Identification of an anti-idiotypic antibody that defines a B-cell subset(s) producing xenoantibodies in primates.

Synthetic anti-idiotypic antibodies represent a potentially valuable tool for the isolation and characterization of B cells that produce xenoantibodies. An anti-idiotypic antibody that binds to a subset of B cells producing antibodies encoded by the variable-region heavy chain 3 (V(H)3) germline genes DP35 [immunoglobulin variable-region heavy chain 3-11 (IGHV3-11)], DP-53 and DP-54 plus a small number of V(H)4 gene-encoded antibodies in humans has recently been identified. These germline progenitors also encode xenoantibodies in humans. We tested whether the small, clearly defined group of B cells identified with this anti-idiotypic antibody produce xenoantibodies in non-human primates mounting active immune responses to porcine xenografts. Peripheral blood B cells were sorted by flow cytometry on the basis of phenotype, and cDNA libraries were prepared from each of these sorted groups of cells. Immunoglobulin V(H) gene libraries were prepared from the sorted cells, and the V(H) genes expressed in each of the sorted groups were identified by nucleic acid sequencing. Our results indicate that xenoantibody-producing peripheral blood B cells, defined on the basis of binding to fluorescein isothiocyanate (FITC)-conjugated galactose alpha(1,3) galactose-bovine serum albumin (Gal-BSA) and the anti-idiotypic antibody 2G10, used the IGHV3-11 germline gene to encode xenoantibodies and were phenotypically CD11b+ (Mac-1+) and CD5-. This novel reagent may be used in numerous applications including definition of xenoantibody-producing B-cell subsets in humans and non-human primates and immunosuppression by depletion of B cells producing anti-Gal xenoantibodies.

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.

Tolerance induction by lentiviral gene therapy with a nonmyeloablative regimen.

Antibodies (Abs) directed at the Gal alpha1,3Gal beta1,4GlcNAc-R (alphaGal) carbohydrate epitope initiate xenograft rejection. Previously, we have shown that bone marrow transplantation (BMT) with lentivirus-mediated gene transfer of porcine alpha1,3 galactosyltransferase (GalT) is able to induce tolerance to alphaGal-expressing heart grafts following a lethal dose of irradiation. Here we show the first demonstration of permanent survival of alphaGal+ hearts following transplantation with autologous, lentivirus-transduced BM using a nonmyeloablative regimen. Autologous BM from GalT knockout (GalT-/-) mice was transduced with a lentiviral vector expressing porcine GalT and transplanted into sublethally irradiated (3 Gy) GalT-/- mice. Chimerism in the peripheral blood cells (PBCs) remained low but was higher in the BM, especially within the stromal cell population. Mice reconstituted with GalT did not produce anti-alphaGal Abs over time. We immunized these mice with alphaGal-expressing cells and assessed humoral immune responses. Anti-alphaGal xenoantibodies were not produced in mice reconstituted with GalT, but normal Ab responses to other xenoantigens were detected. Mice reconstituted with GalT accepted alphaGal+ heart grafts over 100 days. Transduction with lentiviral vectors results in chimerism at levels sufficient to induce long-term tolerance under nonmyeloablative conditions.

Identification of the V genes encoding xenoantibodies in non-immunosuppressed rhesus monkeys.

The major immunological barrier that prevents the use of wild-type pig xenografts as an alternative source of organs for human xenotransplantation is antibody-mediated rejection. In this study, we identify the immunoglobulin variable region heavy (IgV(H)) chain genes encoding xenoantibodies to porcine heart and fetal porcine islet xenografts in non-immunosuppressed rhesus monkeys. We sought to compare the IgV(H) genes encoding xenoantibodies to porcine islets and solid organ xenografts. The immunoglobulin M (IgM) and IgG xenoantibody response was analysed by enzyme-linked immunosorbent assay and cDNA libraries from peripheral blood lymphocytes were prepared and sequenced. The relative frequency of IgV(H) gene usage was established by colony filter hybridization. Induced xenoantibodies were encoded by the IGHV3-11 germline progenitor, the same germline gene that encodes xenoantibodies in humans mounting active xenoantibody responses. The immune response to pig xenografts presented as solid organs or isolated cells is mediated by identical IgV(H) genes in rhesus monkeys. These animals represent a clinically relevant model to identify the immunological basis of pig-to-human xenograft 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.