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. 2000 May;100(1):131–139. doi: 10.1046/j.1365-2567.2000.00999.x

Molecular remodelling of human CD46 for xenotransplantation: designing a potent complement regulator without measles virus receptor activity

N A Begum *,, Y Murakami †, S Mikata *, M Matsumoto *,§, M Hatanaka *, S Nagasawa ‡, T Kinoshita ‡,, T Seya *,§,
PMCID: PMC2326985  PMID: 10809968

Abstract

In pig-to-human discordant xenotransplantation, human complement (C) is a major barrier to long survival of xenografts. The current idea on how to cope with this barrier is that human complement regulatory proteins are forcibly expressed on xenografts to serve as safeguards against host C-induced hyperacute rejection of xenografts. Co-expression of decay-accelerating factor (DAF) (CD55) and membrane cofactor protein (MCP) (CD46) would be the first choice for this trial, because most of the human cells are protected from C-mediated damage by two different modes with these two kinds of C-regulators. Many problems have arisen, however, for MCP expression on grafts. (i) MCP acts as a measles virus receptor, which may function to render donor pigs measles virus (MV) sensitive. (ii) MCP signals immune suppression which causes devastation of the recipient's immune responses. (iii) MCP exerts relatively low self-protective activity against C compared with other cofactors; development of more efficient forms is desirable. (iv) Grafts with a high expression level of MCP are difficult to produce. In this study, we made a number of cDNA constructs of MCP, expressed them on swine endothelial cell lines, and tested cell-protective potency and MV susceptibility. The short consensus repeat 1 (SCR1)-deleted MCP with glycosyl phosphatidylinositol (GPI)-anchored form (Δ1MCP-PI) of MCP was found to be most suitable for the purpose of overcoming these problems. However, it was also found that MV induces two modes of cytopathic effect (CPE) on swine endothelial cells, either MCP-dependent or -independent. Here, we discuss these two points which will be raised through study of MCP-transgenic animals.

Introduction

Human membrane cofactor protein (MCP) plays a protective role in host cells against homologous complement by acting as a protease (factor I)-cofactor for irreversible inactivation of C3b to iC3b.1 The complement (C)-regulatory function of MCP is complementary with that of CD55 (decay-accelerating factor, DAF), a decay-accelerator but not cofactor, and thus cotransfection of human DAF and MCP into discordant graft cells is a potentially useful strategy to extensively suppress non-human-to-human xenograft hyperacute rejection.2 A number of attempts have been made to induce non-human cells to express high copy numbers of human MCP,2–5 which may successfully confer resistance to human complement-mediated lysis on the non-human cells and overcome the first major barrier against xenotransplantation.

Human MCP additionally serves as a receptor for measles virus and probably sustains systemic measles infection.6,7 There have also been many attempts to generate human MCP-expressing animals as model systems for human measles infection.8–11 Mice have been mainly used for human MCP transgenic studies, which is sensible as mice express the MCP homologue only in the testis.12 MCP transgenic mice become susceptible to measles virus (MV) when lacking in type 1 interferon (IFN) responses.11 Thus, a major point is to establish model animals (1) expressing high or excess levels of human MCP for successful transplantation, and (2) circumventing virus infection.

As to the first point, we found that in a specific manner, the levels of human MCP protein production are regulated in non-human cells,13 and discovered silencer elements in the 3′-untranslated region (3′-UT) of human MCP.14,15 On the other hand, the second point has been hardly taken into consideration in transplantation studies. We and others have shown that the short consensus repeat 1 (SCR1)-deleted form of MCP fails to act as an entry receptor for MV without hampering C-regulatory function.16,17 Here, we found that this is true in swine cells expressing human cell-equivalent levels of human MCP. Furthermore, we tested whether this MCP mutant is suitable for a graft-protective molecule in swine-to-human xenotransplantation.

MATERIALS and METHODS

Cells, antibodies and reagents

Two different sources of swine endothelial cells (SEC-E, kindly supplied by Dr Nagai, Eizai Co., ltd.)18 and PAE (a generous gift from Dr Miyazono, Ludwig Institute, Uppsaala, Sweden)19 were used in this study. Cells were cultured in Dulbecco's modified Eagle's minimal essential medium (DMEM) containing 20% fetal calf serum (FCS), 5 mm l-glutamine and penicillin/streptomycin (Gibco, Grand Island, NY) at 37° in an atmosphere of 5% CO2/95% air. Gelatin-coated tissue culture dishes were used for SEC-E cell culture. Chinese hamster ovary (CHO) cells were obtained from American Type Culture Collection (ATCC, Rockville, MD). Vero cells and MV, a modified Nagahata strain,20 which underwent four passages in hamster brain, were obtained from the Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.

Human factor H, factor I and C3 were purified from human serum.21 Methylamine-treated DACM-labelled C3 (DACM-C3ma) were prepared in our laboratory.21

Human MCP cDNA (STc/CYT2 form) was a kind gift from Dr J. P. Atkinson (Washington University, St. Louis, MO).22 The mammalian expression vector pME18S was a gift from Dr J. Miyazaki (Osaka University, Japan).23 pUC18 and pUC19 were purchased from Toyobo Co., Japan.

Monoclonal antibodies (mAbs) against human C3b24 and MCP25 were produced as described previously. mAbs against swine MCP were produced as described previously.19

cDNA construction and ligation into vectors

cDNA encoding a glycosyl phosphatidylinositol (GPI)-anchored form of MCP in pME18S (MCP-PI) was constructed as described previously.26 Briefly, SCRST domain (1–293 residues) of MCP was fused to PI anchor region of DAF (308–347 amino acids) by two successive rounds of polymerase chain reaction (PCR) and finally the desired fragment was cloned into EcoRI and PstI sites of pME18S.

Mutation of the MCP cDNA22 was performed using a T7-GEN In Vitro Mutagenesis Kit (U.S.B., Cleveland, OH). Specific primer sequence, 5′-TGGACATGTTTCTCTGGCATCGGAGAAGGA-3′ was designed for looping out the first SCR domain (amino acids 1–61) of MCP.22 SCR1-deleted MCP (ΔSCR1) fragment was subcloned into EcoRI/PstI site of pME18S.

To generate GPI form of SCR1-deleted MCP, above two constructs were utilized. A restriction site, ScaI was selected from a common region (SCR4 domain) of both the constructs. EcoRI–ScaI fragment of MCP–PI and ScaI–PstI fragment of Δ1MCP were ligated into EcoRI/PstI digested pME18S to obtain Δ1MCP-PI form of MCP.

The nucleotide sequences of the resulting cDNAs were all confirmed by sequencing. Intact and mutated MCP cDNAs, all were subcloned into the EcoRI/PstI site of the mammalian expression vector pME18S.

The conditions for transfection to SEC-E and PAE cells

The manufacturer provided the protocol for Lipofectamine Plus (Gibco BRL, Gaithersburg, MD) which was mainly followed to introduce various constructs of MCP in SEC-E and PAE cells. For each set of transfection, 10 µg of respective cDNA construct and 1 µg of pGKhygro plasmid were used. After 48 hr of transfection, cells were harvested for replating and hygromycin B selection was started at a concentration of 0·7 mg/10 ml and 1·2 mg/10 ml for SEC-E and PAE cells, respectively. After 3 weeks of selection, several resistant colonies were isolated and expanded further to obtain positive colonies for MCP and MCP mutants by flow cytometry.

Flow cytometry

Flow cytometry was performed as described.25 Briefly, aliquots of 106 cells were incubated with primary mAb (5 µg) for 45 min. After 3 washes, fluoroscein isothiocyanate (FITC)-labelled secondary antibody was added and allowed to stand for 45 min. After two washes, cells were fixed with 0·5% paraformaldehyde. Samples were applied to Epics profile II within 3 days.

Reverse transcription (RT)–PCR

Total RNA was isolated from swine cells either MV-infected or uninfected according to the standard procedure using guanidinium–HCl and acid-phenol/chloroform,27 and cDNA was synthesized with SuperScript II RNase H-Reverse Transcriptase (Gibco-BRL). The primers for detection of MV-H (upstream, 5′-CTGGAGTTTGCCATTCAGCCC-3′; downstream, 5′-GAACAGGCGCTGGGTGATATC-3′) were designed to amplify a 261-bp segment. As a control for the presence of amplifiable RNA, hypoxanthine phosphoribosyl transferase (HPRT) primers were utilized to amplify 249 bp segment as previously described.28 Amplified PCR products were analysed by agarose gel (2·0%) containing ethidium bromide.27 Quantitative RT–PCR assay was performed in some experiments for determination of MV-H synthesis as described previously.29

MV infection assay

Cells with or without MCP or its mutants were cultured at 70% confluency in 24-well plates (Corning) for 15 hr, and infected with MV at 0·01–7·5 × 103 plaque-forming units (p.f.u.)/well. Simultaneously, plaque-forming assays were performed in some experiments, and the results confirmed the correlation between CHO cell syncytium formation and plaque formation.16 The syncytia formed were counted, and the cytopathic effects of the pig cell transfectants were observed 2–8 days post-infection. Cells were photographed under an Olympus microscope (IX-60; Olympus, Tokyo, Japan).

Virus production was determined as described previously.28 Briefly, swine endothelial cells were infected with MV (1·5 × 102−7·5 × 103 p.f.u./well) for 2 hr, extensively washed and cultured for 6 hr. The supernatants were removed and the wells were washed again to remove free MV. Four days later, we took the supernatants to determine the MV titres by the standard method using Vero cells.16

C3 fragment deposition assay and C-mediated cytotoxic assay

C3 deposition was assessed by flow cytometry as described previously.25 The cytotoxic assay was performed using a lactate dehydrogenase (LDH) kit (Kyokuto MTX, Tokyo, Japan).18 Briefly, the transfected cells were plated at 2 × 104 per well in 96-well trays 1 day before assay. After 15 hr, the plates were incubated with 20% or 40% normal human pooled serum (NHS) diluted in DMEM for 2 hr at 37°, and the released LDH was then determined. The spontaneous release of LDH activity from target cells was less than 5% of the maximal release of LDH activity, as determined by the addition of 1% sodium dodecyl sulphate (SDS).

Results

Production of cDNA constructs and their expression on swine endothelial cell lines

The cDNA encoding the STc/CYT2 isoform of MCP was used throughout this study. We first produced the cDNA constructs of MCP, MCP with transmembrane (TM, MCP) or a GPI-anchor (MCP-PI) and SCR1-deleted MCP with TM (Δ1MCP) or a GPI-anchor (Δ1MCP-PI). The predicted sequences of all cDNAs were confirmed by the DNA sequencer. The scheme of these constructs and their partial amino acid sequences around the junctions between signal peptide (SP) and SCR2 domains, and between STc and the GPI-anchor domains are shown in Fig. 1.

Figure 1.

Figure 1

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Schematic view of MCP and its mutants used for the present study. All the constructs were produced as described in Materials and Methods, and cloned into mammalian high expression vector pME18S and transfected into two different endothelial cell lines of pig origin to obtain stable clones. The STc/CYT2 isoform was used as a wild-type MCP (WT). In the MCP-PI mutant, four SCRs and STc domain of MCP was fused with DAF sequence (308–347 aa) for GPI-anchor attachment. The constructs of the Δ1MCP and Δ1MCP-PI mutants are schematically shown. The amino acid sequences of each modified junction are shown in the inset. SP, signal peptide.

These cDNAs were placed in a mammalian expression vector pME18S, and protein expression was first tested with CHO cells by the Lipofectamine Plus method as previously described.20 Since expected results were obtained in the CHO system, similar expression trials were next carried out with two swine endothelial cell lines, SEC-E and PAE. These cell lines expressed pig MCP,19 which was detectable only with specific mAbs19,30 Many clones expressing the four human MCP variants were successfully established (Fig. 2). The expression levels were assessed by flow cytometry and shown as mean fluorescence shift (MFS). MFS of each clone was determined by subtraction of fluorescence intensity of control cells (vector only transfected) from that of transfectants. We chose SEC-E clones with similar expression levels of MCP variants (MFS = 6·6∼8·9) and PAE clones with MCP variants (MFS = 8·0∼12·2) for further experiments. These levels were comparable to those on human vascular endothelial cells (HUVEC).18 The flow cytometric profiles of these clones are shown in italics in Fig. 2.

Figure 2.

Figure 2

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Flow cytometric analysis of SEC-E and PAE clones expressing wild-type and mutant MCPs. Cells were transfected with pME18S with cDNA of MCP or various mutants, and screened with hygromycin. The expression levels of MCP on established clones of SEC-E and PAE were assessed by flow cytometry using anti-MCP mAb M177 and FITC-labelled second antibody. Mean fluorescence shifts (MFS) of each clone are indicated in the table. Representative clones having a comparable expression level and used in the following experiments are indicated as italics and shown to the right as flow cytometric profiles. Refer to Reference 18 for the expression level of MCP on HUVEC.

Tests for MV-mediated cytopathic effect on two swine cell lines

A wild-type MV strain Nagahata, which induces severe cytopathic effect (CPE) in macrophages,29 was used in this experiment. First, we tested MV-permissiveness of the SEC-E clone expressing MCP (SEC-MCP) and the PAE clone expressing MCP (PAE-MCP). MV (1·5 × 102 p.f.u.) was inoculated onto the cells in a conventional manner16 and after washing allowed to stand for 5 days. The results are shown in Fig. 3(a). MV induced different CPE on these two swine endothelial cell clones. SEC-MCP exhibited a ‘scatter-like’ profile while PAE-MCP showed typical syncytia secondary to MV infection. MV replication was confirmed with PAE-MCP but not in other cells by amplification of the H-protein message by RT–PCR (data not shown). Thus, MV infects swine cells to induce CPE in variable fashions.

Figure 3.

Figure 3

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MV-mediated cytopathic effect on two swine endothelial cell lines. (a) The naive pig endothelial cell lines, SEC-E and PAE, tested for MV susceptibility. A range of MV concentrations were used and cell morphology was checked at different time points. Five days after infection by MV (Nagahata strain, 1·5 × 102 p.f.u.), two cell lines showed differential response. MV-H was not detected by PCR in the 7 day-cultured SEC-E and PAE (data not shown). Top panel: SEC-E, scatter-like pattern was observed through MV infection. Bottom panel: PAE, no morphological change was induced by MV. (b) SEC-E and PAE expressing MCP were tested for MV susceptibility. Experimental conditions were the same as in (a). Microscopic profiles are shown for the cells 7 days after MV inoculation. MV-H was detected by PCR in PAE cells but not SEC-E cells 7 days after MV inoculation (data not shown). Top panel: SEC-E, scatter-like profile was observed similar to naive cells secondary to MV infection. Bottom panel: PAE, typical syncytia were observed through MV infection.

We next checked whether the observed CPE was directly connected with MCP expression. MV infection studies were performed with control cells expressing no human MCP (Fig. 3b). PAE cells were resistant to MV when MCP was not expressed. Unexpectedly, however, SEC-E cells were changed into scatter-like forms in the absence of MCP expression. Thus, the two types of CPE were induced by MV but only syncytium formation on SEC-MCP cells was MCP-dependent. The other CPE, scatter-like elongation of cells, may be caused by some factors primarily provided on swine cells.

MV-susceptibility was examined with the four clones expressing MCP moderately (6·6–12·2) by the criteria of MFS. Various doses of MV (0·01–7·5 × 103 p.f.u.) were used for this trial. PAE-MCP cells (right panel of Fig. 4) formed syncytia by function of MV within 3 days only when MCP with SCR1 (MCP or MCP-PI) was expressed. The syncytia formed as a result of the expression of human CD46 were confirmed with the mAb against MCP M177 which blocks MV entry.16 Syncytium formation was almost completely suppressed by the addition of M177 (Fig. 5) regardless of the clones with the different expression levels of MCP. Compared to CHO cell system, PAE clones (cells with Δ1MCP or Δ1MCP-PI) were resistant to MV. The results were confirmed with a variety of MV doses (1·5 × 102−7·5 × 103 p.f.u.), suggesting that the even moderate expression levels of MCP and relatively low doses of MV are sufficient for induction of syncytium formation on pig cells. SCR1 deletion was found to be effective for protection of pig cells from MV.

Figure 4.

Figure 4

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MV-mediated cytopathic effect on SEC-E and PAE cells expressing MCP or its mutants. SEC-E (left panel) and PAE cells (right panel) expressing the indicated MCP mutants were infected with MV (1·5 × 102 p.f.u.), and allowed to stand for 3 days (centre lanes in the left panel and right lanes in the right panel) or 7 days (right lanes in the left panel). Microscopic profiles of each clone expressing various MCP derivatives without MV infection are shown to the left lanes in the two panels. Typical syncytia were observed in PAE clones expressing MCP or MCP-PI 3 days after MV inoculation. Scatter-like CPE was observed in all SEC-E clones irrespective of type of MCP expressed.

Figure 5.

Figure 5

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MV-mediated syncytia formation induced in PAE clones expressing MCP is effectively blocked by preincubation with M177. The numbers of virus-dependent syncytia formed in the presence or absence of mAb M177 were plotted as a function of MV concentration on the x-axis (5 units = 40 p.f.u.). Anti-pig MCP #6 and #7 remained ineffective in this system (data not shown).

On the other hand, SEC-MCP cells did not form typical syncytia irrespective of expression of any MCP variants. A representative result is shown in the left panel of Fig. 4 (see left versus centre lanes). Similar results were obtained with a variety of doses of MV and cells with higher expression levels of MCP variants. Again, irrespective of any MCP variant expression, a scatter-like profile is developed after 5 day culture (see left versus right lanes in the left panel of Fig. 4). These results reinforce the fact that MCP-independent CPE occurs on pig cells at relatively low doses of MV.

Effects of MCP expression on human C-mediated cytolysis

As MCP plays an important role both in MV-permissiveness and in host-cell protection from C-mediated cytotoxicity in PAE-MCP, this cell line was used for the model study on cell protection from C. First, the PAE clones with MCP variants were incubated with NHS, and C3 fragment deposition was assessed by flow cytometry. The results are shown in Fig. 6. Surprisingly, GPI-form and Δ1MCP form induced less C3 deposition on PAE cells than the conventional MCP. Statistical analysis suggested that these tendencies are significant, though not marked (Fig. 6). A combination of both, namely Δ1MCP-PI, conferred the most efficient protection against human C on these pig cells.

Figure 6.

Figure 6

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Inhibition of C3 fragment deposition on PAE cells by expression of MCP. PAE cells expressing varous MCP derivatives and control naive PAE cells were incubated with 20% NHS. The degrees of C3 fragment deposition were assessed concerning cells still alive by flow cytometry using mAb against human C3 and FITC-labelled second antibody. Percentage inhibition of C3 deposition in each clone was assessed from these results in respect of C3 deposition in naive PAE.

It has been believed that factor I–cofactor activity of MCP is responsible for host-cell protection from homologous C.31 The solubilized cellular extracts containing almost the same amounts of the MCP variants were prepared and their cofactor activity was assessed by the fluorescent substrate DACM-labelled methylamine-heated C3 (DACM-C3ma).21 All variants showed indistinguishable degrees of cofactor activity (data not shown).

Percent suppression of PAE cytolysis is also tested with NHS (a source of C) using the series of MCP transfectants (Fig. 7). Again, the PI form and the Δ1MCP form conferred effective protection against C on host cells compared to conventional MCP, and the most efficient was the Δ1MCP-PI. The levels of C-mediated cytolysis do not always parallel with those of C3-deposition and cofactor activity in these transfectants; this, however, suggests that most likely the microenvironment around the membrane26 plays a role in the different functional potencies of the GPI-forms versus the transmembrane forms, although the artificial problems such that cells with C3 deposited are lost during analysis could not be ruled out. We fairly conclude from these results that the Δ1MCP-PI is the best candidate of the MCP mutants for swine-to-human xenotransplantation.

Figure 7.

Figure 7

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Percent inhibition of C-mediated cytolysis of PAE cells by function of MCP. Naive PAE and PAE clone expressing Δ1MCP-PI were incubated in either 20% or 40% NHS, and percentage lysis was calculated by measuring the LDH activity in the supernatants. Other PAE clones expressing MCP derivatives other than Δ1MCP-PI showed protection against C-mediated lysis compared to naive PAE but MCP-PI was the strongest protection potency of these mutants (data not shown).

Discussion

The purpose of this study was to establish the most suitable MCP construct for xenotransplantation. A Δ1MCP-PI form was found to be a good candidate for this purpose. We succeeded in expression of this MCP mutant on swine endothelial cells without facilitating MV infection. The expressed protein sustained potent factor I–cofactor activity and more effectively protected swine cells from human C. However, certain swine cell lines per se were found to be susceptible to MV even in the absence of human MCP and forms scatter-like CPE. Thus, there are MCP-dependent and MCP-independent measles CPE in swine endothelial cells, the mechanism of which remains as-yet unknown. These results fit the current concept on MV receptor that in primate cells, MCP and unidentified alternative molecule(s) serve as MV receptors.

What works as an alternative receptor in SEC-E cells is an intriguing issue. Although pigs have their own MCP on most tissue/organs,19,30,32 the pig MCP does not act as a MV receptor (data not shown). It has been reported that ‘scatter-like’ transdifferentiation was observed in endothelial and epithelial cells including hepatic cells treated with hepatocellular growth factor (HGF).33,34 However, to our knowledge, there is no report suggesting that MV induces this kind of cell differentiation. As it takes > 5 days to form the scatter-like pattern on the swine endothelial cells, induction of this unusual cell morphology may be attributable to mediators secreted by SEC-E cells secondary to MV infection. Furthermore, MV are barely replicated in SEC-E cells, suggesting that there is no MV entry receptor on SEC-E cells or MV does not allow replication in these cells. Further studies are needed to clarify the mechanism of this cell alteration in association with MV infection and mediators including HGF.

Settling this point will be important for producing human MCP-transgenic pigs.35,36 Pigs are believed to be insensitive to MV but pigs expressing human MCP may become highly susceptible to MV. Accordingly, the MV-mediated undefined cell differentiation could occur in MCP-transgenic pigs.

Our basic strategy of molecular designing of C-regulatory proteins for xenograft protection is as follows.37 (1) Because most of the SCR proteins act as viral and/or bacterial receptors, the domains responsible for entry by these micro-organisms must be disrupted for protection of pigs from infection. (2) It is desirable to elevate C-regulatory activity through recombination of the molecules by cDNA engineering. (3) Unnecessary signal transduction via stimulation by ligands must be avoided. (4) High expression levels of MCP may be required to fully protect host cells from C.

For item 1, SCR1-deleted MCP does not act as a MV receptor but exerts factor I–cofactor activity indistinguishable with that of the wild-type molecule. Thus, the SCR1-deleted form is recommended for transgene/transfection for graft survival. For item 2, The Δ1MCP-PI form expresses slightly higher protective activity than the conventional form, although the levels of cofactor activity and inhibition of C3 deposition do not parallel with cell protective activity. GPI-anchored proteins, probably including MCP-PI, are likely to be sorted in ‘rafts’ containing caveolae,38 the localization of which differs from the transmembrane forms of MCP.26 It is likely that microenvironment including membrane and MCP critically affect the cell protection against C3b.

Regarding item 3, we discovered that human MCP acts as a signal-transducing receptor on antigen-presenting cells (APC).29 A problem to be settled is what ligand stimuli and membrane domains are responsible for MCP-mediated signalling and its relationship to immune suppression in resident APC. MCP binds C3b, C4b, factor I, measles virus, Streptococcus M proteins, Neisseria gonorrhoeae, heparin and a putative counter-receptor on oocytes.37,39–42 APC might be present in grafts of pig origin and respond to ligand stimuli through human MCP. Thus, the GPI-form would be more convenient than the transmembrane form because of its mobility and little signalling activity.

Item 4 still remains unsettled. The reason is that the mechanisms determining the levels of human MCP protein production in non-human cells have not yet been perfectly determined. Relatively high levels of human MCP expression have been accomplished with an artificial ‘tail-less’ construct ΔCYT by transfection in non-primate mammalian cells such as CHO26 and swine endothelial cells (SEC-E).43 However, mice expressing very high levels of human MCP failed to be produced even using ΔCYT by transgenic techniques.8–10,43 The fact that expression of human MCP is severely suppressed in transgenic mice was also confirmed with a cDNA construct consisting of DAF, MCP and CD59 being tandemly connected (Seya et al. unpublished data). In this experiment, only MCP expression was selectively suppressed. The mechanism whereby the expression of human MCP is relatively suppressed in transfectants and severely suppressed in mice is not understood. Before final trials for producing MCP-transgenic pigs, this point should be settled.

The C system provides potent interspecies immunity which discriminates self from non-self cells and selectively damages the latter.37 Hyperacute rejection is the first barrier for xenotransplantation.44,45 To succeed in graft survival, however, further overcoming of chronic rejection because of cellular immunity is necessary.45 Latent retrovirus infection in swine graft is a serious problem, which has been mentioned in several reviews.46,47 Furthermore, infection may be accelerated by transfection with the currently available SCR proteins.48 Finally, human immune effectors other than C may cause antibody-mediated cellular responses, natural killer (NK) potentiation and subsequent chronic rejection of the graft.37,45 Most of these encompass both innate and acquired immune responses that are essential for elimination of pathogens in the recipients.49 Here, we eliminated the possible incidence of human MCP-mediated pig MV infection in transgenic pigs. Nevertheless, more caution will be needed to adapt xenotransplantation for clinical use.

Acknowledgments

We are grateful to Drs K. Toyoshima and H. Akedo (Osaka Medical Center for Cancer) for support of this work. Thanks are also due to Dr H. Murakami, S. Miyagawa, and R. Shirakura (PROBRAIN) for providing reagents. Provision of SEC-E and PAE cells by Eizai Co., and Ludwig Institute is most gratefully acknowledged. This work was supported in part by Uehara Memorial Foundation.

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