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(The FASEB Journal. 1999;13:1762-1773.)
© 1999 FASEB

Expression of the human {alpha}1,2-fucosyltransferase in transgenic pigs modifies the cell surface carbohydrate phenotype and confers resistance to human serum-mediated cytolysis

CRISTINA COSTA*, LISA ZHAO*, WILLIS V. BURTON*, KENNETH R. BONDIOLI*, BARRY L. WILLIAMS{dagger}, THOMAS A. HOAGLAND{ddagger}, PAUL A. DITULLIO§, KARL M. EBERT§ and WILLIAM L. FODOR*1

* Department of Molecular Sciences, Alexion Pharmaceuticals Inc, New Haven, Connecticut 06511, USA;
{dagger} U.S. Surgical Corporation, North Haven, Connecticut 06473, USA;
{ddagger} Department of Animal Sciences, University of Connecticut, Storrs, Connecticut 06269, USA; and
§ TranXenoGen, Shrewsbury, Massachusetts 01545, USA

1Correspondence: Department of Molecular Sciences, Alexion Pharmaceuticals Inc., 25 Science Park, New Haven, CT 06511, USA. E-mail: fodorw{at}alxn.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hyperacute rejection (HAR) is the first critical immunological hurdle that must be addressed in order to develop xenogeneic organs for human transplantation. In the area of cell-based xenotransplant therapies, natural antibodies (XNA) and complement have also been considered barriers to successful engraftment. Transgenic expression of human complement inhibitors in donor cells and organs has significantly prolonged the survival of xenografts. However, expression of complement inhibitors without eliminating xenogeneic natural antibody (XNA) reactivity may provide insufficient protection for clinical application. An approach designed to prevent XNA reactivity during HAR is the expression of human {alpha}1,2-fucosyltransferase (H-transferase, HT). H-transferase expression modifies the cell surface carbohydrate phenotype of the xenogeneic cell, resulting in the expression of the universal donor O antigen and a concomitant reduction in the expression of the antigenic Gal{alpha}1,3-Gal epitope. We have engineered various transgenic pig lines that express HT in different cells and tissues, including the vascular endothelium. We demonstrate that in two different HT transgenic lines containing two different HT promoter constructs, expression can be differentially regulated in a constitutive and cytokine-inducible manner. The transgenic expression of HT results in a significant reduction in the expression of the Gal{alpha}1,3-Gal epitope, reduced XNA reactivity, and an increased resistance to human serum-mediated cytolysis. Transgenic pigs that express H-transferase promise to become key components for the development of xenogeneic cells and organs for human transplantation.—Costa, C., Zhao, L., Burton, W. V., Bondioli, K. R., Williams, B. L., Hoagland, T. A., DiTullio, P. A., Ebert, K. M., Fodor, W. L. Expression of the human {alpha}1,2-fucosyltransferase in transgenic pigs modifies the cell surface carbohydrate phenotype and confers resistance to human serum-mediated cytolysis.


Key Words: {alpha}1 • 2 • H-transferase • hyperacute rejection • delayed xenograft rejection • xenotransplantation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE FIRST IMMUNOLOGICAL barrier to be overcome in the development of discordant xenogeneic organs for human transplantation is hyperacute rejection (HAR).2 HAR is a humoral immune response characterized by deposition of xenoreactive natural antibodies (XNA) and complement in the vasculature of the donor tissues (1 2 3 4 5 6) . This massive humoral inflammatory response leads to de-endothelialization, blood vessel occlusion, and graft failure within minutes to hours. If HAR is inhibited or delayed by blocking complement, the xenogeneic organ still faces a vigorous antibody response that can mediate type II endothelial activation and acute vascular rejection, which leads ultimately to rejection of the graft (6) . Therefore, reducing both human serum antibody reactivity and complement activation is a prerequisite to inhibit xenograft rejection.

Transplantation of xenogeneic cells and tissues is also in development to treat human diseases that involve cellular loss or dysfunction (6 , 7) . Although the mechanisms of rejection of transplanted xenogeneic cells and tissues are much less well characterized, an intense antibody response has been reported after cell transplantation (20- to 100-fold in human patients receiving fetal porcine islet clusters) (8) . Even if some cells, such as those from pancreatic islets, are not rejected hyperacutely, this humoral response may accelerate the delayed cellular rejection, which prevents the survival and appropriate function of the grafted cells. The great potential of this field encourages us to further explore the immunological hurdles to xenogeneic cell transplantation.

The best approach to prevent humoral-mediated HAR and delayed organ, tissue, or cellular xenograft rejection is to engineer the donor tissue, thereby minimizing the need for immunosuppression and conditioning therapies in the human patient. The species receiving the most consideration for the development of universal donor cells and organs is the pig. The pig has several advantageous qualities, including anatomical and physiological similarities to human cells and organs (5) . Moreover, pigs can be transgenically modified to express molecules that modulate the host immune system (9 10 11 12) . To date, inhibiting complement activation, either systemically or at the xenogeneic cell surface via expression of human complement inhibitors, has been the most successful approach to inhibit xenograft rejection in the pig-to-primate model (13) . Initially, in vitro studies showed that expressing hCD59 or decay-accelerating factor significantly protected pig cells from human serum-mediated cytolysis (14 15 16) . Ex vivo human blood perfusion experiments also demonstrated that transgenic kidneys and hearts from hCD59-expressing pigs functioned longer than control organs (10) . Subsequently, prolonged survival of transgenic pig organs expressing human complement inhibitors was observed in pig-to-primate heterotopic transplant models (11 , 12 , 17 , 18) . However, none of the current engineering approaches sufficiently protect pig tissue from the massive XNA reactivity.

Old World primates, including humans, have high titers of preexisting circulating antibodies that react predominantly with carbohydrate epitopes on the surface of discordant xenogeneic cells. The major xenoepitope recognized by these XNAs is the carbohydrate epitope Gal{alpha}1,3-Gal, which is synthesized by {alpha}1,3-galactosyltransferase ({alpha}1,3-GT) (19 , 20) . This enzyme is nonfunctional in humans and other Old World primates as a result of frameshift and nonsense mutations in the structural gene (21) . Consequently, humans produce high titer anti-Gal{alpha}1,3-Gal antibodies in response to natural flora antigen stimulation (19) . Therefore, eliminating XNA reactivity is essential to abrogate the antibody-mediated activation of complement and the antibody-mediated cellular response that leads to delayed xenograft rejection (DXR).

The generation of pigs deficient in {alpha}1,3-GT by homologous recombination is a goal of different laboratories. However, the production of pigs with targeted genetic modifications of the genome remains to be accomplished. Therefore, an alternative enzymatic strategy was developed to reduce Gal{alpha}1,3Gal epitope expression. We hypothesized that expression of the human {alpha}1,2-fucosyltransferase (H-transferase, HT) might compete for the common acceptor substrate, N-acetyl lactosamine, thereby interfering with the expression of the Gal{alpha}1,3-Gal epitope (22) . Furthermore, HT synthesizes a glycosidic moiety that is universally tolerated, the O blood group antigen (22) . This hypothesis was tested by transfecting LLC-PK1 cells, a porcine kidney fibroblast cell line, with a human HT cDNA expression construct. The HT-transfected pig cells exhibited a high level of H epitope expression that resulted in a concomitant decrease in the level of Gal{alpha}1,3-Gal epitope expression. Moreover, the reduction in cell surface Gal{alpha}1,3-Gal results in decreased human immunoglobulin G (IgG) and IgM antibody reactivity and serum-mediated cytolysis (22) . These experiments demonstrated that HT efficiently competes with {alpha}1,3-GT and reduces the expression of Gal{alpha}1,3-Gal epitope in HT engineered cells (22) . This strategy was recently applied to transgenic mice (22 23 24 25) . In vitro studies of isolated transgenic cells demonstrated reduced Gal{alpha}1,3-Gal expression and XNA reactivity and increased resistance to human serum-mediated cytolysis (22 23 24 25) . Moreover, transgenic mouse hearts expressing human HT exhibited enhanced survival when challenged with human serum (26) . Expression of HT was shown to be as effective as the {alpha}1,3-GT knockout in protecting mouse cells and organs from human serum damage (27 , 28) . The generation of HT transgenic pigs has been described (24 , 29) , but no functional results after cell activation or data from F1 generation pigs have been reported.

We have produced several lines of transgenic pigs expressing HT. We show for the first time expression of HT in different cells, tissues, and organs such as the heart, kidney, liver, lung, pancreas, sciatic nerve, and skin. Expression of H epitope was demonstrated on vascular endothelium by histologic analysis of tissue sections and flow cytometry analysis of isolated porcine aortic endothelial cells (PAEC). HT expression in PAEC was accompanied by reduced Gal{alpha}1,3-Gal epitope expression, resulting in resistance when challenged with high concentrations of human serum. Isolated fibroblasts from different transgenic lines of pigs also showed decreased Gal{alpha}1,3-Gal expression and human serum-mediated cytolysis that inversely correlated with the levels of expression of H-epitope. Moreover, the expression of the transgene was both constitutive and regulated in a manner designed to confer protection in proinflammatory conditions. Thus, the expression of HT singly or in combination with complement inhibitors may be a critical component of engineered xenogeneic cells and organs that are resistant to HAR and DXR.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Gene constructs
The full-length cDNA of human HT was inserted 3' of CMV promoter into BamHI/XhoI sites of APEX-1, a pGEM7Z-derived expression vector (22) . The polyadenylation signal of SV40 was contained in APEX-I and located 3' of HT. A 2.45 Kb fragment with the complete sequence of the chimeric gene was digested with SspI and ClaI, electrophoresed and purified by electroelution and Elutip (Schleicher & Schull, Keene, N.H.) to be used for microinjection. H2Kb-HT construct was generated and isolated as described previously (25) . Both genes were coinjected in equimolar concentrations in order to produce transgenic pigs.

Generation and analysis of transgenic pigs
Transgenic pigs were generated using advanced embryo microinjection. Briefly, two to four cell embryos recovered from mature, hormonally synchronized, ovulation-induced donors were subjected to nuclear microinjection and transferred to the oviducts of norgestomet synchronized recipient gilts. Animals born were tested for the presence of the transgene by polymerase chain reaction (PCR) and Southern blotting (30) analysis of genomic DNA, following standard procedures. Southern blotting of genomic DNA digested with NsiI and hybridized with an HT-specific probe revealed two fragments of 4 and 0.8 Kb, which demonstrated the presence of the H2Kb-HT and CMV-HT transgenes, respectively. Flow cytometry analysis of peripheral blood mononuclear cells (PBMCs) was also performed. Artificial insemination was used in order to obtain F1 and F2 transgenic offspring. Fo (founder) to F2 heterozygous transgenic pigs, as well as control littermates, were used for subsequent experiments.

RNA isolation and RT-PCR analysis
Total RNA was prepared using RNA Isolator (Genosys Biotechnologies, Inc., The Woodlands, Tex.) as described by the manufacturer. Expression of the transgenes was evaluated by reverse transcriptase (RT) -PCR from total mRNA samples of kidney, liver, lung, heart, aorta, lymph node, spleen, pancreas, olfactory bulb, sciatic nerve, skeletal muscle, and skin. First strand cDNA was prepared by reverse transcription using avian myeloblastosis virus reverse transcriptase (Seikagaku America, Inc, Rockville, Md.) and used for polymerase chain reaction using Perkin Elmer reagents (Perkin-Elmer, Norwalk, Conn.). The primers used to detect expression of HT driven by H2Kb promoter were 1) 5'-GGACAGGAGGCTACACCGTGG; 2) 5'-GGTGACGAAATACCTCAGCG for AT20 tissues; 3) 5'-ATGTCGGAGGAGTACGCGG; and 4) 5'-GGCGGTGACGAAATACCTCAGCGGTGTGG for AT21 F1 tissues. Primers 1) and 3) are internal oligonucleotides in HT and primers 2) and 4) hybridize to exon 2 of the H2Kb gene 3' to the HT cDNA. The sizes of the expected PCR products from these sets of primers are 0.4 and 0.8 Kb, respectively. To determine expression driven by CMV promoter, we used the set of primers 3) and 5) 5'-TAAGCTGCAATAAACAAGTTC, which is located 5' to the polyadenylation site. PCR reactions using these primers should produce a 717 bp fragment. The ß-actin primers 6) 5'-CCAACTGGGACGACATGGAG and 7) 5'-AGGTCCAGACGCAGGATGGC were used as a positive control for each tissue RNA, and produce a 300 bp PCR product expected from a correctly processed ß-actin mRNA.

Flow cytometry analysis and immunofluorescence
PBMCs from transgenic and negative littermate control pigs were purified from whole blood by Ficoll gradient centrifugation (Pharmacia Biotech, Uppsala, Sweden) and one-step ACK lysis (Biofluids, Rockville, Md.). PAEC from a control and an AT21 F1 transgenic pig were isolated by scrapping the aorta with a scalpel and culturing in Dulbecco's modified Eagle's medium (DMEM) 10% fetal calf serum (FCS). Primary cultures of fibroblasts from the different animals analyzed were obtained by mincing ear tissue and culturing in DMEM 10% FCS. Direct fluorescence of cell surface carbohydrate epitopes was performed with fluorescein isothiocyanate- (FITC) conjugated lectins: GS-IB4 lectin isolated from Griffonia simplicifolia (EY Laboratories, Inc. San Mateo, Calif.) detects Gal{alpha}-1,3-Gal (31) and UEAI lectin isolated from Ulex europaeus (EY Laboratories) detects H-substance (32) . SLA class I was detected with a murine monoclonal antibody PT85A (VMRD, Inc., Pullman, Wash.). Goat anti-mouse IgG, IgA, and IgM (Zymed Laboratories, Inc. San Francisco, Calif.) FITC-conjugated antisera were used to detect specific antibody binding to the cell surface. Purified human anti-Gal {alpha}-1,3-Gal antibodies (10 µg/ml) were used to detect this xenoantibody reactivity on pig cells. Briefly, anti-Gal {alpha}-1,3-Gal antibodies were purified from human serum by column chromatography using 142a and 142b resins (Glycorex, Lund, Sweden). Total XNA reactivity was determined by incubating pig cells with 5% heat-inactivated human serum. Goat anti-human IgG and IgM were used as secondary antibodies (Zymed Laboratories). Cell surface expression was then measured by flow cytometry on a Becton Dickinson FACSort.

Immunofluorescence was performed on fresh-frozen tissue samples embedded in Tissue-Tek OCT compound (Sakura Finetek USA, Torrance, Calif.). Samples from two different transgenic animals from AT21 line and two controls were examined. Sections (5–8 µM) were stained with UEAI-FITC (1:100 dilution) or IB4-FITC (1:100 dilution) (EY Laboratories Inc.).

Human serum-mediated cell lysis assay
PAEC and fibroblasts were trypsinized, washed twice with Hanks' balanced sale solution 1% bovine serum albumin and exposed to increasing concentrations of human serum for 2 h at 37°C in flat-bottom plates. Cells were subsequently washed and loaded with Calcein AM (Molecular Probes, Inc., Eugene, Oreg.) (10 µg/ml) for 20 min and lysed with 1% sodium dodecyl sulfate. Cell survival was calculated as the relative amount in duplicate samples of fluorescence detected with a cytofluor (Perspective Biosystems, Framingham, Mass.).

Statistical analysis
All values are expressed as the means ± SE. Statistical analysis was carried out using the Student-Newmann-Keuls test. Differences were considered statistically significant at P<=0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation and characterization of HT transgenic pigs
Transgenic pigs expressing H-transferase were obtained by coinjection of two chimeric genes, H2Kb-HT and CMV-HT, by advanced embryo injection. Three transgenic pigs were positive by PCR and Southern blot of genomic DNA, AT20, AT21, and AT22 (data not shown). The presence of both transgenes was detected in AT20 and AT21, whereas AT22 had incorporated only the H2Kb-HT gene. Subsequent studies were performed with AT20 (female), AT21 (male) and their offspring. We estimated that AT20 and AT20 descendants had incorporated ~10 copies of the H2Kb-HT gene and ~10 copies of the CMV-HT transgene (data not shown). AT21 showed by Southern blot analysis a signal below the intensity expected for one copy per haploid genome. However, the transgenic offspring derived from AT21 consistently contained one integrated copy of each transgene (data not shown).

The transgenic pigs were also screened for H-epitope expression by flow cytometry on isolated PBMCs. Control pig cells showed only background staining for H-epitope (Fig. 1 ). Cells from AT20 founder animal expressed high levels of this epitope, whereas AT21 cells showed a mosaic pattern of expression (Fig. 1) . F1 pigs derived from the founder AT20 had levels of H-epitope on PBMCs comparable to those of their progenitor, whereas expression on the AT21 F1 and F2 corresponding cells was higher than on the parental AT21 cells (Fig. 1) . The results from the Southern blot and flow cytometry analyses were consistent with the transmission frequencies of the transgenes derived from the two founders. AT20 transmitted the transgenes in a mendelian fashion to its descendants. By contrast, AT21 was highly mosaic and transmitted the transgenes to only 4.6% of the offspring. We have not observed segregation of copies or transgenes in any of the offspring from AT20 or AT21. Moreover, F1 males derived from AT21 transmitted with 50% efficiency.



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  		Figure 1. Flow cytometry analysis of PBMCs isolated from control, founder (Fo), F1, and F2 transgenic pigs as indicated. Staining with UEA-I lectin (thick line) or GS-IB4 lectin (dotted line) was performed to detect cell surface H-substance and Gal{alpha}-1,3-Gal epitope, respectively. The thin line corresponds to unstained cells.

Expression of Gal{alpha}1,3-Gal epitope and human antibody reactivity was initially evaluated by flow cytometry on PBMCs from control and transgenic pigs (Fig. 1) . Staining with the GS-IB4 lectin revealed a fourfold reduction in mean fluorescence intensity of AT20 HT-expressing cells relative to controls (Fig. 1) . Little effect was observed in the AT21 mosaic cells, but a more pronounced decrease in Gal{alpha}1,3-Gal epitope was observed in cells from the AT21 F1 transgenic offspring (Fig. 1) . Anti-Gal{alpha}1,3-Gal IgM and IgG antibody reactivity was similarly decreased in the transgenic cells when compared with controls (data not shown).

Expression tissue analysis of HT transgenic pigs
RT-PCR analysis was used to determine HT RNA expression in various tissues derived from control (AT20) and an AT21 F1 transgenic pig (Fig. 2 ). Total mRNA was isolated from kidney, liver, lung, heart, aorta, lymph node, spleen, pancreas, olfactory bulb, sciatic nerve, skeletal muscle, and skin. To determine the expression pattern derived from the two different promoter constructs, H2Kb-HT and CMV-HT, we used transgene specific primer sets. Expression driven by the H2Kb promoter was detected by RT-PCR in RNA samples isolated from kidney, liver, lung, lymph node, spleen, skeletal muscle, and skin of AT20 (Fig. 2A ) and heart, lung, lymph node, and spleen of AT21 F1 (Fig. 2B ). A 717 bp DNA fragment was detected in all AT20 tissues analyzed and most of AT21 F1, which corresponds to the mRNA derived from the CMV-HT transgene (Fig. 2A, B ). No specific PCR products were observed in any of the nontransgenic littermate samples. Endogenously expressed ß-actin was used as a control to confirm the integrity of all samples tested by PCR (Fig. 2A, B ).



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  		Figure 2. RT-PCR analysis of HT expression in different tissues from control and transgenic pigs. A) AT20 tissues (1, 1 Kb ladder marker; 2, kidney; 3, liver; 4 lung; 5, heart; 6, aorta; 7, lymph node; 8, spleen; 9, skeletal muscle; 10, skin; 11–12, control (+); 13, control (-). B) AT21 F1 tissues (1, 1 Kb ladder marker; 2, aorta; 3. heart; 4 kidney; 5, liver; 6, lung; 7, lymph node; 8, olfactory bulb; 9, pancreas; 10, sciatic nerve; 11, skin; 12, spleen; 13, control CMV (+); 14, control (-). A, B) i. HT expression driven by H2Kb promoter. ii. HT expression driven by CMV promoter. iii. All samples were tested for ß-actin mRNA.

To further explore the pattern of expression of the HT transgene, we performed immunofluorescence analysis of ear sections isolated from the AT20 founder and heart sections from two AT21 F1 transgenic pigs by staining with the anti-H lectin, UEAI. H-antigen was strongly and broadly expressed in an AT20 ear section (Fig. 3 B), and in arteries, arterioles, veins, capillaries, and myocardium of the AT21 F1 transgenic pig heart (Fig. 3F ). Only background staining was observed in control samples (Fig. 3A, E ). To determine the level of Gal{alpha}1,3-Gal expression, tissue sections were incubated with the GS-IB4 lectin. High expression of this epitope was observed in the ear sections of the control pig, whereas a marked and generalized reduction in Gal{alpha}1,3-Gal antigen was detected in AT20 ear tissue (Fig. 3C, D , respectively). Control heart tissue also expressed high levels of the Gal{alpha}1,3-Gal epitope on the endothelium and tunica adventicia of arteries, arterioles, veins, and capillaries (Fig. 3G ). A less marked, but clearly perceptible, reduction in GS-IB4 staining was observed in the heart vessels of the AT21 F1 transgenic pig (Fig. 3H ).



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  		Figure 3. Immunofluorescence of tissue sections from control and transgenic pigs. Staining with UEA-I lectin (A, B) and GS-IB4 lectin (C, D) is shown on ear tissue sections from a control (A, C) and AT20 Fo transgenic pig (B, D). Staining with UEA-I lectin (E, F) and GS-IB4 lectin (G, H) is shown on tissue sections of heart from a control (E, G) and an AT21 F1 transgenic pig (F, H). Results are representative of two different transgenic animals from AT21 line and two controls examined. Actual magnification, x50 (A–H).

Expression of H-epitope on endothelial cells and functional analysis
Porcine aortic endothelial cells from an AT21 F1 transgenic pig were isolated to quantify the levels of Gal{alpha}1,3-Gal- and H-epitope expression. In accordance with the tissue immunofluorescence analysis, expression of H-epitope was clearly detected by UEA-I staining and flow cytometry analysis of the transgenic cells (Fig. 4 A). No UEA-I staining was shown on control PAEC (Fig. 4A ). Furthermore, expression of Gal{alpha}1,3-Gal epitope was significantly reduced in the transgenic PAEC (50% reduction) when compared with control cells (Fig. 4B ). To assess the functional significance of the modified cell surface phenotype, we evaluated the cell survival of control and HT transgenic PAEC challenged with 20 and 40% human serum (Fig. 4C ). The AT21 F1 transgenic endothelial cells showed a marked and elevated protection from human serum-mediated lysis relative to control cells (Fig. 4C ).



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  		Figure 4. Flow cytometry analysis of PAEC isolated from a control and an AT21 F1 transgenic pig (different to the animals used for histological analysis), as indicated. Staining with UEA-I lectin (A) or GS-IB4 lectin (B) was performed to detect on the cell surface H-substance and Gal{alpha}-1,3-Gal epitope, respectively. The bars represent the mean FL-1 fluorescence intensity of five independent experiments; error bars indicate the standard error of the mean. A Student t test was applied to compare transgenic vs. control PAEC, #P<0.05 *P<0.005. C) Human serum-mediated cell lysis assay of PAEC isolated from a control and an AT21 F1 transgenic pig. A cytolysis assay was performed as described in Materials and Methods. Results represent mean ± SE of three independent experiments. Transgenic cells were significantly protected relative to controls at 40% human serum, *P < 0.005.

Regulation of H-epitope expression and functional analysis
To carry out the functional analysis in another cell type, we isolated fibroblasts from control and transgenic pigs. The H-epitope was highly expressed in fibroblasts from AT20, as observed by flow cytometry, whereas fibroblasts from AT21 showed a mosaic profile of expression (Fig. 5 ). Similar to the expression pattern observed in PAEC, HT expression in AT21 F1-derived fibroblasts was high and homogeneous (Fig. 5) . Moreover, the reduction in Gal{alpha}1,3-Gal epitope expression in the transgenic fibroblasts correlated inversely with the level of H-antigen expression (Fig. 5) . We next analyzed expression of the HT transgenes, regulated by the two different promoter systems, when cells are exposed to proinflammatory cytokines. First, RT-PCR analysis demonstrated that expression of HT was driven by both of the promoters, H2Kb and CMV, in fibroblasts isolated from AT20 and AT21 F1 animals (data not shown). We incubated fibroblasts from the founder pigs AT20 and AT21 for 24 h with DMEM 10% FCS or with DMEM 10% FCS supplemented with phytohemagglutinin- (PHA) conditioned media (CM) derived from porcine PBMCs. The cells were subsequently stained with UEA-I and GS-IB4 lectins to assess H- and Gal{alpha}1,3-Gal epitope expression, respectively (Fig. 6 A, B). SLA class I up-regulation was monitored as a control of the biological activity of the CM (Fig. 6C ). Untreated fibroblasts from AT20 showed high expression of H-epitope with a marked reduction in Gal{alpha}1,3-Gal antigen (Fig. 5 , Fig. 6A, B ). Fibroblasts isolated from AT21 exhibited a mosaic expression of H-epitope that was lower than that of AT20 cells. Nevertheless, Gal{alpha}1,3-Gal epitope expression was reduced to 50% in these cells (Fig. 5 , Fig. 6A, B ). Treatment with CM led to an elevation in Gal{alpha}1,3-Gal epitope in control fibroblasts. A 100% increase in expression of H-epitope was detected in CM-treated cells from AT20, which was accompanied by very low expression of Gal{alpha}1,3-Gal antigen (Fig. 6A, B ). In accordance with lower levels of H-epitope achieved in fibroblasts from AT21, these exhibited an increase in Gal{alpha}1,3-Gal reactivity. However, the level of the Gal{alpha}1,3-Gal antigen remained below the level detected in the control cells (Fig. 6A, B ).



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  		Figure 5. Flow cytometry analysis of fibroblasts isolated from control and transgenic pigs as indicated. Staining with UEA-I lectin (thick line) or GS-IB4 lectin (dotted line) was performed to detect on the cell surface H-substance and Gal{alpha}-1,3-Gal epitope, respectively. The thin line corresponds to unstained cells.



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  		Figure 6. UEA-I (A), GS-IB4 (B), and PT85A (C) staining of fibroblasts from control, AT20, and AT21 transgenic pigs analyzed by flow cytometry. The bars represent the mean FL-1 fluorescence intensity of six independent experiments; error bars indicate the standard error of the mean. UEA-I reactivity was significantly elevated in AT20 and AT21 cells relative to controls in the two conditions assayed, *P<0.005. Inversely, GS-IB4 staining was significantly reduced in AT20 cells, *P<0.005. All samples treated with PHA-CM had a significant increase in PT85A reactivity compared to untreated cells. *P<0.005.

Xenoantibody reactivity was also evaluated by flow cytometry analysis in control and AT20 transgenic fibroblasts with purified anti-Gal{alpha}1,3-Gal antibodies (Fig. 7 A) and with 5% heat-inactivated human serum (Fig. 7B ). Anti-Gal{alpha}1,3-Gal reactivity was clearly detected in control fibroblasts of both IgM and IgG subtypes, whereas very little, if any, antibody deposition was detected in AT20 cells (Fig. 7A ). Treatment with CM led to more than a twofold increase in the mean fluorescence intensity corresponding to IgM reactivity in control cells. Anti-Gal{alpha}1,3-Gal IgM reactivity remained low in the transgenic cells with only a slight increase in IgG reactivity (Fig. 7A ). Similar results were observed when XNA deposition was assessed (Fig. 7B ). At 5% human serum, XNA reactivity was predominantly of IgM subtype in both untreated and CM-treated control cells. The antibody reactivity was markedly reduced in AT20 fibroblasts, which showed low XNA deposition when treated with CM (Fig. 7B ).



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  		Figure 7. Anti-human Gal{alpha}-1,3-Gal antibody (A) and 5% heat-inactivated serum (B) reactivity analyzed by flow cytometry in fibroblasts isolated from control and AT20 transgenic pigs untreated or treated with PHA-CM. The thick line corresponds to the Gal{alpha}-1,3-Gal antibody (A) and total XNA antibody (B) reactivity, the dotted line to the corresponding secondary antibody control. Results are representative of four independent experiments.

To assess whether the reduction in Gal{alpha}1,3-Gal antigen expression and the correspondingly reduced XNA reactivity led to an increased resistance when challenged with human serum, we performed serum-mediated cytolysis assays (Fig. 8 ). Cell survival was determined by the quantity of dye retained by cells incubated with increasing concentrations of human serum relative to cells with no serum added. Control fibroblasts incubated with 40 and 100% serum showed minimal cell survival (Fig. 8 A). No significant differences were observed between control cells that were either untreated or treated with CM at any concentration of serum assayed. AT21 fibroblasts exhibited a twofold increase in cell survival when compared to controls. However, consistent with the levels of expression of H and Gal{alpha}1,3-Gal epitopes, AT21 fibroblasts were less resistant than the AT20 cells (Fig. 8A ). Untreated AT20 cells were highly resistant to human serum-mediated cytolysis, even at 100% human serum (Fig. 8A ).



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  		Figure 8. Human serum-mediated cell lysis assay of fibroblasts isolated from control and transgenic pigs. A cytolysis assay was performed as described in Materials and Methods with fibroblasts from control and HT founder pigs. A) Cells from control, AT20, and AT21 founders were untreated or treated with 60% CM derived from porcine PBMCs as indicated. Results represent mean ± SE of four independent experiments. AT20 cells treated or untreated were significantly protected when compared with control cells from 10 to 100% human serum, P <= 0.005. Untreated AT21 cells were significantly protected from 20 to 100% serum and AT21-CM cells only at 100% serum; P < 0.05. B) Cells from control and AT20 founder were untreated or treated with 100% CM derived from human PBMCs as indicated. Results represent mean ± SE of three independent experiments. AT20 cells treated or untreated were significantly protected relative to control cells from 10 to 100% human serum, P <= 0.001.

Treatment with porcine CM slightly increased the susceptibility to human serum, especially at high serum concentrations. This observation led us to also assay the effect of PHA-CM derived from human PBMCs, as a xenograft would be probably exposed to both porcine and human cytokines. We proved the activity of the human CM by determining the up-regulation of HLA class I on HeLa cells with the specific antibody W6/32 (data not shown). We then incubated fibroblasts from the control and AT20 founder pig with 100% human PHA-CM and assessed cell survival after challenge with human serum (Fig. 8B ). This was carried out in parallel with untreated and porcine CM-treated cells. The treatment with the human CM did not reduce the high protection conferred by the HT transgenic phenotype (Fig. 8B ).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The serious shortage of human organs available for transplantation has resulted in a vigorous research effort to develop a xenogeneic source of organs and tissues for human transplantation. Most groups have focused their studies on the pig, even though pig organs are hyperacutely rejected when transplanted into primates (5 , 7) . Hyperacute rejection has been addressed in pig-to-primate heterotopic transplantation models using systemic treatment regimes as antibody removal therapy (33) , complement inhibition with cobra venom factor or sCR1 (34 , 35) , and combination therapy with immunosuppressive drugs (36 , 37) . Extended organ survival was accomplished using these strategies. However, questions arose as to the clinical viability of such rigorous treatments and the long-term effects of systemic complement inhibition at the C3 convertase step, which would compromise the ability of the patient to opsonize pathogenic microorganisms (38) . Genetic engineering of the donor organ therefore has a greater potential for effectively inhibiting xenograft rejection with reduced risk to the patient. Organs from transgenic pigs expressing human complement inhibitors are not hyperacutely rejected when transplanted into primates (11 , 12 , 39) . The mean survival time of heterotopic heart transplants in baboons is 5 days (39) . However, despite immunosuppression, they still suffer acute vascular rejection or DXR in which natural antibodies play a major role (39) . There is a significant increase in anti-Gal{alpha}1,3-Gal antibody titers after several weeks post-transplantation (8) . This is not inhibited with massive immunosuppression protocols, which abrogate the allogeneic response (8) . Moreover, there is evidence that natural antibodies are partially T cell independent (40) . The role of XNAs in HAR and DXR has been demonstrated in vivo using antibody pheresis experiments that deplete immunoglobulins in the recipient. Expression of human complement inhibitors in transgenic pig xenografts in combination with antibody absorption leads to increased survival time in the pig-to-baboon transplant model (39) . Thus, inhibition of complement and elimination of antibody reactivity is sufficient to prevent HAR and DXR of xenogeneic solid organs.

The development of porcine cell- and tissue-based therapies to human diseases has also gained much attention thanks to several important accomplishments. Examples are the treatment of Parkinson's disease patients with fetal porcine neural cells (7 , 41) or the use of porcine hepatocytes for liver support systems in cases of acute liver failure (7 , 42) . The use of porcine cells presents many advantages over human tissue such as availability, quality control of the tissues obtained, timing of cell harvest, etc. However, the success of these applications is still limited by poor understanding of the mechanisms that in many cases lead to loss of function and rejection of the transplanted tissue. Similarly to endothelial cells, many other porcine Gal{alpha}1,3-Gal positive cell types useful for transplantation, such as cardiomyocytes (43) or olfactory ensheathing cells and Schwann cells for spinal cord repair (44 , 45) , would be expected to trigger a humoral and cellular response.

To address these problems, we have developed a carbohydrate remodeling approach of the donor tissues. We have generated multiple lines of transgenic pigs expressing various levels of HT in cells, tissues, and organs in order to minimize the expression of the Gal{alpha}1,3-Gal antigen. Reduction of the Gal{alpha}1,3-Gal antigen by HT expression decreases natural antibody reactivity and results in significantly reduced complement activation through the classical pathway (22 23 24 25 26) . We show that transgenic expression of HT in different types of pig cells, including endothelial cells, reduces Gal{alpha}1,3-Gal antigen expression. We demonstrate in PAEC and fibroblasts that these effects are accompanied by a marked reduction in cell lysis mediated by human serum. The increased resistance observed in HT transgenic PAEC challenged with human serum is of clear significance in the development of solid organ xenotransplantation. The outcome of pig xenografts in primate models has been shown to correlate well with in vitro human serum-mediated cytolysis assays performed on swine cells (46) . The use of fibroblasts also has relevance, not only as a model for the development of different cell-based therapies, but by itself. These cells are included in protocols of gene therapy, tissue engineering, and tissue repair (47 , 48) . We also show expression of HT in olfactory bulb and sciatic nerve. These tissues are used to isolate olfactory ensheathing cells and Schwann cells, which are currently being used to treat animal models of spinal cord injury (44 , 45) .

We show in fibroblasts derived from different lines of transgenic pigs that the amount of protection to human serum-mediated lysis correlated well with levels of HT expression and reduction of the Gal{alpha}1,3-Gal antigen. These results suggest that increasing the expression of the HT transgene, especially on endothelial cells, may further protect the cells from human serum-mediated damage. We used two promoters with the aim that the CMV promoter would lead to constitutive expression of HT whereas the class I promoter, H2Kb, would drive constitutive as well as regulated expression to further protect the cell in a proinflammatory situation. This strategy proved to be useful, as the up-regulation in Gal{alpha}1,3-Gal epitope observed in cells treated with porcine cytokines was counteracted, at least in part, by the increase in HT expression in the transgenic cells. AT20 cells, which exhibit the highest levels of H-antigen expression, show better protection than AT21 cells when both are similarly activated. Although the Gal{alpha}1,3-Gal epitope was significantly reduced in the transgenic fibroblasts, the porcine cytokine treatment led to an increase in serum susceptibility. Such an effect was not observed when cells were treated with human CM. It is possible that one or a combination of porcine cytokines leads to the appearance or up-regulation of other xenoepitopes distinct from Gal{alpha}1,3-Gal. In fact, we detected an increase in serum XNA deposition in transgenic cells treated with porcine CM compared to anti-Gal{alpha}1,3-Gal antibody reactivity. Nevertheless, we must realize that this is an in vitro model. In vivo, the xenograft will be exposed to both pig and human cytokines (49) . To our knowledge, there are no available studies that describe the specific cytokines, their time course and relative proportion, present in a pig-to-primate xenotransplant setting. Different combinations of pig and human cytokines probably have different effects on the graft. Additional studies will be performed in order to elucidate what cytokines and antigens are responsible for increasing the susceptibility to human serum.

The current strategy may also be beneficial in combating parallel cell activation and downstream rejection events. The deposition of natural antibodies not only plays a role in HAR but is integral to endothelial cell activation (50 , 51) , DXR/acute vascular rejection events (52 , 53) , antibody dependent cell-mediated cytotoxicity (53 , 54) , and probably contributes to chronic rejection (55) . Therefore, reducing the expression of the Gal{alpha}1,3-Gal epitope might lead to a decreased XNA-mediated acute vascular rejection and XNA-mediated chronic rejection. Further studies should be performed to elucidate all these processes. The reduction of the Gal{alpha}1,3-Gal epitope may have additional beneficial effects beyond HAR and antibody-mediated acute vascular rejection. Inverardi et al. (56) demonstrated that carbohydrate epitopes, in particular the Gal{alpha}1,3-Gal epitope, directly mediate NK cell adhesion to xenogeneic cells in an antibody independent manner. Thus, inhibiting Gal{alpha}1,3-Gal epitope expression in the xenograft could also lead to decreased NK cell adhesion and NK cell-mediated cytolysis. Moreover, expression of HT in PAEC has been shown to reduce adhesion and activation of human monocytes (57) .

We have pursued an approach that decreases expression of the Gal{alpha}1,3-Gal epitope in cells and tissues, including the vascular endothelium, of transgenic pigs expressing HT. From the different lines studied, we conclude that expression of the H-epitope correlated inversely with that of Gal{alpha}1,3-Gal antigen. We demonstrate the efficacy of this approach on isolated transgenic endothelial cells and fibroblasts that are highly resistant to human serum-mediated lysis. These results encourage further development of HT transgenic pig cells and organs for xenotransplantation. Expression of HT might suffice to protect certain cell types from HAR and DXR whereas others, particularly vascularized organs, will probably benefit from its combination with other strategies such as the expression of complement inhibitors. Further genetic manipulations of the donor tissues designed to abrogate endothelial cell activation and cellular immune responses may lead to the development of universally accepted xenogeneic cells, tissues, and organs for human transplantation.


   ACKNOWLEDGMENTS
 
We thank Y. Shen for excellent technical assistance in the histology procedures and S. Fidel and L. Matis for critical review of the manuscript. The anti-Gal resins were kindly supplied as a gift from Glycorex Transplantation AB. This work was supported in part by a National Institutes of Standards and Technology-Advanced Technology Program grant to W.L.F. C.C. was temporally supported by a fellowship award from the Spanish Ministry of Education and Culture.


   FOOTNOTES
 
2 Abbreviations: {alpha}1,3-GT, {alpha}1,3-galactosyltransferase; CM, conditioned media; DMEM, Dulbecco's modified Eagle's medium; DXR. delayed xenograft rejection; HAR, hyperacute rejection; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; HT or H-transferase; human {alpha}1,2-fucosyltransferase; Ig, immunoglobulin; PAEC, porcine aortic endothelial cells; PBMC, peripheral blood mononuclear cell; PHA, phytohemagglutinin; RT-PCR, reverse transcriptase-polymerase chain reaction; XNA, xenoreactive natural antibody(ies).

Received for publication January 29, 1999. Revised for publication June 8, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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