Transplantation of discordant xenografts: a challenge revisited

IMMUNOLOGY

TODAY

Transplantation of discordant xenografts: a challenge revisited William

Parker, Soheyla Saadi, Shu S. Lin, Zoie E. Holzknecht, Matilde Bustos and Jeffrey L. Platt

he pre-eminent

challenge

in the

field of transplantation

today

neither

the bio-

to understand

logical

basis

for graft

hnzw allowrd

is

fhe cunceyts

nt that time io

be testerl

siptificnnt

rejection

cdmrmti ltlndirfg

pmp3s

rejection2 or delayed xenograft

to

also Bach and cotk,E;ues,

irr

issue),

looms

as

the

rejection

(see

pp. 379-384,

this

most

immediate

nor to prevent it from occurring 45, happily,

impediment

these challenges

transplantation. Here, \ve review the pro-

met.

Rather,

plantation tissues

at least partly

problem

a source

for nil who need transplant

lack of organ

them.

carried

donors

in trans-

of q+ms

gress

and

Indeed,

for

made

logical

out, Ihere USA as 5-10

outlook

The urgency of this problem

has en-

doing

recent efforts to develop

to USC animal

organs

i.e. xenotransplantntion. as a wurce of organs

of full-sized

primate

number

needed.

organs

organs

While

be preferred

for clinical

nonhuman

transplan-

primates

for xenotransplants,

that could be obtained

Thus, there is an emerging

mates such as the pig should

that

the past

might

the number

for this

to have field

is far short of the

consensus

that nonpri-

be used as a source of organs

for xeno-

responses to xeno&msplantation

greater

advocated.

Rather,

cumb to existing

is whether

therapies,

barriers

That is not to say will be less

than to allotransplantation,

the issue,

detail elsewhereg,

perspective.

which

5%~ have

that cellular

as some studies

In

barriers

as these

that celluIar have

the

50 dramatically.

from a clinical

immune

r7d-

brightened

so we focus on the humoral

to xenotransplantation

in

theimmuna

of xenotransplantation,

seem

seem most daunting a hurdle

of xeno-

six years

and overcoming

to uenotraaccplantation,

the means

in lieu of human

during

hurdles

vances

appkfition

to clinical

understanding

tor as many

couraged tation,

been

the major

is finding

every organ more.

have

as some taken

response

would

up in

will suc-

suggesP+.

transplantation. In 19Y0 we proposed xenograft

rejection’.

of that model

and overcoming

hyperacutr

major challenge However,

for the immunopathogenesis

the following

have been tested and much

in understanding hurdle,

a model

During

another

re/cction

years, progress

these hurdles.

CHAR), which termed

xute

of

elements

has been made Indeed,

was

in 1990, can now be prevented type of rejection,

various

the first

considered

in nearly vascular

the

every case. xenograft

Hyperacute Hyperacute interstitiai upon process

xenograft xenograft

hemorrhage

reperfusion inevitably

Hyperacute

and diffuse

of some destroys

xenograft

rejection

rejection, qan

characterized

by

begins immediately

xenografts.

Once

the graft within

rejection

pathologically

thrombosis,

occurs

minutes nearly

initiated,

this

to a few hours. always

in some

IMP&NOLOGY~

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complement regulatory proteins in the transplant with the complement system of the recipients, allowing uncontrolled activation of complement.

Xenoreoctive natural antibodies It has been the classicaP, but controversial*, view that xenograft rejection is initiated by ‘natural’ antibodies that are present in the circulation of all mammals in the absence of a known sensitizing event. The importance of natural antibodies in initiating HAR of pig-to-primate xenografts has been demonstrated definitively by the followmg observations: (1) organs undergoing HAR invariably contain immunoglobulin of the recipient, co-deposited with components

of the classical complement

pathway”;

(2) depletion

of

XNAs from a recipient prolongs xenograft survival, even when the complement

system of the recipient

remains intact11-‘3; and (3)

transplantation of a porcine organ into a newborn recipient, which lacks XNAs but has an intact complement system, does not result in HAR (Ref. 14). With these observations, emerged as an important question.

the specificity of XNAs

While one might imagine that human XNAs would recognize a wide array of porcine antigens, greater than 80% of the complementfixing XNAs in a human serum recognize a single structure GabI-3Gal (Refs 15,161. The synthesis of Galal3Gal is catalyzed by the enzyme a1,3-galactosyltransferase

(Fig. l), which is present

in the cells of all lower mammals and New World monkeys. Humans, apes and Old World monkeys do not express cy1,3Galactose

galactosyltransferase or the corresponding sugar, and have naturally occurring antibodies specific for that sugar”J*. One very prom-

Gala1 -3Galpl-4GlcNAc-R

0 Fucose

ising approach to diminishing expression of Galal-3Gal is the expression of high levels of H-transferase in the pig. H-transferase

Galpi -4GlcNAc Fucal -2Galpl-4GlcNAc-R

H Fig. 1. Synthesis of G&lJGal

(cY1,2-fucosyltransferase) uses the same acceptor, N-acetyl lactos-

nnrl slrpprcssion by expression of

amine, as does c&3-galactosyltransferase (Fig. 1). Recent studies suggest that H-transferase competes very effectively with a1,3-

H-trunsfe7nsr. (a) In porcine cells, d5-8~lnctos~lirnnsfernse (GT) cnMyzes

galactosyltransferase.

addition of a terminal galactose to a srdbterrninnl N-acetyl Iactosamitle

The importance of GalalJGal as a target of XNAs was first suggested by Good et al.ly, who found that the antibodies in a human

lGalfil4GlcNAc) to produce Gal~~l-3G~l@Z4GlcNAc, the mnjor xenogeneic antigen. (b) The enzyme al,2-fucosyltmnsferase (HT), expressed as a product of a trmsgene, cowpetes z&h the GT for N-acetyl Iactosamirre resulting in synNlesis of blood group H rnlher fhan GaJrvl-3Gal

serum specific for a pig cell line could be blocked by purified [xgalactosyl sugars, such as Galcll-3Ga1, but not by other saccharides. Further evidence for the importance of this epitope was provided by Sandrin et nl. who showed that transfection of COS cells with cDNA for oll,3-galactosyltransferase

causes de novo expression

of

combinations of donor and recipient species that have been investigated’ but rarely in others, these combinations being called ‘discor-

Gahl-3Gal and confers sensitivity to lysis by XNAs and complement20. Collins et al. demonstrated that elimination of Galal-3Gal

dant’ and ‘concordant’, respectively. The susceptibility of a given combination to hyperacute xenograft rejection was thought to have

from porcine cells would render the cells resistant to human XNAs and complement2’. Furthermore, the phylogeny of expression of

a .genetic basis, reflecting at least in part the phylogenetic distance bciwcm the donor and the recipient’*8-10. During the past four

Galal3Gal, as well as the antibodies specific for it, could be used to demonstrate the importance of this sugar in vizm. Thus, the or-

yean, the molecular mechanisms underlying the susceptibility of pJicine organs to HAR by humans or nonhuman primates, and the basis for discordance in these species combinations, have been

gans of New World monkeys, which express the sugar, are rejected hyperacutely by baboons, which have antibodies against Galoll-3Gal

shown to be related to two primary factors: (1) the binding of xenoreactive naiural antibodies (XNAs) of the recipient to antigens on endothelial

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cells in the graft; and (2) the incompatibility

1996

of

- these antibodies account for virtually all of the recipient antibody deposited specifically in such grafts=. While it is now widely accepted that expression of Galal-3Gal is necessary

for the binding

of XNAs to a porcine cell, studies

IMMUNOLOGY

suggest that it is not sufficient. For example, Holzknecht found that porcine integrins with Galal3Gal substitutions block antibody binding far more effectively than some other proteins that have more Galotl-3Gal (Ref. 23). Alvarado rt ni. found that a sevenfold range in binding of human anti-Galrul-3GaI antibodies to cells from a large population of pigs is not related to differences in cell-surface GalalSGal (Ref. 24). These observations are consistent with the concept that the binding of XNAs to a cell surface is influenced to a large extent by the three-dimensional array of epitopes, which depends in turn on the nature of the protein core and its positioning on the cell surface. A more complete characterization of the XNAs that initiate the rejection reaction has been possible since identification of their antigenie target. The original studies on natural anti-Galol-3Gal antibodies focused mainly on IgG (Ref. 15). Yet studies on the rejection of porcine-to-primate xenografts suggested that the offending antibody was of the IgM and not the IgG isotype: IgM antibodies seemed to be deposited before IgG antibodies in rejecting xenografts”, and xenoreactive IgM, but not IgG, would activate comple-

TODAY

transplanting organs from transgenic pigs ex_ pressinghuman DAF and CD59 at levels well belew those m human Recent experiments

cells revealed very striking protection from complement-mediated injury and, in most cases, absence of HAR. These results show very clearly that the species-restriction of complement regulatory proteins is an important factor in the sus_ ceptibility of a xenogeneic organ transplant to HAR and thus to discordance. The results also point out that while HAR is either manifest fully or not at all, the susceptibility to HAR is potentially a continuous function of the level of complement control. Thus, in some animal models, ABO-incompatible allografts never undergo HAR and in humans, transplants across ABO barriers only occasionally undergo HAR (Ref. 33). In xenotransplantation which, as discussed below, is much like ABO-incompatible transplantation, HAR always occurs, the major difference being perhaps comple ment regulatory

proteins. Recent experiments

by White and col-

leagues suggest that when the incompatibility of complement regulatory proteins is more fully undersraod, xenograft survival can be extended over at least a period of months3’.

ment on porcine endothelial cells”. Moreover, infusion of large quantities of human IgG into nonhuman primates that received porcine cardiac-xenografts not only failed to cause rejection of the grafts, but in fact prolonged their function and survival by diverting complement away from the xenogeneic cell surfaces2b. The apparent discrepancy was resolved by two studieslh,“O that showed IgM XNAs, most of which are specific for Galal3Ga1,

initiate the

activation of human complement on porcine cells, whereas IgG XNAs, most of which are specific for structures other than Galal3Ga1, do not.

Complement

and the initiation ofhyperacute

rejection

In every combination of donor and recipient species studied to date, the development of hyperacute xenograft rejection hasproved to depend absolutely on the activation of complement. The mechanism by which complement is activated on xenogeneic endothelimn is a pivotal question and one that has been clarified in recent

The pathogenesis of hyperacute xenografi rejection There is accumulating

complement

evidence that HAR is mediated by terminal components. For example, Brauer et01.recently found

that HAR of guinea-pig hearts does not occur in C6-deficient rats35. The membrane-attack complex or C5b678 complexes might cause lysis of endothelial cells, leading to disruption of the integrity of blood vessels, egress of vascular contents and exposure of platelets to the underlying matrix, followed by thrombus formation. However, while lysis of endothelial cells is seen in advanced lesions, it is not a common feature early in the course of HAR. Consistent with this conclusion is our recent observation that expression of human CDS9 in transgenic pigs does not prevent the very early rejection of xenografts, although the formation of the membrane-attack com$ex may be substantially inhibited3b. Therefore, the initiation of LIAR would seem to be mediated by noncytotoxic effects of the terrrinal complement components, which arguably lead to a global 105s of

tation, such as guinea-pig-to-rat or pig-to-dog organ transplants, complement is activated through the alternative pathway, without

endothelial-celi functions (Fig. 2aIzJ7. One mechanism that might account for the loss of endothelialcell functions is an alteration in cell shape, leading to formation of

the need for XNAs (Ref. 27). However, it is now clear that in porcineto-primate xenografts, complement is activated through the classical

intercellular ‘gaps’ that we recently found to be induced by the assembly of C5b67 complexes on endothelial cells and accelerated by

pathway, initiated by the binding of XNAs (Refs 11,13,28).

the membrane-attack complexXs. The presence of the gaps, which are approximately 5 urn in diameter, abrogates the barrier that an

years. In some experimental

models of discordant

xenotransplan-

In addition to the issue of how the complement system is activated in a xenograft, there is the issue of how the activation of complement is controlled. Miyagawa ef n1J7 and Dalmasso ef ni.‘,” suggested that because cell-associated, complement-regmatory proteins endogenous to a xenograft, such as decay-accelerating factor (DAF), function in a species-specific manner, some aspect of xenograft rejection might be attributed to failure of these proteins to control the recipient’s complement cascade. Based on this concept, several trans genie animal models have been developed in which human complement regulatory proteins are expressed, anticipating that the organs of such animals would resist injmy by heterologous complemenp3’.

intact endxrthelium poses between vascular contents and extravascular components. Thus, the formation of gaps might allow the escape of vascular contents, the adhesion of platelets to intercellular spzcesand activation oi pla&ts through CC??? with +be =-+rix (Fig. 31. Another mechanism that might contribute to HAR involves the loss of heparan sulfate from endothelial cells25.Heparan sulfate is a protein-polysaccharide glycoconjugate that supports many endothelial functions, acting as a barrier to cells and plasma proteins, as an anticoagulant,

and providing

protection

from oxidants

and

IMMUNOLOGY

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rejection’2. This type of rejection is sometimes called ‘delayed’ xenograft

Platelets

rejection,

as if to suggest this type of rejection is a delayed form of hyperacute rejection. However, the clear distinction pathogenetitally from hyperacute rejection, and the close resemblance concordant

to rejection observed

xenografts

in

and in some allo-

grafts, argues against the use of the term ‘delayed’ in this context. Acute vascular xenograft rejection is characterized logically by diffuse intravascular lation

and interstitial

pathocoagu-

inflammation.

We

initially considered that acute vascular xenograft rejection might arise as a consequence of the activation of endothelial in the graft? as activated endothelial

cells cells

promote thrombosis and inflammation

(Fig.

2b). Recent studies by Blakely and colleagues, showing de nova expression of

Fig. 2. (0) P~tlzogemisof hyperncllte xcnograft rejection. Binding ofxenoreacfive antibodies and activation of completnent on xenograft endofheliutn causes loss of heparan sulfatefrom endothelium and formation of intercellular gaps. These changes provide a nidus for adhesion and aggregation of

E-selectin in guinea-pig hearts transplanted into rats, demonstrate that activation of endothelial cells indeed occurs in vascularized xenografts,

lending further support

to this

view”“. Nevertheless, the progressive nature of acute vascular xenograft rejection, in con-

platelets and disrupfion of the endothelial barrier. (b) The pathogenesis ofacutevascular xenograft rejection. ‘Quiescent’ endothelium maintains the homeostatic balance in coagulation, inflnnztnation trast to allograft rejection in which endotheand blood flora. bz response to inflalrrmatorymediators, vascular eudothelium becomes ‘activated’, lial cell activation is also observed, suggests adopting a procoagulant and yroir~flnmrnatory posture by elaborating tissue factor, adhesion n7ol- a more confounding pathogenesis might be ecrtles ~71~17 as E-selectin, qtokines and chernokines, Studies using cultured endothelial cells as a at work. Our current working model is that model for the vasculnr xenograft suggest that binding ofxenoreactive antibodies to donor endothe- the endothelial-cell changes associated with hn7

activafes fhe coruplernent sysferu, leading to assembly of the MAC. MAC does not directly ewiothelial cells but causes incrensed producfion of IL-lo; which, in turn, mediates the

activate

changes in ettdothehku characteristic of activatiorr. Vasoconstriction mediated by vasoconstrictors (ET-I and TxAJ hinders the blood floio to ensure availability of&la and propagation ofendothelial-cell activation. Abbreviations: C, confplement; ET-I, endothelin 1; HSPG, heparan sulfate proteoglycan; IL-lot, irtterleukiu Zol;MAC, tnanbrane-attack complex; PMN, poIymorphonwZcar leukocyte; TF, tissnefactor; TxA,, thromboxane A,.

acute vascular xenograft rejection are compounded by alterations in organ physiology that cause the tissue lesions to be propagated rather than resolved, as discussed below.

The initiation of acute vascular xenografi rejection

complemenP9. A substantial amount of heparan sulfate is lost from endothelial cells within minutes of exposure to anti-endothelial-cell antibodies and complement. This process also occurs in viva at a pace that could account for some of the alteration in vascular function observed in HAR. The loss of heparan sulfate cannot by itself account for HAR since it is mediated by C5a and not terminal complement components. However, it might confer heightened susceptibility to complement-mediated injury by impairing control of complement and increasing sensitivity to oxidant-mediated injury.

There is increasing evidence for the importance

of XNAs in acute

vascular xenograft rejection, perhaps with complement as an initiating factor. First, the lesions of acute vascular xenograft rejection generally arise under conditions in which the complement system is inhibited but antidonor antibodies remain, as in recipients treated with complement inhibitors such as cobra-venom factor or soluble CR1 (Ref. 41). Second, the onset of acute vascular rejection coincides with an increase in the synthesis of XNAs in individuals exposed to a porcine organ 42.Third, the physical removal of XNAs from a xenograft recipient delays the onset of acute vascular rejection from days

Acute vascular xenograft rejection If HAR of a xenograft is averted successfully, a xenograft is subject, over the following days to weeks, to a type of rejection that was first identified by Lever&ha1 et al. and termed ‘acute vascular xenograft

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1996

to weeks* and treatment of recipients with agents that suppress antibody synthesis can delay rejection for months or indefinitely”‘. The specificity of XNAs provides some clues to the pathogenesis of acute vascular xenograft rejection. Our own studiesa and those of Satake et a1.43 indicate

that immunosuppressed

individuals

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exposed to porcine cells synthesize XNAs predominantly directed against Galoll3Gal. These antibodies, which might include high levels of IgG and IgM, might damage the graft by one or more of four mechanisms: (1) activating complement as described above; (2) perturbing the function of the cell-membrane antigens that bear the Galal-3Gal epitopes, which consist mainly of integrin@; (3) delivering signals via the integrins, leading to activation of endothelial cells45; or (4) serving as a ligand for receptors on natural killer or other effector cells4‘j.

Pivotal role of interleukin Ia in the pathogenesis rejection

of humoral

Originally, it was conceived that XNAs and complement

might act

on endothelial cells similarily to cytokines or endotoxin, mediating activation of the cells with associated procoagulant and proinflamnratoi-f changes2. However, we recently discovered that complement activates endothelial cells through a novel pathway”. Instead of directiy mediating

endothelial

procoagulant

activity, complement

triggers the synthesis and release of interleukin la (IL-lo) and it is this cytokine acting as a paracrine or autocrine factor that stimulates the expression of tissue factor (Fig. 2b). The availability of IL-lo in this model might depend in part on local blood flow. Thus, vasoconstriction and endothelial thickening, the earliest changes observed in acute vascular xenograft rejection**“, might promote activation of endothelial

Fig. 3. Endothelial

changes in xenofransplarlf rejection. (a) A normal

blood vessel Ilas an intacf, nol;adkesiue

endothelial surface. (b) P&lets

are fallnd to be aftached to xenograft endothelial ceils, particularly along the jm”tions

where

the cells have zozdergone

of reperfusion by the recipih’s

retraction, zoithin minutes

blood.

be expressed on endothelium at similar concentrations as determined in lectin-binding studies. Third, if human complement regulatory proteins are expressed in a xenogeneic organ, the frequency of hyperacute rejection of xenotransplants in untreated recipients is -25%, the same as that observed in unmodified recipients of ABOincompatible organ transplants.

cells by reducing local flow and allowing

IL-la to stimulate the endothelium downstream from the point of constriction. IL-la can itself stimulate production ot thromboxane A2 (Ref. 48) or endothelin-1 (Ref. 49) which, in turn, amplifies this response and the ischemia that follows.

Accommodation

Therapeutic strategies and prospects for xenotrarssplantation Some years ago, Auchincloss reviewed the field of xenotransplantation comprehensively’. At that time, it was clear that intervening in the events that initia:e rejection of discordant xenografts would promote the survival of these grafts. However, therapies aimed at

sponse; rather, under some conditions, a transplant and the host can

the resulting symptoms, such as inflammation and coagulation, are generally less effective, presumably because the effector mechanisms are, to a certain extent, redundant. Based on this concept, we and others have focused on identifying and overcoming the fundamental

undergo ‘accommodation’, a term coined in 1990 (Ref. 1) to refer to a condition in which a graft appears to be accustomed to the hu-

immunological hurdles to discordant xenografting. While a vast array of such hurdles might be envisioned, the re-

moral immune reactions that, under other conditions,

sults of basic investigations have thus far disclosed two fundamental barriers - the interaction of XNAs with GaIrY13Gal and the in-

Fortunately, acute vascular rejection is not the only outcome of vascularized organ transplants confronted with a humoral immune re-

would de-

stroy it. The concept of accommodation emerged from the analysis of ABO-incompatible kidney allografts, where we observed that removal of antidonor antibodies from the graft recipient could lead to

compatibility of complement regulatory proteins of the donor with the compIement system of the recipient. The expression of human

lasting engraftment, even if antidonor antibodies later returnecP. A similar phenomenon was observed in xenografts, albeit quite infrequently’,“, and we postulated that it might result horn a change

complement regulatory proteins in the pig has dealt successfully with the second barrier and the challenge now is to prevent, in a clinically acceptable way, the interaction of XNAS with the graft.

in XNAs, a change in the antigens they recognize or an acquired

Recent studies by Sandrinsz and by Sharmas3 suggest that by expressing H-transferase in the pig the antigenic barrier might be

resistance of endothelium

to humoral injury.

ncompatibiity of complement regulatory proteins, xenografts might resemble ABO-incompatible allografts. First, XNAs and antibodies

overcome (Fig. I), perhaps addressing the hurdles posed by both hyperacute and acute vascular xenograft rejection. One might conceive that there exist other ‘systemic’ incompatibilities that would

against blood group A or B exhibit remarkably similar functional

stand in the way of successful xenotransplantation.

properties and concentrations in the serum, suggesting that they might be members of a common family of antibodies5’. Second, the

found that porcine thrombomodulin functions very poorly with human coagulation proteins (J. Lawson, unpublished). However, the

ABO antigens and GalulJGal

recent studies by White, demonstrating prolonged survival of porcine

Several lines of evidence suggest that, with the exception of the

are related biosynthetically

and can

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Indeed, we have

1996

~~

IMMUNOLOGY

hearts

in baboons”,

TODAY

suggest that these hurdles might not have the

impact that in vitro studies suggest. This perhaps leaves the cellular and elicited humoral immune responses to a discordant xenograft and physiological compatibility of the various porcine organs with the recipient as prominent questions to be addressed.

23 Platt, J.L. and Holzknecht, Z.E. (1994) Ttnttsp/~~~ttu”fiott 57, 327-335 24 Alvarado, C.G., Cotterell, A.H., McCurry, K.R. et RI. (1995) Ttwtwplnt~tnfiotz 59,1589-1596 25 Platt, J.L., Vercellotti, G.M., Lindman, BJ., Oegema, T.R., Jr, Bach, EH. and Dalmasso, Al? (1990) J. Exp. Med. 171,1363-1368 26 Magee, J.C., Collins, B.H., Harland,

R.C. et rd. (1995) I. Crirf. Inz~esf.96,

2404-2412

We thank T. Coffman and C. Smith for thoughtful comments on the manu-

27 Miyagawa,

script and the National Institutes of Health and Nextran for supporting

825-830

our

S., Hirose, H., Shirakura,

R. et nl. (1988) Tmusplnilrntioir 46,

28 Platt, J.L. (1995) Hypernctcfc Xrttogtzft Rcjrcfiotl, R.G. Landes

research in this area.

Company

William Parker, Soheyla Saudi, Shu Lin, Zoie Holzknecht, Matilde Bustos and Jeffrey Platt ([email protected]#) are nt the Dept of Surgery, Pediatrics

and Immunofogy,

Dtlke University,

Durhn,

NC

27710, USA.

29 Dalmasso, Al’., Vercellotti, G.M., Platt, J.L. and Bach, F.H. (1991) Trmtsplutttutiott52,530-533 30 White, D. and Wallwork, J. (19931 Lnnccf 342,879-880 31 Fodor, W.L., Williams, B.L., Matis, L.A. cf al. (1994) Proc. Nnfl. Acnd. Sci. U. S. A. 91,11153-11157 32 McCurry, K.R., Kooyman, D.L., Alvarado, C.G. ~‘fnl. (1995) Nut. Med. 1,

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