Workshop G Transplantation

WORKSHOP G

Transplantation

Institute of Immunology, University of Vienna, Vienna, Austria

G.l Interference with a late event of Tcell activation by n-butyrate results in the induction of alloantigen-specific hyporesponsiveness G. A. BOHMIG, K. WENHARDT, P. ALAEI, M. SAEMANN, and G. J. ZLABINGER Recently, n-butyrate has been shown to induce antigen-specific functional inactivation in a human T cell clone when added during antigen-contact. Selective blockade of the G la phase of the cell cycle was proposed to be the underlying mechanism. Testing the efficacy of n-butyrate in freshly isolated human T cells, we were ab le to demonstrate that pretreatment of a primary mixed lymphocyte culture (MLC) w ith n-butyrate also induces specific hyporesponsiveness to alloantigen. Since specific downregulation of T cell reactivity is described to be achieved by blocking the costimulatoty activity of antigenpresenting cells, we wanted to investigate the involvement of the CD28 / B7 pathway, which is suggested to be critical in determining the outcome of a T cell response. First, we examined the effect of n-butyrate on the expression of this receptor-ligand pair. The agent did not affect CD28 expression on T cells. Interestingly, partial inhibition of spontaneous as well as stimulated up regulation of B7-1 on monocytes was observed. In contrast, n-butyrate profoundly increased B7-2 expression on these cells. To investigate if differential regulation of B7-1 and B7-2 prevents costimulation via CD28, bivalent anti-CD28 mAb was added to primary culture. However, artificial CD28 costimulation did neither restore proliferation in primary MLC nor prevent the induction of specific hyporesponsiveness. Furthermore, increased B7-2 expression argues against impaired CD28 costimulation. Time kinetic experiments revealed, that addition of n-butyrate up to 5 days after culture initiation effectively downregulates an established primary proliferative T cell alloresponse and is still effective in inducing alloantigen-specific hyporesponsiveness. Finally, specific downregulation of T cell reactivity required at least four days continuous presence of n-butyrate in primary culture. In summary, our results indicate, that n-butyrate interferes with a late stage of T cell activation. A mechanism different from cell cycle blockade, however, cannot be excluded.

Joint annual meeting 1995 OGAI/GFI . 121 Departments of 1Cardiology and 2Cardiac Surgery, University of Heidelberg, and 3German Cancer Research Center, Heidelberg, Germany

G.2 C02 directed immunosuppression in rat heart transplantation is synergistic with cyclosporine but not with C048 (C02 ligand) antibodies T. J. DENGLERl, B. SID02, P. MULLER 3, R. ZIMMERMANNl, G. OTTd, and S. C. MEUER 3 The CD2 antigen on T cells and NK cells represents an accessory receptor capable of modulating the T cell immune response. In a heterotopic rat heart transplantation model, inhibitory CD2 monoclonal antibody OX34 prolonged median graft survival time (mGST) dose-dependently to a maximum of 49.5 days (range: 35-81) versus 7 days (range: 6-8) in controls (p < 0.001) by induction of CD2 antigen down modulation without significant depletion of CD2+ cells. In contrast, CD2 antibody OX54, inducing peripheral T cell depletion for 40 days, prolonged mGST only to 14 days (range: 10-20, p < 0.001 versus controls). Combined use of subtherapeutic doses of cyclosporine (4 x 1.5 mg/kg) and OX34 antibody (2 x 0.075 mg) increased mGST to 20 days (range: 16-27, p < 0.001 versus controls). A monoclonal antibody against the CD2 ligand CD48 did not prolong mGST versus controls, when used alone, and had no synergistic effect in combination with CD2 antibody OX34. Thus, CD2 directed immunotherapy prolongs allograft survival in a dose-dependent manner without induction of tolerance. Suggested mechanisms are CD2 antigen downmodulation and blockade, whereas depletion of T cells appears not to be relevant. CD2 mediated immunosuppression is synergistic with cyclosporine but cannot be enhanced by modulation of CD2 ligand CD48 in a rat model. The complete lack of effect of an anti-CD48 antibody may suggest the existence of other CD2 ligands than CD48 in the rat system.

Christian Albrechts University at Kiel, 2nd Medical Clinic, Kiel, Germany

G.3 Autologous transplantation of peripheral blood progenitor cells in a patient with lymphoma and systemic lupus erythematosus S. FASTENRATH, H. H.

EULER,

G. SCHROEDER, P. DREGER, and N. SCHMITZ

Autologous transplantation of bone marrow or peripheral blood progenitor cells (PBPC) is being intensively discussed as a possible option in the treatment of severe autoimmune diseases (Lancet 345: 878, 1995). The rationale of this treatment is based on the 2-fold hypothesis that myeloablation can drastically reduce autoaggressive cell clones and that newly developing lymphocytes with acquired self-tolerance towards former auto antigens suppress or eliminate surviving autoaggressive cells. It is not yet known what type of myeloablation optimally eliminates autoaggressive cell clones or whether purging of proliferating lymphocytes from the graft is necessary. This is the first report on the autologous transplantation of unpurged PBPC in a patient with a severe autoimmune disease. A 28-year-old female patient with a 14-year history of systemic lupus erythematosus (SLE) (discoid rash, malar rash, photosensitivity, oral ulcers, leukopenia, and elevated

122 . Joint annual meeting 1995 OGAIIGFI titers of antinuclear antibodies (ANA), anti-ds-DNA, anti-SS-A and anti-SS-B) received an autologous graft of unpurged PBPC for treatment of a relapsing high-grade nonHodgkin's lymphoma in 6/94. Myeloablation was performed according to the BEAMprotocol: Carmustin 2 x 150 mg/m2, Etoposid 8 x 150 mg/m2, Cytarabin 8 x200 mg/m2, Melphalan 140 mg/m 2. The graft contained 10.7xl06 CD34+-cells per kg BW. Hematopoietic reconstitution was uncomplicated. The lymphoma is currently in complete remission. Since transplantation there have been no clinical symptoms of SLE; she is off all drugs. WBC is still reduced at 3300/ml. However, ANA have returned to pretransplant levels (IgG 1:1280). Anti-ds-DNA have increased to 13.75 U/ml. SS-A/52 kD (157 U/ml pre-transplant) and SS-B (39 U/ml pre-transplant) have decreased to normal levels. Thus, autologous transplantation of unpurged PBPC led to a temporary remission of SLE in our patient but not to a deletion of autoaggressive lymphocyte clones. Possibly, the chances of achieving the latter goal could be increased by a more highly lymphocytotoxic type of myeloablation (e.g. with cyclophosphamide) and/or purging of the autologous graft (e.g. of CD34+ stem cells).

Institute of Immunology, University of Heidelberg, Heidelberg, Germany

G.4 Inhibition of hyperacute xenograft rejection by soluble human complement regulators: analysis in a pig-to-human in vitro model B. HECKL-OSTREICHER and M. KIRSCHFINK Hyperacute graft rejection triggered by the activation of the complement (C') system represents the major obstacle to xenotransplantation. Inhibition of C' activation is therefore considered as a prerequisite for xenograft survival. The application of physiological soluble as well as membrane-bound C' inhibitors appears to be a logical approach. Recently, we could show that the transfer of the human membrane-associated C' regulator CD59 to aortic porcine endothelial cells (PEC) mediates protection from lysis by human serum. In an in vitro cell culture model for xenotransplantation we tested a possible protective function of the C' regulators Cl-inhibitor and soluble recombinant human C' receptor 1 (sCRl). In the presence of Cl-inhibitor as well as of sCRI PEC were dose-dependently protected from C' -mediated cytotoxicity. The effect of Clinhibitor was augmented by heparin. In addition, soluble C' inhibitors in suboptimal concentrations were tested on PEC which were previously transfected with CD59eDNA. In PEC-clones expressing only small amounts of CD59, thereby showing only limited protection, the addition of soluble C' regulators, significantly suppressed C'mediated cell destruction. The combination of soluble and membrane-bound human C' regulators interferring at various sites of the C' reaction provides optimal protection of xenogeneic cells from C' -mediated cytotoxicity.

Joint annual meeting 1995 OGAIIGFI . 123 1Klinik fur Abdominal- und T ransplantationschirurgie, Medizinische Hochschule Hannover, Hannover, Germany; 2Second Department of Surgery, Kyoto University Faculty of Medicine, Kyoto, Japan

G.S Development of microchimerism in pediatric patients after living-related liver transplantation J. HUNDRIESER!, M. UEDA2, K. TANAKA2, R. PICHLMAYER!, H.J. SCHLITT!, Y. YAMAOKA 2, and K. WONIGEIT 1 Microchimerism is supposed to play an important role in the long-term acceptance of allogeneic organ grafts by transplant patients and for the maintenance of a state of donorspecific low responsiveness. In order to elucidate the kinetics of the development of chimerism we have performed a follow-up analysis in 10 pediatric patients with livingrelated liver transplantation (LRLTx). Blood samples obtained during the first 6 months post transplant and skin biopsies taken at one month were analysed for the presence of donor cells by peR using donor-specific HLA-DRBI primer pairs or primers for a Y chromosome-specific sequence. Furthermore 13 long-term patients more than two years after LRL Tx were studied. In the follow-up studies donor cells could be demonstrated in the blood of all patients immediately after transplantation. After a gradual decline all patients became chimerism-negative for several weeks or months. At 6 months, however, in 5 of 8 patients tested donor cells had reappeared probably leading to long-term chimerism. This biphasic pattern in the development of chimerism is proposed to reflect the occurrence of different donor-derived cell populations in the recipient. The population giving rise to the first wave of chimerism probably represents matured cells with a limited lifespan which are released from the graft into the circulation of the recipient during the first weeks after transplantation. The population of cells occurring with the second wave of chimerism is likely to have been generated by donor-derived cells with stem cell potential located either in the graft or in the hematopoietic organs of the recipient after emigration from the graft. This model may be able to explain fluctuations in the incidence and degree of micro chimerism described in other patient populations during the first year post transplant. Of the 13 long-term patients chimerism could be demonstrated in 11. In 6 patients it was detected in both blood and skin, in 4 patients the results obtained for blood and skin were discordant. There was no clear correlation of the presence or absence of chimerism with the clinical course.

124 . Joint annual meeting 1995 OGAI/GFI 1Institute of Experimental Surgery and 2Department of Biochemistry, Albert SzentGyorgyi Medical University, and 3City Hospital, Szeged, Hungary; 4Department of Immunology, Erasmus University, Rotterdam, The Netherlands

G.6 Hyperimmunoglobulinemia, autoimmunity and Iymphoproliferative disorders in mice following neonatal induction of transplantation tolerance T.JANOSSY!' c. VIZLER!, M. BOSKO!, S. MOOOK!, I. OCSOVSZKI2, Z. HmVEGI 3, H. F. J. SAVELKOUL \ A. VREEDENDAAL \ R. BENNER\ and P. VEGH 1 In A mice, neonatal i.v. injection of 2 x 10 7 (B10 X A)Fl spleen cells (SC) induces partial transplantation tolerance (TT) to B 10 skin allografts and 60 % (mostly lethal) lymphoproliferative disorders (LPD) of host origin. At 1 month of age, autoimmune antibodies (autoAb) and hypo reactivity to T cell mitogens have been shown. In this study, a marked hyperimmunoglobulinemia was detected. Peak levels of IgM, IgE, IgG1, and IgG2a were 8 x, 140 x, 6 x, and 4 x higher, than in nontolerized A mice. IgM and IgG autoAb appeared in the sera of mice between 1 and 2 weeks and slowly waned after 3 months. The sera reacted with thymocytes, splenic T cells, non-T-non-B lymphoid cells, and platelets. No strict correlation was found among these reactivities. Thrombocytopenia (TP) and splenomegaly developed at 2 weeks of age. In A mice tolerized with (CBA x A)Fl SC that display > 40 % permanent TT and 2 % LPD, only IgE was transiently elevated; autoAb and TP were not found. These results indicate that, in A mice neonatally tolerized to B10, polyclonal B cell activation develops preceding the LPD. The autoAb might playa role in TP and T cell deficiency. Autoimmunity, LPD and TT seem to be associated with different immunogenetic factors, thus, TT is not necessarily complicated by these disorders.

Medizinische U niversitatsklinik, Abteilung fur T ransplantationsimmunologie und Immunhamatologie, Tubingen, Germany

G.7 Analysis of CMV-specific T cell reconstitution following allogeneic BMT: patients with sibling or unrelated donor transplants H. KRAUSE, H. EINSELE, I. WAGNER, and C. A. MOLLER 30 patients undergoing allogeneic bone marrow transplantation and their marrow donors were screened for CMV -specific T cell proliferation and for CMV infection by virus culture assays. 21 of these patients received a marrow transplant from an HLA-matched sibling donor, 9 patients a transplant from an HLA-matched unrelated donor. All these patients were either CMV -seropositive and/or received a transplant from a CMVseropositive donor. Culture screening was performed weekly until day +100, during the later post-transplant period CMV screening was performed only if CMV infection was clinically suspected. CMV -specific T cell proliferation was determined at regular monthly intervals from day +30 after BMT until two positive results were obtained. On 120d post transplantation 50 % of the patients of a sibling HLA-identical donor showed a proliferative capacity against HCMV compared to 30 % of the patients with an

Joint annual meeting 1995 0GAIIGFI . 125 unrelated donor. All patients showing CMV -specific proliferation did not develop CMV disease afterwards. Following transplantation from an unrelated donor the CMV -specific T cell reconstitution was markedly delayed, occuring not before day +83 in contrast to day +37 in a patient after transplantation of a sibling donor. In two patients from an unrelated donor T cell proliferation was observed only 2 years after BMT. Four of these 9 patients had CMV disease documented more than 100 days after BMT when still lacking CMV-specific proliferation. Thus, screening for CMV-specific T cell proliferation might help to identify patients at risk to develop CMV disease in the later post-transplant period. These patients might benefit from sensitive screening even after day +100 after allogeneic BMT.

I Medizinische U niversitatsklinik, Abteilung fur T ransplantationsimmunologie und Immunhamatologie, Tubingen; 2Institut fur Klinische und Molekulare Virologie, Erlangen; 3GBF, Braunschweig; 4Biotest AG, Dreieich; sMedizinisch-Naturwissenschaftliches Forschungszentrum, Tubingen, Germany

G.B Epitope-mapping of HCMV pp65-specific T cell clones before and after allogeneic BMT H. KRAUSEl, R. HOLOCHERI, H. HEBARTI, M. MACH2, w. LINDENMAIER 3, R. VORNHAGEN 4 , H. KALBACHER S, H. EINSELFl, and C. A. MOLLER! In order to compare the cellular immune response against HCMV before and after BMT we established CMV-specific T cell clones from a patient 123 days after BMT as well as from his seropositive donor. T cell clones were generated after initial restimulation of PBMC from the donor or from the patient by CMV whole antigen for 4 days and subsequently adding IL-2 for 6 days. The limiting dilution was performed by addition of autologous irradiated PBMCs from the donor or from the patient and an optimal dilution of whole CMV-antigen. After 10 days the T cell clones were restimulated with PHA and pooled irradiated PBMC as APC adding a suboptimal concentration of CMV-antigen. Afterwards the T cell clones were restimulated every week in this way until 40 to 50 days after initial stimulation. The specificities of the predominantly CD4+ T cell clones were tested in proliferation assays with HCMV antigen and three recombinant proteins of the virus: pp65, gB and gH. We obtained 15 HCMV-specific T cell clones of the donor and 22 specific clones of the patient. Interestingly in both cases about 50 'Yo of the clones showed high proliferative capacity for pp65 (donor: 8/15, patient 11122) suggesting a dominant proliferative cellular reactivity against this late structural protein in the CMVspecific immune response. None of the T cell clones revealed proliferation to stimulation with the recombinant envelope proteins gB or gH. Using 6 different recombinant fusion proteins of pp65 the stimulatory T cell epitope of all investigated pp65-specific T cell clones was mapped to a 52 AA region near the carboxy terminus of the 563 AA protein. Using irradiated allogeneic PBMC of known HLA-class II phenotypes as APC the T cell clones were found to be preferentially restricted by one of the HLA-DR alleles of the donor (HLA-DRBl':·0301), only one clone was found to proliferate in response to pp65 presented by APC expressing HLA-DRBFOI01. Computerized epitope mapping predicted several T helper cell epitopes represented in the 52 AA peptide-sequence of pp65 sequence with a single optimal peptide-motif for HLA-DRBF0301. Currently all T cell clones are evaluated for their precise epitope specificity with different relevant synthetic peptides of pp65.

126 . Joint annual meeting 1995 OGAI/GFI Department of Clinical Immunology, Medizinische Hochschule Hannover, Hannover, Germany

G.9 NK allorecognition of hematopoietic progenitor cells D. MEYER, S. BRAUN, and R. E. SCHMIDT NK cells play an important role in graft-versus-leukemia-reaction as well as in engraftment and failure of bone marrow transplants. The MHC-I antigens are believed to be involved in allorecognition of NK cells. In the present studies we examined the role of MHC-I in allorecognition of hematopoietic progenitor cells by NK cells. An in vitro model using 51Cr-cytotoxicity assays was established with bone marrow cells (BMC) as targets for IL2-activated murine splenocytes. BMC were derived from the ~rmicro­ globulin-deficient mouse strain, CID t m lacking MHC-I expression, and from the C57B1/6 (H-2K b)- and the Balb/c (H-2K d)-inbred mouse strains. BMC were coincubated with monoclonal antibodies (mAb) against H-2K antigen (AF6-88.5 3/anti-H2Kb and SFl-1.1.1/anti-H-2Kd) or their Fab-fragments. After 51Cr-incorporation BMC were added to IL2-activated splenocytes from the C57B1I6 strain at various effectorl target ratios. NK cells do not kill syngeneic BMC. Allogeneic BMC were recognized by IL2activated NK cells weakly. BMC from the CID t m strain exhibit a significant increase in cytotoxic susceptibility compared to the wild type control. The same results were obtained after coincubating resistant syngeneic BMC with mAb against H-2Kb. Also masking of the allogeneic BMC with anti-H-2Kd mAb results in an increase of NK cytotoxicity. ADCC could be excluded by using Fab-fragments of the respective mAb. Thus the MHC-I complex is an important structure in NK allorecognition of BMC, and mediates resistance of NK-specific lysis. Masking of the H-2K antigen results in lysis of syngeneic BMC suggesting that this MHC-I antigen is essential to protect against NK killing. The results obtained for allogeneic BMC indicate that this phenomenon is not restricted to the specific haplotype of the effector cell. Our in vitro-model enables the investigation of direct NK-BMC-interactions (e.g. in hybrid resistance or the involvement of specific NK antigens for BMC-recognition).

Clinical Department Blood Group Serology, University of Vienna, Vienna, Austria

G.10 Xenorecognition of porcine antigen by human T helper cell precursors S. PANZER and K. LINDENBAUER The shortage of human donors for organ transplantation has induced a resurgence of interest in xenotransplantation. T cell responses to xenoantigen from discordent species differ from allorecognition. We estimated human anti-pig T cell response to evaluate 1) helper T cell precursor frequency (HTLp), 2) differentiate direct versus indirect xenoantigen recognition. Human PBMNC and purified human T cells were cultured in limiting dilution assays (1-105 cells) with porcine PBMNC (lOs cells/well) for 3 days; responsiveness was detected by IL-2 production. Allorecognition of unrelated human antigens served as a positive control.

Joint annual meeting 1995 OGAIIGFI .

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In all donors HTLp frequencies in PBMNC and with purified human T cells against pig were higher than against alloantigens (1/9827-1/47169 versus 1/33689-1/77499) indicating that T cell-mediated acute graft rejection is expected in pig-to-human organ transplantation. The response of purified T cells followed an assumption of single hit kinetics, but not if bulk PBMNC were used. Thus, xenorecognition occurs both directly and after procession of xenoantigen by human APC.

Institute of Immunology, University of Kiel, Kiel, Germany

G.ll Lack of in vitro synergy between clinically applied monoclonal antibodies against LFA1, ICAMl and IL-2R~ K. ROMER, C. HERW ARTZ, and J. STEINMANN Tolerance to heart transplants can be achieved in mice by the combination of LFAl and ICAM1 antibodies but not by one of these antibodies alone. In rats tolerance to liver transplants can be induced by a combination of low doses Ciclosporin A and the combination of antibodies against ICAM1 and the IL-2R. Antibodies against all three antigens are available in man and currently under clinical investigation in organ transplantation (LFAl: ANTILFA, ICAM1: BIRRl and IL-2R: BT563). These antibodies were tested alone and in combination in vitro using allogeneic endothelia cells (from V. umbilicalis and V. saphena), the lymphoblastoid cells line MSAB or peripheral blood mononuclear cells as stimulators. DNA synthesis and IL-2 production were measured for T cell response. ANTILF A was slightly stronger immunosuppressive than BIRR. The combination was not better than ANTILF A alone. BT563 was comparably immunosuppressive as ANTILF A. BT563 had an additive effect in combination with the adhesion molecule antibodies but no synergism was seen.

Departments of 1 Ophthalmology and 2Biostatistics, University of Innsbruck, Innsbruck, Austria

G.12 Penetrating keratoplasty for herpes simplex keratitis. Prognostic factors and beneficial effects of immunosuppression with cyclosporine J. STOIBER', W. GOTTINGERt, E. POTTINGER!, H. ULMER2, K. PPEIHER2, and E. U. IRSCHICK 1 Herpes simplex virus (HSV) often causes severe lesions of the cornea with following neovascularization and the need for transplantation. In a retrospective study 85 high-risk keratoplasties after HSV -ulcers were followed up between 1979 and 1994. Graft survival was calculated by the Kaplan-Meier method. The overall survival rate of clear grafts was 82.2 % after one and 79.8 % after two years. Vascularization and previous graft failures were important prognostic factors for graft survival. 52 cases were considered as «highrisk-keratoplasties» (> 2 quadrants of the cornea vascularized or regrafts), 25 patients received immunosuppression with systemic and topic corticosteroids, 22 underwent

128 . Joint annual meeting 1995 OGAI/GFI systemic cyclosporine (CyA) for three months in addition to topical corticosteroids. Patients receiving Cy A had a significant better survival (p = 0,02) than the group with systemic corticosteroids, Transplantations performed it chaud (corneas either perforated or with a descemetocele) resulted in a lower survival rate: 75 % after one year, 56.3 % after two years. Neither HLA-matching nor ABO incompatibility or ABO phenotype did influence graft survival. This high-risk transplantation group showed differences to other previously investigated patients with corneal traumata or alcalic burns. There were no significant differences in graft survival after HSV-keratitis associated with graft diameter, age and sex of host or donor.

INSERM U430, Paris, France

G.13 A novel synthetic dextran derivative inhibits complement activation in an in vitro model of xenogeneic porcine aortic endothelial cells H. THOMAS, F. MAILLET, M. D. KAZATCHKINE, and E. FISCHER Complement activation is initiated through the binding of xenoreactive natural antibodies to the vascular endothelium plays a critical role in the pathophysiology of hyperacute xenograft rejection. We demonstrate that a novel synthetic soluble dextran derivative bearing carboxylic and benzylamide sulfonate groups (CMDBS) inhibits complement activation and complement-mediated damage in an in vitro model where porcine aortic endothelial cells (P AEC) were incubated with normal human serum (NHS). Incubation for 4 h at 37"C of PAEC with NHS resulted in 50 % decrease of CH50 correlating with generation of 30 fig/ml of C3a. The addition of 20 to 100 mg/ml of CMDBS inhibited complement activation and C3a generation from 56 % to 90 %. Complement activation was associated with deposition on P AEC of complement cleavage fragments C3bi, C5, C5b-c; and 51Cr release by PAEC. Addition of 100 mg/ml of CMDBS decreased fixation of complement fragments and complement-dependent cytotoxicity by 90 % and 50 % respectively. Unsubstituted dextran derivatives with anticomplementary properties may represent a novel class of potential therapeutic agents for the prevention of complement-dependent hyperacute xenograft rejection.

Institute of Immunology, University of Heidelberg, Heidelberg, Germany

G.14 Determination of a threshold level of human CD59 on porcine endothelial cells for protection against complement-mediated cytotoxicity in a pig-to-human in vitro model A. WOSNIK, M. KIRSCHfINK, and B. HECKL-OSTREICHER Xenotransplantation is considered as a possible strategy to overcome the worldwide shortage of donor organs for transplantation. So far, hyperacute xenograft rejection mediated by activation of the recipient's complement (C') system represents the major

Joint annual meeting 1995 OGAIIGFI . 129 barrier to xenotransplantation. The transfer of human membrane-bound C regulatory proteins offers new chances to protect the xenograft from cytolytic complement attack. Recently, we have demonstrated a protective function of the human membrane-associated C inhibitor CD59 in a pig-to-human in vitro model for hyperacute graft rejection consisting of porcine aortic endothelial cells (PEC) incubated with human serum as the source of natural antibodies and C. We now wanted to correlate the level of human CD59 expressed on porcine aortic endothelial cells with the degree of protection in order to determine a threshold that is necessary for the protective function. For this purpose we have cloned the CD59-expressing porcine endothelial cell line by limiting dilution and have isolated clones expressing varying numbers of CD59. The observed sensitivity of the PEC-clones to human serum was proportional to the amount of human inhibitor expressed. Only clones expressing similar numbers of CD 59 molecules as human umbilical vein endothelial cells (HUVEC) were completely protected from the cytolytic activity of human serum. Our results demonstrate that protection of xenogeneic cells requires a number of regulatory proteins which are in the range of that of the corresponding human cell type.