Christian LeGuern

The mouse Igh-1a and Igh-1b H chain constant regions are derived from two distinct isotypic genes.

Genetic and structural analyses of the mouse genes encoding constant region of immunoglobulin subclasses (Igh-C) have shown that recombination is rare within this cluster which is inherited as a set designated the Igh haplotype. Recent molecular analyses have demonstrated that either DNA exchanges or gene duplications have probably occurred during the evolution of this set of genes. In order to assess the generality of the duplication processes, the presence and expression of two allelic forms of the Igh-1 (γ2a) gene (Igh-1a and Igh-1b) were examined in a large panel of wild mice belonging to Mus musculus domesticus and Mus musculus musculus species. Our data indicate that certain M. m. domesticus animals and most animals in the M. m. musculus group coexpress the two allelic forms of Igh-1. Moreover, genetic studies show that these two immunoglobulin types are encoded by tandemly arranged genes. We propose that wild mice, from which laboratory mice are derived, carry three isotypic γ2 genes (Igh-1a, Igh-1b, Igh-3), and these have given rise to the two isotypes seen in laboratory strains by a deletion/insertion mechanism.

Tolerance to solid organ transplants through transfer of MHC class II genes

Donor/recipient MHC class II matching permits survival of experimental allografts without permanent immunosuppression, but is not clinically applicable due to the extensive polymorphism of this locus. As an alternative, we have tested a gene therapy approach in a preclinical animal model to determine whether expression of allogeneic class II transgenes (Tgs) in recipient bone marrow cells would allow survival of subsequent Tg-matched renal allografts. Somatic matching between donor kidney class II and the recipient Tgs, in combination with a short treatment of cyclosporine A, prolonged graft survival with DR and promoted tolerance with DQ. Class II Tg expression in the lymphoid lineage and the graft itself were sequentially implicated in this tolerance induction. These results demonstrate the potential of MHC class II gene transfer to permit tolerance to solid organ allografts.

Immortalized bone-marrow derived pig endothelial cells.

Abstract: Primary cultures of porcine endothelial cells (EC) can only be maintained for a limited number of passages. To facilitate studies of xenogeneic human anti‐pig immune responses in vitro, pig microvascular bone‐marrow (BM) and macrovascular aortic EC were obtained from our herd of partially inbred miniature swine, homozygous for the major histocompatibility locus, and immortalized with a modified SV40 large T vector. The resulting BM‐derived (2A2) and aortic (PEDSV.15) immortalized EC lines showed unlimited growth and EC phenotype as indicated by expression of von Willebrand Factor (vWF) and low density lipoprotein (LDL) receptors as well as by formation of typical cobblestone monolayers. Ultrastructural studies revealed morphological similarities in primary and immortalized EC. Flow cytometry analysis demonstrated constitutive SLA class I expression by all lines whereas SLA class II was only expressed after stimulation with porcine IFNγ. Furthermore, pig CD34 mRNA was detected by Northern blot analysis in primary and immortalized aortic EC but not in 2A2. Both EC lines expressed a number of myeloid markers, adhesion molecules and xeno‐antigens, the latter being determined by binding of human natural antibodies. Gene transfer into the porcine EC lines was successfully performed by electroporation or calcium‐phosphate transfection, as well as by adenoviral infection. Finally, the functional similarity between primary and immortalized EC was demonstrated in adhesion and cytotoxicity assays. Together, these results suggest that 2A2 and PEDSV.15 represent valuable tools to study both human cellular and humoral immune responses in vitro against pig EC derived from microvascular and large vessels.

TRANSFER OF SWINE MAJOR HISTOCOMPATIBILITY COMPLEX CLASS II GENES INTO AUTOLOGOUS BONE MARROW CELLS OF BABOONS FOR THE INDUCTION OF TOLERANCE ACROSS XENOGENEIC BARRIERS

BACKGROUND The present study examined the potential role of gene therapy in the induction of tolerance to anti-porcine major histocompatibility complex (SLA) class II-mediated responses after porcine renal or skin xenografts. METHODS Baboons were treated with a non-myeloablative or a myeloablative preparative regimen before bone marrow transplantation with autologous bone marrow cells retrovirally transduced to express both SLA class II DR and neomycin phosphotransferase (NeoR) genes, or the NeoR gene alone. Four months or more after bone marrow transplantation, the immunological response to a porcine kidney or skin xenograft was examined. Both the renal and skin xenografts were SLA DR-matched to the transgene, and recipients were conditioned by combinations of complement inhibitors, adsorption of natural antibodies, immunosuppressive therapy, and splenectomy. RESULTS Although the long-term presence of the SLA transgene was detected in the peripheral blood and/or bone marrow cells of all baboons, the transcription of the transgene was transient. Autopsy tissues were available from one animal and demonstrated expression of the SLA DR transgene in lymphohematopoietic tissues. After kidney and skin transplantation, xenografts were rejected after 8-22 days. Long-term follow-up of control animals demonstrated that high levels of induced IgG antibodies to new non-alphaGal epitopes developed after organ rejection. In contrast, induced non-alphaGal IgG antibody responses were minimal in the SLA DR-transduced baboons. CONCLUSIONS Transfer and expression of xenogeneic class II DR transgenes can be achieved in baboons. This therapy may prevent late T cell-dependent responses to porcine xenografts, which include induced non-alphaGal IgG antibody responses.

Expression of an allogeneic MHC DRB transgene, through retroviral transduction of bone marrow, induces specific reduction of alloreactivity.

BACKGROUND Transfer of MHC class II genes, through allogeneic bone marrow (BM) transplantation, induced long-lasting acceptance of renal allografts in miniature swine. To adapt this approach to the clinic, we have now examined whether somatic transfer of allogeneic class II DR genes, into otherwise autologous bone marrow cells (BMC), can provide the matching required for inducing immune tolerance. METHODS Autologous BMC were transduced ex vivo with recombinant retroviruses for allogeneic DRB followed by BM transplantation. The recipients were then challenged with kidney allografts solely matched to the DRB transgene. RESULTS Five miniature swine received autologous BMC conditioned with growth factors and transduced with recombinant retrovirus vectors containing allogeneic (n=4) or syngeneic (n=1) class II DRB genes and a drug-resistance marker. Expression of retrovirus-derived products in BM-derived cells was demonstrated by the detection of drug-resistant colony-forming progenitors and the presence of DRB retrovirus transcripts in peripheral cells. Analysis of selective mixed lymphocyte reaction responses to DR or DQ antigens indicated decreased reactivity toward the transduced DR gene product. Among all of the animals receiving fully mismatched kidney allografts, but with DRB matched to the transduced DRB, the one with the highest gene transduction rate showed stable allograft function and essentially normal renal histology for 2.5 years. A control animal, which received a syngeneic DRB gene, rejected its kidney allograft in 120 days after an earlier rejection crisis. CONCLUSIONS These studies demonstrate that allogeneic MHC gene transfer into BM provides a new strategy for inducing tolerance across MHC barriers.

Donor exosomes rather than passenger leukocytes initiate alloreactive T cell responses after transplantation

Early T cell activation by recipient antigen-presenting cells cross-dressed with donor MHC antigens may initiate acute allograft rejection. Transplant ride along Despite the successful use of organ transplantation in the clinic, the mechanisms behind early rejection of transplants remain unclear. The passenger leukocyte theory suggests that graft leukocytes that express donor major histocompatibility complexes (MHCs) migrate to recipient lymphoid organs, where they activate host T cells. In multiple mouse models of transplantation, Marino et al. found few donor leukocytes in lymph nodes; rather high numbers of recipient antigen-presenting cells (APCs) were present that were cross-dressed with donor MHC. The donor MHC was derived from allogeneic exosomes, which could induce proinflammatory alloimmune responses even without transplantation. These data suggest that cross-dressed recipient APCs rather than passenger leukocytes may contribute to early T cell activation and transplant rejection. Transplantation of allogeneic organs and tissues represents a lifesaving procedure for a variety of patients affected with end-stage diseases. Although current immunosuppressive therapy prevents early acute rejection, it is associated with nephrotoxicity and increased risks for infection and neoplasia. This stresses the need for selective immune-based therapies relying on manipulation of lymphocyte recognition of donor antigens. The passenger leukocyte theory states that allograft rejection is initiated by recipient T cells recognizing donor major histocompatibility complex (MHC) molecules displayed on graft leukocytes migrating to the host’s lymphoid organs. We revisited this concept in mice transplanted with allogeneic skin, heart, or islet grafts using imaging flow cytometry. We observed no donor cells in the lymph nodes and spleen of skin-grafted mice, but we found high numbers of recipient cells displaying allogeneic MHC molecules (cross-dressed) acquired from donor microvesicles (exosomes). After heart or islet transplantation, we observed few donor leukocytes (100 per million) but large numbers of recipient cells cross-dressed with donor MHC (>90,000 per million). Last, we showed that purified allogeneic exosomes induced proinflammatory alloimmune responses by T cells in vitro and in vivo. Collectively, these results suggest that recipient antigen-presenting cells cross-dressed with donor MHC rather than passenger leukocytes trigger T cell responses after allotransplantation.

Class II genes of miniature swine. III. Characterization of an expressed pig class II gene homologous to HLA-DQA.

This report presents an analysis of the SLA-DQA gene organization, the isolation of two SLA-DQA allelic cDNA clones, as well as sequence comparisons between the SLA-DQA clones and other DQA-related genes

Molecular and cellular events implicated in local tolerance to kidney allografts in miniature swine.

Long-term tolerance to class I-mismatched renal allografts can be induced in miniature swine by treatment with a short course of cyclosporine (CsA). Kidney recipients treated with CsA and untreated control kidney recipients both demonstrated infiltration of the transplanted kidney by mononuclear cells, which reached a maximum between postoperative days 8 and 11. Recipients that did not receive the tolerizing regimen rejected their grafts between postoperative days 8 and 12 in this model. The kinetics of cytokine gene expression, including interleukin (IL)-1alpha, IL-1beta, IL-2, IL-6, IL-10, tumor necrosis factor, and interferon-gamma (IFN-gamma), within the grafted kidney of rejector and acceptor animals, were determined using Northern blot hybridization. A strong correlation between rejection and up-regulation of the IFN-gamma gene was observed, whereas animals with long-term tolerance showed low levels of IFN-gamma, but high levels of IL-10 gene transcription. None of the other cytokine genes demonstrated a reproducible pattern of expression that correlated with acceptance/rejection of allografts. Analysis of transcription patterns of cytokine genes in mononuclear cells purified from renal grafts confirmed the initial observations made on biopsies. The phenotype of graft-infiltrating cells (GIC) showed a dominance of CD8+ cells, with an average of 66% single-positive cells and 19% CD4/CD8 double-positive cells, compared with 30% and 14%, respectively, for peripheral cells. Predominance of CD8+ GIC was dictated neither by the MHC antigen disparity nor the rejector/acceptor status. These results, therefore, suggest that GIC represent a regulated combination of mononuclear cells producing local immune mediators that, in part, control the fate of allografts in this large animal model.

Gene therapy and transplantation.

Advances in molecular biology and in techniques of gene transfer have resulted in the development of practical approaches to human gene therapy. Many applications are of relevance to manipulation of the immune system and have potential in organ and cell transplantation. For example, gene therapy approaches may facilitate the induction of immunological tolerance to a donor organ or protect it locally against the hosts immune response. Based on a comprehensive review of the world literature, examples of current research efforts in both allogeneic and xenogeneic transplantation are presented and discussed.

Sequence determination of a transcribed rabbit class II gene with homology to HLA-DQ α

The human HLA-DQα probe was used to screen genomic and cDNA libraries constructed from a rabbit T-cell line. Clones containing highly homologous sequences were obtained from both libraries and their sequences were determined. The organization of the RLA-DQ gene was determined by comparison of the nucleotide sequences of the genomic clone to that of the corresponding cDNA clone. This analysis allowed assignment of the complete structure of the RLA-DQα chain. Comparisons with human and mouse class II products revealed that RLA-DQ is more closely related to HLA-DQ/DX than to H-2 A. In contrast to the DQ/DX region of man, which contains at least two distinct alpha genes, the rabbit genome contains a single DQ gene which is equally distant from the HLA-DQ or -DX genes. The rabbit DQαgene, like human HLA-DQ, is transcribed in T cells.