Heart Xenografts Research Articles

Ostéites multifocales et Propionibacterium acnes

Solid organ xenotransplantation is a potential solution to current organ shortages in allotransplantation. We performed four heart transplantations from alpha1, 3-galactosyltransferase gene-knockout (GT-KO) pigs to cynomolgus monkeys and monitored immunological parameters before and after transplantation.After blood typing of the cynomolgus monkeys, we assessed the binding activity of immunoglobulin G (IgG) and IgM of monkey serum and serum toxicity toward porcine peripheral blood mononuclear cells (PBMCs) using flow cytometry. Immunosuppressive protocols consisted of anti-thymocyte globulin (25 mg/kg), rituximab (20 mg/kg), anti-CD154mAb (20 mg/kg), cobra venom factor (0.05 mg/kg), tacrolimus, and steroid. Cynomolgus monkeys with A or AB blood type with the lowest antibody binding and serum toxicity activity on porcine PBMCs were selected as recipients.Absolute numbers of CD3+ T cells, CD20+ B cells, and CD3+CD95+ memory T cells in the peripheral blood were suppressed upto 24 days after transplantation. Interferon gamma production of T cells in response to porcine antigens were also significantly suppressed. Heart xenografts from GT-KO pigs survived for upto 24 days without pathologic evidence of rejection.We successfully performed 4 heart xenotransplantations using GT-KO pigs. We overcame hyperacute rejection by using GT-KO pigs, and all of the heart xenografts from the GT-KO pigs survived between 11 and 24 days without pathologic evidence of rejection, disseminated intravascular coagulation, or consumptive coagulopathy; however, we need to optimize protocols for immune modulation and postoperative care to attain long-term survival of solid organ xenografts.

Mycophenolate sodium: tolerability and efficacy in transplantation in the rat

Inhibition of inosine monophosphate dehydrogenase by mycophenolate compounds results in potent immunosuppression, as demonstrated by the efficacy of the marketed prodrug mycophenolate mofetil (MMF) in clinical allotransplantation. Side effects are well-known and include bone marrow depression and gastrointestinal intolerability. Mycophenolate sodium (MPS) is in clinical development as an enteric-coated formulation to alleviate this gastrointestinal adverse effect. Accompanying this development, MPS and MMF were evaluated in a tolerability study in rats and in efficacy studies in rat allo- and xeno-transplantation models. The compounds were given either singly or in combination with cyclosporine A and were efficacious in the prevention of allo- or xeno-graft rejection, but with a rather narrow window between optimal immunosuppression and adverse side effects. For instance, the minimal effective dose to prevent rejection of a kidney or heart allograft or a hamster heart xenograft is a daily dose of 10–20 mg/kg MPS, at which dose the first adverse side effects can be observed: the compound at 40 mg/kg is not tolerated. This window is even narrower for MMF than for MPS, and in most models, a minimal effective MMF dose could not be established. The window between optimal immunosuppression and adverse side effects is larger when the compounds are given in combination with cyclosporine A: in all models investigated combinations were established yielding long-term survival without histologic signs of rejection and without signs of side effects. Thus, the combination of an IMPDH inhibitor (MPS, MMF) and a calcineurin inhibitor (cyclosporine A) enables fine-tuning in achieving optimal immunosuppression avoiding drug side effects.

Survival of heterotopic heart xenografts from Helisoma duryi, Planorbula armigera, and Planorbarius corneus in Biomphalaria glabrata (Pulmonata, Basommatophora, Planorbidae): evidence for phylogenetic relatedness?

Abstract. Heart xenografts from the pulmonate snail Helisoma trivolvis survive in Biomphalaria glabrata, whereas xenografts from most other pulmonate snail genera are rejected within 3 to 15 days. To test whether xenografts from snails closely related to H. trivolvis were also accepted, specimens of an albino strain of B. glabrata were implanted with hearts from H. duryi, Planorbula armigera, and Planorbarius corneus. The fate of implants was monitored for 180 days by measuring heartbeat and by histological analysis. All 3 types of implants survived beyond the 3 to 15 days required for the complete destruction of incompatible xenografts, suggesting a degree of physiological and immunological compatibility between B. glabrata and these donors. Among the 3 types of xenografts, all from H. duryi survived for the entire 180 days. Fewer grafts from P. armigera and P. corneus survived for prolonged periods, although some still were beating at 60 and 180 days post implantation (DPI) respectively, suggesting that a range of histocompatibility, and perhaps phylogenetic relatedness, with B. glabrata occurs within this group. Paradoxically, the initial hemocytic response by the recipient was strongest against grafts from H. duryi, the most compatible donor, while responses to grafts from P. armigera and P. corneus were mild or did not occur during the first 7 DPI. We used 2 albino strains of B. glabrata in these studies, and the data suggest slight differences in recipient strain compatibility.

A soluble chimeric inhibitor of C3 and C5 convertases, complement activation blocker-2, prolongs graft survival in pig-to-rhesus monkey heart transplantation.

Complement plays a critical role in many pathologic processes and in xenograft rejection. Therefore, effective complement inhibitors are of great interest. In pig-to-primate organ transplantation, hyperacute rejection results from antibody deposition and complement activation. Complement activation blocker-2 (CAB-2), a recombinant soluble chimeric protein derived from human decay accelerating factor (DAF) and membrane cofactor protein, inhibits C3 and C5 convertases of both classical and alternative pathways. CAB-2 reduces complement-mediated tissue injury of a pig heart perfused ex vivo with human blood. Therefore, we studied the efficacy of CAB-2 when a pig heart is transplanted heterotopically into rhesus monkeys receiving no immunosuppression. Graft survival in three control monkeys was 1.26 +/- 0.2 h; it was markedly prolonged in eight monkeys that received CAB-2. Of the six monkeys that received a single dose of CAB-2 (15 mg/kg i.v.), four had graft survivals of 21, 95, 96, and 108 h, and two died at 7 to 11 h post-transplant with a beating graft, as a result of technical complications. The two monkeys given multiple doses of CAB-2 had graft survivals of 95 and 96 h. CAB-2 markedly inhibited complement activation, as shown by a strong reduction in generation of C3a and SC5b-9. At graft rejection, tissue deposition of iC3b, C4 and C9 was similar or slightly reduced from controls, and deposition of IgG, IgM, C1q and fibrin did not change. Thus, complement inhibition with CAB-2 abrogates hyperacute rejection of pig hearts transplanted into rhesus monkeys, but does not prevent delayed/acute vascular rejection. These studies demonstrate that the beneficial effects of complement inhibition on survival of a pig heart xenograft in rhesus monkeys are similar to those in other primate species and that CAB-2 may be useful in xenotransplantation and other complement-mediated conditions.

Late xenograft rejection: comparison between liver and heart xenografts under low-dose tacrolimus

Late xenograft rejection: comparison between liver and heart xenografts under low-dose tacrolimus

The structure of anti-Gal immunoglobulin genes in naïve and stimulated Gal knockout mice.

Naturally occurring antibodies (Nabs) that bind to terminal galactose alpha1,3-galactose carbohydrate structures (Gal) are present in humans and Old World monkeys but are negatively regulated in other mammalian species because they express Gal epitopes on their cell surfaces. A Gal knockout mouse (Gal-/-) model, generated by homologous disruption of alpha1,3-galactosyltransferase gene, is capable of producing natural anti-Gal Abs. To study the genetic control of the anti-Gal response, we have generated anti-Gal hybridomas from Gal-/- mice and analyzed VH genes of anti-Gal Abs from naïve animals and from mice stimulated by rat heterotopic heart transplantation. Six immunoglobulin (Ig)M anti-Gal hybridomas derived from naïve Gal-/- mice exhibited anti-Gal binding activity with some cross-reactivity to related carbohydrate structures. These naïve anti-Gal Abs used five different VH genes in a germline configuration. Anti-Gal IgM hybridomas isolated after a rat heterotopic heart xenograft (4 days) utilized germline VH gene segments from the VH7183 family and exhibited less cross-reactivity. In contrast to mice 4 days after xenograft, we have predominantly isolated IgG anti-Gal hybridomas from mice 21 days after rat heterotopic heart xenografts, indicating an isotype switch. Nine of the IgG anti-Gal hybridomas secreted IgG3 subclass and one produced IgG1. Sequence analysis of the VH gene usage from the induced anti-Gal IgG antibodies demonstrated a restricted gene utilization (VHJ606-V14A). Our results demonstrate that the anti-Gal response in naïve Gal-/- mice is encoded by multiple germline progenitors. In response to a xenograft, the induced anti-Gal Abs exhibited a restricted gene usage and somatic mutations, indicating a positive selection.

Suppression of T cells results in long-term survival of mouse heart xenografts in C6-deficient rats.

The present study aimed to investigate the role of cellular immune response in the absence of membrane attack complex (MAC) formation in the concordant mouse-to-rat heart xenografting. Hearts from BALB/c mice were transplanted into the neck vessels of C6-competent (C6(+)) and C6-deficient (C6(-)) PVG rats. Liposome-encapsulated dichloro-methylene diphosphonate (Lip-Cl2MDP) was administered at a dose of 10 ml/kg 2 days before transplantation and every 5 days thereafter. Cyclosporine (CsA) was administered intramuscularly (i.m.) at a dose of 15 mg/kg per day. The heart xenografts were harvested for immuno-histological analysis at the time of rejection and the functioning grafts were removed at 70 days after transplantation. In untreated C6(+) rats, xeno-grafts survived for 2.3 +/- 0.5 days. Treatment with CsA or Lip-Cl(2)MDP in C6(+) rats did not significantly affect graft survival (2.5 +/- 0.6 and 2.3 +/- 0.4 days, respectively). In untreated C6(-) rats, xenografts survived for 5.0 +/- 0.6 days. However, Lip-Cl(2)MDP in C6(-) rats resulted in a prolongation of graft survival to 11 +/- 2.3 days (P < 0.05 vs. untreated C6(-) rats), while treatment with CsA alone in these rats led to more than 70 days' survival in four out of six grafts (61 +/- 16 days). In untreated C6(+) rats, immunohistology showed a severe myocardial necrosis and thrombosis with a scarce cellular infiltrate in the rejected xenografts. By contrast, in untreated C6(-) rats, xenografts were heavily infiltrated by macrophages and T cells. The number of macrophages, but not T cells, was markedly reduced in Lip-Cl(2)MDP-treated rats. In CsA-treated C6(-) rats, the grafts harvested at 70 days after transplantation had a normal morphology, with a minimal cellular infiltrate. Our data indicate that MAC-mediated injury plays an essential role in concordant xenograft rejection. Once this mechanism has been prevented, suppression of T cells allows for long-term xenograft survival.

T cell and B cell tolerance to GALalpha1,3GAL-expressing heart xenografts is achieved in alpha1,3-galactosyltransferase-deficient mice by nonmyeloablative induction of mixed chimerism.

We have previously demonstrated that mixed xenogeneic chimerism and donor-specific T-cell tolerance can be induced in the rat-to-mouse species combination by using a relatively nontoxic, nonmyeloablative conditioning regimen. However, natural antibodies (NAbs) against Galalpha1,3Gal (Gal) pose an additional major barrier to pig-to-human vascularized xenograft acceptance. To determine whether the mixed chimerism approach could also overcome this humoral barrier, T cell-depleted rat (GalT+/+) bone marrow cells (BMC) were transplanted to alpha1,3-galactosyltransferase deficient (GalT-/-) mice conditioned with a nonmyeloablative regimen, consisting of transient T cell and natural killer (NK) cell depletion, 3 Gy whole body irradiation, and 7 Gy thymic irradiation. By giving a high dose (180x106) of rat BMC, persistent mixed chimerism could be induced in GalT-/- mice, although the level of donor-type hematopoietic repopulation declined over time. Induction of mixed chimerism was associated with a rapid disappearance of anti-Gal and anti-rat NAb in the sera. Both anti-Gal Ab-producing cells and B cells with receptors recognizing Gal were undetectable in mixed chimeras, even when the chimerism levels declined, suggesting that a very low level of chimerism could effectively maintain B-cell tolerance to Gal, probably by clonal deletion and/or receptor editing. Mixed chimeras accepted subsequently transplanted donor-type rat hearts (>100 days) without immunosuppressive therapy, whereas delayed vascular and even hyperacute rejection of rat hearts occurred in conditioned control GalT-/- mice. Cellular rejection occurred by 5-6 days in conditioned control wild-type mice. These findings demonstrate that induction of mixed chimerism with a nonmyeloablative regimen can prevent vascularized xenograft rejection by cellular and anti-Gal Ab-dependent pathways in GalT+/+-to-GalT-/- species combinations.

LEWIS RAT PANCREAS, BUT NOT CARDIAC XENOGRAFTS, ARE RESISTANT TO ANTI-GAL ANTIBODY MEDIATED HYPERACUTE REJECTION 1

The objective of this study was to evaluate the role of anti-Gal Abs and non-anti-Gal Abs in hyperacute rejection (HAR) of concordant pancreas xenografts compared with heart xenografts. In addition, we tested whether rejection of Lewis rat pancreas grafts was T-cell dependent and could be prevented by anti-T-cell treatment. To determine the role of anti-Gal Abs in the induction of HAR, Lewis rat pancreas and heart xenografts were transplanted into alpha1,3Galactosyltransferase knockout (GT-Ko) mice treated with normal human serum (NHS) or hyperimmune serum, or into presensitized GT-Ko mice. To investigate whether rejection of pancreas xenograft was mediated by a T-cell dependent response, Lewis rat pancreas grafts were transplanted into streptozotocin (STZ)-induced diabetic GT-Ko mice treated with FK506, anti-CD4 mAbs (GK1.5), and thymectomy. Antidonor-specific IgM and IgG and anti-Gal Abs were analyzed by flow cytometry. Rejected and long-term surviving pancreas xenografts were assessed by functional (blood glucose) and histopathological examination. HAR of Lewis rat pancreas xenografts could not be induced by NHS (0.4 ml), whereas NHS (0.2 ml) resulted in HAR of Lewis heart xenografts. Infusion of Lewis rat-specific hyperimmune serum (0.2 ml) resulted in HAR of Lewis rat pancreas xenografts. In addition, second Lewis rat pancreas grafts were hyperacutely rejected by presensitized GT-Ko mice. Immunohistochemical staining showed a low expression of Galalpha1,3Gal antigen in the endocrine tissue compared with that in the cardiac grafts. The levels of anti-Gal Abs in pancreas xenograft transplantation did not increase in GT-Ko mice after pancreas xenograft transplantation that was significantly increased after heart transplantation. FK506 treatment induced long-term survival of Lewis pancreas xenografts (mean survival time (MST) >90 days). Anti-CD4 treatment delayed rejection of Lewis rat pancreas xenografts with MST of 34.3 days, whereas anti-CD4, in combination with thymectomy, synergistically prolonged survival of pancreas xenograft (MST=70.4 days). Pancreas xenograft is resistant to anti-Gal Abs-induced HAR but is susceptible to anti-donor specific Abs. Rejection of Lewis pancreas xenograft in STZ-induced, diabetic, GT-Ko mice is T-cell dependent.

Porcine heart xenograft function and hyperacute rejection: the role of thrombin inhibition

Porcine heart xenograft function and hyperacute rejection: the role of thrombin inhibition

Prolonged survival of guinea-pig-to-rat heart xenografts following complement depletion and B-cell-directed immunosuppression by malononitrilamide

Prolonged survival of guinea-pig-to-rat heart xenografts following complement depletion and B-cell-directed immunosuppression by malononitrilamide

Serum protein immunogenicity: Implications for liver xenografting

The objectives of this study were threefold: (i) assess immunogenicity of donor plasma proteins following hepatic xenotransplantation, (ii) identify potential immunogens, and (iii) consider the implications of antibody formation against these plasma proteins in xenograft survival. We studied liver and heart xenografts in a concordant combination, hamster to rat. All grafts were examined at necropsy for evidence of rat immunoglobulin G (IgG) deposition. Cardiac xenografts were placed in recipients who had, or had not, been sensitized with hamster serum. Hepatic xenografts were placed in naive recipients to see if antibodies to hamster serum proteins could be eluted from the rejecting organ. Sera of immunized rats were examined for the presence of anti-hamster antibodies by immunoelectrophoresis and by Western blotting following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of hamster serum. Antibodies in sera of immunized rats were compared with those eluted from rejecting livers. Candidate antigens were identified by tandem mass spectrometry, sequence analysis, and reference to protein databases. Results showed that sera of immunized rats recognized a minimum of four different antigens in hamster serum by immunoelectrophoresis, and a minimum of seven by the more sensitive SDS-PAGE Western blot. IgG eluted from rejecting livers bound three of seven candidate antigens recognized by sera of the immunized animals. Sequence analysis searches revealed proteinase inhibitors in each of the three SDS-PAGE bands common to the above samples. All of these candidate proteinase inhibitor immunogens share a common catabolic fate, uptake via the lipoprotein-related protein (LRP/alpha 2-macroglobulin receptor (CD91). Sensitization to hamster serum proteins hastened cardiac xenograft rejection in 30-50% of recipients (depending on sensitization protocol). Vascular deposition of rat IgG occurred in all rejecting xenografts. Antibody binding to proteinase inhibitors could disturb their functional activity and contribute to the pathogenesis of delayed xenograft rejection.

Prolongation of xenograft survival using monoclonal antibody CD45RB and cyclophosphamide in rat-to-mouse kidney and heart transplant models.

Intrigued by the finding that a monoclonal antibody (mAb) directed against the B exon of restricted CD45 (CD45RB mAb) induced renal allograft tolerance in the mouse model, we hypothesized that CD45RB mAb may prevent xenograft rejection. We explored the role of CD45RB mAb in preventing xenograft rejection in rat-to-mouse kidney and heart transplant models. Mice with rat kidney and heart xenografts were treated with a short course of mAb, cyclosporine, cyclophosphamide, or mAb + cyclophosphamide combination therapy. Untreated heart and kidney xenografts served as controls. Untreated controls developed acute vascular and cellular rejection rapidly with a median survival time of only 6 days. Long-term kidney (median survival time = 70 days) and heart xenograft survival (median survival time = 65 days) was achieved using the combination therapy of mAb + cyclophosphamide. One-third of the kidney recipients with combination therapy survived 100 days. Immunohistochemistry and xenospecific-antibody analysis demonstrated that combination therapy remarkably reduced IgG and IgM deposition and also inhibited CD4+, CD8+, and Mac-1+ cell infiltration at early stages. This therapy, however, did not induce tolerance in this model as evoked xenoreactive antibodies and cellular responses may be the cause of late xenograft failure. A short course of CD45RB mAb combined with cyclophosphamide effectively inhibits cellular and humoral immunoresponses and remarkably prolongs xenograft survival in rat-to-mouse heart and kidney transplant models.

Evidence against a pivotal role of preformed antibodies in delayed rejection of a guinea pig–to–rat heart xenograft

Introduction: Whereas the involvement of elicited xenoantibodies in delayed xenograft rejection is currently being substantiated, this study focuses on the role of the preformed fraction of xenoantibodies. Methods: To check the influence of the latter, we combined pretransplant complement inactivation (cobra venom factor) and antibody reduction (plasmapheresis) in a guinea pig–to–rat heart transplant model. Results: Antibody reduction on plasmapheresis before xenografting did not prolong delayed xenorejection in decomplemented rats, although the immunohistologic pattern lacked the immunoglobulin deposits along endothelial walls found in xenografts of merely decomplemented recipients. Astonishingly, plasmapheresis, if carried out 2 days before transplantation, almost tripled xenograft survival, although preformed antibody levels were completely restored and even rebounding at the time of grafting. The pattern and number of infiltrating cells did not differ in dependence of the timing of plasmapheresis nor did the proliferative response of lymphocytes in the mixed lymphocyte reaction differ. However, plasmapheresis led to a retarded decrease of the mononuclear cell tumor necrosis factor α secretory potential, which correlated well with a diminished immunohistologic staining of tumor necrosis factor α secreted by graft-infiltrating mononuclear cells. Conclusion: These findings argue against a pivotal role of preformed xenoantibodies in the pathomechanistic process of delayed xenograft rejection and challenge the therapeutic strategy to reduce preformed xenoantibody levels before xenotransplantation in complement-inactivated recipients. (J Thorac Cardiovasc Surg 2000;119:477-87)

Effect of surrogate tolerogenesis on the vascular rejection of pig heart xenografts.

Organ xenografts are fulminantly rejected by antibody-mediated vascular rejection. Surrogate tolerogenesis (ST), the induction of tolerance within the donor, is effective with aorta xenografts. This preliminary study assesses the effect of ST on preformed antibodies and rejection of porcine heart xenografts. Tolerance to the donor pig was induced by infusing recipient marrow into fetal pigs. Later, pig splenocytes were transfused and heterotopic pig hearts transplanted using chimeric or nonchimeric pigs. Anti-pig antibodies were assessed. With ST alone, xenografts developed cellular rejection at 4-6 days, whereas control grafts developed vascular rejection at 3-4 days (cellular vs. vascular, P<0.03). There was a reduction in preformed antibodies (P<0.03). ST combined with moderate cyclosporine prevented rejection at 9+ and 25 days in sensitized recipients compared with vascular rejection at 0.5-2 days for controls (P<0.07). ST seems to provide protection against vascular rejection. The cellular rejection seems sensitive to cyclosporine.

Failure of anti-Forssman antibodies to induce rejection of mouse heart xenografts.

The Forssman antigen has been proposed to be a target for the xenograft reaction in selected species combinations, including the rat and mouse, which are Forssman-negative and -positive species respectively. The mouse represents an important experimental model for a variety of immune-mediated disease processes, and the availability of a simple, inexpensive target antigen could provide an important tool for studying a selected portion of the immunologic basis for the rejection of xenografts. We have examined the potential that antibodies directed against mouse Forssman antigen could cause the hyperacute rejection of mouse heart xenografts in naive rat recipients. The Forssman antibodies tested included rat anti-mouse (R-anti-M) antiserum, R-anti-M antiserum depleted of anti-Forssman (anti-F) antibodies, rat anti-sheep red blood cell (SRBC) antiserum containing anti-F antibodies and a rat monoclonal anti-F IgM antibody. Our results demonstrate that the R-anti-M antiserum at day 4 post transplantation displayed significant titers (1:512-4096) of hemagglutinating antibodies for SRBC and mild to moderate levels of IgM that specifically binds to Forssman glycolipid (GalNAcalpha1-3GalNAcbeta1-3Galalpha1-4Galbeta1- 4Glcbeta1-1ceramide) as measured by an enzyme-linked immunosorbent assay (ELISA). Passive transfer of the R-anti-M serum to rats receiving mouse cardiac grafts immediately after transplantation caused hyperacute rejection of the xenografts. Sequential immunoabsorption of R-anti-M sera with SRBCs resulted in total removal of the anti-Forssman activity (as defined by negative hemagglutination titer and minimal binding to Forssman glycolipid in ELISA). The anti-F Ab-depleted R-anti-M antisera, however, retained the capacity to induce hyperacute rejection of the mouse hearts [n = 6, median survival time (MST) 13 min] when passively transferred to rat recipients. Anti-Forssman antibodies induced by immunization of LEW rats with SRBCs or a rat anti-Forssman monoclonal antibody, mAb M.1.22.25, exhibited substantial anti-Forssman activity (hemagglutinating titer 1:512-4096 and moderate-to-strong binding to Forssman glycolipid in ELISA respectively). These antibodies also failed, however, to trigger hyperacute rejection of mouse cardiac xenografts. In conclusion, our results suggest that the rat anti-Forssman antibodies, including those stimulated by mouse cardiac xenografts, do not appear to play a role in the immediate (hyperacute) rejection of mouse heart xenografts.

INHIBITION OF COMPLEMENT-MEDIATED XENOGENEIC INJURY BY THE MEMBRANE ATTACK COMPLEX IS SUFFICIENT TO ALLOW INDUCTION OF ACCOMMODATION IN THE HAMSTER-TO-RAT MODEL

55 Accommodation is long-term graft survival in the presence of xenoreactive antibodies and complement. Accommodation can be obtained in the hamster-to-rat cardiac model by a combination of complement inactivation with cobra venom factor (CVF) and daily administration of cyclosporin A (CSA). Since CVF inactivates both C3 and subsequent components following C3, experiments with CVF suggest that several complement activation fragments, in addition to the membrane attack complex (MAC), may play a role in preventing accommodation in this model. To define which complement-mediated process needs to be inhibited to allow induction of accommodation, we investigated accommodation of a hamster heart xenograft in C6-deficient (C6D) PVG rats and in normocomplementemic PVG rats treated with antibodies against C6. Golden Syrian hamster hearts were transplanted heterotopically into normal or C6D rats. C6D rats had complete deficiency of C6 hemolytic activity; addition of purified C6 to the serum fully restored CH50 total complement activity, confirming that other complement components were intact. Graft survival in untreated rats was 3-4 days (n=4) and in untreated C6D rats was 5 days (n=5). Experimental groups received either CVF (60 U/Kg at day-1 pre-tx. and 20U/day × 9 days) or polyclonal goat anti-C6 antiserum (Cappel) (140 mg IgG/kg/day × 5 days) with or without CSA. The table shows the number of grafts performed in each group and grafts surviving >100 days. C6D rats bearing hamster hearts had no reconstitution of hemolytically active C6. Histopathologic examination was performed on rejected hearts (<100 day survivors) or after sacrifice (>100 day survivors).TableC6D rats treated with CSA or CSA and CVF had 90% long-term graft survival. Normal rats treated with CSA and CVF, or CSA and anti-C6 antibodies had >60% long-term graft survival. Histologically, rejected hearts had myofiber necrosis, interstitial hemorrhage, acute inflammation and vascular damage. In contrast, hearts from >100 day survivors lacked these findings and had preservation of normal architecture; however, a mononuclear cell infiltrate was present. These features are characteristic of accommodated hearts in the hamster-to-rat model. In conclusion, the finding that C6D rats and normal rats treated with anti-C6 antibodies develop accommodation without CVF treatment to inactivate C3 and C5 demonstrates that xenogeneic injury due to the MAC is responsible for preventing the development of accommodation in the hamster-to-rat model. Accommodation occurred in the face of unabated formation of fragments derived from C3 and C5.

IFN-γ MAY PLAY A "PROTECTIVE" ROLE IN A RAT-TO-MOUSE HEART TRANSPLANT MODEL

449 We previously reported that transplanting a Lewis rat heart into a BALB/c mouse produced vigorous rejection (MST: 6.0±0.6 days), while transplanting a Lewis heart into a C57BL/6 mouse produced "weak" rejection (MST: 20.6±4.9 days). The present study was undertaken to explore cellular and molecular mechanisms attributing the different pattern of rejection between these two recipient strains. Sequential routine and immunopathology, and cytokine expression measured by northern blotting were studied in both BALB/c and C57BL/6 mice with a Lewis heart xenograft. Vascular rejection characterized by vasculitis, infarction, thrombosis and hemorrhage was dominated in Lewis xenografts transplanted in BALB/c mice, while cellular rejection was dominant in those of C57BL/6 mice. Immunopathology studies further revealed that these infiltrating cells in C57BL/6 mice were CD4+ and CD8+ T cells, NK cells and macrophages. Notably, strong intragraft IFN-γ expression measured by northern blotting and immunopathology was found in heart xenografts in C57BL/6 mice compared to relatively weak expression in BALB/c mice. To further define the role of IFN-γ in xenograft rejection, Lewis heart grafts were transplanted into IFN-γ knock-out (IFN-γ−/−) mice. Interestingly, the heart xenograft survival was significantly shortened in IFN-γ−/− C57BL/6 mice (MST: 9.0±1.0 days) as compared with that of wild-type C57BL/6 mice (MST: 20.6±4.9 days, P <0.001). In contrast, there was no difference between wild-type BALB/c mice and IFN-γ−/− BALB/c mice (MST: 6.0±0.6 days vs. 6.1±1.5 days). Furthermore, the rejection pattern was shifted from cellular rejection to vascular rejection when IFN-γ was knocked out from C57BL/6 mice. We conclude that 1) predominant cellular rejection in C57BL/6 mice produces "weak" xenograft rejection; 2) high expression of IFN-γ is present in these grafts and 3) depleting IFN-γ gene shifts cellular rejection to vascular rejection, thereby accelerating xenograft rejection. These data indicate that IFN-γ may play a "protective" role in xenograft rejection. Studies are currently underway to see whether manipulating cytokine network could attenuate xenograft rejection.

THE AVOIDANCE OF FORSSMAN-LIKE REACTION IN HAMSTERS RECEIVING RAT ANTISERUM IS DUE TO ACCOMMODATION TO HETEROLOGOUS COMPLEMENT

532 Forssman reaction can be induced in guinea pigs by injecting anti-Forssman Ab, but this reaction can be avoided if the same is given in progressively increasing doses. Shedding of Forssman antigen-antibody complexes from endothelium has been postulated as the mechanism of desensitization from this reaction and it has been hypothesized that organ xenografts might become less vulnerable to hyperacute rejection, if exposed to tolerable levels of xenoantibodies prior to transplantation (Tx). A Forssman-like reaction can be produced in hamsters (Forssman +) by injecting into them a lethal dose of rat (Forssman −) antihamster Ab. We examined whether desensitizing hamsters to this reaction could render their hearts less susceptible to humoral rejection when transplanted into rats. Anti-hamster Ab was prepared by implanting hamster hearts into rats and collecting the sera 7 days after Tx. The titer of the anti-hamster Ab was 1/4000 and 0.2 cc of this unmodified antiserum was determined to be the lethal dose (LD) required to induce fatal Forssman-like reaction. Hamsters were placed in 3 groups according to these treatments: (1) a single LD of anti-hamster Ab (n=5); (2) desensitization by injecting hamsters (i.v.) with unmodified anti-hamster antiserum in progressively increasing doses over a period of 48 h for a total of 25 LD (n=5); and (3) a single injection of 10 LD of antiserum that had been heat-inactivated (56°C for 30 min) to deplete complement (n=5). The hearts from the desensitized hamsters in Group 2 were harvested and transplanted into rats. Samples from lungs, liver and heart xenografts were taken at death or sacrifice for H&E and staining for rat IgM and C3. All hamsters in Group 1 died of lung edema within 5 min of Ab infusion. Histology revealed severe congestion of the lungs, perivascular edema and hemorrhage. Moreover, strong binding of rat IgM and C3 in the lungs were observed. On the contrary, hamsters that had received 25 LD in Group 2 appeared unharmed. However, the hearts taken from these "adapted" hamsters suffered hyperacute rejection in 2-9 min when transplanted into rats (normal rejection time: 3 d). Adapted hamsters showed moderate lung congestion and arteritis, the liver was infarcted with evidence of multifocal hepatocyte necrosis. Rat IgM was bound to the lungs, heart and kidney, indicating that the xenoantigen was still present. However, there was no evidence of C3 deposits in the lungs. Hamsters in Group 3 who received a single injection of 10 LD of decomplemented antiserum did not undergo the Forssman-like reaction and all survived. At sacrifice, their lungs and livers were mildly congested with strong binding of IgM but little or no C3. In conclusion, adaptation of lung endothelium to heterologous complement seems to be the main mechanism underlying this phenomenon and not the antigenic modulation on endothelial cells. Moreover, this adaptation is not conferred to other organs in the same animal.

LATE XENOGRAFT REJECTION INVOLVES A STRONG T CELL RESPONSE

1009 Both hyperacute and acute vascular rejections have not yet allowed for the analysis of T cell responses in vivo in vascularized xenografts. We used a model of late xenograft rejection in order to assess the role of T cells in this process. Long term survival hamster heart xenografts can be obtained in LEW.1A rat recipients treated with a combination of Cobra Venom Factor (CVF) and Cyclosporin A (CsA). In this study, all recipients with long term xenografts rapidly rejected their grafts (4+/−1 days) after withdrawal of CsA on day 40 after transplantation. In comparison, LEW.1A recipients of a LEW.1W heart allograft having received the same treatment rejected their graft 18+/−2 days after withdrawal of CsA. We subsequently analysed T cell infiltration and Vβ TCR usage at the complement determining region 3 (CDR3) level, in rejected xeno- and allografts. Four days after withdrawal of CsA, a large number of infiltrating T cells, a large proportion of which were positive for the activation markers IL2 α receptor and MHC class II, were detected in xenografts only. Both types of graft were similarily heavily infiltrated with α/β positive T cells at rejection time, with a substantial deposit of T cell dependant IgG antibodies. However, CVF did not prolong xenograft survival after CsA withdrawal and no vascular lesion were detected, suggesting a predominant role of T cells in this rejection process. The Immunoscope and Reperturb software® and the sequencing of junctional regions were used to identify, quantify and compare the nature of alterations of CDR3 segments of each Vβ family. Surprisingly, rejected xenografts revealed a conserved Gaussian distribution of CDR3 lengths within all Vβ families tested. A similar pattern was observed in vitro, using purified rat T cells stimulated with hamster dendritic cells. These Gaussian patterns contrasted with the strongly altered patterns of most Vβ famillies exhibited in rejected allografts (p<0.001). Our study suggests that, after withdrawal of CsA, the under-represented T cell clones expanded more rapidly in xeno- than in allografts, leading to a more rapid rejection in xeno- (4+/−1 days) than in allografts (18+/−2 days). These results show that under these specific experimental conditions, αβT cells are the predominant cellular species within a rejected xenograft and that most of the T cells are stimulated. It is thus likely that xenografts provide large quantities, combined with a high diversity of peptides and that important TCR/MHC peptide cross reactions occurs. Our data favour the concept of a strong T cell response in xenografts.