Characterization of xenoreactive natural secreting cells in rats

Abstract: IgM xenoreactive natural antibodies (XNA) and the activation of complement are thought to be the main components involved in the hyperacute rejection (HAR) of discordant xenografts. Nevertheless few studies have focused on the B cell population responsible for the production of XNA. Using the guinea pig to rat discordant xenograft model we have developed a xenoreactive ELISA-spot technique allowing the specific detection of XNA secreting cells in rats. In order to detect rat XNA-secreting cells, guinea pig platelet membrane extracts, which have been shown to share major xenoantigens as recognized by XNA, with endothelial cells, were used as targets for rat XNA. Adult LOU/C rats lymphocytes from spleen, mesenteric lymph nodes, Peyer's patches, bone marrow, and the peritoneal cavity were used as source of XNA secreting cells. The number of IgM, IgG2a, Ig k light chain XNA, and IgM XNA secreting cells was measured using standard and xenoreactive ELISA-spot techniques respectively. Data presented here show that IgM XNA secreting cells are almost exclusively located in the spleen. Moreover, IgM XNA secreting cells represent around 0.0001% of spleen lymphocytes corresponding to 0.1% of the IgM secreting cells in the spleen. Although circulating IgG2a XNA are detectable, by cellular ELISA, in normal LOU/C rats, IgG2a secreting cells were not found in any of the cell suspensions analyzed. In contrast, Ig k light chain XNA were found in the spleen, mesenteric lymph nodes, and the bone marrow but not in the peritoneal cavity. Taken together these results suggest that extremely low numbers of secreting cells producing isotypes other than IgM and IgG2a might exist in different rat organs but are not detectable using the xenoreactive ELISA-spot technique. In conclusion, our data show that only 0.0001% of spleen lymphocytes were found to be IgM XNA secreting cells. Moreover this IgM XNA secreting cells population is located almost exclusively in the spleen further illustrating the role of the spleen in the production of XNA.     

Immunoselection of human lymphocytes producing xenoreactive natural antibodies against Galocl,3Gal terminal residues

Abstract: In the pig-to-human xenograft combination, human xenoreactive natural antibodies (XNA) bind to carbohydrate moieties, especially those with Galα1,3Gal terminal residues, expressed on the surface of most cells. The aim of this study was to select the cells that produce XNA by functionally characterizing a subset of cells for future studies on immunosuppression of XNA formation. We especially addressed the question whether XNA could be produced by CD5+ B lymphocytes, a subset of cells producing antibodies of high connectivity and polyreactivity, possibly involved in autoimmune processes. For that purpose we used porcine thyroglobulin (PTg), which expresses several Galα,3Gal terminal residues, for the immunoselection of XNA-producing cells. On FACS analysis, biotinylated PTg appeared to react with 5% of the B lymphocytes but the proportion of CD5+ B lymphocytes was not enriched in the PTg-reactive population. Similarly, magnetic beads coated with PTg were used to sort lymphocytes with Ig receptors recognizing PTg. Two and one-half percent of cells reacted, mainly B lymphocytes (89%), but they were not enriched for CD5+ B lymphocytes. When PTg-reactive lymphocytes were cultivated in presence of irradiated T cells and stimulated with PWM, the synthesis of Galα1,3Gal reactive antibodies, mainly of the IgG class, was demonstrated. The results of this study suggest that a high percentage of B lymphocytes react with Galα1,3Gal terminal residues. Such XNA-producing cells are not particularly common in the CD5+ subset.     

Human xenoreactive natural antibodies of the IgM isotype activate pig endothelial cells

Abstract: Preformed, xenoreactive natural antibodies (XNA) and complement (C) are involved in the initiation of vascular rejection of organs transplanted between discordant species, presumably by stimulating donor organ endothelial cells (EC). Although C is known to play a role in the activation of EC, it has not been clear whether the antibodies serve only to anchor the initial components of C, and thus permit the C cascade to proceed, or whether the antibodies themselves deliver a signal to the EC. We have tested affinity-purified human IgM containing XNA (IgM-XNA) for its ability to stimulate in vitro the up-regulation of genes in pig EC. Northern blot analysis shows that IgM, which contains XNA, stimulates mRNA accumulation for certain genes (including IL-8, PAI-1, and ECI-7, a new gene that we have found is associated with EC activation), but not others known to be up-regulated in response to TNF, IL-1 or LPS. Our results show that XNA provide a signal to EC, and thus may themselves participate in activation of EC and consequent vascular rejection.     

Human IgM xenoreactive natural antibodies can induce resistance of porcine endothelial cells to complement-mediated injury

Abstract: It is thought that human IgM xenoreactive natural antibodies (nAbs) can induce activation of porcine endothelial cells independent of complement. Therefore we hypothesized that pretreatment of porcine endothelial cells with anti-pig nAbs may affect the ability of the endothelial cells, when subsequently incubated with a source of nAbs and complement, to bind antibodies and complement components and to undergo complement-mediated cytotoxicity. We preincubated porcine endothelial cells at 37°C for 1 hr or 40 hr with a source of nAbs. We then incubated these pretreated endothelial cells with a complement source that contained a normal complement level and a low level of IgM nAbs for 1 hr to measure bound IgM and IgG and complement components, and for 4 hr to measure cytotoxicity. We found that preincubation for as long as 40 hr did not impair the binding of IgM and IgG, implying no antibody-induced loss of membrane antigens from the endothelial cells, or the binding of C3bi, C4d, C6, and C9 upon complement activation. In contrast, preincubation for 40 hr with a nAb source induced in the endothelial cells marked resistance to complement-mediated killing. Resistance could be induced with purified human IgM but not with purified IgG or IgM-depleted human serum. The ability of purified IgM to induce resistance was abrogated by removal of anti-pig xenoreactive nAbs by absorption with pig endothelial cells, and induction of resistance required protein synthesis. We conclude that prolonged incubation of human anti-pig nAbs with pig endothelial cells does not cause loss of endothelial cell membrane antigens or impairment in binding of nAbs or complement components; instead, it induces marked resistance to complement-mediated cytotoxicity. These observations may be of value to develop strategies that enhance survival of a xenograft.     

Endothelial cell antigens recognized by xenoreactive human natural antibodies.

Hyperacute rejection of vascularized, discordant xenografts is generally though to be initiated when natural antibodies of the recipient bind to endothelial cells of the donor organ. While rejection of such xenografts always occurs, the molecular targets of natural antibodies have not been elucidated. The aim of the experiments reported herein was to identify the molecules on porcine endothelial cells that would be recognized by human natural antibodies if a porcine organ were to be transplanted into a human (or rhesus). Toward the end, it was shown that the major components recognized by human serum on porcine endothelial cells are glycoproteins of 115kDa, 125kDa, and 135kDa (gp115/135). Reactivity with these glycoproteins was abrogated by enzymatic cleavage of N-linked oligosaccharides or of subterminal beta-D-gal residues suggesting that the determinants are located on oligosaccharides rather than on the polypeptide cores. The biological relevance of gp115/135 was suggested by experiments in which a similar series of components was shown to be recognized by rhesus natural antibodies and by the absorption of such antibodies by perfusion of porcine kidneys. The gp115/135 antigens were present on porcine platelets but not porcine RBC or lymphocytes. Nevertheless, purified RBC and lymphocytes absorbed human anti-gp115/135, suggesting that human natural antibodies recognize the same or crossreactive carbohydrate determinants expressed on the surface of a variety of cells.     

Polyreactivity and antigen specificity of human xenoreactive monoclonal and serum natural antibodies

Naturally occurring antibodies that react with xenogeneic antigens are a clinically important subset of antibodies because they initiate hyperacute rejection of organs transplanted between disparate species. This currently precludes the use of nonprimate organs for human transplantation. Most antibodies that arise after immunization are monoreactive, i.e., bind only to the immunogen. Similarly, some "natural" antibodies, e.g., isohemagglutinins, bind in a monoreactive manner. In contrast, other natural antibodies, e.g., those that bind to actin, are polyreactive (i.e., bind to multiple ligands). Such polyreactive antibodies may be derived predominantly from CD5+ B cells. In this study, we demonstrate that the majority of xenoreactive natural antibodies in human serum are polyreactive, as indicated by the ability of ssDNA and thyroglobulin (ligands commonly used as targets of polyreactive antibodies) to block the binding of the antibodies to xenogeneic antigens, whereas these ligands could not block the binding of antitetanus antibodies to tetanus toxoid. Furthermore, we compared the ability of 8 polyreactive and 7 monoreactive human mAb to bind to porcine antigens. All of the polyreactive mAb reacted with porcine antigens at mAb concentrations less than 3 micrograms/ml, while none of the monoreactive mAb reacted at concentrations less than 3 micrograms/ml. Each polyreactive mAb reacted with partially overlapping, but distinct sets of porcine cell surface moieties. These results indicate that human polyreactive mAb can bind to multiple xenogeneic antigens in a selective manner and that xenoreactive natural antibodies in human serum are largely polyreactive.     

Role of platelet-activating factor in functional alterations induced by xenoreactive antibodies in porcine endothelial cells

BACKGROUND: Platelet-activating factor (PAF) is a phospholipid mediator of inflammation which has been implicated in rejection. The interaction of anti-alpha-galactosyl natural antibodies (anti-alpha gal Abs) with endothelial cells is the initial step for the development of xenograft rejection. In our study, we stimulated porcine aortic endothelial cells (PAEC) with anti-alpha gal IgG to investigate the synthesis of PAF from PAEC and its biological consequences. METHODS AND RESULTS: PAF was extracted and chromatographically purified from cultured PAEC stimulated with baboon anti-alpha gal Abs. The Abs induced a dose-dependent synthesis of PAF peaking after 30 min of incubation, and decreasing thereafter. Concomitant cell shape change, motility, and cytoskeleton redistribution were observed. These events were prevented by addition of a panel of PAF-receptor antagonists. An SV40 T-large antigen-immortalized PAEC line was engineered to express PAF acetyl-hydrolase (PAF-AH) cDNA, the major PAF-inactivating enzyme. These transfected cells exposed to anti-alpha gal Abs showed reduced cell contraction and motility compared with empty vector-transfected cells. Moreover, in PAEC stimulated with anti-alpha gal Abs, the synthesis of PAF promoted the adhesion of a monocytic cell line as shown by the inhibitory effect of PAF-receptor antagonists and of PAF-AH expression. Finally, studies on cell monolayer demonstrated an enhanced permeability 48 hr after exposure to anti-alpha gal Abs, and this increase was prevented by PAF-inactivation and by PAF-receptor blockade. CONCLUSIONS: These results demonstrate that on stimulation with anti-alpha gal Abs, PAEC synthetize PAF which can contribute to several vascular events involved in xenograft rejection.     

Differential effects of injections of anti-mu and anti-delta monoclonal antibodies on B-cell populations in adult mice: regulation of xenoreactive natural antibody-producing cells

BACKGROUND: The depletion of differential B cell and xenoreactive natural antibodies (XNA) by anti-delta and anti-mu injections was analyzed in adult mice. Sequential treatment with anti-delta and then anti-mu induces a complete depletion of B cells and XNA and represents a potential approach to induce xenograft tolerance. METHODS: Adult mice were injected with anti-mu, anti-delta, anti-delta then anti-mu, or control isotype monoclonal antibodies from day 0 to day 14. The different B-cell populations were analyzed by FACS and immunohistology. Ig production was tested by ELISA. XNA were analyzed by FACS. RESULTS: Anti-mu injections induced a depletion of IgMhigh, immature B cells, marginal zone B cells, and B1 cells and an increase of IgG-XNA production. Anti-delta injections induced mature conventional IgDhigh B-cell depletion and increased IgM-XNA production. Interestingly, sequential injections of anti-delta then anti-mu induced a depletion of immature B cells, mature B cells (MZ, B2, and B1), and XNA. CONCLUSIONS: These results demonstrate that mature B-cell depletion in adult mice can be obtained by mAb injections and depends on the surface immunoglobulin cross-linking threshold. Indeed, anti-mu mAb depleted IgMhigh B cells (MZ and B1) and anti-delta, IgDhigh B cells (B2). The differential B-cell suppression shows that conventional B cells are responsible in the IgG-XNA production and MZ and B1 cells in the IgM-XNA production. Sequential repeated injections of anti-delta then anti-mu mAb depleted all B-cell populations and suppressed the whole XNA production.     

Acute vascular rejection of xenografts: roles of natural and elicited xenoreactive antibodies in activation of vascular endothelial cells and induction of procoagulant activity

BACKGROUND: Hyperacute rejection of vascularized discordant xenografts can now be effectively managed. However, acute vascular rejection (AVR) then ensues, resulting in graft destruction, coagulopathy, or both within weeks. The aim of this study was to determine associations between humoral responses to the xenograft and the induction of AVR, coagulopathy, or both. METHODS: In vitro, heat-inactivated, naive or sensitized baboon sera containing xenoreactive natural or elicited antibodies were used to activate porcine aortic endothelial cells (PAEC) in vitro. Tissue factor expression on PAEC was determined as an index of heightened procoagulant activity. In vivo, porcine renal xenografts were transplanted into immunosuppressed baboons, and at the time of rejection or the development of a consumptive coagulopathy, biopsy specimens were obtained for studies of xenoreactive antibody binding and tissue factor expression. RESULTS: In vitro, incubation of PAEC with naive baboon sera containing natural anti-Galalpha1,3Gal (Gal) antibodies resulted in minimal tissue factor induction; the addition of complement boosted procoagulant responses. Elicited xenoreactive antibodies, and to non-Gal epitopes alone, induced high amounts of procoagulant activity on PAEC; the addition of complement resulted in overt cytotoxicity. In vivo, AVR was associated with xenoreactive antibody deposition in the graft. When vascular endothelial binding of xenoreactive antibody was combined with the expression of tissue factor, consumptive coagulopathy developed irrespective of histopathologic features of AVR. CONCLUSIONS: Our in vitro results indicate that elicited antibodies, potentially to non-Gal epitopes, induce endothelial cell activation and tissue factor expression; in vivo, a consumptive coagulopathy occurred when there was xenoreactive antibody deposition and increase of tissue factor.     

Removal of xenoreactive antibodies by protein-A immunoadsorption: experience in 22 patients

Abstract: The presence of naturally occurring anti‐Galα1–3Gal (antiαGal) Ab in human serum is believed to be a major factor in the pathogenesis of hyperacute rejection of discordant organ xenografts such as the pig‐to‐human combination. Galα1–3Gal epitopes are expressed on pig tissues and the binding of anti‐Galα1–3Gal leads to endothelial cell activation and complement‐mediated hyperacute graft rejection. Several strategies have been suggested in donor animals or in the xenograft recipient to overcome the anti‐αGal barrier. Protein‐A immunoadsorption (PAIA) was developed for the in vivo removal of circulating Ab and it has been shown to be effective in cases where pathogenic auto or alloAb are present. The aim of our study was to analyze the effect of PAIA on total and xenoreactive serum anti‐αGal immunoglobulin levels in a group of patients treated with this technique for different diseases. After three consecutive sessions of PAIA, total and xenoreactive IgG and IgM immunoglobulin levels were decreased by more than 50% of pre‐treatment levels. So we conclude that PAIA is an effective method to significantly reduce circulating Ab, including xenogeneic IgM and IgG Ab. This mode of therapy might be considered as a tool to overcome hyperacute xenograft rejection. PAIA combined with other therapeutic approaches may well protect the xenograft.     

Characterization of circulating osteoblast lineage cells in humans

We recently identified circulating osteoblastic cells using antibodies to osteocalcin (OCN) or alkaline phosphatase (AP). We now provide a more detailed characterization of these cells. Specifically, we demonstrate that 46% of OCN positive (OCN(pos)) cells express AP, and 37% also express the hematopoietic/endothelial marker CD34. Using two different anti-OCN antibodies and forward/side light scatter characteristics by flow cytometry, we find that OCN(pos) cells consist of two distinct populations: one population exhibits low forward/side scatter, consistent with a small cell phenotype with low granularity, and a second population has higher forward/side scatter (larger and more granular cell). The smaller, low granularity population also co-expresses CD34, whereas the larger, more granular cells are CD34 negative. Using samples from 26 male subjects aged 28 to 68 years, we demonstrate that the concentration of circulating OCN(pos) cells increases as a function of age (R=0.59, P=0.002). By contrast, CD34(pos) cells tend to decrease with age (R=-0.31, P=0.18); as a consequence, the ratio of OCN(pos):CD34(pos) cells also increase significantly with age (R=0.54, P=0.022). These findings suggest significant overlap between circulating cells expressing OCN and those expressing the hematopoietic/endothelial marker CD34. Further studies are needed to define the precise role of circulating OCN(pos) cells not only in bone remodeling but rather also potentially in the response to vascular injury.     

Characterization of B cell antibodies in kidney transplant recipients

Antibodies against B lymphocytes were found in the serum of the majority of 59 kidney transplant recipients and of 22 eluates obtained from kidney allografts undergoing rejection. To characterize these B cell lymphocytotoxins we have used a mouse monoclonal anti-DR antibody (L227) that inhibits cytotoxicity of antibodies against HLA-DR antigens and a chicken serum against human Ia-like antigens that also inhibits antibodies against DR-related supertypic determinants and other Class II histocompatibility antigens. Three types of B cell cytotoxins were defined: antibodies against HLA-DR, antibodies against Ia-like antigens other than DR, and antibodies against non-Ia-related B cell antigens. Before transplantation, B cell antibodies were detected in about a third of the patients studied. They were inhibited by monoclonal anti-DR more often in recipients who ultimately rejected a kidney allograft (67%) than in those in whom the graft was successful (44%, P less than 0.03). After transplantation, antibodies inhibited by L227 were found in 56% of the patients with functioning grafts and in 94% of the recipients whose grafts had been removed because of rejection (P less than 0.001). B cell antibodies inhibited by monoclonal anti-DR were found in the majority of kidney eluates. However, although 85% of the B cell reactions of kidney eluates were blocked by this antibody, only 55% of the B cell reactions of sera obtained from the same recipients after nephrectomy were similarly inhibited. Thus it appears that antibodies against HLA-DR were bound and concentrated in the transplanted organ and other B cell antibodies were not. These results indicate that anti-DR antibodies blocked by the monoclonal antibody L227 are the most common type of B cell lymphocytotoxins formed in kidney transplant recipients. Their role in kidney allografts undergoing rejection, where they are bound in high concentration, needs to be determined.     

Characterization of natural human anti-non-gal antibodies and their effect on activation of porcine gal-deficient endothelial cells

BACKGROUND: The generation of Galalpha1-3Gal (Gal) transferase deficient pigs has increased the interest in non-Gal antigens potentially representing important targets for xenoreactive antibody binding leading to xenograft rejection. The present study addressed the levels and immunoglobulin isotypes of preformed human anti-non-Gal antibodies and their potential to activate porcine endothelial cells. METHODS: Porcine endothelial cells lacking the Gal epitope (Gal-/-) were used to measure immunoglobulin (Ig) M and IgG subclass anti-non-Gal antibodies, using sera from 80 blood donors and pooled human AB serum. Antibodies specific for the non-Gal Hanganutziu-Deicher (HD) xenoantigen were measured by enzyme-linked immunosorbent assay. Activation of Gal-/- and Gal+/+ endothelial cells by human serum was measured, in the presence or absence of complement inhibitors, by E-selectin cell-surface expression using flow cytometry. RESULTS: Anti-non-Gal antibody levels varied considerably among individual sera and comprised approximately 10% of total anti-porcine antibodies without sex or age differences. Among the IgG subclasses only IgG1 and IgG2 were detected. Human serum-induced E-selectin expression on Gal-/- cells was less than 20% compared with Gal+/+ cells, correlated with anti-HD IgM and IgG antibody levels (P=0.027 and 0.032, respectively), and was largely complement-independent in accordance with the lack of IgG3 anti-non-Gal antibodies. In contrast, E-selectin upregulation on Gal+/+ cells was reduced in complement blocking experiments. CONCLUSION: Preformed anti-non-Gal antibodies, in particular anti-HD antibodies, were present in all human sera samples, activated porcine endothelial cells, and may therefore play a role in xenograft rejection using organs from GalT-/- pigs.     

异种反应性天然抗体在豚鼠至大鼠肝移植中的作用

目的　研究异种反应性天然抗体 (XNA)在豚鼠至大鼠异种肝移植超急性排斥反应(HAR)中的作用。方法　将实验鼠随机分成A、B、C、D组 ,每组 2 0只 ,分别为对照组、术前输注豚鼠肝细胞 (HC)组、术前连续肌注山地明 (CsA)组和术前输注HC合用CsA组。采用流式细胞仪和免疫组织化学方法检测受体体内XNA含量 ,观察了受体存活时间和移植肝组织学改变。结果　移植肝组织发生了HAR。受体体内存在XNA ,以IgM为主。术前输注HC使受体体内的抗体明显升高 ,A组IgM(单位 :平均荧光强度 ,下同 )为 74.58± 31 .75 ,B组为 40 6 .42± 1 0 8.0 2 (P <0 .0 1 ) ,而使用CsA能预防抗体爆发反应 ,C组为 48.82± 1 1 .0 4 (同B组比较 ,P <0 .0 1 )。术前输注豚鼠肝细胞合并使用CsA能延长受体存活时间 ,A组为 (1 2 4 .1 0± 33 .42 )min,D组为 (1 83 .70± 2 6 .85)min(P <0 .0 1 )。移植肝表现为肝细胞水样变性 ,肝血窦和血管扩张瘀血 ,但小叶结构完整。结论在豚鼠至大鼠异种肝移植中发生的HAR是一种强烈的免疫反应 ,其中XNA所起作用有限 ;术前输注豚鼠肝细胞合并使用CsA能延长受体存活时间     

Characterization of anti-Gal antibody-producing cells of baboons and humans

BACKGROUND: Anti-Gal antibodies cause hyperacute and delayed xenograft rejection in pig-to-primate transplantation. The cell populations producing anti-Gal and other natural antibodies in primates are unknown. METHODS: Cells from different lymphoid compartments of na?ve or sensitized baboons were examined for anti-Gal and total Ig production by ELISPOT. B and plasma cells from humans and baboons were purified by FACS sorting and characterized for anti-Gal and total Ig production and cytology. RESULTS: In na?ve baboons, the spleen was the major source of anti-Gal IgM-secreting cells. Two months after sensitization with porcine tissues, high frequencies of anti-Gal IgM- and IgG-secreting cells were detected in the spleen, lymph nodes, and bone marrow. Six months after antigen exposure, anti-Gal IgM- and IgG-secreting cells were preferentially localized in the bone marrow. Cells from human spleen, bone marrow, and blood were also analyzed and anti-Gal IgM-secreting cells were detected mainly in the spleen. Sorting of baboon and human cells showed that anti-Gal IgM-secreting cells were mainly splenic B cells (CD20+, CD138-, and Ig+). Although low in percentage, sorted CD20-CD138+ plasma cells in spleen and bone marrow secreted large quantities of anti-Gal IgM. Most anti-Gal IgG-secreting cells were plasma cells (CD138+) at both early (Ig+) and late (Ig-) stages of differentiation. CONCLUSIONS: Similar to Gal knockout mice, natural anti-Gal IgM antibodies in primates are produced mainly by splenic B cells. After antigen exposure, anti-Gal IgM and IgG were secreted by both B and plasma cells. These results suggest strategies to remove xenoreactive antibody-secreting cells prior to transplantation.     

Identification of porcine membrane antigens involved in the cytotoxic response mediated by human xenoreactive antibodies

Abstract: The identification of xenoantigens on the surface of endothelial cells is important for understanding the mechanism of hyperacute rejection and development of abrogating methods. The objective of our study was to identify the porcine antigens that, when bound by xenoreactive antibodies in human serum, result in cytotoxicity of porcine cells. Human AB and O sera were adsorbed with porcine aortae, erythrocytes, platelets, and a broad spectrum of immobilized carbohydrate moieties (Synsorbs). Aortae and erythrocytes were able to adsorb the xenoreactive antibodies that were cytotoxic to porcine cells (LLC-PK1), determined using an MTT cytotoxicity assay. Only carbohydrates having the αGal(1–3)βGal(1–4) moieties (Synsorbs 90, 115) were able to significantly reduce cytotoxicity with both types of sera. Western blots of porcine aortic endothelial cells (PAEC) and LLC-PK1 cell membrane extracts probed with unadsorbed sera indicate the binding of xenoreactive IgM to approximately 17 and 11 antigen bands, respectively, having molecular weights ranging from 20–133 kDa. Anti-IgG development showed 8 and 11 antigen bands on PAEC and LLC-PK1 membrane preparations, respectively. When blots were performed using adsorbed AB sera, the binding to all antigens was still observed. When Synsorb 90 bound antibodies were used to probe the blots, the majority of antigens were still detected. This suggests that the binding of xenoantibodies to the most prominent antigens, as detected by Western blot procedures may not be the ones to which cytotoxic xenoreactive antibodies bind. Alternative approaches are required to identify such antigens.     

Characterization of porcine endothelial cell determinants recognized by human natural antibodies

Abstract: Organs transplanted from pigs to primates are subject to hyperacute rejection. This immunologic reaction is initiated by the recipient's natural antibodies that bind to endothelial cell antigens of the organ, resulting in the activation of the complement system and rapid destruction of the graft. Various lines of evidence, particularly blocking studies, using purified carbohydrates have suggested that the endothelial cell determinant recognized by human natural antibodies is a terminal galactose in an α configuration (α-Gal). Although these studies are compelling, they fall short of proof because xenoreactive natural antibodies, being polyreactive, might bind to structures other than those used for blocking. Moreover, in vivo evidence that anti-α-Gal antibodies participate in hyperacute rejection has not been reported. Here we report that an enzyme specific for α-Gal, α-galactosidase, removes α-galactose residues from both intact porcine aortic endothelial cells and immobilized porcine aortic endothelial cell membrane extracts and that as a result of this, xenoreactive natural antibody binding is lowered by 70 to 80%. This decrease in binding of human IgM causes a corresponding decrease in complement activation. Similar results were obtained using the sera of other Old World primates. Digestion of immobilized porcine aortic endothelial cell membrane extracts with α-galactosidase produced a similar reduction in human IgM binding to gp 115/135. These results indicate that a terminal α-galactose is an important component of the gp 115/135 porcine endothelial cell antigen. We also report that perfusion of porcine organs I with primate serum removes anti-α-Gal IgM from the serum.     

Evidence for polyreactive xenoreactive antibodies in the repertoire of human anti-swine antibodies: the 'next' humoral barrier to xenotransplantation?

The xenoreactive nature of anti-Galalpha1-3Gal antibodies, and to a lesser extent, polyreactive antibodies, has been characterized by a number of investigators. With the advent of therapies that avoid hyperacute xenograft rejection due to anti-Galalpha1-3Gal antibodies coupled with the possible development of Galalpha1-3Gal deficient swine, the Galalpha1-3Gal antigen may soon cease to be a barrier to xenotransplantation. With this in mind, the potential xenoreactive nature of polyreactive antibodies was investigated using several approaches. The levels of polyreactive antibodies from the serum of newborn (n = 2) and adult (n = 4) baboons undergoing pulmonary xenotransplantation were evaluated. Depletion of 95% and 94% of total serum IgM, without any decrease in albumin levels, was observed in the newborn baboons. This finding indicates that the IgM present at birth and germ line polyreactive IgM was adsorbed by the xenografts. The depletion of polyreactive antibodies (43-83% reduction of anti-DNP IgM) from adult baboons was also observed following pulmonary xenotransplantation or immunoadsorption therapy plus pulmonary xenotransplantation. Additional experiments using human cord serum indicated that most human polyreactive IgM were adsorbed by pig lung homogenate and that the human polyreactive IgM bound approximately two-fold more to immobilized pig lung antigens than to immobilized human lung antigens. These findings indicate that germline polyreactive antibodies are, for the most part, xenoreactive. These data suggest that polyreactive antibodies, although autoreactive, may be more xenoreactive than autoreactive.