Xenotransplantation and other means of organ replacement.

Exciting new technologies, such as cellular transplantation, organogenesis and xenotransplantation, are thought to be promising approaches for the treatment of human disease. The feasibility of applying these technologies, however, might be limited by biological and immunological hurdles. Here, we consider whether, and how, xenotransplantation and various other technologies might be applied in future efforts to replace or supplement the function of human organs and tissues.

Cryptic B cell response to renal transplantation.

Transplantation reliably evokes allo-specific B cell and T cell responses in mice. Yet, human recipients of kidney transplants with normal function usually exhibit little or no antibody specific for the transplant donor during the early weeks and months after transplantation. Indeed, the absence of antidonor antibodies is taken to reflect effective immunosuppressive therapy and to predict a favorable outcome. Whether the absence of donor-specific antibodies reflects absence of a B cell response to the donor, tolerance to the donor or immunity masked by binding of donor-specific antibodies to the graft is not known. To distinguish between these possibilities, we devised a novel ELISPOT, using cultured donor, recipient and third-party fibroblasts as targets. We enumerated donor-specific antibody-secreting cells in the blood of nine renal allograft recipients with normal kidney function before and after transplantation. Although none of the nine subjects had detectable donor-specific antibodies before or after transplantation, all exhibited increases in the frequency of donor-specific antibody-secreting cells eight weeks after transplantation. The responses were directed against the donor HLA-class I antigens. The increase in frequency of donor-specific antibody-secreting cells after renal transplantation indicates that B cells respond specifically to the transplant donor more often than previously thought.

Human complement regulatory proteins protect swine-to-primate cardiac xenografts from humoral injury.

The susceptibility of xenografts to hyperacute rejection is postulated to reflect in part failure of complement regulatory proteins (CRPs) to control activation of heterologous complement on graft endothelium. To test this concept, transgenic swine expressing the human CRP decay accelerating factor and CD59 were developed using a novel expression system involving transfer of the proteins from erythrocytes to endothelial cells. Hearts from transgenic swine transplanted into baboons had markedly less vascular injury and functioned for prolonged periods compared to hearts from nontransgenic swine. These results indicate that expression of human CRPs in xenogeneic organs may contribute to successful xenografting and suggest that intercellular protein transfer might be a useful approach for expression of heterologous proteins in endothelial cells.

Transient perturbation of endothelial integrity induced by natural antibodies and complement.

The barrier function of blood vessels is though to be regulated at least in part by endothelium. This concept is supported by the dramatic loss of barrier function occurring in the hyperacute rejection of vascularized grafts mediated by anti-endothelial cell (EC) antibodies and complement. In this process, the endothelium is not destroyed but instead loses the ability to retain blood cells and plasma proteins within capillaries. The noncytotoxic mechanism that allows this change in EC function has been unknown. Here we report that within 10 to 20 min of exposure to human xenoreactive natural antibodies and complement, porcine EC undergo alterations in cell shape and in the cytoskeleton that disrupt monolayer integrity and lead to formation of intercellular gaps. Gap formation is not associated with cell death but requires the complement complex C5b67. The gaps induced by anti-EC antibodies and complement are transient; gap closure requires formation of C5b-9 complexes on the cells and the rate of recovery depends on the release of cellular products into the medium. Preincubation of EC with dibutyryl cAMP (0.5 mM) prevents gap formation and disruption of the cytoskeleton caused by antibodies and complement. These results provide evidence that the integrity of endothelium is regulated by components of the complement system and suggest a mechanism that may explain the prominent loss of endothelial integrity seen in humoral immune responses.

Stages of renal ontogenesis identified by monoclonal antibodies reactive with lymphohemopoietic differentiation antigens.

Differentiation antigens of T and B lymphocytes were sought in human fetal and adult kidney tissues with monoclonal antibodies by indirect immunofluorescence. Antibodies that identify B cells (BA-1 and anti-B1) and leukemia-associated antigens (BA-2, BA-3, and J5) reacted with renal glomerular and tubular epithelium at characteristic stages of nephron development. BA-1 and BA-2 identified primitive epithelium of the glomerulus, and ureteral bud and nephron development was characterized by loss of BA-1 and BA-2 binding by visceral glomerular and proximal tubular epithelium. In contrast, J5 and BA-3 did not react with primitive epithelium but identified visceral and proximal tubular epithelium after appearance of the glomerular basement membrane and throughout subsequent nephron differentiation. Anti-B1 reacted with ureteral bud and distal nephron epithelium in more mature fetal tissues. Monoclonal antibodies that identify populations of T cells and thymocytes did not react with parenchymal cells of fetal or adult kidneys. They did identify interstitial mononuclear cells whose size and relative numbers appeared gestationally related. Monoclonal antibodies that recognize a human monomorphic HLA-DR determinant reacted with glomerular and peritubular capillaries as early as 11 wk of gestation. The distribution and density of HLA-DR expression appeared more related to gestation than nephron development. The relationship between renal parenchymal expression of lymphohemopoietic antigens and glomerular acquisition of C3b receptor activity was determined using C3b-coated fluoresceinated Escherichia coli. In fetal tissues, C3b receptor activity appeared developmentally related to the loss of determinants recognized by BA-1 and BA-2 and to the appearance of J5 and BA-3 reactivity with visceral glomerular epithelium. Tissue binding and comparative avidity of J5 and BA-3 antibodies was studied in a series of experiments, the results of which suggest that these antibodies are directed against the same epitope or closely related epitopes of the common acute lymphoblastic leukemia antigen. The common expression of differentiation antigens and C3b receptors by cells of lymphohemopoietic lineage and renal epithelia suggests the possibility of heretofore unrecognized commonality of function or developmental experience.

Interstitial mononuclear cell populations in renal graft rejection. Identification by monoclonal antibodies in tissue sections.

The interstitial mononuclear cell populations of 22 renal grafts with interstitial rejection (IR), 6 grafts with interstitial nephritis without rejection (IN), and 5 kidneys without infiltration (3 donor kidneys, 2 grafts) were identified and quantitated by monoclonal antibodies recognizing T cells (TA-1, OKT3), helper inducer cells (OKT4), cytotoxic/suppressor cells (OKT8), B cells (BA-1), and monocytes and null cells (OKM1). Double-layer fluorochrome enhancement using F(ab')(2) reagents and nuclear counter staining with ethidium bromide enabled quantitation of the number of positive mononuclear cells, interstitial cells, and total cells on each of 30-55 microscopic fields per tissue section. T cells were the most abundant infiltrating cell in tissues with IR (35 +/- 9.8 percent), significantly higher than that seen in IN (21 +/- 16 percent) or in kidneys without infiltration (5.0 +/- 3.9 percent). The percentage of T cells identified by TA-1 or OKT3 was approximately equivalent to the summation of OKT4 plus OKT8. Although no differences were observed in the percentage of OKT4 cells, the percentage of OKT8 was significantly higher in IR (26 +/- 7.7 percent, P {less than} 10(-4)) than in IN (9.3 +/- 6.2 percent) or in kidneys with normal interstitium (3.0 +/- 2.4 percent). The ratio of OKT8/OKT4-positive T cells in 22 graft tissues with IR (3.2 +/- 1.4) was greater (P {less than} 0.0007) than 6 graft tissues with IN without rejection (0.82 +/- 0.39) and the 5 kidney tissues without interstitial infiltration (0.75 +/- 0.25). There was no significant difference between the groups in the relatively low percentage of interstitial cells identified as B cells reacting with BA-1 or containing S(IgD,M). The percentage of interstitial cells recognized by OKM1 was similar in rejection and interstitial nephritis, with both being greater than controls (P {less than} 0.02). The relative numbers of blood mononuclear cells identified by the monoclonal antibodies was generally not predictive of the proportions present in kidney tissue, although OKT4-positive blood cells were less numerous and OKMI+ blood cells were more numerous than in controls (P {less than} 0.002). Quantitative analysis of identifiable interstitial cells in graft rejection reveals that most infiltrating cells are T cells, the greater proportion of which are recognized by OKT8. OKT8-positive cells may play an important role in mediating renal graft rejection.

Complement-mediated regulation of tissue factor activity in endothelium.

Inflammation and immunity may be associated with endothelial cell (EC) injury and thrombus formation. We explored the mechanisms through which a humoral immune response directed against the endothelium might promote coagulation. Using the interaction of anti-EC antibodies and complement (C) with cultured EC as a model, we studied the expression and function of tissue factor, a cofactor for factor VIIa-mediated conversion of factor X to Xa. Exposure of EC to anti-EC antibodies and C in sublytic amounts stimulated the synthesis of tissue factor over a period of 16-42 h. Cell surface expression of tissue factor activity required activation of C and assembly of the membrane attack complex, because expression was inhibited by soluble CR1 and was not detected in the absence of C8. Elaboration of tissue factor messenger RNA was observed over a period of 8-30 h and required protein synthesis. Expression of tissue factor was not a direct consequence of the action of C on the EC but was a secondary response that required as an intermediate step the release of interleukin 1 alpha, an early product of the EC response to C activation. These findings suggest that, after the assembly of membrane attack complex on EC, the production of tissue factor and initiation of coagulation in a blood vessel depend on the production of interleukin 1 alpha and on its availability to stimulate affected EC.

Immune cell populations in cutaneous delayed-type hypersensitivity.

Delayed-type hypersensitivity (DTH) is a prototypic T lymphocyte-mediated response to antigenic challenge. In this study, mononuclear cells infiltrating the skin during cutaneous response to tuberculin in presensitized human subjects (responders) and nonimmune controls were identified using monoclonal antibodies by indirect immunofluorescence. In both responders and controls the infiltrate consisted mainly of T lymphocytes (T11+ and OKT3+) and monocytes (OKM1+, 63D3+, Mo2+) which initially accumulated in proximity to small blood vessels and later infiltrated the interstitial dermis and epidermis. More T lymphocytes reacted with OKT4 than with OKT8. 6 h after tuberculin the ratio of OKT4/OKT8 in tissue from responders exceeded that in blood, whereas in tissues studied at 15-48 h and in all control tissues those ratios in blood and tissue were similar. Evidence of T lymphocyte activation was sought using monoclonal antibodies anti-Tac, OKT9, and OKT10. In responders but not in controls the proportion of infiltrating cells reactive with these antibodies increased during the course of DTH. The presence of activated T lymphocytes in tissue was not associated with a comparable increase in peripheral blood cell populations identified by anti-Tac and OKT10. Studies using anti-B1, Leu-7, and anti-IgD/IgM revealed comparatively few reactive cells. Dual-labeling studies demonstrated that most Leu-7--reactive cells also bound T11 while fewer bound OKM1 or OKT8 and that cells reactive with OKIa1 and T11 constituted largely nonoverlapping populations. Specific patterns of reactivity were not observed when tissues were stained with anti-human C3, or poly C9-MA, a monoclonal antibody reactive with a neoantigen on polymerized C9 of the membrane attack complex of complement. The number of epidermal Langerhans cells identified by OKT6 was similar in responders and controls. Thus, the cutaneous response to tuberculin in sensitized individuals is characterized by early enrichment of the OKT4 subpopulation of T lymphocytes in tissue infiltrates and subsequent (15-48 h) evidence of T lymphocyte activation.

Release of heparan sulfate from endothelial cells. Implications for pathogenesis of hyperacute rejection.

Heparan sulfate proteoglycan associated with endothelial cells in normal blood vessels inhibits intravascular coagulation and egress of blood cells and plasma proteins, key features of hyperacute rejection. It was shown herein that exposure of cultured porcine endothelium to human serum as a source of natural antibodies and complement caused cleavage and release of 5% of endothelial cell proteoglycans within 4 min and greater than 50% within 1 h. Proteoglycan release depended on activation of the classical complement pathway and preceded irreversible cell injury. These findings suggest that loss of endothelial cell proteoglycan may be a critical step in the pathogenesis of hyperacute rejection and in diseases involving humoral injury to endothelial cells.

Differential roles of Mac-1+ cells, and CD4+ and CD8+ T lymphocytes in primary nonfunction and classic rejection of islet allografts.

The high rate of persistent hyperglycemia, termed primary nonfunction, after islet allotransplantation in C57BL/6 mice recipients of B10.BR strain islets, as compared with B10.BR recipients of C57BL/6 islets, led to a series of experiments to determine whether islet allograft primary nonfunction was attributable to technical aspects of the transplant procedure or whether it was a consequence of alloimmunity. Primary nonfunction was prevented by systemic pharmacologic immunosuppression of the host with cyclosporine. Selective immunodepletion of host CD4+ and CD8+ T lymphocytes significantly extended the time of classic rejection but did not significantly affect the rate of primary nonfunction. However, modulation of macrophages by administration to the host of silica completely abolished primary nonfunction. These observations, in conjunction with the immunohistological findings of intense macrophage infiltration in islet allografts from recipients exhibiting persistent post-transplant hyperglycemia, support the hypothesis that primary nonfunction results from a cell-mediated host-immune response of rapid onset that is dependent on macrophages or macrophage byproducts as the main effectors.

Absence of donor-specific anti-HLA antibodies after ABO-incompatible heart transplantation in infancy: altered immunity or age?

Specific B-cell tolerance toward donor blood group antigens develops in infants after ABO-incompatible heart transplantation, whereas their immune response toward protein antigens such as HLA has not been investigated. We assessed de novo HLA-antibodies in 122 patients after pediatric thoracic transplantation (28 ABO-incompatible) and 36 controls. Median age at transplantation was 1.7 years (1 day to 17.8 year) and samples were collected at median 3.48 years after transplantation. Antibodies were detected against HLA-class I in 21 patients (17.2%), class II in 18 (14.8%) and against both classes in 10 (8.2%). Using single-antigen beads, donor-specific antibodies (DSAs) were identified in six patients (all class II, one additional class I). Patients with DSAs were significantly older at time of transplantation. In patients who had undergone pretransplant cardiac surgeries, class II antibodies were more frequent, although use of homografts or mechanical heart support had no influence. DSAs were absent in ABO-incompatible recipients and class II antibodies were significantly less frequent than in children with ABO-compatible transplants. This difference was present also when comparing only children transplanted below 2 years of age. Therefore, tolerance toward the donor blood group appears to be associated with an altered response to HLA beyond age-related effects.

In vivo transfer of GPI-linked complement restriction factors from erythrocytes to the endothelium.

Many proteins are associated with the outer layer of the cell membrane through a posttranslationally added glycosyl phosphatidylinositol (GPI) anchor. The functional significance of this type of protein linkage is unclear, although it results in increased lateral mobility, sorting to the apical surface of the cell, reinsertion into cell membranes, and possibly cell signaling. Here evidence is presented that GPI-linked proteins can undergo intermembrane transfer in vivo. GPI-linked proteins expressed on the surface of transgenic mouse red blood cells were transferred in a functional form to endothelial cells in vivo. This feature of GPI linkage may be potentially useful for the delivery of therapeutic proteins to vascular endothelium.

Two novel assays of alloantibody-secreting cells demonstrating resistance to desensitization with IVIG and rATG.

Donor-specific alloantibody presents a major barrier to the successful transplantation of kidneys and hearts. However, the study of alloantibody production has been hampered by both an inadequate source of antibody-secreting cells (ASCs) and a paucity of assays to determine their function. We describe two new assays that allow for the determination of the frequency and specificities of allo-ASCs in humans using purified HLA as targets. These assays demonstrated allo-ASCs in the CD138(+) fraction of the bone marrow, but not in peripheral blood. Alloantibody specificities in these assays correlated well with those detected in the serum suggesting that bone marrow-derived ASCs are indeed a major source of alloantibody in vivo. However, ASCs for a specific HLA antigen were rare with an estimated frequency of only 1/2 x 10(6) marrow cells. Pretransplant treatment in vivo with multiple plasmaphereses and low-dose IVIG alone or in combination with rATG had no effect on ASC number or alloantibody production. These techniques allow for the study of allospecific ASCs and provide a method to test the potential efficacy of agents on alloantibody production in vivo.

Durable targeting of B-lymphocytes in living mice.

Transfer to and enduring expression of genes in B cells has proved a vexing challenge. We report here a novel method for the specific and durable targeting of B lymphocytes in living mice. The method involves generation of lentiviruses pseudotyped with an anti-CD19 antibody. CD19 targeting viruses injected in the spleen of living mice efficiently transduced B cells and plasma cells detected by flow cytometry analysis of GFP expression. Expression of the reporter gene could be detected in the intact animal by external imaging for more than a year and was enhanced by booster immunization. Our method thus enables the specific delivery, expression and localization by external imaging of exogenous genes to the B cells and plasma cells of living individuals.

Modulation of eicosanoid metabolism in endothelial cells in a xenograft model. Role of cyclooxygenase-2.

Lipid inflammatory mediators are thought to play a critical role in the pathogenesis of vascular injury. Among the events which might cause the synthesis of eicosanoids in blood vessels is activation of the complement. To evaluate how complement might influence eicosanoid metabolism, we investigated endothelial cells exposed to xenoreactive antibodies and complement, as might occur in rejecting xenografts where severe vascular injury is a typical feature. While resting porcine aortic endothelial cells released only prostaglandin (PG) I2, endothelial cells stimulated with xenoreactive antibodies and complement released PGE2 and thromboxane A2 (TXA2), in addition to increased amounts of PGI2. This alteration in eicosanoid metabolism was associated with induction of cyclooxygenase (Cox)-2 and thromboxane synthase, but not Cox-1. Unlike results seen in other systems, the upregulation of Cox-2 and the subsequent release of eicosanoids by endothelial cells was not directly induced by complement but rather required production of IL-1alpha, which acted on endothelial cells as an autocrine factor. Since eicosanoids have a potent effect on inflammation, vascular tone and platelet aggregation, we postulated that the abnormalities in eicosanoid release induced by xenoreactive antibodies and complement might provide one explanation for the vascular injury, focal ischemia, and thrombosis observed in acute vascular rejection and other vasculitides mediated by complement.

Immunoglobulin prevents complement-mediated hyperacute rejection in swine-to-primate xenotransplantation.

Immunoglobulins regulate the complement system by activating complement on foreign surfaces and diverting reactive complement proteins away from autologous cell surfaces. Based on this model, we explored the ability of Ig to balance complement activation versus control in a pig-to-primate cardiac xenotransplantation model in which the binding of xenoreactive antibodies of the recipient to graft blood vessels and the activation of complement cause hyperacute rejection. Human IgG added to human serum caused a dose-dependent decrease in deposition of iC3b, cytotoxicity, and heparan sulfate release when the serum was incubated with porcine endothelial cells. This decrease was not caused by alteration in antibody binding or consumption of complement but presumably reflected decreased formation of C3 convertase on the endothelial cells. Infusion of purified human IgG into nonhuman primates prevented hyperacute rejection of porcine hearts transplanted into the primates. As expected, the transplants contained deposits of recipient Ig and C1q but not other complement components. The inhibition of complement on endothelial cell surfaces and in the xenotransplantation model supports the idea that IgG regulates the classical complement pathway and supports therapeutic use of that agent in humoral-mediated disease.

The role of antibodies in acute vascular rejection of pig-to-baboon cardiac transplants.

Long-term success in xenotransplantation is currently hampered by acute vascular rejection. The inciting cause of acute vascular rejection is not yet known; however, a variety of observations suggest that the humoral immune response of the recipient against the donor may be involved in the pathogenesis of this process. Using a pig-to-baboon heterotopic cardiac transplant model, we examined the role of antibodies in the development of acute vascular rejection. After transplantation into baboons, hearts from transgenic pigs expressing human decay-accelerating factor and CD59 underwent acute vascular rejection leading to graft failure within 5 d; the histology was characterized by endothelial injury and fibrin thrombi. Hearts from the transgenic pigs transplanted into baboons whose circulating antibodies were depleted using antiimmunoglobulin columns (Therasorb, Unterschleisshein, Germany) did not undergo acute vascular rejection in five of six cases. Biopsies from the xenotransplants in Ig-depleted baboons revealed little or no IgM or IgG, and no histologic evidence of acute vascular rejection in the five cases. Complement activity in the baboons was within the normal range during the period of xenograft survival. In one case, acute vascular rejection of a xenotransplant occurred in a baboon in which the level of antidonor antibody rose after Ig depletion was discontinued. This study provides evidence that antibodies play a significant role in the pathogenesis of acute vascular rejection, and suggests that acute vascular rejection might be prevented or treated by therapies aimed at the humoral immune response to porcine antigens.

Xenotransplantation: recent progress and current perspectives.

A severe shortage of human transplant donors has sparked interest in the use of animals as a source of organs and tissues for transplantation. Clinical application of xenotransplantation is limited in large part by the severe immunological reaction of the recipient against the graft. This immunological reaction is mediated initially by components of natural immunity such as xenoreactive antibodies, complement and natural killer cells and later by elicited humoral and cellular immune responses which act in concert to disrupt the function of the endothelial lining of blood vessels. The past few years have brought considerable progress in elucidating the molecular and cellular basis of xenograft rejection and in developing strategies to overcome xenograft rejection.

Discordant xenografting: challenges and controversies.

The transplantation of organs between individuals of disparate species is being considered as a potential solution to a serious shortage of donor organs for clinical transplantation. We summarize recent papers concerning the immunologic barrier to xenotransplantation, the pathogenesis of hyperacute rejection and reports on efforts made to prevent or mitigate hyperacute xenograft rejection.