to be far more significant than that to avascularised graft, perhaps due to differences in susceptibility to the rejectionreaction by the different tissues or to the affinity of lymphocytes involved in thereaction, as discussed in a previous paper {Fabre & Morris 1975).

Enhancement of adult isolated pancreatic islets in the rat has proved possiblebut only when the Fl hybrid donor is used (Finch and Morris 1976). In thismodel syncrgism with ALS was not seen (Finch & Morris 1977). linhanccmentof foetal pancreatic tissue was unsuccessful, perhaps due to antibody mediateddamage of this tissue (Morris et al. 1980), while some minimal effect is seen withvascularised segmental pancreata in the rat (Poole - unpublished).

VI. ENHANCEMENT IN SPECIES OTHER THAN IN THE RODENT

Suppression of rejection of renal allografts in the rabbit by donor specificalloantibody has been achieved, but antibody-mediated damage of the kidneys,resulting in hyperacute rejection, was seen in about half of the animals(McDowall et al. 1973). This effect was dose dependent just as in the DA toLewis combination in the rat. Relatively unsuccessful attempts have been madeto induce suppression of rejection of renal allografts by donor specificiilioantibody in the dog and in the monkey.

In our laboratories hyperimmune antisera in the dog have been prepared byusing mesenteric lymph node lymphocytes as an immunogen in unrelatedmongrel dogs. Several pools of antisera were prepared. Kidneys were trans-planted into recipient dogs without immunosuppression, using either third partykidneys or kidneys from the immunising donors. In both groups of animalsantisera was chosen on the basis of specific cytotoxic and binding activity withthe potential donor's lymphocytes and non-reactivity with recipient lympho-cytes. Donor specific antiserum was given in a dose of 5 ml/kg intravenouslyshortly after transplantation and on day 1. In two of the 15 animals so treated

116 MORRIS

there was some modest evidence of graft prolongation in that both animals hadnormal blood ureas at day 10 and prolongation of .survival of these allogruftsby several days over that of the contro Group (Figure 6). However in 7 dogs thekidneys failed to function presumably due to antibody mediated damage, thusunderlining the need for caution in the use of passive enhancement in speciesother than the rat, including man.

Jeekel et al. (1975) produced antisera by immunising one of a pair of DLAidentical beagles over 6 weeks with unrelated beagle lymphocytes and complete-Freund's adjuvant. The kidney of the unrelated immunising donor was thentransplanted into the other one of the pair of DLA identical beagles without

101102110H I116132m626370

119220

121

1Z2123

125127

124128

129130135139

UO142

138141

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Days

No t r e a t m e n t

E n h a n c e m e n t ( I m m u n i s i n g d o n o r - k i d n e y d o n o r )

E n h a n c e m e n t ( t h i r d p a r t y k i d n e y d o n o r )

}20 anti 19

6 a n i i 11

}3 ant i 14

}lO anti 12. 125

}21 anti 7.124.128

\b an t i n i

10 anti 1346 anti 1346 anti 136

21 anti 136} 3 anti 137

Figure 6. Survival of dog renal allografts between mongrel dogs. The first group received notreatment. The second group received a kidney from the same donor as used for theimmunisation, while the third group received a kidney from a third party donor. Possibleenhancement was produced in dogs 119 and 121. The dogs treated with antisera weretransplanted in pairs with the donor dog providing kidneys for each of the pair of recipients.The sf)ccificity of the antiserum was directed against donor antigens only, with the exception ofdogs 125. 124. and 128 where ihere was activity against recipient antigens also.

SUPPRESSION OF REJECTION BY ALLOANTIBODY 1 17

immunosuppression, and 20 ml of donor specific antiserum was given IV on theday before transplantation and daily thereafter. No effect was seen if the donorand recipient differed by two DLA haplotypes. but two of 6 dogs where donorand recipient differed by one haplotype survived for more than 50 days, onesurviving indefinitely. No mention was made of antibody mediated damage inany of the kidneys, but no data concerning the serological characteristics of the;mtiserum with donor lymphocytes was reported. However much smaller dosesof antisera were given in these experiments in comparison to our own.

In the Rhesus monkey Balner and his colleagues (Marquet et al. 1978) haveused both a polyspecific anti SD IgG which killed 60-80% of lymphocytes fromeach of a panel of 25 monkeys, and a donor specific la antiserum prepared bycross-imtnunisations of SD-identical monkeys, in monkeys receiving a renalallograft. The group given anti-la sera were also given Azathioprine andPrednisolone. Once again there was some evidence of a mild suppression of therejection reaction in monkeys receiving anti la sera. No evidence of antibodymediated damage was seen. Thus at present no striking evidence of suppressionof rejection of renal allografts in the higher species has been obtained, althoughadmittedly a great deal more work in this area still has to be performed.

VIII. PREVENTION OF ANTIBODY MEDIATED DAMAGE

Antibody mediated damage of an allograft by the passive administration ofdonor specific alloantibody is very uncommon in the rat, the species in whichmost of the studies of passive enhancement have been performed. As discussedin an earlier section the absence of immediate graft damage after administrationof antibody may be due to an in vivo defect in rat complement or to inadequatetlxation of rat complement by antigen-antibody complexes in the rat. Certainlyhyperacute rejection may be seen after the administration of guinea pigcomplement (French 1972). or after large doses of antiserum in the DA to Lewisstrain combination (Fabre & Morris 1974a). In the latter instance the effect isdo.se dependent. However in higher species there is little doubt that antibodymediated damage to an organ allograft will occur, and indeed this has been seenin the rabbit (McDowall et al. 1973) and the dog as described in section VI.Furthermore it is probable that antibody-mediated damage occurred in therenal allografts in man after attempts to enhance with impure F(ab')2 (French &Batchelor 1972). Thus before any further clinical attempts at passive enhance-ment of renal allografts are undertaken, methods must be designed that willallow enhancement to occur in the absence of immediate antibody-mediateddamage to the graft itself.

As mentioned above, both in the rat and the rabbit, and probably in the dog,the occurrence of antibody mediated damage is dose dependent. However asany dose selection in man will have to be an arbitrary one in the first instance.

118 MORRIS

further steps will be necessary to ensure that antibody mediated damage doesnot occur. We have explored other possible approaches in our laboratory, usinga reproducible model of hyperacute rejection of renal allografts in the rat. A DArenal allograft is implanted in a Lewis recipient, and Lewis anti-DA serum isadministered IV immediately after the vascular anastomoses are completedfollowed 5 minutes later by 2 ml guinea pig complement. This producesimmediate anuria with interstitial haemorrhage, polymorphonuclear infiltra-tion, and glomerular and tubular necrosis in the kidney allograft. Guinea pigcomplement alone produces no effect (Winearls et al. 1979b). As mentioned inSection III, we have shown already that F(ab')2 which does not fix complementand therefore would not result in antibody-mediated damage, does not enhancerenal allografts but on the other hand it does not prevent enhancement (Table IIIand Winearls et al. 1979a). However, in the above model the administration ofdonor specific (Fab')2 in equivalent amounts to the IgG in whole serum beforethe donor specific antiserum was given, prevented antibody mediated damage,while still allowing enhancement to occur (Table XII). Thus this might be onepossible approach to the prevention of antibody-mediated damage in the clinicalapplication of passive enhancement.

A second approach is based on the observation that antibodies directedagainst la antigens will enhance heart and renal allografts in the rat as discussedin detail in Section III. Nevertheless in view of the possible existence of la onvascular endothelium (Hirschberg et al. 1975), and the demonstration of large-amounts of la in rat and human kidney (Hart and Fabre, 1979; Williams et al.1979), the strong possibility of antibody mediated damage with such antiseramust be considered. We have explored this in the above model of hyperacuterejection, using an antiserum exhaustively absorbed with erythrocytes to leavejust anti-la activity, and failed to see any evidence of antibody-mediateddamage, although the absorbed serum still did exhibit enhancing activity(Table XII). It is indeed puzzling that anti-la antisera do not produce antibody-mediated damage in view of the expression of so much la on kidney. Thisobservation needs further study, but does suggest another approach to theclinical problem.

Other approaches to this potential clinical problem might be the use of non-complement fixing IgG, which Jansen et al. (1975) have shown may enhance, ortemporary decomplementation of the recipient with an agent such as cobravenom factor during the inductive phase of enhancement.

Vin. EXPERIENCE IN MAN

There have been only two systematic attempts to produce passive enhancementof renal allografts in the human. Batchelor's group have attempted to passivelyenhance several recipients of both living related and cadaver kidneys. Three of


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120 MORRIS

the living related transplants have been reported {Batchelor et al. 1970. French &Batchelor 1972) but the total experience of both living related and cadavertransplants does not provide any convincing evidence of better graft outcome{Batchelor, personal communication). A relatively impure preparation ofF{ab')2 was used in these patients, and some evidence of antibody-mediateddamage was seen in that some of the recipients of living related grafts wereoliguric for several days. Solheim et al. {1976) have reported attempts to enhancesix living related transplants where one HLA haplotype was shared betweendonor and recipient. F{ab')2 was used, but again there was nothing to suggestimproved graft function, although no evidence of graft damage was seen. Olcourse as we know now that F(ab')2 has little enhancing activity, neither of thesestudies can be said to show that passive enhancement was unsuccessful in humanrenal transplantation.

Perhaps one of the more encouraging observations in human renal transplan-tation in terms of possible enhancement are those showing that a renaltransplant may be performed in the presence of a positive crossmatch betweenrecipient and donor, where the donor-specific antibody giving rise to the positivelymphocytotoxic complement-dependent crossmatch is directed against donorB lymphocytes only (Ettengeret al. 1976. Morris etal. 1977). Although such a Hcell positive crossmatch is now known to be caused by several types of antibodiesincluding those directed against la {HLA-DR), there is no doubt that some aredue to anti-la or HLA-DR activity, but yet hyperacute rejection does not occur.Furthermore some groups {d'Apice et al. 1979) have suggested that such graftshave a better outcome than otherwise (perhaps due to enhancement), althoughour own data does not support this suggestion {Ting & Morris 1979).

IX CONCLUSIONS

Alloantibody mediated suppression of rejection {passive enhancement) of tissueallografts can be achieved consistently in the rodent, but has yet to be achieved inhigher species. The mechanisms by which a stable state of donor specificsuppression is induced are not known other than that la antigen andalloantibody to la play a key role, nor indeed the mechanism by which it ismaintained, although here indirect evidence points to an active suppressormechanism probably mediated by suppressor cells. Although antibody medi-ated damage is uncommon in the rat. it is seen in higher species such as the dog.and must be expected to be a problem in any clinical application, unless steps aretaken to avoid it. Passive enhancement remains an alternative approach to thatofantigen pretreatment for the induction of specific suppression of rejection inman, but it must be realised that it is a long step from rat to man. Until themechanisms by which passive enhancement both induces and maintains thesuppression of rejection are better defined, it seems unlikely that this step will betaken with success.


ACKNOWLEDGMENTS

Many people have contributed to the work of the department described in thisreview, but I am especially grateful to Drs. John Fabre, Christopher Winearisand Derek Hart and Miss Judith McKenzie.

REFERENCES

\bbas. A. K., Corson. J. M., Carpenter. C. B.. Gaivanek. E. G., Merrill. J. P. Dammin.G. J. (1974a) Immunologic enhancement of rat renal altografts. 1. Comparativemorphology of acutely rejecting and passively enhanced grafts. Amer. J. Path. 75. 255.

Abbas, A. K., Corson. .1. M.. Carpenter, C. B., Gaivanek E, G.. Merrill. J. P.. Dammin, G, J.(1974b) Immunotogic enhancement of rat renal allografts. 11. Immunohistology ofacutely rejecting and passively enhanced grafts. Amer. J. Path. 75. 271.

d'Apice. A. J. F, Tait. B. D. (1979) The positive B-cell crossmatch: A marker of activeenhancement? Transplant. Proc. 11. 854.

Balchelor. J. R.. Brent, L. & Kilshaw. P. J. (1977a) Absence of suppressor cells from ratsbearing passively enhanced kidney allografts. Nature 270, 522.

llatchelor. J. R.. Ellis. F. French. M, F.. Bewick, M.. Cameron, .1. S.. OGG. C. S.(1970) Imniunologicai enhanccmcEit of a luinmn kidney graft. Lancer ii. 1(H)7.

Matchelor, J. R.. Fabre, J. W. & Morris, P. J. (1972) Passive enhancement of kidneyallografts: potentiation with antithymocyte serum. Transplantation 13. 610.

liatchelor, J. R. & Welsh, K. I. (1976) Mechanisms of enhancement of kidney allograft survi-val. Brit- med Bull- 32. 1 LV

Batchelor. J. R., Welsh. K. 1. & Burgos, H. (1977b) Immunological enhancement. Transplant.Proc. 9,931.

Batchelor. J. R., Welsh, K. 1. & Burgos, H. (1978) Transplantation antigens per se arepoor immunogens within a species. Nature 273. 54.

Batchelor. J. R., Welsh. K. I.. Maynard. A. & Burgos, H. (1979) Failure of long surviving.passively enhanced kidney allografts to prove T-dependent alloimmunity. 1. Retrans-planiation of (.AS X AIJG)F1 kidneys into secondary as recipients. J. CA'/J. .Mt'd150,455.

Uieseckcr. .1. I... Filch, f-. W., Rowley, D. A. & Stuart. F. P. (1973) Cellular and Humoralimmunity after allogeneic transplantation in the rat III. The effect of passive antibodyon cellular and bumoral immunity after allogeneic renal transplantation. Transplantation16, 432-440.

Binz. H., Lindemann, J. & Sigzell, H. (1973) Inhibition of local graft-versus-host reactionby alloantibodies. Nature 245, 146.

liinz, H. & Wigzell. H. (1976) Specific transplantation tolerance induced by autoimmunisationagainst the individual's own naturally occurring idiotypic. antigen-binding receptors../. exp. Med 144. I43ii.

Borel. J.. Feurer. C . Magnee, D. & Stakelin. H. (1977) Effects of new anti-lymphocyticpeptide Cyclosporin A in animals. Immunology 32. 1017.

Bowen, .1. E-. Batchelor, J. R., French, M. E.. Burgos, H. & Fabre. J. W. (1974) Failureof adoptive immunisation of parabiosis with hyperimmune syngeneic partners loabrogate long-term enhancement of rat kidney ailografts. Tan.'iplantation 18, 322.

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Suppression of Rejection of Organ Allografts by Alloantibody