Use of molecular modeling and site-directed mutagenesis to define the structural basis for the immune response to carbohydrate xenoantigens.

BACKGROUND: Natural antibodies directed at carbohydrates reject porcine xenografts. They are initially expressed in germline configuration and are encoded by a small number of structurally-related germline progenitors. The transplantation of genetically-modified pig organs prevents hyperacute rejection, but delayed graft rejection still occurs, partly due to humoral responses. IgVH genes encoding induced xenoantibodies are predominantly, not exclusively, derived from germline progenitors in the VH3 family. We have previously identified the immunoglobulin heavy chain genes encoding VH3 xenoantibodies in patients and primates. In this manuscript, we complete the structural analysis of induced xenoantibodies by identifying the IgVH genes encoding the small proportion of VH4 xenoantibodies and the germline progenitors encoding xenoantibody light chains. This information has been used to define the xenoantibody/carbohydrate binding site using computer-simulated modeling. RESULTS: The VH4-59 gene encodes antibodies in the VH4 family that are induced in human patients mounting active xenoantibody responses. The light chain of xenoantibodies is encoded by DPK5 and HSIGKV134. The structural information obtained by sequencing analysis was used to create computer-simulated models. Key contact sites for xenoantibody/carbohydrate interaction for VH3 family xenoantibodies include amino acids in sites 31, 33, 50, 57, 58 and the CDR3 region of the IgVH gene. Site-directed mutagenesis indicates that mutations in predicted contact sites alter binding to carbohydrate xenoantigens. Computer-simulated modeling suggests that the CDR3 region directly influences binding. CONCLUSION: Xenoantibodies induced during early and delayed xenograft responses are predominantly encoded by genes in the VH3 family, with a small proportion encoded by VH4 germline progenitors. This restricted group can be identified by the unique canonical structure of the light chain, heavy chain and CDR3. Computer-simulated models depict this structure with accuracy, as confirmed by site-directed mutagenesis. Computer-simulated drug design using computer-simulated models may now be applied to develop new drugs that may enhance the survival of xenografted organs.

The xenoantibody response and immunoglobulin gene expression profile of cynomolgus monkeys transplanted with hDAF-transgenic porcine hearts.

BACKGROUND: Recent work has indicated a role for anti-Gal alpha 1-3Gal (Gal) and anti-non-Gal xenoantibodies in the primate humoral rejection response against human-decay accelerating factor (hDAF) transgenic pig organs. Our laboratory has shown that anti-porcine xenograft antibodies in humans and non-human primates are encoded by a small number of germline IgV(H) progenitors. In this study, we extended our analysis to identify the IgV(H) genes encoding xenoantibodies in immunosuppressed cynomolgus monkeys (Macaca fascicularis) transplanted with hDAF-transgenic pig organs. METHODS: Three immunosuppressed monkeys underwent heterotopic heart transplantation with hDAF porcine heart xenografts. Two of three animals were given GAS914, a poly-L-lysine derivative shown to bind to anti-Gal xenoantibodies and neutralize them. One animal rejected its heart at post-operative day (POD) 39; a second animal rejected the transplanted heart at POD 78. The third monkey was euthanized on POD 36 but the heart was not rejected. Peripheral blood leukocytes (PBL) and serum were obtained from each animal before and at multiple time points after transplantation. We analyzed the immune response by enzyme-linked immunosorbent assay (ELISA) to confirm whether anti-Gal or anti-non-Gal xenoantibodies were induced after graft placement. Immunoglobulin heavy-chain gene (V(H)) cDNA libraries were then produced and screened. We generated soluble single-chain antibodies (scFv) to establish the binding specificity of the cloned immunoglobulin genes. RESULTS: Despite immunosuppression, which included the use of the polymer GAS914, the two animals that rejected their hearts showed elevated levels of cytotoxic anti-pig red blood cell (RBC) antibodies and anti-pig aortic endothelial cell (PAEC) antibodies. The monkey that did not reject its graft showed a decline in serum anti-RBC, anti-PAEC, and anti-Gal xenoantibodies when compared with pre-transplant levels. A V(H)3 family gene with a high level of sequence similarity to an allele of V(H)3-11, designated V(H)3-11(cyno), was expressed at elevated levels in the monkey that was not given GAS914 and whose graft was not rejected until POD 78. IgM but not IgG xenoantibodies directed at N-acetyl lactosamine (a precursor of the Gal epitope) were also induced in this animal. We produced soluble scFv from this new gene to determine whether this antibody could bind to the Gal carbohydrate, and demonstrated that this protein was capable of blocking the binding of human serum xenoantibody to Gal oligosaccharide, as had previously been shown with human V(H)3-11 scFv. CONCLUSIONS: DAF-transgenic organs transplanted into cynomolgus monkeys induce anti-Gal and anti-non-Gal xenoantibody responses mediated by both IgM and IgG xenoantibodies. Anti-non-Gal xenoantibodies are induced at high levels in animals treated with GAS914. Antibodies that bind to the Gal carbohydrate and to N-acetyl lactosamine are induced in the absence of GAS914 treatment. The animal whose heart remained beating for 78 days demonstrated increased usage of an antibody encoded by a germline progenitor that is structurally related, but distinct from IGHV311. This antibody binds to the Gal carbohydrate but does not induce the rapid rejection of the xenograft when expressed at high levels as early as day 8 post-transplantation.

Rat chemokine CXCL11: structure, tissue distribution, function and expression in cardiac transplantation models.

CXCL11 is thought to play a critical role in allograft rejection. To clarify the role of CXCL11 in the rat transplantation model, we cloned CXCL11 cDNA from rat liver tissue and used it to study CXCL11 structure, function and expression. The rat CXCL11 gene encodes a protein of 100 amino acids and spans approximately a 2.8 kb DNA segment containing 4 exons in the protein coding region. Tissue distribution of rat CXCL11 was analyzed by quantitative RT-PCR and showed that rat CXCL11 mRNA is expressed in various tissues and, in particular, at high levels in the spleen and lymph nodes. COS-1 cells were transfected with a plasmid vector encoding rat CXCL11 and used to study CXCL11 effects on cell migration and internalization of CXCR3, the CXCL11 receptor. The recombinant CXCL11 showed chemotactic properties and induced CXCR3 internalization in CD4(+) T cells. Expression of CXCL11 mRNA also was measured in rat acute (ACI to LEW) and chronic (LEW to F344) heart transplant rejection models. CXCL11 mRNA expression in allografts increased in both models, compared with controls, and was primarily observed in infiltrating macrophages and donor endothelial cells. These results indicate that, like the other CXCR3 chemokines, rat CXCL11 seems to have a role in the homing of CD4(+) T cells in both acute and chronic rejection models of heart allotransplantation.

Protection against H1, H5, H6 and H9 influenza A infection with liposomal matrix 2 epitope vaccines.

The recent emergence of multiple avian influenza A subtypes that cause human disease (i.e., H5N1, H9N2 and H7N7), coupled with the fear that one of these strains might precipitate a new pandemic, underscores the need to develop new technological approaches to immunization which elicit protective immune responses against multiple subtypes of influenza A. In response to this demand, several matrix 2 protein ectodomain segments (M2eA) corresponding to the H1N1, H5N1 and H9N2 influenza strains were formulated using a novel liposome-based vaccine technology and evaluated as potential immunogens for developing a "universal" influenza vaccine. Mice immunized with liposomal M2eA survived homologous challenges with H1N1 (100% survival) or H9N2 (80% survival) influenza strains. There were significant reductions in their lung viral load as well as in immunized mice challenged with the H5N1 subtype. The mice vaccinated with an M2eA segment corresponding to the H1N1 and H6N2 (a reassortant influenza A virus carrying the M2eA from PR8/34) strains elicited elevated IgG ELISA antibody titers to this M2eA epitope segment and antiserum from these immunized mice provided passive protection (100% survival) to naïve mice receiving a lethal dose of H6N2 influenza virus. These results provide the first evidence that recombinant M2eA epitopes to multiple subtypes elicited immune protection against a homologous challenge and provides further evidence in favor of the development of a "universal" influenza vaccine based on M2eA.

Similarities in the immunoglobulin response and VH gene usage in rhesus monkeys and humans exposed to porcine hepatocytes.

BACKGROUND: The use of porcine cells and organs as a source of xenografts for human patients would vastly increase the donor pool; however, both humans and Old World primates vigorously reject pig tissues due to xenoantibodies that react with the polysaccharide galactose alpha (1,3) galactose (alphaGal) present on the surface of many porcine cells. We previously examined the xenoantibody response in patients exposed to porcine hepatocytes via treatment(s) with bioartficial liver devices (BALs), composed of porcine cells in a support matrix. We determined that xenoantibodies in BAL-treated patients are predominantly directed at porcine alphaGal carbohydrate epitopes, and are encoded by a small number of germline heavy chain variable region (VH) immunoglobulin genes. The studies described in this manuscript were designed to identify whether the xenoantibody responses and the IgVH genes encoding antibodies to porcine hepatocytes in non-human primates used as preclinical models are similar to those in humans. Adult non-immunosuppressed rhesus monkeys (Macaca mulatta) were injected intra-portally with porcine hepatocytes or heterotopically transplanted with a porcine liver lobe. Peripheral blood leukocytes and serum were obtained prior to and at multiple time points after exposure, and the immune response was characterized, using ELISA to evaluate the levels and specificities of circulating xenoantibodies, and the production of cDNA libraries to determine the genes used by B cells to encode those antibodies. RESULTS: Xenoantibodies produced following exposure to isolated hepatocytes and solid organ liver grafts were predominantly encoded by genes in the VH3 family, with a minor contribution from the VH4 family. Immunoglobulin heavy-chain gene (VH) cDNA library screening and gene sequencing of IgM libraries identified the genes as most closely-related to the IGHV3-11 and IGHV4-59 germline progenitors. One of the genes most similar to IGHV3-11, VH3-11cyno, has not been previously identified, and encodes xenoantibodies at later time points post-transplant. Sequencing of IgG clones revealed increased usage of the monkey germline progenitor most similar to human IGHV3-11 and the onset of mutations. CONCLUSION: The small number of IGVH genes encoding xenoantibodies to porcine hepatocytes in non-human primates and humans is highly conserved. Rhesus monkeys are an appropriate preclinical model for testing novel reagents such as those developed using structure-based drug design to target and deplete antibodies to porcine xenografts.

Transfer of regulatory T cells generated ex vivo modifies graft rejection through induction of tolerogenic CD4+CD25+ cells in the recipient.

Certain CD4+CD25+ T cells can induce and maintain T-cell non-responsiveness to donor alloantigens and have therapeutic potential in solid organ transplantation. Peripheral CD4+CD25- cells alloactivated with IL-2 and transforming growth factor beta (TGF-beta) ex vivo express the transcription factor FoxP3, and become potent antigen-specific CD4+CD25- suppressor cells. Here we report that the transfer of TGF-beta-induced regulatory CD4+ and CD8+ T cells (Tregs) co-incident with transplantation of a histoincompatible heart resulted in extended allograft survival. To account for this result, we injected non-transplanted mice with a single dose of CD4+ and CD8+ Tregs and transferred donor cells every 2 weeks to mimic the continuous stimulation of a transplant. We observed increased splenic CD4+CD25+ cells that were of recipient origin. These cells rendered the animals non-responsive to donor alloantigens by an antigen-specific and cytokine-dependent mechanism of action. Both the increased number of CD4+CD25+ cells and their tolerogenic effect were dependent on continued donor antigen boosting. Thus, Tregs generated ex vivo can act like a vaccine that generates host suppressor cells with the potential to protect MHC-mismatched organ grafts from rejection.

Transforming growth factor-beta/interleukin-2-induced regulatory CD4+ T cells prolong cardiac allograft survival in rats.

BACKGROUND: Treatment of naive CD4+ T cells in vitro with transforming growth factor-beta (TGF-beta) or TGF-beta/interleukin-2 (IL-2), combined with stimulation in a mixed lymphoid culture (MLC), has been shown to generate CD4+ CD25+ regulatory T cells. However, little is known about the effect of these regulatory T cells on cardiac allograft survival in vivo. METHODS: CD4+ CD25+ T cells were generated from Lewis (LEW) rat spleen through a primary MLC with TGF-beta (10 ng/ml) or TGF-beta/IL-2 (10 U/ml). The effect of adoptive transfer of the CD4+ CD25+ T cells (5.0 x 10(7)) was evaluated using an animal model of ACI rat cardiac allograft survival in LEW recipients. RESULTS: The MLC with TGF-beta or TGF-beta/IL-2 generated CD4+ CD25+ regulatory T cells, which suppressed the cytotoxic activity of LEW spleen T cells against irradiated ACI spleen cells in vitro. Adoptive transfer of the CD4+ CD25+ regulatory T cells intravenously to naive syngeneic recipients significantly prolonged the ACI cardiac allograft survival (N = 6, 13.5 +/- 3.4 days) compared with the control group (N = 6, 5.0 +/- 0.6 days). CONCLUSIONS: Intravenous administration of CD4+ CD25+ regulatory T cells, successfully generated by TGF-beta/IL-2 treatment, had a significant effect on cardiac allograft survival in this rat model. Adoptive transfer of regulatory T cells may represent a novel approach for preventing allograft rejection.

Use of lentiviral vectors to induce long-term tolerance to gal(+) heart grafts.

BACKGROUND: Tolerance to organ grafts has been achieved by establishing a state of stable mixed-cell chimerism after bone marrow transplantation. Gene therapy has been applied to establish chimerism for cells expressing galactose alpha 1,3 galactose in alpha 1,3 galactosyltransferase deficient (gal knockout) mice using retroviral vectors. Limitations to the success of this methodology include short-term expression of the introduced gene and rejection of gal hearts transplanted into these animals within a month. METHODS: Autologous bone marrow from gal knockout mice was transduced with a lentiviral vector expressing porcine alpha 1,3 galactosyltransferase and transplanted into lethally irradiated gal knockout mice. Chimerism was monitored by flow cytometry. Hearts from wild type mice (gal/) were transplanted into these animals and palpated daily. Xenoantibodies directed at the gal carbohydrate or porcine xenoantigens were detected by enzyme-linked immunosorbent assay. RESULTS: Hearts from wild-type gal/ donors were permanently accepted in all mice receiving autologous, transduced bone marrow before heart transplantation. Control mice rejected gal hearts within 12 to 14 days. Histologic analysis demonstrated classical signs of rejection in controls and normal myocardium with no evidence of rejection in mice chimeric for the gal carbohydrate. Anti-gal xenoantibodies were not produced in gal chimeras, but normal antibody responses to other xenoantigens were detected. Specific tolerance for the gal carbohydrate was achieved by this procedure. CONCLUSIONS: These experiments report the first demonstration of permanent survival of gal hearts after transplantation with autologous, transduced bone marrow. Transduction with lentiviral vectors results in long-term, stable chimerism at levels sufficient to induce long-term tolerance to heart grafts in mice.

Identification, functional analysis and expression in a heterotopic heart transplant model of CXCL9 in the rat.

CXCR3 chemokines are of particular interest because of their potential involvement in a variety of inflammatory diseases, including the rejection of organ transplants. Although the rat is one of the most appropriate animals for using to study transplantation biology, the structural and functional characteristics of CXCL9 [monokine induced by interferon-gamma (Mig)] in this experimental model have not been described. Therefore, we recently conducted a series of experiments to identify and characterize the rat CXCL9 gene. Accordingly, we isolated rat CXCL9 cDNA and genomic DNA. The rat CXCL9 gene encodes a protein of 125 amino acids and spans a 3.5 kbp DNA segment containing four exons in the protein-coding region. We then analysed mRNA expression in various tissues. Transcripts for the gene were found to be expressed at high levels in the lymph nodes and spleen. Then, to confirm the function of the identified gene, rat CXCL9 was transiently expressed in COS-1 cells. Rat recombinant Mig displayed chemotactic properties and induced CXCR3 internalization in CD4+ T cells. Lastly, we analysed the expression of rat CXCL9 in a heterotopic heart allograft model. Both mRNA and protein levels of intragraft CXCL9 were significantly increased following transplantation of ACI to LEW hearts when compared with syngeneic controls. These findings indicate that rat CXCL9 has an in vivo role in the infiltration of CD4+ T cells in the transplanted graft.

Evidence for recipient derived fibroblast recruitment and activation during the development of chronic cardiac allograft rejection.

BACKGROUND: Allograft fibrosis is a prominent feature of chronic rejection. Although intragraft fibroblasts contribute to this process, their origin and exact role remain poorly understood. METHODS: Using a rat model of chronic rejection, LEW to F344, cardiac fibroblasts were isolated at the point of rejection and examined in a collagen gel contraction assay to measure fibroblast activation. The allograft microenvironment was examined using immunohistochemistry for fibrogenic markers (transforming growth factor [TGF]-beta, platelet-derived growth factor [PDGF], tissue plasminogen activator [TPA], plasminogen activator inhibitor [PAI]-1, matrix metalloproteinase [MMP]-2, and tissue inhibitor of matrix metalloproteinase [TIMP]-2). The origin of intragraft fibroblasts was studied using female to male allografts followed by polymerase chain reaction [PCR] and in situ hybridization for the male sry gene. RESULTS: The cardiac fibroblasts isolated from allografts with chronic rejection exhibited higher gel contractibility (50.9% +/- 6.1% and 68.2% +/- 3.8% at 4 and 24 hr) compared with naive cardiac fibroblasts (30.7% +/- 3.5% and 55.3% +/- 6.6% at 4 and 24 hr; P<0.05 and <0.05, respectively). Immunostaining for TGF-beta, PDGF, TPA, PAI-1, MMP-2 and TIMP-2 was observed in all allografts at the time of rejection. In situ hybridization demonstrated the presence of sry positive cells in female allografts rejected by male recipients. Sixty-five percent of fibroblast colonies (55 of 85) isolated from female heart allografts expressed the male sry gene. CONCLUSION: Cardiac fibroblasts are activated and exist in a profibrogenic microenvironment in allografts undergoing chronic rejection. A substantial proportion of intragraft fibroblasts are recruited from allograft recipients in this experimental model of chronic cardiac allograft rejection.

Predominant expression of the Th2 response in chronic cardiac allograft rejection.

Chronic rejection is the main cause of late allograft failure in patients. CD4+ T cells activated by indirect recognition of alloantigens are implicated in this rejection reaction. However, the type of T cell response (Th1 vs Th2) that contributes to chronic rejection has not been fully investigated. The purpose of this study is to examine whether chronic rejection is associated with a polarized T-cell response in a rat cardiac allograft model, where long-term graft survival is achieved by intrathymic immunomodulation with donor class I, RT1.Aa, allopeptides. All long-surviving allografts showed histological evidence of chronic rejection. Chronic rejection was associated with high levels of intragraft Th2 cytokines and the Th2-regulated alloantibodies. The Th2 response was systemic, since long-surviving allografts with chronic rejection had high levels of serum IL-10. The predominance of the Th2 cytokines demonstrates that the Th2 response was not sufficient for the prevention of chronic rejection in this model. The predominant expression of Th2 cytokines, together with the presence of Th2-regulated alloantibodies, suggests that the Th2 response may play a role in the development of chronic rejection.

Migration of mesenchymal stem cells to heart allografts during chronic rejection.

BACKGROUND: Mesenchymal stem cells (MSC) are pluripotent progenitors for a variety of cell types, including fibroblasts and muscle cells. Their involvement in the tissue repair of allografts during the development of chronic rejection has been hypothesized, but not yet substantiated, by experimental evidence. METHODS: Rat MSC were isolated from circulation using an aortic pouch allograft as a trapping device. The plasticity of these cells was examined in differentiation cultures. One of the resulting MSC lines was immortalized and transduced to express a marker gene. The -labeled cells were then transferred to F344 rats bearing Lewis (LEW) cardiac allografts to measure their localization and contribution to graft tissue repair. RESULTS: The MSC isolated from circulation exhibited multipotential for differentiation in culture, developing into various lineages including osteoblasts, lipocytes, chondrocytes, myotubes, and fibroblasts. Intravenous engraftment of the -labeled cells into recipients of heart transplant resulted in migration of the beta-gal+ cells into the lesions of chronic rejection in the cardiac grafts and homing of the cells to the bone marrow. The majority of beta-gal+ cells present in the allografts exhibited fibroblast phenotypes, and a small number of the cells expressed desmin, indicative of myocyte differentiation. CONCLUSION: MSC vigorously migrated into the site of allograft rejection. This data suggests that they may be attracted to this site to actively participate in tissue repair during chronic rejection. In addition, given the robust migration, the inhibition of MSC differentiation toward fibroblast progeny and induction toward the myocyte lineage may serve as a new strategy for treatment of chronic rejection and allograft tissue repair.

Vascular endothelial cell apoptosis induced by anti-donor non-MHC antibodies: a possible injury pathway contributing to chronic allograft rejection.

BACKGROUND: Non-major histocompatibility complex (non-MHC) alloantibodies may play a pathogenic role in chronic rejection but remain poorly characterized. METHODS: The kinetics of alloantibody production and the mechanism by which non-MHC alloantibodies cause graft injury were investigated in a Lewis-to-Fischer 344 (LEW-to-F344) rat model of cardiac transplantation. RESULTS: Flow cytometry detected that all the F344 recipients of LEW allografts produced anti-donor immunoglobulin G (IgG) antibodies reactive with LEW lymphocytes and endothelial cells. A sub-group of recipients that rejected their grafts in 30 to 60 days exhibited markedly increased levels of anti-donor IgG antibodies (n = 6, mean fluorescence intensity [MFI]:23.85 +/- 2.7) than recipients with long-surviving allografts (n = 4, MFI:11.23 +/- 0.81; p = 0.00058). Passive transfer of anti-donor sera induced chronic rejection of LEW heart allografts in an immune non-responsiveness model of F344 rats induced by intrathymic inoculation of donor-specific lymphocytes. Immunoglobulin G antibodies purified from the anti-LEW sera exhibited complement-dependent cytotoxicity against LEW vascular endothelial cells in flow-cytometric cytotoxicity assay. The targeted endothelial cells displayed early (annexin V+) and late (TUNEL+) evidence for programmed cell death. Western blot analysis of poly (ADP-ribose) polymerase (PARP) demonstrated that the 25-kD PARP-cleavage fragment was present at the lysates of the vascular endothelial cells treated with anti-donor IgG antibodies, indicating apoptosis-associated caspase activity in these cells. In situ teminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining demonstrated that vascular endothelial cell apoptosis was consistently present in all LEW heart allografts with chronic rejection. CONCLUSIONS: Non-MHC alloantibodies are pathogenic and capable of causing chronic graft injury through an antibody-induced cell apoptosis mechanism. The results emphasize the importance of non-MHC antibodies as a common predisposing factor in the development of chronic rejection.