The anti-non-gal xenoantibody response to xenoantigens on gal knockout pig cells is encoded by a restricted number of germline progenitors.

Antibodies directed at non-gal xenoantigens are responsible for acute humoral xenograft rejection when gal knockout (GalTKO) pig organs are transplanted into nonhuman primates. We generated IgM and IgG gene libraries using peripheral blood lymphocytes of rhesus monkeys initiating active xenoantibody responses after immunization with GalTKO pig endothelial cells and used these libraries to identify IgV(H) genes that encode antibody responses to non-gal pig xenoantigens. Immunoglobulin genes derived from the IGHV3-21 germline progenitor encode xenoantibodies directed at non-gal xenoantigens. Transduction of GalTKO cells with lentiviral vectors expressing the porcine alpha1,3 galactosyltransferase gene responsible for gal carbohydrate expression results in a higher level of binding of 'anti-non-gal' xenoantibodies to transduced GalTKO cells expressing the gal carbohydrate, suggesting that anti-non-gal xenoantibodies cross react with carbohydrate xenoantigens. The galactosyltransferase two gene encoding isoglobotriaosylceramide synthase (iGb3 synthase) is not expressed in GalTKO pig cells. Our results demonstrate that anti-non-gal xenoantibodies in primates are encoded by IgV(H) genes that are restricted to IGHV3-21 and bind to an epitope that is structurally related to but distinct from the Gal carbohydrate.

Gene therapy to inhibit xenoantibody production using lentiviral vectors in non-human primates.

Xenoantibodies to the gal alpha1,3 gal (gal) epitope impede the use of pig tissues for xenotransplantation, a procedure that may help overcome the shortage of human organ donors. Stable gal chimerism and tolerance to gal(+) hearts could be achieved in alpha1,3-galactosyltransferase (alpha1,3GT)(-/-) mice using lentiviral vectors expressing porcine alpha1,3GT, the enzyme that synthesizes the gal carbohydrate. In this study, we evaluated whether chimerism sufficient to inhibit anti-gal xenoantibody responses can be achieved using lentivectors in non-human primates. Rhesus macaques were transplanted with autologous, alpha1,3GT-transduced bone marrow (BM) following sublethal irradation. Simian immunodeficiency virus (SIV)- and human immunodeficiency virus (HIV)-1-derived lentiviral constructs were compared. Chimerism was observed in several hematopoietic lineages in all monkeys. Engraftment in animals receiving SIV-based alpha1,3GT constructs was similar to that achieved using the HIV-1-derived lentivector for the first 2 months post-transplantation, but increased thereafter to reach higher levels by 5 months. Upon immunization with porcine hepatocytes, the production of anti-gal immunoglobulin M xenoantibody was substantially reduced in the gal(+) BM recipients compared to controls. This study is the first to report the application of gene therapy to achieve low-level, long-term gal chimerism sufficient to inhibit production of anti-gal antibodies after immunization with porcine cells in rhesus macaques.

Expression changes in tolerant murine cardiac allografts after gene therapy with a lentiviral vector expressing alpha1,3 galactosyltransferase.

Comparison of intragraft gene expression changes in tolerant cardiac allograft models may provide the basis for identifying pathways involved in graft survival. Our laboratory has previously demonstrated that tolerance to the gal alpha1,3 gal epitope, the major target of rejection of wild-type pig hearts in human cardiac transplantation, can be achieved after transplantation with bone marrow transduced with a lentiviral vector expressing alpha1,3 galactosyltransferase. We now present intracardiac gene expression changes associated with long-term tolerance in this model. Biotin-labeled cRNA was hybridized to Affymetrix GeneChip 430 2.0 Mouse Genome Arrays. Data were subjected to functional annotation analysis to identify genes of known function in which expression was increased or decreased by at least 2-fold (t-test, P < .05) in tolerant gal+/+ wild-type hearts as compared to transplanted syngeneic controls. Tolerant hearts demonstrated increased expression of genes associated with the stress response, modulation of immune function and cell survival (HSPa9a, CD56, and Akt1s1), and decreased expression of several immunoregulatory genes (CD209, CD26, and PDE4b). These data suggest that tolerance may be associated with activation of immunomodulatory and survival pathways.

Characteristics of immunoglobulin gene usage of the xenoantibody binding to gal-alpha(1,3)gal target antigens in the gal knockout mouse.

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.

The human antibody response to porcine xenoantigens is encoded by IGHV3-11 and IGHV3-74 IgVH germline progenitors.

Preformed and induced Ab responses present a major immunological barrier to the use of pig organs for human xenotransplantation. We generated IgM and IgG gene libraries established from lymphocytes of patients treated with a bioartificial liver (BAL) containing pig hepatocytes and used these libraries to identify IgVH genes that encode human Ab responses to pig xenoantigens. Genes encoded by the VH3 family are increased in expression in patients following BAL treatment. cDNA libraries representing the VH3 gene family were generated, and the relative frequency of expression of genes used to encode the Ab response was determined at days 0, 10, and 21. Ig genes derived from the IGHV3-11 and IGHV3-74 germline progenitors increase in frequency post-BAL. The IGHV3-11 gene encodes 12% of VH3 cDNA clones expressed as IgM Abs at day 0 and 32.4-39.0% of cDNA clones encoding IgM Abs in two patients at day 10. IGHV3-11 and IGHV3-74 genes encoding IgM Abs in these patients are expressed without evidence of somatic mutation. By day 21, an isotype switch occurs and IGHV3-11 IgVH progenitors encode IgG Abs that demonstrate somatic mutation. We cloned these genes into a phagemid vector, expressed these clones as single-chain Abs, and demonstrated that the IGHV3-11 gene encodes Abs with the ability to bind to the gal alpha (1,3) gal epitope. Our results demonstrate that the xenoantibody response in humans is encoded by IgVH genes restricted to IGHV3-11 and IGHV3-74 germline progenitors. IgM Abs are expressed in germline configuration and IgG Abs demonstrate somatic mutations by day 21.

Genetic control of the humoral responses to xenografts. III. Identification of the immunoglobulin V(H) genes responsible for encoding rat immunoglobin G xenoantibodies to hamster heart grafts.

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.

Characterization of human xenoreactive antibodies in liver failure patients exposed to pig hepatocytes after bioartificial liver treatment: an ex vivo model of pig to human xenotransplantation.

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.

The humoral response to xenografts is controlled by a restricted repertoire of immunoglobulin VH genes.

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.

Xenoantibodies to pig endothelium are expressed in germline configuration and share a conserved immunoglobulin VH gene structure with antibodies to common infectious agents.

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.

EBV binds to lymphocytes of transgenic mice that express the human CR2 gene.

Epstein Barr virus (EBV) is unable to bind to or infect normal mouse lymphocytes. A construct containing the human complement receptor type 2 (CR2) gene, the receptor for EBV, was placed under the control of the IgH/c-fos enhancer/promoter and microinjected into single cell embryos. A total of five transgenic mouse lines were established and four expressed hCR2 mRNA. Flow cytometry and immunostaining revealed that approximately 15-30% of the lymphocytes from the thymus, spleen and lymph nodes expressed hCR2 protein on their surface and bound EBV. Despite this binding, less than 1% of the cells showed evidence that the virus was internalized or replicated. Transgenic mouse lymphocytes, expressing hCR2, could not be immortalized with EBV. It is concluded that the simple expression of hCR2 receptor on mouse lymphocytes is not sufficient for efficient infection.

Human serum reactivity to porcine endothelial cells after antisense-mediated down-regulation of GpIIIa expression.

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.

Pig aortic endothelial cell antigens recognized by human IgM natural antibodies.

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.

Transgenic mouse lines that are immunologically tolerant and nontolerant to herpes simplex virus glycoprotein D.

A construct containing the gene for glycoprotein D of herpes simplex virus (HSV-gD), under the control of the simian virus 40 early promoter, was microinjected into single-cell embryos, and four transgenic mouse lines were established. Three were homozygous (lines 75, 111, and 113) and one was hemizygous (line 108) for the HSV-gD gene. Examination of sera revealed that only one of the lines (line 75) spontaneously produced antibody to HSV-gD. Immunization of the other three lines with vaccinia virus-HSV-gD showed that one of them (line 113) responded by making antibody to HSV-gD, whereas the other two (lines 108 and 111) appeared to be immunologically tolerant. Evidence that tolerance was not absolute was obtained by immunization with infectious HSV, which resulted in an antibody response to HSV-gD in some of the animals from line 111. Examination of organs for HSV-gD mRNA revealed transcripts in the tolerant line (line 108) and in the partially tolerant line (line 111), but not in the nontolerant line (line 113), suggesting that the development of immunological tolerance requires active expression of the HSV-gD gene.