Cross‐species reactivity of a panel of antibodies with monkey and porcine tissue

The continuing shortage of organs available for transplantation limits the number of patients able to benefit from this highly successful form of therapy. Interest in alternative sources of organs has now turned towards the pig because of its physiological similarity to human. There is a requirement therefore for reagents not only for research purposes but possibly for studying xenotransplants in the clinical situation in the future. In this study, we have concentrated on determining the cross-species reactivity of a large panel of antibodies directed against human leukocyte markers, testing peripheral blood leukocytes and also including renal tissue to determine non-leukocyte cross-reactivity. A total of 63 out of 127 antibodies cross-reacted with cynomolgus monkey cells. Twenty of these antibodies stained similar populations of leukocytes to human, whereas the remaining 43 reacted with clearly different populations. The majority of antibodies (108/127) were unreactive with porcine leukocytes, reflecting the evolutionary differences between pig and man. Of the 19 antibodies cross-reactive with porcine cells, seven reacted with similar proportions of leukocytes to human, whereas the remaining 12 antibodies stained entirely different populations. The most interesting, and potentially most useful, antibodies were four that reacted with human, cynomolgus monkey and porcine tissue in a similar manner, suggesting that the epitopes recognized are present on similar molecules. These antibodies were directed against CD29 (MEM101A, K20) and CD18 (BU87, 7E4), the common β₁- and β₂-integrin subunits respectively. This study demonstrates that there are antigens common to cynomolgus monkey, pig and man that react with currently available antibodies. Nevertheless, when determining cross-species reactivity of human antibodies, it is important to consider the possibility that there may be additional non-leukocyte reactivity in other tissues.     

CD97-decay-accelerating factor interaction is not involved in leukocyte adhesion to endothelial cells

BACKGROUND: Effective improvement in xenograft survival is achieved using transplants from transgenic pigs expressing human complement (C) regulatory proteins, including decay-accelerating factor (DAF), CD59, and CD46 on endothelial cells (ECs). The aim of this study was to investigate whether human DAF expression in porcine ECs, as well as regulating C activation, can modify intercellular events through its interaction with its receptor, CD97, on human leukocytes. METHODS: Cellular interactions between human leukocytes and porcine ECs were investigated in vitro using ECs from either wild-type or DAF-transgenic pigs. Static leukocyte adhesion and T cell activation assays were performed using porcine ECs as target or effector cells, respectively. The role of the DAF-CD97 interaction was investigated using specific blocking monoclonal antibodies (mAbs) against human DAF and its receptor, CD97, in adhesion assays. RESULTS: Adhesion of U937 or Jurkat T cells, both expressing human DAF and CD97, was quantitatively similar for wild-type and transgenic-DAF-expressing pig ECs. Furthermore, blocking the CD97-DAF interaction did not inhibit xenogeneic leukocyte-endothelium adhesion, whereas blocking the very late antigen 4-vascular cell adhesion molecule-1 pathway reduced this adhesion by 50-80%. Furthermore, DAF and CD97 expression was not up-regulated during tumor necrosis factor-alpha- or lipopolysaccharide-mediated EC activation, unlike the adhesion molecules E-selectin, vascular cell adhesion molecule-1, and intracellular adhesion molecule (ICAM)-1. CONCLUSION: We found that high levels of human DAF expressed on ECs abrogates C-mediated cell damage but did not affect the in vitro adhesive properties or antigen-presenting cell function of genetically modified porcine ECs.     

Human platelets activate porcine endothelial cells through a CD154-dependent pathway.

BACKGROUND: Delayed xenograft rejection is associated with endothelial cell activation, platelet sequestration, and subsequent thrombosis. We evaluated whether human platelets could directly activate porcine endothelium (PEC), and if so, whether this was mediated by an interaction between platelet-bound CD154 and PEC CD40. METHODS: Platelet activation was achieved by thrombin exposure and confirmed by evaluation of up-regulated CD62P and CD154. Co-incubation of platelets or D1.1 cells with PEC was performed, and PEC activation was evaluated by up-regulation of CD62E. RESULTS: Co-incubation of resting platelets that lacked significant expression of CD62P and were void of CD154 did not activate PEC. In contrast, thrombin-activated human platelets expressing considerable amounts of both CD62P and CD154 induced PEC activation. This activation could be completely inhibited by coincubation with a humanized monoclonal antibody directed at human CD154 (hu5c8). Similarly, human D1.1 cells expressing CD154 were shown to activate PEC in a CD154-dependent manner. CONCLUSION: Human CD154 expressed on activated human platelets or on T cells interacts with CD40 expressed on PEC leading to PEC activation. This interaction can be inhibited by a monoclonal antibody directed against CD154, suggesting that an interaction between human CD154 and PEC CD40 is at least in part responsible for PEC activation seen in delayed xenograft rejection. These data strengthen the rationale for the use of CD154-directed therapy in discordant xenotransplantation.     

Co-stimulatory molecules in islet xenotransplantation: CTLA4Ig treatment in CD40 ligand-deficient mice

Previous work has demonstrated that short-term systemic administration of cytotoxic T lymphocyte antigen-4 (CTLA-4) Ig blocks human pancreatic islet xenograft rejection in mice and induces long-term, donor-specific tolerance, whereas studies on pig pancreatic islet rejection in mice have failed to demonstrate a role for CTLA4Ig in preventing rejection. Treatment with anti-CD40 ligand (L) monoclonal antibodies alone is somewhat effective in prolonging the survival of islet xenografts, but ineffective when applied to skin xenografts. However, simultaneous blockade of the CD28 and CD40 co-stimulatory pathways prolongs the survival of pig skin on recipient mice. To evaluate the role of CD28 and CD40 co-stimulatory pathways in pig islet-like cell cluster (ICC) xenograft rejection in mice, CD40L-deficient mice transplanted with fetal porcine ICCs were given posttransplant treatment with human (h) CTLA4Ig or a human IgG1 chimeric mAb (hL6). Xenografts were evaluated 6 or 12 days after transplantation. Fetal porcine ICC xenografts were protected from rejection in hCTLA4Ig-treated CD40L-deficient mice, whereas xenograft rejection persisted in untreated CD40L-deficient mice. Simultaneous blockade of the CD28 and CD40 co-stimulatory pathways is mandatory to inhibit ICC xenograft rejection in the pig-to-mouse model, because the CD28 and CD40 co-stimulatory pathways seem capable of efficiently substituting for one another.     

Induction of swine major histocompatibility complex class I molecules on porcine endothelium by tumor necrosis factor-alpha reduces lysis by human natural killer cells

BACKGROUND: Natural killer (NK) cells have been implicated in a process of delayed xenograft rejection occurring in pig-to-primate organ transplants. As tumor necrosis factor-a (TNF-a) induces expression of both adhesion receptors and major histocompatibility complex class I molecules on porcine endothelium, we investigated the effects of TNF-alpha on human NK cell adherence to and cytotoxicity of porcine aortic endothelial cell (PAEC) monolayers. METHODS: Adherence of human NK cells was measured after PAEC treatment with increasing concentrations of TNF-alpha. Monoclonal antibodies (mAbs) against adhesion molecules on NK cells and PAEC were used in inhibition studies. Resting or TNF-alpha-treated PAEC were used as targets for NK lysis. Increasing titers of anti-swine leukocyte antigen (SLA) class I antibodies or pooled human immune globulin (IVIg) were used to reverse the effects of TNF-alpha on NK lysis. RESULTS: NK cell adhesion to TNF-a-treated PAEC increased in a dose-dependent manner by a maximum of 44%, and was inhibited by mAbs against CD49d, CD11a, CD11b, CD18, and CD2, as well as porcine vascular cell adhesion molecules. In contrast, TNF-alpha treatment of PAEC reduced human NK lysis in a dose-dependent manner. Preincubation of TNF-a-treated PAEC with increasing concentrations of anti-SLA class I mAb increased NK lysis in a titer-dependent manner, and reversed the protective effect on human NK lysis by 77%. Treatment with IVIg, containing antibodies against an a-helical region of HLA class I molecules, had a similar effect. CONCLUSIONS: These results imply that SLA class I molecules can bind to inhibitory receptors on human NK cells, and that these interactions can be augmented by increasing the level of SLA class I molecule expression on porcine endothelium. Strategies that can increase porcine endothelial cell expression of either swine or human major histocompatibility complex class I molecules may reduce human NK activity against porcine xenografts.     

Alpha-galactosyl-mediated activation of porcine endothelial cells: studies on CD31 and VE-cadherin in adhesion and signaling.

BACKGROUND: Ligation of alpha-galactosyl epitopes on endothelial cells by naturally occurring human antibodies causes hyperacute rejection in porcine-to-human xenotransplantation. The alpha-galactosyl-specific lectin Bandeiraea simplicifolia isolectin B4 (IB4) has been reported to trigger endothelial "gap" formation and tyrosine phosphorylation of an unidentified 130-kDa protein. We have studied two 130-kDa junctional adhesion molecules, CD31 and VE-cadherin, in porcine aortic endothelial cells (PAECs) during IB4-mediated activation. The cellular distribution of these molecules, their susceptibility to tyrosine phosphorylation, and their capacity to bind IB4 or natural human antibodies have been determined. METHODS: Porcine CD31 and VE-cadherin were cloned. Recombinant proteins and monoclonal antibodies were prepared. The distribution and phosphorylation of CD31 and VE-cadherin in confluent PAECs activated with IB4 or human serum were studied by confocal microscopy and Western blotting, respectively. RESULTS: IB4 caused rapid redistribution of CD31 and VE-cadherin away from cell junctions and tyrosine-phosphorylation of CD31 but not VE-cadherin. A monoclonal antibody to CD31 also triggered tyrosine phosphorylation of this molecule, but brief exposure of PAECs to normal human serum did not. Tyrosine-phosphorylated CD31 complexed with SHP2 and other unidentified phosphoproteins. Both IB4 and natural human antibodies bound to porcine CD31 but not to VE-cadherin. Cell adhesion tests showed that porcine and human CD31 are functionally incompatible. CONCLUSIONS: Endothelial cell retraction during IB4-mediated activation of PAECs is associated with rapid loss of CD31 and VE-cadherin from cell junctions. CD31 becomes strongly tyrosine-phosphorylated and forms a cell signaling complex, which may have a significant role in the response of the xenograft vascular endothelium.     

Antibodies to human adhesion molecules and their ligands: Cross-species reactivity and potential application in xenotransplantation

Abstract: Given the renewed interest in the possibility of using animals as an alternative source of organs for transplantation, it would be useful to have tools to study and manipulate the inflammatory response to a xenograft. With this in mind, we have screened cynomolgus monkey and porcine tissue with 37 antibodies specific for human leukocyte differentiation antigens and adhesion molecules. The cynomolgus monkey, an old world primate, is a concordant species with respect to humans, in contrast to the pig, which is a discordant species. The cross-species reactivities of the antibodies tested fall into three groups: i) no crossreactivity; ii) crossreactivity with a tissue distribution similar to that in the human; iii) crossreactivity, but with a distribution different from that in the human. Those antibodies directed against E- and P-selectin, ICAM-1 (intercellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1), which were reactive with monkey tissue, had a similar distribution to that seen in human tissue, but were unreactive with the porcine tissue tested. While an anti-CD31 antibody detected a conserved epitope on endothelium, species differences were apparent in leukocyte reactivity. One of the antibodies directed against CD 18 reacted with leukocytes in all three species, whereas the other antibody detected an epitope present on porcine muscle/connective tissue. While antibodies to VLA-4 (Very Late Antigen-4) detected a small number of leukocytes in the kidney, they also reacted with the Bowman's capsule in the kidney and matrix protein/connective tissue in the lymph node. This study indicates that when antibodies react across species, some epitopes recognized in the old world nonhuman primates may have a distribution similar to those detected in human tissue, whereas in more distant species such as the pig, in many instances the epitope is present on entirely different structures. Nevertheless, the absence of crossreactivity of human reagents with porcine tissue may allow targeting of molecules in a species-specific manner, allowing their use for diagnostic or therapeutic purposes.     

Identification of swine and primate cellular adhesion molecules (CAM) using mouse anti-human monoclonal antibodies

Abstract: A series of adhesion molecules of swine and Cynomolgus monkeys were identified by screening for cross-reactivity with a panel of monoclonal murine anti-human adhesion molecule antibodies obtained from the 5th International Workshop and Conference on Human Leukocyte Differentiation Antigens (Boston, MA, USA, 1993). Of 162 antibodies tested, 25 were found that cross-react significantly with swine cells, while 67 were found to react strongly with cells of Cynomolgus monkeys. Cross reactivities to swine were found with antibodies to CD 18 (β2 integrin), CD29 (β1 integrin), β7 integrin, CD49d (α4 integrin) CD49b(α2 integrin), and to a lesser extent to CD62p(P-selectin), CD62L(L-selectin), CD102(ICAM-2), CD11b, and CD49c (α3 integrin). Cross-reactivities to primate cells also included CD18, CD29, CD49d, and CD49b. In addition, reactivities to Cynomolgus monkey cells were detected with antibodies to CD11 (a, b, and c), CD31, CD44, CD49e, CD49f, CD50, CD54, CD56, CD62p, CD 102 and CD56. The tissue distribution and molecular weight of the swine antigens are similar to their human counterparts. These findings provide a spectrum of monoclonal antibodies that react with shared epitopes on homologous adhesion molecules of human, swine, and monkey cells, thus facilitating study of the role of these molecules in the immunobiology of monkeys and swine. These reagents may be useful to dissect the role of adhesion molecules in both alio- and xenoreactivity.     

The serum level of xenoantibodies, and hDAF or alphaGAL expression on pig cells, modulate in vitro the protection given by hDAF to primate complement-mediated damage

The ability of human complement regulatory molecules to prevent xenograft rejection following pig-to-primate xenotransplantation is limited. We assayed the efficacy of transgenic human decay accelerating factor (hDAF) expressed on porcine cells to inhibit the in vitro complement activity of primate sera. We measured the cytotoxic activity of baboon or human sera against peripheral blood lymphocytes (PBLs) from hDAF or nontransgenic pigs using a flow cytometry complement-mediated cytotoxicity assay (FCCA). We also analyzed the anti-Galalpha1-3Gal (alphaGal) antibody titer of the baboon sera by ELISA and the expression of hDAF and alphaGal on the PBL surface by immunofluorescence. Transgenic hDAF expression was capable of protecting pig cells against injury produced by both baboon and human serum. However, the hDAF molecule was more efficient against human than baboon sera. The humoral cytotoxicity capacity correlated with the level of both IgG and IgM anti-alphaGal antibodies. In addition, inhibition of complement-mediated cytotoxicity of hDAF pig cells correlated with the expression of hDAF and alphaGal molecules on target cells. These results confirm in vitro the protective role of hDAF in pig cells to heterologus complement mediated damage, but they also suggest that protection decreases in the presence of high levels of anti-porcine antibodies in serum, low expression of hDAF, or high expression of alphaGal on pig cells.     

Effect in vitro and in vivo of a rat anti-CD2 monoclonal antibody (LO-CD2b) on pig-to-baboon xenogeneic cellular (T and natural killer cells) immune response

Abstract: Although hyperacute rejection of discordant xenogeneic grafts can be prevented, baboon or human anti-pig cellular response may lead to acute xenograft rejection. Among the immune cellular actors participating in such a xenograft rejection are both T and natural killer (NK) cells. In the pre-clinical model of pig-to-baboon discordant xenograft, there is however, a lack of specific immunological therapeutic agent, in particular antibaboon T-cell monoclonal antibodies do not exist. We therefore developed a rat anti-CD2 monoclonal antibody (LO-CD2b) that recognizes both baboon and human CD2 + cells. In this study, we show that in vitro LO-CD2b inhibits a pig-to-baboon mixed lymphocyte reaction, the direct cytotoxicity of baboon peripheral blood lymphocytes to pig aortic endothelial cells, as well as the baboon NK activity against K562 cell line. In vivo, LO-CD2b produces a strong depletion of all peripheral CD2+ cells including NK CD2+ cells. In summary, LO-CD2b represents an important immunological tool that can be used in the preclinical model of discordant pig-to-baboon vascularized xenograft.     

Kinetics and character of xenoantibody formation in diabetic patients transplanted with fetal porcine islet cell clusters

Abstract: Porcine fetal islet-like cell clusters (ICC) were transplanted to 10 renal transplant patients suffering from long-standing type I diabetes. Since they had renal grafts, they were given immunosuppression with cyclosporine, prednisolone and azathioprine. Eight patients had the ICC injected intraportally and two had the ICC placed under the renal graft capsule. At the time of the xenotransplantation, ATG or 15-deoxyspergualin was given as an adjunct. Evidence of engraftment, as reflected by excretion of small amounts of porcine C-peptide into the urine, was observed in four of the patients. In all patients, irrespective of the type of immunosuppression given and whether graft function was established or not, specific xenoantibodies were formed. Titer increases in lymphocytotoxic antibodies occurred after 10 to 14 days with peak reactivity after 30 to 50 days. High titers of ADCC activity measured against porcine lymphoblasts were found in all patients. Titers were maintained at a high level for at least 100 days. Antibody titers were monitored against pig lymphocytes, erythrocytes and against pig membrane antigens solubilized from peripheral blood mononuclear cells, platelets and erythrocytes. Both IgM and IgG antibodies were formed with identical kinetics. There was no increase in the titers of alloantibodies, as evidenced by panel-reactive lymphocytotoxic antibodies. In all patients an increase in titers of isohemagglutinins was recorded, especially against blood group B antigens. Absorption studies showed that xenoreactivity present in healthy individuals could not be blocked by absorption with human RBC. However, in all transplanted patients, xenoreactivities against pig antigens were inhibited by absorption with human RBC, in particular with B-type RBC. These data show that the increase in isohemagglutinins was probably due to cross-reactivity with xenogeneic antigens. Using an ELISA assay, increased antibody titers were also recorded against purified pig MHC class I antigens. However, as is shown in the accompanying paper (next issue), these antibodies were probably not directed against protein determinants on the MHC molecules but rather against glycosylated side chains of these molecules.     

Transplantation of organ cultured fetal pig pancreas in non-obese diabetic (NOD) mice and primates (Macaca fascicularis)

Abstract: Xenografts of organ cultured fetal pig pancreas in prediabetic NOD mice can survive for prolonged periods (>20 weeks) in recipients treated with anti-T cell monoclonal antibodies (MAb) directed against host CD4 and CD3 cell surface molecules. Anti-CD4 MAb treatment alone is only partly effective and xenograft rejection occurs over a period of many weeks. In diabetic recipients, by contrast, recurrence of autoimmune disease in isografts is rapid (<28 days) despite similar depletion of CD4+ve T cells. In spontaneously diabetic NOD mice immunosuppressed with anti-CD3 and anti-CD4 MAbs xenograft function occurs and the recipient's blood glucose levels fall into the pig range. Organ cultured fetal pig pancreas transplanted into cynomolgus monkeys is rapidly but not hyperacutely rejected when azathioprine-cyclosporin A-prednisolone immunosuppression is used. Anti-T-cell MAb treatment is now being studied in this primate model.     

CD4+ cells play a major role in xenogeneic human anti-pig cytotoxicity through the Fas/Fas ligand lytic pathway

BACKGROUND: In this study, the role of cell-mediated cytotoxicity by human leukocytes against pig endothelial cells was examined in vitro. The aim was to determine which cell subsets were responsible for this phenomenon and which pathways were involved in cell lysis. METHODS: Primed human peripheral blood mononuclear cells (PBMC) or purified CD4+ or CD8+ T cells were used in a cell-mediated cytotoxicity assay in which cytotoxicity of an SV40 transformed porcine endothelial cell (EC) line (SVAP) was determined by Annexin V binding. RESULTS: Human PBMC demonstrated specific lysis of porcine EC that was proportional to the effector: target ratio. CD4+ T cells accounted for >60% of this lysis, whereas CD8+ T cells accounted for <20%. CD4+ T cell-mediated lysis depended on direct recognition of porcine major histocompatibility complex class II molecules as inhibition of swine leukocyte antigen class II on porcine EC-inhibited CD4+ T cell cytotoxicity. This lysis was mediated through the Fas/FasL pathway as addition of anti-Fas and/or anti-FasL antibody profoundly inhibited antiporcine lysis. In addition, FasL gene expression was detected in primed PBMC and CD4+ T cells by RT-PCR, whereas granzyme B gene expression was not. Primed CD4+ T cells demonstrated high level FasL protein by Western blotting and two-color FACS analysis, whereas NK cells and CD8+ T cells did not. Finally, recombinant human FasL induced apoptosis in Fas expressing porcine EC cells, demonstrating that human FasL interacted with and activated Fas on porcine EC cells. CONCLUSIONS: In conclusion, human to pig cell-mediated cytotoxicity was mediated predominantly by CD4+ T cells through the Fas/FasL pathway of apoptosis. These results suggest that direct cytotoxicity by xenoreactive CD4+ T cells may be one of several effector mechanisms involved in cellular xenograft rejection.     

Classical pathway complement destruction is not responsible for the loss of human erythrocytes during porcine liver perfusion

BACKGROUND: Porcine livers perfused with human blood destroy 85% of human erythrocytes (red blood cells [RBC]) during prolonged extracorporeal perfusion, raising the possibility of a complement-mediated graft-versus-host effect. METHODS: Isolated porcine livers were perfused with fresh human blood. Plasma samples were analyzed for complement production by reverse CH50 analysis and porcine immunoglobulin class and specificity by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Anti-CD59 and anti-decay accelerating factor (DAF) monoclonal antibody were used to investigate whether human complement regulatory proteins inhibit porcine complement. RESULTS: After 64 hr of perfusion of porcine livers with human blood, mean complement activity in the perfusate was 95% of the starting value and increasing, whereas perfusion in the absence of a liver showed a falling complement activity of 28.7%. ELISA demonstrated porcine immunoglobulin (Ig) G and IgM in the xenoperfused human plasma. Whereas in a previous study flow cytometry demonstrated porcine antibodies specific for antigens on human T lymphocytes, in this study, anti-human RBC antibodies were not found. Xenoperfused human plasma did not lyse fresh human RBC. Human complement was consistently more efficient at lysing porcine RBC than was porcine complement at lysing human RBC, and human plasma inhibited the ability of porcine plasma to lyse human RBC, raising the possibility of cross-species complement regulation. Complement regulatory proteins on human RBC were blocked using mouse monoclonal anti-human CD59 and DAF. Blocking CD59, but not DAF, augmented lysis of human RBC by porcine complement. CONCLUSIONS: Human CD59 inhibits porcine complement. The production of porcine complement from xenoperfused porcine livers is unlikely to result in clinically significant injury mediated through the classical pathway of complement activation.     

CD8-interaction mutant HLA-Cw3 molecules protect porcine cells from human natural killer cell-mediated antibody-dependent cellular cytotoxicity without stimulating cytotoxic T lymphocytes

BACKGROUND: CD56+ human natural killer (NK) cells are the principal anti-pig cytotoxic effectors in vitro. Expression of certain human leukocyte antigen (HLA) class I molecules in porcine cells can inhibit NK cell-mediated natural cytotoxicity in serum-free medium, but had not been shown to inhibit antibody-dependent cellular cytotoxicity (ADCC) by CD16+ NK cells in the presence of human xenoreactive immunoglobulin G. Moreover, expression of HLA molecules might amplify the previously weak CD8+ cytotoxic T-lymphocyte (CTL) response against porcine cells. METHODS: A novel porcine B-lymphoblastoid cell line (13271) was stably transfected with HLA-Cw*0304 gene constructs encoding wild-type (wt) Cw3 or genetically modified Cw3 unable to interact with CD8 (Cw3-D227K). The Cw3 transfectants were used in limiting dilution assays to estimate the CTL precursor frequency in CD56-depleted human peripheral blood mononuclear cells (PBMC) obtained from eight unrelated donors. The 13271 transfectants were also used as targets for clonal and polyclonal NK cells in the presence and absence of human serum, to measure inhibition of ADCC. RESULTS: Expression of Cw3-wt in 13271 cells significantly increased the human CTL response compared with the empty-vector control transfectant, whereas no significant increase resulted from expression of CD8-interaction mutant Cw3-D227K molecules. The Cw3-D227K mutant was indistinguishable from Cw3-wt in its ability to inhibit both natural cytotoxicity and ADCC mediated by human NK clones that have the appropriate CD158b inhibitory receptor. CONCLUSIONS: Transgenic expression of HLA molecules in pig cells will likely amplify the CD8+ CTL response against the xenograft. Disruption of HLA-CD8 interaction could minimize this amplification without compromising NK-cell inhibition.     

High sequence homology between human and pig CD40 with conserved binding to human CD154

BACKGROUND: Understanding the molecular interactions between pig tissues and human immune cells is fundamental to achieving long-term pig to human xenograft survival. CD40 has been shown to be central in the interaction of T cells with many antigen-presenting cells including B cells, and dendritic cells. It has been clearly shown in vitro that human T cells can effectively recognize pig major histocompatibility complex proteins, and that various accessory molecule interactions are compatible between these species, including human CD28 with pig B7 family members (CD80/CD86). The importance of CD40 in transplantation has been established using blocking antibodies to its ligand, CD154, which prolong allograft survival in mouse and primate models. METHODS: Pig CD40 was cloned from a porcine spleen cDNA library and subsequently sequenced. Expression of pig CD40 was detected by flow cytometry using soluble human CD154 (hCD154-Ig). Results. Comparison of the derived amino acid sequence of pig with human shows 74% identity. Significantly, there is conservation between pig and human at 5 residues shown by mutagenesis studies to be essential for binding of human CD40 to CD154. hCD154Ckappa was shown to bind pig B cell lines and a proportion of human and pig lymphocytes and further confirmed by staining of COS cells transfected with pig CD40. Conclusions. Recipient human cells bearing CD154 will, therefore, be able to bind donor pig CD40, and these interactions might modulate effector functions and hence influence xenograft survival. Further investigation is necessary to ascertain the exact nature of these interactions and their implications for xenograft survival.     

Immortalized bone-marrow derived pig endothelial cells

Abstract: Primary cultures of porcine endothelial cells (EC) can only be maintained for a limited number of passages. To facilitate studies of xenogeneic human anti-pig immune responses in vitro, pig microvascular bone-marrow (BM) and macrovascular aortic EC were obtained from our herd of partially inbred miniature swine, homozygous for the major histocompatibility locus, and immortalized with a modified SV40 large T vector. The resulting BM-derived (2A2) and aortic (PEDSV.15) immortalized EC lines showed unlimited growth and EC phenotype as indicated by expression of von Willebrand Factor (vWF) and low density lipoprotein (LDL) receptors as well as by formation of typical cobblestone monolayers. Ultrastructural studies revealed morphological similarities in primary and immortalized EC. Flow cytometry analysis demonstrated constitutive SLA class I expression by all lines whereas SLA class II was only expressed after stimulation with porcine IFNγ. Furthermore, pig CD34 mRNA was detected by Northern blot analysis in primary and immortalized aortic EC but not in 2A2. Both EC lines expressed a number of myeloid markers, adhesion molecules and xeno-antigens, the latter being determined by binding of human natural antibodies. Gene transfer into the porcine EC lines was successfully performed by electroporation or calcium-phosphate transfection, as well as by adenoviral infection. Finally, the functional similarity between primary and immortalized EC was demonstrated in adhesion and cytotoxicity assays. Together, these results suggest that 2A2 and PEDSV.15 represent valuable tools to study both human cellular and humoral immune responses in vitro against pig EC derived from microvascular and large vessels.     

Alloantibody and xenoantibody cross-reactivity in transplantation

The recent availability of pigs homozygous for alpha1,3-galactosyltransferase gene knockout, and improved immunosuppressive regimens that prevent an elicited antibody response, are expected to contribute to significantly increased survival of pig organs transplanted into primates, bringing clinical trials of xenotransplantation closer. Patients highly sensitized to human leukocyte antigens, who may be precluded from obtaining a human donor organ, would be one group that might benefit from xenotransplantation. However, there have been few studies on whether there is cross-reactivity of anti-human leukocyte antigen antibodies with pig antigens. What data there are suggest that such cross-reactivity exists and that this may be detrimental to the outcome after transplantation of a pig organ. Neither is it known whether sensitization after a pig xenograft would preclude subsequent allotransplantation, although the data available suggest that this will not be the case. Further investigation on allo- and xenoantibody cross-reactivity is required.     

Cross-species costimulation: relative contributions of CD80, CD86, and CD40

BACKGROUND: The response of human CD4+ T cells against porcine cells is of comparable magnitude to that induced by human leukocyte antigen-mismatched allogeneic cells. This reflects productive interactions between key costimulatory molecules across the species barrier. Inhibition of these molecular interactions will be crucial in overcoming CD4+ T-cell-mediated rejection of xenografts. We have performed a detailed investigation to determine the expression profiles and relative contributions of the three key costimulatory molecules in the porcine-human xenogeneic response. Whereas only porcine CD86 is constitutively expressed on resting endothelial cells, both CD40 and CD80 are rapidly expressed after activation. All three costimulatory molecules are expressed by professional antigen-presenting cells. METHODS: We have isolated full-length cDNA clones for human and porcine CD80, CD86, and CD40. Human fibroblast cell lines (M1) coexpressing DR1 were transfected with these cDNAs and used in mixed lymphocyte reactions and flow cytometric studies in vitro. RESULTS: These data provide the first characterization of the expression profile and functional role of porcine CD80. Functional assays demonstrate that pCD40, pCD80, and pCD86 are independently capable of costimulating human CD4+ T cells, albeit with differing kinetics. Proliferative responses were of comparable magnitude to those obtained when costimulation was provided by human CD40, CD80, and CD86. CONCLUSIONS: These data have implications for therapy targeting the direct pathway of xenorecognition; costimulatory molecule blockade must be directed against both the B7/CD28 and CD40/CD40L pathways.     

Porcine endothelial cell activation in hDAF pig hearts transplanted in baboons with prolonged survival and lack of rejection

Depletion of anti-alphaGal antibodies before and after transplantation with GAS 914, a polylysine containing alphaGal epitopes, together with immunosuppression, has been shown to prevent acute humoral xenograft rejection (AHXR) in hDAF pig-to-baboon xenotransplantation. This therapy was associated with low levels of serum anti-alphaGal antibodies and lack of antipig hemolytic antibodies (APA) during the entire transplant course. In the present study we investigated the condition of xenograft endothelial cells and the presence of other antipig antibodies. No xenograft failed because of AHXR. However, endothelial cell markers of activation, such as CD62, CD106, ET-1, and particularly 5A6/8, were detected at necropsy, along with a lack or scarce deposits of IgM and total absence of complement and fibrin. The endothelial cell markers were negative or slightly positive at biopsy obtained 30 minutes after transplantation. At the time of animal death serum xenoantibodies against pig aortic cells were also detected by immunochemistry whereas anti-alphaGal and APHA were almost absent, suggesting that the presence of non-anti-alphaGal and noncytotoxic xenoantibodies may cause endothelial activation.