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Background: We previously characterized a facilitating cell (FC) in mouse marrow that enables engraftment of allogeneic hematopoietic stem cells (HSCs) without causing graft-versus-host disease (GVHD). The FC shares some cell surface molecules with T cells (Thy1+, CD3epsilon+, CD8+, CD5+, and CD2+) but is T-cell receptor (TCR) negative. Historically, depletion of CD3+ or CD8+ cells from rat marrow was associated with an increased rate of failure of engraftment. In this study, we evaluated whether depletion of alphabeta- and gammadelta-TCR(+) T cells from donor marrow would retain engraftment potential yet avoid GVHD., Methods: Wistar-Furth rats were conditioned with 950 cGy of total body irradiation and transplanted with ACI bone marrow processed to remove either alphabeta-TCR(+), gammadelta-TCR(+), or alphabeta- plus gammadelta-TCR(+) T cells. Recipients were typed for chimerism at 28 days and monthly thereafter., Results: Recipients of marrow depleted of alphabeta- (group A), gammadelta- (group B), or alphabeta- and gammadelta-TCR(+) T cells (group C) engrafted and had an average chimerism level of 73.0+/-8.3%, 92.3+/-9.2%, and 46.3+/-32.8%, respectively. Aggressive T-cell depletion did not remove the FC population (CD8+/CD3+/TCR(-)). Group A and group B both developed GVHD, with a higher incidence of GVHD in group B compared to group A. None of the recipients in group C developed GVHD., Conclusions: These data demonstrate that depletion of T cells from rat marrow does not impair engraftment of HSCs, indirectly supporting the existence of FCs in rat marrow. Moreover, donor alphabeta- and gammadelta-TCR(+) T cells contribute to GVHD in a nonredundant fashion, although alphabeta-TCR(+) T cells are more potent as the effector cells. Finally, the level of donor chimerism is influenced by the composition of the graft, because recipients of marrow that contain alphabeta-TCR(+) T cells exhibited significantly higher donor chimerism compared to recipients of marrow depleted of both alphabeta- and gammadelta-TCR(+) T cells.

Background: The clinical development of liver-support devices based on perfusion of either pig hepatocytes cartridges or whole pig livers has been hampered by the ability to use sufficient liver cell mass to provide adequate metabolic support, limited perfusion times, and the potential for patient exposure to pig zoonotic diseases., Methods: We designed an original system in which an isolated intact pig liver was perfused extracorporeally under physiological conditions in a closed loop circuit with allogeneic pig blood and constant monitoring of major physiological and functional parameters. The perfusion circuit further included an interface membrane to provide for separation of patient and liver perfusion circulation., Results: Prolonged (6-21 hr) liver perfusion did not produce significant liver damage as reflected by modest rises in the levels of the serum transaminases, stability of main biochemical parameters (including potassium), and the maintenance of normal cellular morphology. Optimal liver function was documented as measured by lactate consumption, control of glycemia, and the results of clotting studies and functional assays. The perfused liver cleared 82% and 79% of peak bilirubin and ammonia concentrations with clearing kinetics identical throughout perfusion. Indocyanine green clearance was identical to that observed in the living donor before explant surgery., Conclusions: In conclusion, the extracorporeal pig liver perfusion apparatus described here allows optimal pig liver function for prolonged periods of time. The microporous membrane to provide separation of donor organ and recipient and the high level of functional activity suggest that this form of liver metabolic support may have important clinical applications.

Background: Natural antibodies that react with galactose-alpha(1,3)galactose [galalpha(1,3)gal] carbohydrate epitopes exist in humans and Old World primates because of the inactivation of the alpha1,3-galactosyltransferase (alpha1,3GT) gene in these species and the subsequent production of antibodies to environmental microbes that express the galalpha(1,3)gal antigen. The Gal knockout (Gal o/o) mouse, produced by homologous disruption of the alpha1,3GT gene, spontaneously makes anti-galalpha(1,3)gal antibodies and can be used to study the genetic control of humoral immune responses to this carbohydrate epitope., Methods: Six hybridomas that produce monoclonal antibodies (mAbs) to galalpha(1,3)gal were generated in Gal o/o mice. The mAbs were tested to characterize the binding activity with flow cytometry using pig aortic endothelial cells and ELISA with galalpha(1,3)gal carbohydrates. The VH and VK genes of these hybridomas were cloned, sequenced, and analyzed., Results: The mAbs showed distinct patterns of antibody binding to galalpha(1,3)gal antigens. The VH genes that encode the mAb binding activity were restricted to a small number of genes expressed in their germline configuration. Four of six clones used closely related progeny of the same VH germline gene (VH441). Comparison of the mouse gene VH441 to the human gene IGHV3-11, a gene that encodes antibody activity to galalpha(1,3)gal in humans, demonstrates that these two genes share a nonrandom distribution of amino acids used at canonical binding sites within the variable regions (complimentary determining regions 1 and 2) of their immunoglobulin VH genes., Conclusions: These results demonstrate the similarity of the Gal o/o mice and humans in their immune response to galalpha(1,3)gal epitopes. Gal o/o mouse can serve as a useful model for examining the genetic control of antibody/antigen interactions associated with the humoral response to pig xenografts in humans.

Background: We have previously reported that the early phases of the immune response of rats to hamster xenografts are characterized by the production of IgM xenoantibodies encoded by a restricted group of Ig germline V(H) genes (V(H)HAR family). In the later phases of the reaction, an IgM to IgG isotype switch occurs and our study examines the structure of the rearranged V(H)HAR genes used to encode IgG antibodies after this isotype switch., Methods: A quantitative polymerase chain reaction was used to investigate the changes in the levels of V(H)HAR+ IgG mRNA seen after xenotransplantation. cDNA libraries specific for V(H)HAR+ Iggamma chain were established from total RNA extracted from splenocytes of naive rats and xenograft recipients of hamster hearts at days 4, 8, 21, and 28 posttransplantation. Colony filter hybridization was used to estimate the relative frequency of the use of individual V(H)HAR+ IgG subclasses. Selected IgG clones from day 21 cDNA libraries were sequenced and analyzed for VH-D-J(H) gene usage and antibody combining site structure., Results: The level of mRNA for V(H)HAR+ IgG increased 6-fold in xenograft recipients at day 21 post-transplantation when compared with naive animals. The relative frequency of isotype usage for V(H)HAR+ IgG1 antibodies alone increased from 22.3% at day 0 to 37.4% at day 21 PTx. Ten IgG clones from the day 21 cDNA libraries have been sequenced for the rearranged V(H)-D-J(H) genes. Thirty percent (3/10) of these IgG clones used V(H)HAR genes for the coding of heavy chain variable region with limited numbers of nucleic acid substitutions (>98% identity with their germline progenitors) although others demonstrated increased variation in nucleotide sequences (95-97% identity) when compared with germline V(H) genes. Analysis of the canonical binding site structure from the predicted amino acid sequences demonstrated that the majority of IgG clones (9/10) displayed a similar pattern of conserved configurations for their combining sites., Conclusions: The change in IgM to IgG antibody production in the later stages of the humoral immune response of rats to hamster xenografts is associated with an IgM to IgG isotype switch and an increased production of antibodies of the IgG1 isotype. Rat anti-hamster IgG xenoantibodies continue to express the V(H)HAR family of V(H) genes, many in their original germline configuration, to encode antibody recognition of the hamster target antigens. There are, however, a majority of antibodies for which the V(H) genes express evidence of increased nucleic acid sequence variation when compared to currently available germline sequences. The source of this variation is not known but may represent the expression of as yet unidentified germline genes and/or the introduction of T cell-driven somatic mutations. Despite the appearance of this variation, the unusual level of conservation in key antigen binding sites within the V(H) region suggests the variation, independent of its origin, may have a limited influence on the restricted nature of the host antibody response to xenografts.

Background: There are limited experimental data on the nature of the humoral response elicited in humans against pig antigens. In this study, we have examined the xenoantibody (XAb) response in eight patients with acute liver failure exposed to pig hepatocytes after treatment with the bioartificial liver (BAL)., Methods: Patients' plasma samples obtained before and after BAL treatment were tested for IgM and IgG XAbs, IgG subclasses, and XAb cytotoxicity, using enzyme-linked immunosorbent assay and flow-cytometric assays. The characterization of pig aortic endothelial cell (PAEC) surface xenoantigens was analyzed by immunoprecipitation., Results: We observed by day 10, a strong anti-pig IgG and IgM XAb response in patients undergoing two or more BAL treatments, with a significant increase in all the IgG subclasses; in contrast, XAb titers did not change if the patients received only one BAL treatment. The majority of the XAbs produced to porcine antigens were primarily specific for the alphaGal epitope. Both IgG and IgM XAbs were cytotoxic to PAECs, and the cytotoxic activity of IgG was associated with high levels of IgG1 and IgG3 subclasses, known to be efficient on complement activation. The characterization of porcine surface antigens demonstrated that IgM human XAbs, before and after BAL exposure, recognized xenoantigens on PAECs with similar molecular weights, suggesting that the same population of XAbs were present in the patients before and after exposure to pig antigens., Conclusions: Repetitive exposure of humans to porcine antigens after BAL treatment, results in a strong IgG and IgM XAb responses that are primarily directed against the alphaGal epitope. These XAbs are cytotoxic to PAECs and the IgG toxicity correlates with high IgG1 and IgG3 levels. Our data also suggest that no new XAb specificity emerges after porcine exposure.

Background: The early phases of the host immune response to xenografts are dominated by anti-donor antibodies. The immunological pathways responsible for mediating the host humoral responses to xenografts are largely unknown, and this report addresses the nature of the immunoglobulin genes controlling the host antibody response to xenografts., Methods: cDNA libraries established from rat anti-hamster monoclonal antibodies and splenic lymphocytes from LEW rats rejecting hamster heart xenografts were used to clone, sequence, and identify the immunoglobulin genes responsible for encoding rat xenoantibodies to hamster heart grafts. Libraries for germline variable region heavy chain (VH) genes encoding the anti-hamster xenograft antibodies were established by genomic DNA cloning and analyzed by nucleotide sequencing. The frequency of Ig VH gene usage for controlling the antibody responses to hamster xenografts was examined by colony-filter dot hybridization. The nucleic acid structure of these genes was then compared to their genomic progenitors to identify the number and structural diversity expressed by the Ig VH genes used to mediate the response., Results: Rat monoclonal antibodies selected for their ability to precipitate the rejection of hamster xenografts exclusively use a closely related group of VH genes. The VH genes used by these antibodies are restricted to a single family of germline genes (VHHAR) for which 15 family members have been identified. The frequency of VHHAR gene usage in splenic IgM-producing B cells from LEW rats rapidly expands from 0.8% in naive animals to 13% in recipients 4 days after xenotransplantation. cDNA libraries expressing VHHAR genes were established from splenic lymphocytes derived from naive or xenograft recipients at 4 and 21 days after transplantation. Examination of 20 cDNA clones revealed that the majority (75%) of these clones express VHHAR genes displaying limited somatic mutation., Conclusions: The use of a closely related group of Ig VH genes in a germline configuration to control the early humoral response to xenografts suggests that this response may represent the utilization of a primitive, T cell-independent pathway of antibody production by the graft recipients.

Background: The rejection of pig xenografts in humans is initiated by preformed antibodies that may be related to the natural antibodies that formulate a first line of defense against infectious agents. Immunoglobulin gene variable domains encoding the antibodies that react with similar epitopes expressed on xenoantigens and bacteria may share structurally similar antigen-binding site configurations., Methods: We sequenced the VH immunoglobulin genes and germline progenitors of two rat monoclonal antibodies that recognize pig xenoantigens. Nucleic and amino acid sequences of these xenoantibodies were compared with immunoglobulin genes encoding antibodies that react with bacteria or viruses., Results and Conclusions: VH genes encoding rat anti-pig xenoantibodies are expressed in germline configuration and share structural similarities, including identical amino acids in key antigenic contact sites that define antibody canonical structural groups, with antibodies to infectious agents.

We have recently demonstrated that cardiac allograft rejection in the PVG.R8-to-PVG.1U rat strain combination involves the recognition of a isolated class I (RT1.Aa) molecules as peptides in the context of the recipient MHC molecules. Three synthetic peptides (P1, P2, and P3) corresponding to the alpha-helices of the RT1.Aa molecule served as T-cell epitopes for graft rejection. In this study, we demonstrate that two of these peptides (P2 and P3) are sufficient to induce immune nonresponsiveness (median survival time >237 days) to cardiac allografts when presented to the recipient immune system in the thymus 7 days before transplantation. This effect was time dependent, as intrathymic inoculation 60 days before transplantation did not prolong graft survival (median survival time=12 days). Previous studies have demonstrated a critical role for alloantibody responses in mediating graft rejection in this rat strain combination. We, therefore, studied the role alloantibody responses may play in the observed immune nonresponsiveness. The titers of alloantibody in serum samples harvested from graft recipients at different times after transplantation were measured. We used recipient primary aortic endothelial cells genetically manipulated to express the donor RT1.Aa molecule as targets in an enzyme-linked immunosorbent assay. High titers of anti-RT1.Aa IgM antibody were detected in unmanipulated controls at the time of graft rejection. The IgM antibody switched to high IgG titers in intrathymically inoculated rats with accelerated or delayed rejection. Graft rejection in intrathymically manipulated recipients that had achieved a transient state of immunological nonresponsiveness correlated with higher titers of the IgG2b alloantibody. In marked contrast, the long-term graft survivors expressed undetectable or low levels of the IgG2b antibody and moderate to high levels of the IgG1 and IgG2a subclasses. These data suggest that the IgG2b alloantibody may contribute to the rejection reaction, whereas IgG1 and IgG2a may be involved in active enhancement of graft survival.

We have recently demonstrated that three synthetic peptides corresponding to the donor class I RT1.Aa molecule induce long-term survival of cardiac allografts in the PVG.R8-to-PVG.1U rat strain combination (disparate for one isolated class I, RT1.A, molecule) when presented to the recipient immune system in the thymus. Long-term graft survivors had measurable levels of donor-reactive alloantibodies in their serum. In this study, we examined long-term allografts for the presence of chronic rejection and donor microchimerism to assess whether this regimen of immune modulation establishes true tolerance and whether this tolerance is dependent upon the presence of donor-recipient microchimerism. Histological examination of long-term heart grafts (>100 days) demonstrated chronic rejection, including a mild degree of myocardial infiltration by mononuclear cells, mild to moderate myocardial fibrosis, and various vascular changes ranging from focal intimal thickening to total vascular lumen blockade due to smooth muscle cell proliferation. In contrast, long-term syngeneic hearts transplanted under similar experimental conditions lacked these pathological manifestations. Donor microchimerism was analyzed using the polymerase chain reaction with a pair of oligonucleotides specific for the donor class I RT1.Aa gene and genomic DNA harvested from various tissues from graft recipients. We detected high levels of donor microchimerism in the heart, kidney, liver, skin, bone marrow, thymus, and lymph nodes of long-term graft recipients. Donor microchimerism was also detected in unmanipulated control graft recipients at rejection (7 days) and in intrathymically manipulated recipients that rejected allografts in a delayed fashion (12-82 days). These data clearly demonstrate that intrathymic inoculation of donor class I allopeptides induces long-term graft survival but does not prevent chronic rejection. Allograft rejection occurred despite high levels of donor microchimerism, providing direct evidence that donor-recipient microchimerism is not sufficient for the prevention of acute or chronic rejection in this model.

The hyperacute rejection of vascularized grafts exchanged between discordant species is a result of the binding of preformed natural antibodies to the endothelium of the donor organ, and the subsequent activation of the complement system. Human natural antibodies to pig endothelial cell antigens appear to be predominantly directed at carbohydrate epitopes expressed by a variety of porcine integrins, including GpIIIa. The identification of porcine xenoantigens whose recognition by human natural antibodies results in hyperacute rejection would allow for the development of strategies to genetically modify the xenograft reaction. We have used antisense technology to down-regulate the expression of one of seven recently identified xenoantigens from the surface of pig aortic endothelial cells. Down-regulation of GpIIIa on endothelial cells resulted in a 20.8% decrease in the mean channel shift (MCS) of IgM natural antibody binding from pooled human sera, and a 28-35% decrease in the MCS of IgM binding from two high-titer individuals. The MCS for human IgG natural antibody binding to the surface of pig cells decreased by 27%. Natural antibody-mediated cytotoxicity to pig endothelial cells was not significantly altered, as indicated by a 2.5-6% decline in complement-mediated cytotoxicity. These results indicate that down-regulation of GpIIIa alone may not be sufficient to significantly alter xenograft rejection. Our results also suggest, however, that antisense-mediated regulation of a functionally important target antigen is technically feasible and may represent a strategy to prevent the xenograft reaction.

Donor-recipient microchimerism has recently been suggested to play a critical role in the induction and maintenance of allograft tolerance. In this study we sought evidence for this hypothesis using the LEW-to-ACI cardiac allograft as a model system. Donor-specific tolerance to cardiac allografts was induced by intravenous or intraportal injection of graft recipients with donor peripheral blood, T cells, or B cells 7 days before transplantation. All the graft recipients injected with donor antigens accepted donor heart grafts indefinitely when compared with control recipients that rejected donor allografts in 12 days. Long-term graft survivors rejected third-party BN heart allografts in 14 days without an adverse effect on the survival of the first LEW heart allografts, demonstrating the specificity of the tolerance. Tissue lysates prepared from heart, kidney, liver, bone marrow, thymus, lymph nodes, and spleen of tolerant (>120 days) graft recipients were analyzed for the presence of donor DNA using LEW T cell receptor C beta gene-specific primers for polymerase chain reaction that detects donor DNA at > or = 1:10,000 dilution. Donor DNA was detected in 77% of tolerant graft recipients. Chimeric recipients showed variations in the levels and presence of donor DNA in different tissues. The status of donor microchimerism, with respect to its presence and tissue distribution, was dependent upon the donor cell type and route of injection used for the induction of tolerance. Intraportal injection of the graft recipients with donor peripheral blood resulted in the highest degree of chimerism, whereas intravenous injection with donor B cells did not induce detectable microchimerism in this group of recipients. These data clearly demonstrate that the presence of microchimerism is common following administration of donor cells, but that its presence is not an absolute requirement for the long-term survival of allografts.

The early phases of the rejection of xenografts exchanged between closely related species are dominated by a vigorous humoral immune response. We have recently used a linker-mediated polymerase chain reaction (LM-PCR) to generate Ig heavy and light chain-specific cDNA libraries to examine the Ig gene control of a prototypic IgM monoclonal antibody, HAR-1, that causes the hyperacute rejection of hamster xenografts. Recombinant clones from the library were screened directly from bacterial colonies by PCR and the nucleic acid sequences of the clones established. Our results demonstrate that the HAR-1 hybridoma is encoded by Ig VH and JH genes in a germline configuration. Comparison of the cDNA sequence for HAR-1 VH with the germline equivalent of this gene isolated from newborn LEW liver (provisionally designated VHHAR-1) showed that the two VH sequences share a nucleic acid identity of 99.3%. Similarly, the HAR-1 monoclonal uses a Ig JH gene that is 98.2% identical with the JH1 nucleic acid sequence available in the GeneBank. The use of Ig VH and JH genes in a germline configuration is similar to that seen with polyreactive natural antibodies to infectious agents and autoantibodies. These humoral responses are thought to be the result of the stimulation of a T cell-independent subset of B cells, the B-1a/B-1b subset, that is responsible for producing antibodies that serve as a primitive humoral (natural antibody) defense mechanism against infectious diseases. Our results suggest that the humoral component of the rejection of xenografts in the hamster-to-rat model may represent the stimulation of this type of B cell antibody response by xenogeneic target antigens that share antigenic epitopes with bacteria and other infectious agents.

The rejection of cardiac xenografts in the hamster-to-rat combination is characterized by the production of IgM antibodies that result in the rapid loss of the graft. We have recently produced rat monoclonal antibodies (mAb) to hamster heart xenografts in an attempt to develop reagents for use in identifying the target antigens for this reaction and to study the nature of the genetic control of the humoral response. The monoclonals were created by the fusion of myeloma cells with splenic lymphocytes from LEW rat recipients of hamster cardiac xenografts. The hybridomas were screened for antibody production, reactivity to hamster cell surface antigens, and the ability to mediate hyperacute rejection of hamster heart xenografts. A panel of monoclonal antibodies has been identified that are capable of inducing hyperacute rejection. All of these mAbs are IgM and bind strongly to hamster vascular endothelium. None of the mAbs were lymphocytotoxic or bound to hamster lymphocytes or erythrocytes. Immunopathologic studies demonstrated that these mAbs react specifically with hamster vascular endothelium and mediate a complement-dependent humoral reaction leading to the destruction of the cardiac xenografts. One of the mAbs (designated as HAR-1) has been characterized in detail. HAR-1 detects antigens distributed in the vascular endothelium, epithelium of bronchi in the lung, small intestine, tubules of kidney, and selective components of lymphoid organs--e.g., the stromal cells of the spleen and thymic medullary epithelium. Western blot analysis of hamster heart proteins with HAR-1 showed multiple bands with two major bands migrating at 80 kDa and 48 kDa. Absorption of the HAR-1 antibody with 48 individual carbohydrate molecules demonstrated that the strongest reactivity of the antibody is with a sialyl-Lea carbohydrate antigen.

A 26-year-old female patient with fulminant hepatic failure and a history of autoimmune hepatitis was heterotopically transplanted with a pig hepatic xenograft to provide temporary metabolic support prior to transplantation with a human donor organ. Circulating natural antipig antibodies were removed prior to transplantation by plasmapheresis and ex vivo en bloc perfusion of the donor pig kidneys. The liver xenograft functioned after transplantation as measured by active bile production, stabilization of prothrombin levels, and reduction in the circulating levels of lactic acid and the enzymes AST and ALT. Despite the removal of greater than 90% of the recipient's natural xenoantibodies prior to transplantation, the levels of antibody rapidly returned and were associated with antibody and complement-mediated rejection of the donor graft. Immunohistochemical evidence of graft rejection could be detected by the deposition of antibody, complement components including properdin, and endothelial swelling as early as 3 hr posttransplantation. These lesions progressed in severity and were accompanied by evidence of thrombosis and ischemic necrosis of the liver xenograft by 34 hrs posttransplantation. The main portal vein, hepatic artery, and vena cava were patent. The placement of the liver graft did not result in any improvement in the neurological status of the patient and she died 34 hr after xenografting due to irreversible brain damage. The information derived from this case has renewed interest in the clinical use of bioartificial devices and whole organ perfusion using xenogeneic tissue for temporary bridging of patients prior to allografting.

We have previously demonstrated that the immune response to cardiac allografts in the ACI-to-LEW rat strain combination involves a limited use of the TCR V beta gene repertoire. In the present study we analyzed the expression of V beta genes by T cells infiltrating kidney and liver allografts to test whether a limited use of the T cell receptor (TCR) repertoire is a common denominator for immune responses to allografts. Graft-infiltrating lymphocytes (GIL) were isolated from allografts on different days after transplantation and analyzed for the expression of V beta genes using a semi-quantitative polymerase chain reaction (PCR) without manipulations in tissue culture. We detected a limited expression of the V beta gene repertoire in fresh GIL harvested from both kidney and liver allografts early in graft rejection. The level of TCR repertoire usage, however, was influenced by the type of graft. The rejection of heart and kidney allografts was associated with more limited use of the V beta gene repertoire when compared with that seen for the rejection of liver allografts. The limited use of the V beta gene repertoire was only apparent when analyzed early in graft rejection; as the rejection reaction progressed T cells using a more diverse V beta repertoire infiltrated the graft. The limited use of TCR repertoire of the early T cell response to allografts may provide the opportunity to therapeutically disrupt the rejection reaction by targeting selected T cell populations for elimination at the time of organ transplantation.

We studied the rejection of cardiac allografts in a rat strain combination (PVG.R8 to PVG.1U) disparate for a single class I MHC antigen (RT1.Aa) to test the extent by which this molecule is recognized as peptides in association with recipient MHC molecules during graft rejection and the contribution of this recognition process to the rejection reaction. Three synthetic peptides that correspond to the portions of alpha-helices of the alpha 1 (P1, P2) and alpha 2 (P3) domains of the donor RT1.Aa molecule were used in this study. Splenocytes from heart allograft recipients at rejection responded in a proliferation assay to all 3 peptides and in a cytotoxic assay to peptides P1 and P2. The peptide-mediated proliferation and cytolytic reactions were blocked by antibodies against CD4/class II MHC and CD8 molecules. Serum from graft recipients at rejection contained significant titers of antibodies to peptides. Presensitization of graft recipients with the peptides resulted in a marked increase in peptide-mediated T cell and antibody responses. Although all 3 peptides were effective in eliciting active immune responses, the P3-mediated response was minimal when compared with those mediated by P1 and P2. Recipients presensitized with the peptides rejected their grafts in 5 days compared with 6 days for unsensitized animals. Recipients presensitized with donor-irradiated splenocytes and aortic endothelial cells, on the other hand, rejected their grafts in 1 and 3 days, respectively, which suggests that immunization with the whole RT1.Aa molecule is required to stimulate accelerated rejection of the graft. This rejection was associated with high titers of donor cell-specific antibodies that exhibited moderate cross-reactivity with the peptides. Our results clearly demonstrate that (1) the donor RT1.Aa molecule is recognized as peptides in the context of recipient class I and class II MHC molecules during the rejection of heart allografts, and (2) peptides derived from this molecule are highly immunogenic in that they contain epitopes recognized by CD4+ and CD8+ T cells and alloantibodies. Immune responses elicited by these peptides, however, did not significantly affect the rate of rejection. These results suggest that acute rejection of allografts may be mediated primarily by the direct recognition of intact MHC molecules.

Brequinar sodium (BQR) is a new immunosuppressive drug that is highly effective in preventing graft rejection in several different experimental settings, including primary allografts and xenografts. A short course of BQR treatment during the onset of allograft rejection can induce the permanent survival of liver and kidney allografts in rats. To study the molecular basis of BQR-induced prolongation of allograft survival, we analyzed the intragraft pattern of IL-1 alpha, IL-2, IL-2R, IL-4, IL-6, IL-10, and TNF gene expression in the ACI-to-LEW liver allograft model. A semiquantitative polymerase chain reaction was developed to measure cytokine gene expression in control and BQR-treated liver graft recipients at various days after transplantation. Untreated control liver allografts expressed all of the cytokines analyzed. There was a marked increase in the steady state level of transcripts for each cytokine as graft rejection proceeded. The treatment of liver graft recipients with 12 mg/kg/day of BQR on days 6, 7, and 8 after transplantation suppressed the expression of all these cytokines within 24 hr of administration. The early suppression of cytokine expression was associated with a modest but distinct reduction in the infiltration of inflammatory cells into the liver grafts. The reduction in the level of transcripts for IL-4, IL-6, and IL-10 persisted in long-term survivors (30 days after transplantation). In contrast, there was a significant increase in the level of transcripts for IL-1 alpha, IL-2, and IL-2R in these long-term survivors. Our results demonstrated clearly that the pattern of cytokine gene expression during allograft rejection is significantly altered by a 3-day course of therapy with BQR. The temporary down-regulation of cytokine gene expression may be responsible for an altered immunological state that results in the prolonged survival of liver allografts.

Liver regenerative processes are associated with enhanced expression of alloantigens. Accordingly, we tested the hypothesis that such enhanced surface expression of alloantigens during regeneration of reduced-size liver grafts is associated with accelerated rejection. Our OLT model was LEW (RT1) to BN (RT1n), with donor liver resected by 50%. The study group consisted of reduced-size allografts. Control groups were syngeneic reduced-size isografts and full-size allografts. Reduced-size isograft recipients survived indefinitely. Both isografts and allografts regenerated to their prereduction size within 12 days. Recipients of reduced-size allografts died of accelerated rejection within 12.2 +/- 0.8 days, significantly earlier than recipients receiving full-size allografts (36.2 +/- 4.1 days, P < 0.01). The accelerated rejection in the regenerating allografts was mediated both by cellular and humoral mechanisms, evidenced by earlier lymphocytic invasion of the graft, enhanced donor MHC class II expression, and the emergence of IgM antibodies, directed specifically against donor endothelial antigens. These data suggest that regeneration of reduced-size allografts is accompanied by accelerated cellularly and humorally mediated alloreactivity. Recipients of reduced-size allografts may, therefore, benefit from more potent immunosuppression during the period of active liver regeneration.

Brequinar sodium (BQR) is a novel immunosuppressive agent that is highly effective in preventing B lymphocyte-mediated antibody production. We have examined the effects of BQR treatment in sensitized recipients on graft survival, donor-specific antibody responses (IgM and IgG), and the appearance of immunopathological lesions present in the grafts. LEW rat recipients were sensitized with single ACI skin graft on day 7 and received heterotopic ACI cardiac grafts on day 0. The recipients rejected the cardiac grafts in an accelerated fashion at day 2.5 post-transplantation, compared to day 7.0 in unsensitized recipients. The animals were treated with low (3 mg/kg/day) or high (12 mg/kg/3x weekly) doses of BQR during skin graft sensitization and/or after challenge with ACI heart allografts. All groups treated with BQR showed significant prolongation of graft survival in the sensitized recipients. The best survival was observed following high-dose BQR therapy during both sensitization and effector phases (median survival time = 40.0 days, P << 0.001). Daily treatment with BQR (3 mg/kg/day) prevented IgM (but not IgG) antibody responses. Treatment with higher doses of BQR (12 mg/kg/3x weekly) before and after skin graft sensitization was effective in preventing both IgM and IgG production. In general, BQR treatment resulted in effective suppression of anti-donor antibody responses, stable graft function, and a reduction in the severity of the acute vascular lesions in the graft. The effectiveness of BQR in preventing accelerated graft rejection when used at 12 mg/kg/3x weekly was comparable to that seen with treatment of sensitized animals with CsA at 15 mg/kg/day for 30 days. Daily treatment with cyclophosphamide at 5 or 15 mg/kg/day was ineffective for preventing graft rejection in sensitized recipients. These results indicated that BQR may provide an important addition to treatment protocols designed to prevent transplantation rejection in presensitized patients. BQR has the ability to significantly inhibit host cellular and humoral immune responses to the donor graft and this facet of the immunosuppressive activity of the drug may be responsible for preventing this aggressive form of rejection.

Human-to-pig xenoantibodies may constitute a major obstacle to the successful use of pigs as xenograft donors for human transplantation. Our studies demonstrate that normal human serum contains antibodies, primarily IgM, that are cytotoxic for pig aortic endothelial cells (PAECs). These antibodies bind to several antigens isolated from PAECs, lymphocytes, platelets, red blood cells, and the kidney. Absorption of human serum with pig lymphocytes removes the cytotoxic activity to PAECs and some, but not all, of the IgM antibodies capable of binding in an ELISA assay to the PAECs. The cytotoxic antibodies are inactivated by 2-mercaptoethanol, suggesting that they are primarily IgM. Whole cell extracts of PAEC, lymphocytes, platelets, red blood cells, and kidney were prepared and analyzed by Western blots to establish the cellular distribution of the xenoantigens that react with human IgM in pooled human serum. Results showed that several of the most intensely stained bands migrated between 24 and 66 kDa. High molecular weight bands (> 100 kDa) were observed only in kidney, platelet, and PAEC preparations. Human IgM xeniantibodies also reacted strongly in Western blots to endothelial cell membranes proteins with molecular weights of 62, 48, 42, 36, 34, 28, and 26 kDa. Absorption of human serum with pig lymphocytes removes IgM binding to all bands except for a 34-kDa Treatment of the PAEC membrane proteins with proteinase K disrupts the binding of the human IgM antibodies. Similar treatment with glycosidase F) resulted in a decrease in molecular weight of the 28- and 26-kDa bands, suggesting that these xenoantigens are glycoproteins and that antibody binding to some xenoantigens may not require glycosylation.

Brequinar sodium (BQR) is a novel immunosuppressive drug that inhibits cell proliferation by virtue of its disruption of the de novo pyrimidine biosynthesis. The basis of the immunosuppressive activity of BQR is distinctively different from that of cyclosporine (CsA), and we have recently evaluated in vivo and in vitro the efficacy of the two drugs when used in combination. Subtherapeutic doses of BQR and CsA were tested for their ability to prolong heterotopic cardiac allograft survival in the MHC- and non-MHC-mismatched ACI-->LEW rat strain combination. The graft survival data derived from these experiments were analyzed using the median-effect analysis to establish the immunosuppressive interaction of both drugs. The administration of BQR 3 mg/kg three times weekly or CsA 2.5 mg/kg daily moderately prolonged cardiac allograft survival, with a mean survival of 10 +/- 0.5 and 16 +/- 5.3 days, respectively. The use of the two drugs in combination with the same dose schedule exerted a synergistic effect on graft survival, prolonging the graft function to a mean of 31 +/- 5.7 days. Sera from animals treated with the two drugs displayed, when compared with single treatment groups (BQR 3 mg/kg and CsA 2.5 mg/kg), significantly (P < 0.01) increased in vitro inhibition of lymphocyte proliferation following stimulation with PHA. Finally, a clear correlation between the mean survival time and BQR plasma levels of animals treated with BQR alone or in combination with CsA was seen. Those treatment groups with BQR levels below 2 micrograms/ml (1.5 and 3.0 mg/kg/3x/week) had a mean graft survival of less than 10 days). In contrast, recipients treated with a combination of low doses of BQR and CsA displayed higher drug plasma levels (> 2 micrograms/ml) and longer mean graft survival times. These observations suggest that BQR and CsA may be highly effective when used in combination to prevent organ allograft rejection for clinical transplantation.

We have examined the nature of the binding of human xenoantibodies to pig liver and kidney vascular endothelium. Our results demonstrate that human serum contains IgM and IgG xenoantibodies that bind to pig vascular endothelium, and that the pattern of antibody binding is similar for both livers and kidneys. Immunohistochemical analysis of pig kidneys after perfusion with human blood demonstrated the binding of both IgM and IgG xenoantibodies, complement (C3), and fibrinogen to the vascular and glomerular endothelium. An ELISA assay of the perfusate after perfusion of 500 ml of human blood through a single pig kidney for 60 min demonstrated a significant reduction in the amount of human IgM (67%) and IgG (55%) binding to pig aortic endothelium. Similar perfusion experiments conducted with pig livers were associated with minimal immunohistochemical evidence of the binding of human xenoantibodies to liver vascular endothelium. Immunofluorescence staining for IgM, IgA, C3, and C1q was negative or minimally positive in the liver vascular endothelium. Sinusoidal endothelium were weakly positive for IgG and fibrinogen. The perfusion of the pig liver with human blood was, however, associated with a significant reduction in the subsequent binding of IgM and IgG to pig kidney vascular endothelium. Pig liver perfusion was also responsible for the removal of both IgM and IgG xenoantibodies capable of reacting with pig aortic endothelium, as measured by an ELISA assay of the perfusate. These results suggest that both pig kidney and livers are capable of absorbing the xenoantibodies that may be responsible for mediating a hyperacute rejection of pig xenografts and that the distribution of the target antigens for these antibodies is similar in the two organs.

Heterotopic hamster hearts transplanted to unmodified LEW rats underwent humoral rejection in 3 days. Survival was prolonged to a median of 4 days with 2 mg/kg/day FK506. As monotherapy, 15 mg/kg/day cyclophosphamide greatly prolonged graft survival--far more than could be accomplished with RS-61443, brequinar (BQR), mizoribine, methotrexate, or deoxyspergualin. However, when FK506 treatment, which was ineffective alone, was combined with a short induction course (14 or 30 days) of subtherapeutic BQR, RS-61443, or cyclophosphamide, routine survival of heart xenografts was possible for as long as the daily FK506 was continued. In addition, a single large dose of 80 mg/kg cyclophosphamide 10 days preoperatively allowed routine cardiac xenograft survival under FK506. The ability of these antimetabolites to unmask the therapeutic potential of FK506 correlated, although imperfectly, with the prevention of rises of preformed heterospecific cytotoxic antibodies immediately postoperatively. As an adjunct to FK506, azathioprine was of marginal value, whereas mizoribine, methotrexate, and deoxyspergualin (DSPG) were of intermediate efficacy. After orthotopic hepatic xenotransplantation, the perioperative survival of the liver with its well-known resistance to antibodies was less dependent than the heart on the antimetabolite component of the combined drug therapy, but the unsatisfactory results with monotherapy of FK506, BQR, RS-61443, or cyclophosphamide were changed to routine success by combining continuous FK506 with a short course of any of the other drugs. Thus, by breaking down the antibody barrier to xenotransplantation with these so-called antiproliferative drugs, it has been possible with FK506 to transplant heart and liver xenografts with consistent long-term survival of healthy recipients.

Brequinar sodium (BQR) prevents cell proliferation by virtue of its inhibition of de novo pyrimidine biosynthesis. The immunosuppressive activity of BQR is highly effective in prolonging heart, liver, and kidney allograft survival in the rat. In these experiments, we have tested the ability of BQR to prevent the rejection of concordant cardiac xenografts. LEW inbred rats transplanted with heterotopic hamster hearts were treated orally with brequinar sodium as a single agent. The survival of the cardiac xenografts was significantly prolonged with a variety of treatment regimens. The most effective treatment was the daily oral administration of BQR at 3 mg/kg. At this level, the median graft survival was approximately 25 days. Four animals had hamster heart xenografts that functioned for more than 90 days. The prolonged survival of the xenografts was associated with relatively constant plasma drug levels of approximately 1 to 3 micrograms/ml and a marked suppression of IgM production. At rejection, there was a significant rise in IgM levels compared with those of recipients with stable xenografts. In vitro MLR responses were effectively inhibited by BQR, with an IC50 of 0.08 microgram/ml. The results of these experiments demonstrate that BQR is a new immunosuppressive agent that is highly effective as a single agent in prolonging the survival of hamster-to-rat cardiac xenografts. The prolonged xenograft survival is associated with effective suppression of rat antihamster antibody production, suggesting that brequinar sodium may be an important addition to multidrug immunosuppressive regimes designed to prevent B and T lymphocyte-mediated immune responses.

Brequinar sodium (BQR) prevents cell proliferation by virtue of its inhibition of de novo pyrimidine biosynthesis. BQR is capable of inhibiting immune responses in vitro and is effective in suppressing the development of contact sensitivity and adjuvant arthritis in rodent models. Based on the antiproliferative and immunosuppressive capacity of BQR, we have evaluated the efficacy of BQR in preventing allograft rejection utilizing experimental models of heterotopic heart and kidney and orthotopic liver transplantation in an MHC and non-MHC mismatched ACI----LEW rat strain combination. The immunosuppressive activity of BQR is illustrated by its ability to inhibit the development of delayed-type hypersensitivity to DNFB in mice. When BQR was administered orally throughout the sensitization and elicitation phases of the DNFB contact sensitivity response, it was found to be a potent immunosuppressant with an ED50 value of 0.5 mg/kg. This immunosuppressive activity is also seen in vitro, where BQR is capable of inhibiting the mixed lymphocyte response between allogeneic ACI and LEW rat strains with an IC50 of 150 ng/ml. The immunosuppressive activity of BQR is highly effective in prolonging heart, liver, and kidney allograft survival in the rat. Cardiac allografts are not rejected during the period of drug treatment at dosage levels of 12 to 24 mg/kg orally three times weekly. The grafts survive until the drug is discontinued (30 days posttransplantation), and the grafts are then rejected approximately 14 days later. Liver and kidney allografts are permanently accepted by approximately 50 to 90% of the recipient rats following 30 days of treatment with BQR at 12 mg/kg. The tolerance that is induced to the liver grafts extends in the majority of animals to greater than 250 days and is specific for the donor ACI strain. Challenge of long-term liver graft survivors with donor cardiac grafts is associated with permanent survival of donor, but not third-party, heart grafts. Combination therapy consisting of suboptimal doses of BQR and CsA demonstrates that the combination of these two immunosuppressive drugs results in an increased efficacy in prolonging graft survival. The results of these allograft experiments demonstrate that this new immunosuppressive agent is highly effective in preventing allograft rejection in the rat. The antiproliferative activity of BQR is effective for inhibiting T-lymphocyte-mediated immune responses, and Brequinar sodium should be an important addition to a polytherapeutic approach in the treatment of organ graft rejection.

Lewis recipients of orthotopic ACI livers had permanent graft acceptance induced with 3 doses of i.m. FK506 in the early postoperative period. They were studied 100 and 300 days posttransplantation. The recipients rejected ACI as well as Brown Norway (BN) (third-party) skin grafts, and had lymphocytes with substantial reactivity by mixed lymphocyte culture testing against ACI and third-party (BN) alloantigens. Lymphocyte subset redistribution had not occurred in the peripheral blood or spleens of these animals, and there was no evidence of suppressor cell activation by in vitro and in vivo tests. Graft-versus-host reactivity in splenic lymphoid tissues of these recipients was demonstrated with the popliteal lymph node assay. Attempts at adaptive transfer with recipient lymphocytes were unsuccessful. Heart graft acceptance was far more difficult to accomplish than liver graft acceptance, and probably was never permanent. ACI heart graft prolongation in LEW recipients after a brief induction with FK506 lasted for no more than 3 months in most animals. The temporary heart graft acceptance was specific for hearts of the original ACI donor strain but not for ACI skin. Results of studies of lymphocyte subsets and suppressor cell activity were similar to those in the liver recipients. These studies illustrate how poorly graft acceptance is understood and how badly further work is needed to clarify its mechanism.

Cardiac transplantation between inbred rat strains that differ for weak histocompatibility antigens is associated with the development of arteriosclerosis in the arteries of the donor graft myocardium. The lesions are seen in donor/recipient pairs that differ for both MHC and non-MHC histocompatibility antigens that apparently stimulate a low-level, chronic rejection of the donor heart graft. The arteriosclerosis associated with this chronic rejection consists of a diffuse, concentric proliferation of the intima and pathologically resembles the lesions observed in the coronary arteries of long-term human cardiac graft recipients. We have recently examined the influence of positive and negative manipulation of the host immune response on the development of the graft arteriosclerosis. Our results demonstrate that delayed harvest of the cardiac grafts or immunization with donor skin grafts or splenic lymphocytes increases the sensitivity of the recipient to the donor heart grafts--and, under conditions that allow for the long-term survival of the graft--increases the severity of the arteriosclerotic lesions. Alternatively, suppression of the host immune responses with cyclosporine or FK506, substantially reduces the arteriosclerotic changes. These results suggest that control of accelerated graft arteriosclerosis in long-term human cardiac recipient may require more careful and effective immunosuppression of the allograft reaction.

The pathogenesis of hyperacute transplantation reactions includes the activation of a cascade of nonspecific inflammatory reactions that precipitates the destruction of the target organ. Platelet-activating factor (PAF) represents an important component of these inflammatory cascades, and we have examined the influence of a specific PAF receptor antagonist (SRI 63-441) on the inhibition of hyperacute rejection in two experimental models, the rejection of rat cardiac allografts by presensitized recipients and guinea pig-to-rat and mouse-to-rat cardiac xenografts. Our results demonstrate that inhibition of PAF function by SRI 63-441 has a variable effect on the survival of cardiac allografts in presensitized rat recipients. In the ACI to sensitized BN cardiac allograft model, the use of SRI 63-441 alone, or in combination with CsA, FK506, or prostaglandin E2 (PGE2), does not prolong graft survival. As we have previously reported, SRI 63-441 does act as a single agent to prolong the survival of ACI to sensitized LEW grafts, and this survival effect is synergistic when combined with CsA. Here we extend these results to demonstrate that this survival is also extended when FK506 is used in the ACI-to-LEW model. Concordant mouse-to-rat cardiac xenografts are also relatively resistant to prolongation of graft survival following treatment with SRI 63-441 alone or in combination with CsA or FK506. Discordant xenografts appear to be more susceptible to inhibition of the rejection reaction with SRI 63-441. When either donor or recipient animals were treated with SRI 63-441 alone, or in combination with CsA or FK506, there was significant prolongation of guinea pig-to-rat cardiac xenograft survival. These results are consistent with our earlier description of the effectiveness of SRI 63-441 in preventing the rejection of cat-to-rabbit kidney xenografts. We believe that these results demonstrate that the use of the SRI 63-441 to specifically interfere with the function of PAF has the effect of prolonging graft survival in those systems in which performed antibody and/or complement activation are important components of the hyperacute reaction. This synthetic drug is representative of a family of compounds whose structure can be modified to balance their therapeutic and toxicity activities, and may prove to be important components of a polytherapeutic approach to the control of graft rejection in sensitized patients or following discordant xenografting.

Heterotopic heart and orthotopic liver grafts from ACI donors were transplanted to Lewis rat recipients that were treated with a 3 (or 4) day course of FK506 IM that was started on postoperative day 0, 2, 3, 4, 5, or 6. Hearts, which rejected after a median of 6 days in untreated controls, always had prolonged survival (median 91 days) when treatment was started on postoperative day 4. The results were inferior when treatment was started earlier or later than this, but even when the first dose of FK506 was on postoperative day 5, one day before rejection was imminent in controls, the median survival was 50 days. The poorest results with a median graft survival of only 36 days were obtained when injections were on days 0-3. Results were similar with liver grafts that rejected after a median time of 10 days in nontreated controls but that usually survived permanently after a 3 (or 4) day FK506 course starting on day 0, 2, 3, or 4. Therapy started on day 6 was too late.

We have studied the effect of using lymphokine-activated killer (LAK) cells as an in vitro means for eliminating leukemic cells from normal bone marrow prior to transplantation of experimental animals. Rat LAK cells exhibit broad cytolytic activity against a variety of hematopoietic neoplasms, but do not kill normal bone marrow cells or lectin-stimulated blasts. Bone marrow was harvested from normal Fischer 344 rats, combined with increasing numbers of CRNK-16 tumor cells, and then incubated with LAK cells. The BM/tumor/LAK mixture was then administered to untreated Fischer rats, and the ability of the LAK cells to purge the bone marrow of neoplastic cells and prevent the transmission of the leukemia to recipient animals monitored. Our results demonstrate that LAK cells are capable of efficiently purging the bone marrow of neoplastic cells. Treatment of the BM/tumor mixtures with LAK cells is associated with significant prolongation of survival in the higher tumor doses (10(5) tumor cells/recipient) and complete elimination of the tumor in a high percentage of recipients at lower tumor levels (10(3)-10(4) tumor cells/recipient). At levels of BM transfer comparable to that used in humans, there was no evidence of a failure of LAK-treated bone marrow to reconstitute lethally conditioned recipient animals. However, with lower numbers of BM cells, there was an increased mortality in animals receiving LAK-treated BM, suggesting a minimal inhibition of pluripotent hematopoietic stem cell function when suboptimal numbers of BM cells are used for reconstitution. These experiments demonstrate that LAk cells are capable of eliminating neoplastic cells in bone marrow without significant destruction of immature syngeneic stem cells. LAK cells display a broad range of cytolytic activity against hematopoietic and solid tissue tumors, and are therefore capable of eliminating small numbers of tumor cells from a wide variety of neoplastic diseases of the marrow. The ability to detect and eliminate malignant cells, without interfering with reconstitution with donor marrow, suggests that immune therapy with LAK cells can be a relatively simple and efficient method to purge bone marrow prior to autologous transplantation in patients following high-dose chemotherapy for neoplastic diseases.

The development of arteriosclerosis is the most serious and common complication in long-term survivors of cardiac transplantation. We have used a variety of inbred rat strains with selected histocompatibility differences to examine the influence of prolonged, mild rejection reactions on the development of pathological changes in long-term cardiac allografts. Heterotopic cardiac allografts were exchanged between rat strains that differed for MHC class I (RT1.A and/or RT1.E) antigens or groups of minor, non-MHC antigens in MHC-compatible congenic combinations. Our results demonstrate that in strain combinations in which the allograft reaction is mild and prolonged, the donor hearts exhibit pathological changes that include a diffuse, interstitial myocardial fibrosis, perivascular fibrosis, and intimal proliferation in arteries of the graft myocardium. The lesions were less prominent in animals with more active rejection and infrequent in strains that differ for class I histocompatibility antigens or syngeneic controls. These results suggest that the comparable pathological changes seen in long-term human cardiac survivors may reflect low-level, persistent allograft reactions rather than factors associated with graft anoxia or effects of immunotherapy to prevent graft rejection.