Alloreactivity and apoptosis in graft rejection and transplantation tolerance

Nicholas Zavazava and Dietrich Kabelitz Institute of Immunology, University of Kiel, Michaelisstr. 5, 24105 Kiel, Germany

Abstract: Weissmann wrote as early as 1889 that higher organisms contain within themselves the germs of death [1]. However, the term, programmed cell death, or apoptosis as it is now known, was dened much later [2]. Thus, it was long recognized that damaged and old cells are eliminated within the body, but the underlying mechanisms are only now beginning to emerge. Apoptosis appears central to the process of negative selection of developing T-cells in the thymus. In regard to organ transplantation, apoptosis contributes to graft rejection and the establishment of graft tolerance. Thus, understanding the regulatory mechanisms of apoptosis may help establish a new protocol for the induction of transplantation tolerance. J. Leukoc. Biol. 68: 167174; 2000.
Key Words: programmed cell death soluble MHC peripheral tolerance Fas/CD95 Fas-ligand(FasL)/CD95-L

DEVELOPMENT AND SPECIFICITY OF ALLOREACTIVE T-CELLS

Recognition of donor-type MHC antigens by responder T-cells occurs by direct or indirect antigen presentation. Direct antigen presentation, i.e., recognition of allogeneic major histocompatibility complex (MHC) molecules, is unique to transplantation and contributes to the exceptionally high cytolytic T lymphocyte precursor (pCTL) frequency measured after allogeneic organ transplantation [3]. Responding T-cells are predominantly CD8 and mediate acute rejection. However, membrane-bound MHC molecules are also shed by the allograft into the circulation [4, 5] and are detectable in serum of transplant recipients, in particular during the rejection process. These molecules can be processed and presented by antigen-presenting cells (APC) of the recipient. The MHC antigenic peptides presented by class II molecules result in the stimulation of CD4 T-cells. This so-called indirect presentation leads to a delayed response that, however, appears critical for the longterm survival of allografts. Although immune deviation generally referred to as the T-helper 1 (Th1)/T-helper 2 (Th2) paradigm has been questioned [6], some studies have demonstrated that Th2-type cytokines such as interleukin-4 (IL-4) favor tolerance induction [7]. In particular, He et al. [8] were able to induce tolerance to cardiac allografts after intraperitoneal injection of recombinant IL-4. Recipient animals showed markedly prolonged survival of cardiac allografts. Interest-

ingly, increased levels of IL-4 mRNA were found in the tolerant allografts. Although such experiments clearly indicate a role for Th2-type cytokines in long-term graft survival, the dichotomy between Th1 and Th2 cytokines in the process of tolerance induction remains controversial, because both types of cytokines are detectable during rejection as well as in a tolerant state [6]. The molecular basis of alloreactivity is still poorly understood. The T-cell repertoire of each individual is selected in the thymus to become tolerant to self-peptide/MHC complexes and to react against foreign peptides presented by self-MHC molecules. CD8positive T-cells activated by direct presentation of alloantigen are MHC-specic but not necessarily peptide-specic. Why the MHC molecule itself appears to dominate during the alloresponse and not the endogenous peptide has been indirectly shown in in vitro studies. CTL generated by coculturing stimulator cells with responder peripheral blood lymphocytes are specic for the mismatched MHC molecules, independent of the type of cells on which they are expressed. For example, CTL generated against human leukocyte antigen B7 (HLA-B7) tumor-derived stimulator cells will lyse HLA-B7 Epstein-Barr virus (EBV)-transformed B-cell lines and vice versa, although presumably the presented peptides are different in cells of different tissue origin. More recently, Rammensee and colleagues [9] have used MHC molecules loaded with peptide libraries and dened self- and viral peptides. They showed nicely that the closer the foreign MHC molecule is related to the T-cells MHC, the higher the proportion of peptide-specic, alloreactive (allorestricted) T-cells vs. T-cells recognizing the foreign MHC molecule without regard to the peptide in the groove. Nonetheless, the role of endogenous peptides in allorecognition is not yet well-dened. Until now, only two peptides have been dened as natural HLA ligands for human alloreactive CTL [10, 11]. Nonetheless, endogenous peptides are required for the stability of MHC molecules and for their expression on the cell surface. Although a limited subset of alloreactive CTL might be peptide-independent [12, 13], X-ray diffraction studies show that about 75% of the surface area contacted by the T-cell receptor (TcR) corresponds to the MHC molecules [14, 15]. However, these studies were performed in peptide-specic T-cells. In cases where donor and recipient MHC are structur-

Correspondence: Nicholas Zavazava, MD, and Dieter Kabelitz, MD, Institute of Immunology, University of Kiel, Michaelisstr. 5, 24105 Kiel, Germany. E-mail: MACROBUTTON HtmlResAnchor zavazava@immunologie.uni-kiel. de and kabelitz@immunologie.uni-kiel.de Received April 14, 2000; accepted April 14, 2000.

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ally similar within their polymorphic regions, the endogenous peptide presents the dominant epitope for the allogeneic immune response [16]. Conversely, if the structural difference within the polymorphic region between donor and responder MHC is small, the alloresponse is directed against epitopes on the endogenous peptide. Thus, the pool of T-cells responding to alloantigen is heterogenous, consisting of T-cells recognizing (1) endogenous or exogenous peptides uniquely presented by the allo-MHC; (2) epitopes made up of the peptide and the MHC itself; and (3) epitopes on the mismatched MHC molecules themselves [16] (Fig. 1). This diversity in antigenic epitopes explains why the frequency of alloreactive T-cells is 10- to 100-fold higher than the frequency to nominal antigen. Finally, differences in minor histocompatibilty antigens between donor and recipient, especially after bone-marrow transplantation or small bowel transplantation, are immunologically apparent as a result of peptide presentation and recognition [17]. Indeed minor histocompatibility antigenic peptides have been isolated, including a peptide that provokes graft vs. host disease [18]. More recently, an association between minor histocompatibility antigens hemagglutinin (HA)-1, -2, -4, and -5 and graft vs. host disease was shown [19]. Using tetrameric HLA class I minor histocompatibility HA-1 and male antigen HY peptide complexes, an increase in HA-1- and HY-specic CTL during acute and chronic graft vs. host disease was observed. Thus, a role for minor histocompatibility antigens in graft survival appears well established [20].

MECHANISMS OF TARGET CELL LYSIS BY CYTOTOXIC T-CELLS AND APOPTOSIS IN GRAFT REJECTION
Cytotoxic T-cells are morphologically characterized as large granular lymphocytes expressing perforin, granzymes, and

other granule proteins. Alloreactive T-cells recognize and destroy their target cells via three major mechanisms [21, 22]. Upon TCR-mediated recognition of target cells, signaling including Ca2 inux occurs in CTL. These signals result in the orientation of T-cells to their targets, followed by exocytosis of granules containing perforin and granzymes. Perforin is a poreforming protein with structural similarity and sequence homology with factors of the membrane attack complex (MAC, i.e., C6-C9). Binding of perforin to the cell surface of target cells results in pore formation that can lead to cell death via homeostatic imbalance of the cell. Evidently, however, target-cell killing by granzymes and perforin is more complex. The target cell begins to initiate a repair process that involves endocytosis of granzymes released by the CTL. Granzymes, especially granzyme B, initiate DNA fragmentation by activating caspases that ultimately lead to the demise of a cell [23]. In the acute phase of rejection, allografts consistently show evidence of massive apoptosis independent of the transplanted tissue. However, apoptosis is not restricted to transplanted tissue but has also been demonstrated in graft-inltrating T-cells themselves [24, 25]. The apoptosis observed in some graft-inltrating T-cells might indicate a down-regulatory process of activated T-cells eliminating potentially self-damaging T-cells. The second mechanism by which T-cells recognize and kill target cells involves Fas/FasL (CD95/CD95L) engagement. Programmed cell death controls the homeostasis of multicellular organisms, during embryogenesis, metamorphosis and normal tissue turnover. The Fas molecule belongs to the tumor necrosis factor receptor (TNF-R) and nerve growth factor (NGF) receptor family. This is a group of type I membrane proteins characterized by three to six cysteine-rich repeats in the extracellular domain [22]. Among others, the two TNF receptors, TNF-R1 and TNF-R2, and CD40, OX40, and CD30 belong to this family [26]. Many tissues weakly express Fas,

Fig. 1. Antigenic epitopes for alloreactive T-cells. Donor-derived allo-MHC antigens are processed and presented by APC of the recipient (a). In addition, donor APC directly present allo-MHC antigens to the recipient T-cells. In this case, the bound endogenous peptide together with the allogeneic MHC form the epitope recognized by alloreactive T-cells (b) or the MHC molecule alone, independent of the peptide being immunogenic (c).

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but abundant expression was found in mouse thymus, liver, heart, lung, kidney, and ovary [27]. Although resting T-cells are Fas-negative, Fas expression is rapidly induced upon activation. Contrarily, FasL is a member of the TNF family, which includes TNF, CD40L, CD30L, and OX40L [22]. It has a domain of hydrophobic amino acids in the middle of the molecule, indicating that it is a type II protein. For a long time, it was assumed that FasL is expressed only on activated T-lymphocytes [28]. However, FasL has now been identied in other tissues. The evidence for a role of the Fas/FasL system for immune regulation is best illustrated by observations in mice decient in Fas (lpr/lpr strain) [27] or FasL (gld/gld strain) [29]. The animals are characterized by abnormal accumulation of lymphocytes. In the rst weeks of life, the mice have normal T-cell subsets but gradually lose mature T-cells and accumulate CD4CD8 cells in the periphery. Later on, the animals develop splenomegaly, lymphadenopathy, and autoimmune disease. Thus, Fas/FasL expression is required for normal development of the immune system. Similar symptoms have been observed in patients with genetic defects in Fas/FasL expression [30]. The patients studied had a large deletion in the gene encoding Fas and suffered from lymphoproliferative syndrome and autoimmune disease in agreement with observations made in lpr and gld mice. In striking contrast, perforindecient mice [31, 32] and mice decient in granzyme A or B [33] develop normally and lack any alterations in the T-cell subsets or their activation. Patients with autoimmune lymphoproliferative disease (ALPS) can now be classied into four groups [34, 35]: 1) Type Ia, ALPS with mutant Fas; 2) Type Ib, lymphadenopathy and mutation in FasL; 3) Type II, ALPS with mutant caspase 10; and 4) Type III, ALPS, as yet, without any dened genetic cause. Untreated organ recipients generally lose their allografts by acute rejection. Surprisingly, in some immune priveleged sites, such as the eye, testis, and brain, foreign antigen remains free from immunological damage. Medawar [36] had explained this phenomenon by arguing that immunological ignorance was responsible for protection from rejection, because privileged sites were isolated behind blood-tissue barriers and lacked lymphatic drainage. The assumption was that these sites remained invisible to the immune system. Recent data, however, demonstrate that the mechanism is an active process and might be exploited to combat disease and promote graft acceptance. For example, Griffth et al. [37] revealed that ocular tissue expressed mRNA of FasL that is expressed during inammation. Inammatory cells entering the anterior chamber of the eye in response to viral infection were shown to become apoptotic-dependent on Fas/FasL engagement. In contrast, viral infection of gld mice lacking FasL led to inammation and invasion of the eye by inammatory cells. Morever, Fas-positive but not Fas-negative tumor cells were eliminated by apoptosis when placed in segments of the anterior chamber of the eye of normal mice but not that of FasL-negative mice. In further studies, it was revealed that T-cell death and tolerance required that lymphoid cells be Fas and the eye, FasL [38]. These studies emphasized that immune privilege is not a passive process based on physical barriers but is an active process that involves cell-death receptors and ligands. In studies on the testis, another privileged site, testicular grafts de-

rived from mice that expressed functional FasL survived indenitely when transplanted under the kidney capsule of allogeneic animals, whereas grafts derived from mutant gld mice were rejected [39]. Sertoli cells constitutively express FasL mRNA and upregulate FasL expression in allogeneic recipients repelling invading T-cells. Altogether, the data on the anterior chamber of the eye and testicular tissue implied that tissue expression of FasL confers protection against rejection mediated by Fas-expressing inammatory cells, especially Tcells. The third mechanism of target-cell lysis by CTL involves the secretion of cytotoxic cytokines such as TNF- and interferon (IFN)-. Indeed both cytokines have been shown to induce apoptosis. More recently, stimulation of cytolytic cells with IL-2 and IL-12 has been shown to lead to TNF- and IFN- release [40]. Apoptosis was increased in these natural killer (NK) cells by the two cytokines. The mechanisms of target-cell lysis by CTL, as discussed above, generally depend on the contact of T-cells with target cells. In the literature, these mechanisms were initially classied as calcium-dependent or -independent [41]. The calcium-dependent pathway has now been well characterized and involves the interaction of FasL on activated CTL with Fas on target cells [42].

APOPTOSIS AND TOLERANCE INDUCTION

The majority of developing thymocytes die within the thymus by apoptosis and never reach the periphery [43]. The death of thymocytes is caused by engagement of their TCR/CD3 complexes with MHC molecules expressed by cortical or stromal cells. Developing thymocytes with high afnity for thymic MHC molecules undergo apoptosis, thereby resulting in negative selection mediated by apoptosis [44]. Susceptibility of T-cells to apoptosis is not restricted to immature or transformed T-cells but can be triggered in vivo and in vitro in mature T-cells through TCR engagement. This raises the possibility that apoptosis might be involved in the induction of peripheral tolerance and, thus, contribute to the fate of the allograft [45]. The conditions under which apoptosis can be triggered in mature T-cells depend on their activation state and cytokine microenvironment. For example, engagement of the TCR/CD3 complex of primary resting T-cells by antigen or antibodies against the TCR or CD3 triggers activation, whereas stimulation of activated cells induces apoptosis [46]. The apoptotic process by which activated T-cells are eliminated following TCR stimulation is generally termed activated induced cell death (AICD) [47], which depends on macromolecular synthesis, consistent with the need for de novo synthesis of Fas and FasL. Cyclosporin A, glucocorticoids, and retinoids inhibit AICD [48]. Clinically, OKT3 (an anti-CD3 antibody) is widely used during rejection episodes to eliminate T-cells. It has been shown that OKT3 induces apoptosis in activated T-cells [46]. Interestingly, resting or naive T-cells are resistant to AICD. This resistance has been linked to their inability to recruit caspase-8 [49]. The need for induction of antigen-specic tolerance is not only a requirement for successful organ transplantation but remains a challenge in the management of autoimmune and
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T-cell-mediated diseases. Thus, antigen that causes disease is an important entity to the process of tolerance induction. More recently, the molecular requirements for costimulation of Tcells have been well dened [50]. CD28 has been identied as a major receptor for costimulatory ligands such as CD80 (B7.1) and CD86 (B7.2). The use of antibodies directed against CD80 and CD86 for tolerance induction has indicated that T-cells are anergized if confronted with antigen in the absence of costimulation [51]. Lenschow and colleagues [52] showed down-regulation of the T-cell response when anti-CD80 or anti-CD86 antibodies were added to cell cultures. In combination, the antibodies signicantly prolonged survival of pancreatic allografts in a murine transplantation model. Thus, blocking the binding of ligands to the CD28 molecule signicantly reduces immune responses. CTLA4 is another receptor for CD80 and CD86. In contrast to CD28, signals delivered through CTLA4 engagement appear to downregulate the immune response. A CTLA4-immunoglobulin (Ig) fusion protein consisting of the extracellular domain of CTLA4 fused to an Ig has been shown to bind to CD80 with a 20-fold higher afnity than a similar CD28 construct [53]. When tested in vitro, CTLA4-Ig inhibited CD80-dependent T-cell costimulation and allogeneic mixed lymphocyte reactions. Another promising approach to achieve indenite survival of allografts has been demonstrated in murine models of skin and cardiac allograft transplantation [54]. In these experiments, recipient animals were treated with CTLA4-Ig and an antiCD40L-antibody. The CD40/CD40L (CD154) system constitutes another potent costimulation pathway. Both types of allografts were well-preserved and remained healthy indenitely. More recently, a humanized anti-CD154 (CD40L) antibody effectively protected renal allografts in primate monkeys for over 10 months without any additional immunosuppression [55]. Unfortunately, preliminary clinical data on the use of the anti-CD40L antibody have been disappointing mainly because of the rapid development of thrombosis in treated patients. Nonetheless, the experiments summarized above support the idea that stimulation of T-cells via the TCR in the absence of obligatory costimulation anergizes T-cells and allows the induction of tolerance. Although more work is required to verify the efcacy of this approach in large animals and in humans, indeed there is hope for achieving tolerance after clinical transplantation with some of these biological reagents. In this respect, the combination of various approaches might reveal new perspectives in the future. Delineation of the Fas/FasL system stimulated work and raised hope that transfection of whole allografts or cellular allografts with FasL might prolong graft survival and eventually induce tolerance. Subsequently, allogeneic pancreatic islets were transfected with FasL and transplanted under the renal capsule in mice. Paradoxically, FasL-transfected allografts underwent accelerated neutrophilic rejection. Rejection was Tand B-cell-independent but Fas-dependent [56]. Similar observations were made in FasL-transgenic mice expressing FasL in pancreatic- cells [57]. The transplants developed strong neutrophilic inltrates, and the recipients became diabetic. These results were rather puzzling and in contrast to the observations made in privileged sites, such as the eye [37] or the testis [39], where T-cell inltration was blocked by the
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ability of FasL-expressing tissues to induce apoptosis. Similarly, locally produced FasL in a Fas-negative tumor cell line elicited rapid elimination of the tumor in vivo [58], a situation much desired for treatment of cancer diseases but not for tolerance induction of allografts. More recently, Chen et al. [59] showed that this recruitment of granulocytes after local overexpression of FasL could be blocked by transforming growth factor- (TGF-). Thus, overexpressing FasL in allografts to protect them from T-cell-mediated rejection promotes rapid graft rejection but not tolerance induction. Although the role of the Fas/FasL system in allograft tolerance is not entirely clear, increasing evidence supports the idea that apoptosis is an essential requirement for tolerance induction in transplantation. In a recent study, Wells et al. [60] demonstrated that intact T-cell-apoptosis pathways are required for the induction of tolerance across MHC barriers. Mice decient in passive (Bcl-xL transgenics) or active T-cellapoptosis pathways (IL-2 knockout mice) were found to be resistant to induction of transplantation tolerance by costimulation blockage or rapamycin treatment, indicating an essential requirement for T-cell apoptosis. In a recent study, Li and colleagues [61] investigated the role of Fas in organ transplantation using Fas-mutant lpr mice as recipients. In those experiments, Fas-decient recipients rejected islet and cardiac allografts, and Fas-decient T-cells were able to undergo AICD in vitro. However, graft tolerance was inducable in these mice by costimulation blockage or rapamycin, indicating that tolerance induction can proceed in the absence of Fas-mediated apoptosis.

ANTIGEN-INDUCED TOLERANCE
The observation that vascularized liver allografts spontaneously induce tolerance without any accompanying immunosuppression has remained puzzling and yet fascinating [62]. The explanation for this phenomenon is still controversially discussed in the literature. It was speculated that the liver produces large amounts of donor-type, soluble MHC and that these molecules tolerize T-cells. Indeed donor-specic class I molecules have been readily detected after liver transplantation; in contrast, it has remained difcult to detect donor-type, soluble MHC class I molecules after kidney or heart allografting [5, 63, 64]. The major component of serum MHC molecules in healthy or diseased individuals is contributed by liver tissue [65]. Therefore, it could be speculated that because of the large size of liver allografts, high levels of donor-type, soluble MHC are released into the circulation and might mediate graft tolerance. In support of the possible role of donor-antigen load are the studies by Bishop et al. [66, 67], showing that high-antigen dosage leads to tolerance induction. In rats, for example, liver allografts weigh 9 g compared with 1 g for a heart or kidney allograft. Bishop and colleagues [68] transplanted rat-strain PVG-derived two hearts and two kidneys and infused 1.5 108 cells into a single DA rat-strain recipient. These animals did not receive any further treatment. The organs were accepted and survived 200 days. When these organs were transplanted singly, however, they were acutely rejected. This
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experiment demonstrated high-dose exhaustion of the immune system by donor-derived MHC antigen. Bishop et al. [67] propose a scheme, whereby at low concentration, antigen fails to elicit stimulation and allogeneic responsea state of immunological ignorance. When antigen load is further increased, an immunological response is achieved that leads to graft rejection. At even higher antigen levels, such as in the case of liver transplantation or that of multiple organ transplantation, immunological exhaustion is accomplished that allows allografting. Another interesting approach to reveal the role of antigen concentration was provided by Liblau et al. [69], using a transgenic mouse expressing a TCR specic for a 13 amino acid inuenza virus HA peptide. After injection of 750 g but not 75 g of the peptide into the transgenic animal, a state of hyporesponsiveness was achieved. Deletion of T-cells was observed in the thymus and in all peripheral lymphoid organs. Thus, high doses of antigen appear to tolerize antigenspecic T-cells and induce systemic peripheral tolerance. More recently, Huang et al. [70] demonstrated TCR-mediated internalization of peptide-MHC complexes acquired by T-cells. Within minutes of interaction with APC, peptide-MHC complexes on APC formed clusters at the site of T-cell contact. These were internalized by the T-cells and made them sensitive to fractricide killing. These data presented a possible explanation for antigen-driven death of T-cells, as observed in many different systems, including viral antigen [71, 72], alloantigen [73], and ovalbumin [69]. Interestingly, Qian and colleagues [25] demonstrated apoptosis of T-cells in liver allografts after allogeneic liver transplantation. They suggested that apoptosis of graft-inltrating T-cells contributes to the development of spontaneous tolerance after liver transplantation. However, these experiments did not address the donorspecic components involved in apoptosis induction. It has become evident that the liver serves as a sink for activated T-cells, implying that T-cells activated in the periphery move to the liver where they eventually perish. Mehal et al. [74] infused the liver with a pool of lymphocyte mixtures and showed retention of activated but not resting or apoptotic T-cells. This T-cell trapping was specic for CD8 T-cells that later became apoptotic. The idea that donor-derived antigen produced in the liver may induce apoptosis in activated recipient T-cells is appealing and consistent with the speculation that antigen released by the liver confers protection against rejection.

SOLUBLE MHC MODULATE ALLOREACTIVE T-CELLS BY INDUCING APOPTOSIS

Three decades ago, serum of healthy and diseased individuals was shown to contain soluble MHC (sMHC) class I molecules with the same serological allotypes as present on the cell surface of the same individuals. Charlton and Zmijewski [75] and van Rood et al. [76] showed independently that sMHC bind and neutralize allogeneic sera. Many experiments were initially designed for the application of soluble MHC to generate allo-antibodies for tissue typing [77, 78] but failed, because soluble MHC are poor immunogens. Similarly, experiments aimed at studying the possible immunoregulatory ca-

pacity of T-cells by soluble MHC remained fruitless for almost two decades. Finally, McCluskey et al. [79] demonstrated that puried class I molecules on their own were ineffective in inuencing T-cell reactivity; however, when immobilized on beads or coupled to dextran, they effectively stimulated Tcells. In addition, soluble MHC class I molecules in the mouse [80] successfully blocked the ability of a T-cell hybridoma to proliferate or produce IL-2 after antigenic stimulation. Later, our own group [81, 82] established that afnity-puried class I molecules abrogate the cytotoxicity of alloreactive T-cells in a dose-dependent fashion. However, the underlying mechanism for T-cell inhibition remained unclear for quite some time. Three different possibilities could be discussed. First, because inhibition of T-cells was allospecic, the data suggested that recognition of sMHC occurred via the TCR. Experiments to prove this directly are rather difcult, in particular because the association/dissociation times of MHC molecules on TCRs are very short. Secondly, signaling through the TCR rather than physical masking might lead to the development of T-cell anergy that initiates T-cell nonresponsiveness. Indeed, it has been well established that ligation of the TCR induces T-cell anergy and that a second signal is required to trigger T-cell activation. Blockage of the costimulatory signal by antibodies or soluble receptor molecules has been shown to induce antigen-specic anergy. In transplantation models, this approach has been exploited by several groups to achieve donor-specic tolerance [51, 54, 83]. Finally, T-cells might be driven into apoptosis upon recognition of allogeneic MHC molecules. In fact, it has been shown that CD8 and CD4 T-cells are susceptible to AICD triggered through TCR-mediated recognition of allogeneic MHC class I [84, 85] or class II molecules [86, 87]. More recently, our own group revealed that soluble MHC class I molecules initiated AICD of alloreactive T-cells during in vitro culture [73]. This observation was consistent with observations by Lenardo [88], where V8 T-cells activated in mice by the bacterial superantigen Staphylococcus aureus enterotoxin B were driven into apoptosis upon reexposure to superantigen in the presence of IL-2. In our own studies [73], incubation of alloreactive T-cells with soluble MHC antigen led to T-cell death after a few hours. IL-2 was required for the induction of apoptosis. These results were rather unexpected, because it had been assumed in previous studies that soluble MHC-induced signaling was too weak to initiate regulation of the T-cell function. However, they conrmed the obervation that lack of costimulation leads to cell death and emphasized that soluble donor-type MHC molecules deliver a regulatory signal through the TCR/CD3 complex. Indeed simultaneous ligation of the CD28 molecule rescued T-cells from cell death and led to signicant stimulation of the alloreactive T-cells. sMHC did not modulate the expression of Fas, the TCR, or that of CD8 but rather signicantly upregulated FasL. Thus, sMHC-mediated cell death was initiated by upregulation of FasL that interacts with Fas on activated T-cells, leading to suicide or fractricide killing. Induction of apoptosis by sMHC is not a unique property of soluble MHC class I molecules but can be similarly initiated through MHC class II molecules [89, 90] or allopeptides [91]. Recombinant class II molecules loaded with relevant peptides also efciently induce apoptosis in antigen-specic T-cells.
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Rhode et al. [89] produced class II single chains covalently linked to an ovalbumin-derived peptide or without peptide and showed that peptide-loaded MHC molecules stimulated T-cells and induced T-cell apoptosis. This technology allows rapid production of large amounts of sMHC. Similarly, Nag and colleagues [90] successfully loaded single chains of class II molecules with an immunodominant peptide derived from the myelin basic protein (MBP)-83-102. These molecules interacted with TCRs of MBP-specic T-cells and induced release of IFN- and ultimately apoptosis. Thus, interaction with antigen appears to be a specic approach for the elimination of activated T-cells. Therefore, apoptosis is one of several consequences of T-cell interaction with soluble MHC molecules. Alternatively, recognition of sMHC by T-cells can turn these cells into an anergic state without induction of FasL expression or apoptosis. Thirdly, as discussed above, simultaneous ligation of CD28 results in T-cell activation. These possibilities are schematically depicted on Figure 2. Animal models are now required to prove the efcacy of soluble antigen-induced nonresponsiveness. This approach may nd application not only in the context of transplantation medicine but more generally in T-cell-mediated diseases.

CONCLUDING REMARKS
Apoptosis is a general phenomenon required for the regulation of cellular homeostasis and development of multicellular organisms. Recent studies have clearly dened the molecular requirements of cell death via apoptosis. Apart from the death receptors that have been cloned and studied in great detail, more recently the mitochondrial pathway has emerged as a second major pathway for apoptosis induction [92, 93]. In the context of allograft transplantation, it should be recognized that apoptosis observed in allografts per se does not allow prediction of whether rejection or tolerance induction are occurring but is nonetheless central to both processes. Thus, a better understanding of the mechanisms by which activated antigen-specic T-cells can be eliminated provides the molecular basis for new tolerance-induction protocols. The observation that donor-type soluble MHC induce apoptosis in alloreactive T-cells opens a new therapeutic modality that requires in vivo testing. To avoid the need for purifying large amounts of donor-type soluble MHC molecules, it may be feasible to perform gene transfer of donor-type MHC into the recipient [94]. In T-cell-medi-

Fig. 2. Interaction of alloreactive T-cells with donor-derived soluble MHC. Activated T-cells express Fas on their surface. Upon interaction with soluble MHC, they upregulate FasL and undergo apoptosis (a). However, some T-cells fail to upregulate FasL and become anergic (b). In this state, they can be reactivated if stimulation is accompanied by costimulation. Finally, if binding of soluble MHC to T-cells is accompanied by ligation of CD28, the cells are activated and proliferate (c).

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ated disorders, peptide/MHC complexes, especially as single-chain molecules, might nd application in a wide range of diseases.

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