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 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.

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.

Acceleration of human myoblast fusion by depolarization: graded Ca2+ signals involved.

We have previously shown that human myoblasts do not fuse when their voltage fails to reach the domain of a window T-type Ca(2+) current. We demonstrate, by changing the voltage in the window domain, that the Ca(2+) signal initiating fusion is not of the all-or-none type, but can be graded and is interpreted as such by the differentiation program. This was carried out by exploiting the properties of human ether-à-go-go related gene K(+) channels that we found to be expressed in human myoblasts. Methanesulfonanilide class III antiarrhythmic agents or antisense-RNA vectors were used to suppress completely ether-à-go-go related gene current. Both procedures induced a reproducible depolarization from -74 to -64 mV, precisely in the window domain where the T-type Ca(2+) current increases with voltage. This 10 mV depolarization raised the cytoplasmic free Ca(2+) concentration, and triggered a tenfold acceleration of myoblast fusion. Our results suggest that any mechanism able to modulate intracellular Ca(2+) concentration could affect the rate of myoblast fusion.

Notch signaling is involved in the regulation of Id3 gene transcription during Xenopus embryogenesis.

During Xenopus embryogenesis, XId3, a member of the Id helix-loop-helix protein family, is expressed in a large variety of differentiating tissues including epidermis, cement gland, brain, neural tube, neural crest cell derivatives, somites, and tailbud. Transcription of XId3 is mediated by several cis-regulatory elements including an enhancer of 440 bp located 870 bp upstream from the transcription initiation site. The enhancer activity in embryos was studied using transgenic methodology. A galactosidase reporter gene, driven by a regulatory element composed of the enhancer and a minimal promoter derived from the XId3 gene, was expressed in transgenic embryos with a profile that faithfully reproduced that of the endogenous XId3 gene. The pattern resulted from a synergistic effect between the enhancer and the promoter, and in vitro transactivation assays showed that transcription can be stimulated by Notch signaling. The presence of potential Su(H) binding sites, in both the enhancer and the promoter, suggests that these represent candidates for in vivo cis-regulatory elements. The data presented here suggest that Notch control of differentiation may involve activation of transcription of Id, a negative regulator of bHLH transcription factors.

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.