Tolerance to solid organ transplants through transfer of MHC class II genes (original) (raw)
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Proceedings of the National Academy of Sciences, 1991
As a first step in assessing the efficacy of a gene transer approach to the induction of transplantation tolerance in our miniature swine model, double-copy retroviral vectors engineered to express a drug-resistance marker (neomycin) and a swine class II DRB cDNA were constructed. Infectious particles contaning these vectors were produced at a titer of >1 x 10 G418-relstant colony-forming units/ml using both ecotropic and amphotropic p ing cell lines. Flow cytometric analysis of DRA-trmnsfected'urine fibroblsts subsequently transduced with virus-containing superna
American Journal of Transplantation, 2009
We studied the effects of indirect allorecognition on the induction and maintenance phases of tolerance in miniature swine cotransplanted with heart and kidney allografts. MHC class Imismatched heart and kidney grafts were cotransplanted in recipients receiving CyA for 12 days. Recipients were unimmunized or immunized with a set of donor-derived or control third-party MHC class I peptides either 21 days prior to transplantation or over 100 days after transplantation. T cell proliferation, delayed type hypersensitivity reaction (DTH) and antibody production were assessed. All animals injected with donor MHC class I peptides developed potent indirect alloresponses specific to the immunizing peptides. While untreated recipients developed stable tolerance, all animals preimmunized with donor allopeptides rejected kidney-heart transplants acutely. In contrast, when peptide immunization was delayed until over 100 days after kidney/ heart transplantation, no effects were observed on graft function or in vitro measures of alloimmunity. Donor peptide immunization prevenedt tolerance when administered to recipients pretransplantation but did not abrogate tolerance when administered to long term survivors posttransplantation. This suggests that the presence of T cells activated via indirect allorecognition represent a barrier to the induction but not the maintenance of tolerance.
Recombinant retrovirus vectors for the expression of MHC class II heterodimers
1999
Class II antigens are critical in determining the fate of vascularized allografts across major histocompatibility differences. We have recently developed a new approach to induce transplantation tolerance in miniature swine by creating MHC class II antigen "molecular chimerism" in bone marrow cells of potential recipients through retrovirus-mediated gene transfer. As part of this project, the ability of a recombinant double-expression vector (ZQ32N) to express MHC class II DQA and DQB was investigated. Flow cytometry analyses of ZQ32N transfected virus-producer cells demonstrated the cell surface expression of DQa/DQb heterodimers, thus suggesting a correct transcription, translation, and transport of the swine polypeptides to the cell surface. The analyses of RNA isolated from virus particles produced from ZQ32N transfected virus-producer cells indicated the DQ sequences to be correctly packaged. However, the DQ-negative cells transduced with the ZQ32N retrovirus did not show any DQretrovirus surface expression. Southern and Northern blot analyses of ZQ32N transfected and transduced cells strongly suggested DNA rearrangements and deletions which could account for transgene expression loss. An analysis of transduced cell genomes suggested DNA recombinations targeted to homologous sequences within the recombinant provirus. The implications of the sequence instability in designing vectors for gene therapy of organ transplantation are discussed.
This study tested the hypothesis that vascularized composite allografts (VCA) could be accepted in a robust model of hematopoietic chimerism by injecting allogeneic bone marrow cells (BMC) into swine fetuses. Outbred Yorkshire sows and boars were screened to ensure the absence of the major histocompatibility (MHC) allele SLA cc of inbred MGH miniature swine and then mated. Bone marrow harvested from an SLA cc swine donor was T-cell depleted and injected intravenously into the fetuses between days 50–55 of gestation. After birth, the piglets were studied with flow cytometry to detect donor cells and mixed lymphocyte reactions (MLR) and cell-mediated lympholysis (CML) assays to assess their response to donor. Donor-matched VCAs from SLA cc donors were performed on four chimeric and two nonchimeric swine. The results showed donor cell engraft-ment and multilineage macrochimerism after the in utero transplantation of adult BMC, and chimeric animals were unresponsive to donor antigens in vitro. Both control VCAs were rejected by 21 days and were alloreactive. Chimeric animals accepted the VCAs and never developed antidonor antibodies or alloreactivity to donor. These results confirm that the intravascular, in utero transplantation of adult BMC leads to donor cell chimerism and donor-specific tolerance of VCAs across a full MHC barrier in this animal model.
Xenotransplantation, 2002
We recently established that molecular chimeras of major histocompatibility complex (MHC) class II molecules, created via retroviral transfer of allogeneic class II cDNAs into bone marrow cells (BMCs), alleviated complications associated with mixed BMC chimeras while leading to T cell tolerance to renal grafts sharing the transferred class II. Initially demonstrated for allogeneic transplants in miniature swine, this concept was extended to T-dependent antibody (Ab) responses to xenogeneic antigens (Ags) in the pig ® baboon combination. Successful down-regulation of T cell responses appeared, however, to be contingent on a tight lineage-speci®c expression of transferred class II molecules. The present studies were, therefore, designed to evaluate the in¯uence of construct design and cellular environment on expression of retrovirally transferred xenogeneic class II cDNAs. Proviral genomes for pig class II SLA-DR expression, diering only at the marker neo(r) or enhanced green¯uorescent protein (EGFP) gene, showed increased membrane SLA-DR density on HLA-DR ± ®broblasts as well as HLA-DR + , TF-1 erythroleukemia cells. More importantly, HLA-DR + human B cell lines, although ef®ciently transduced with pig DR retroviruses, exhibited unstable surface pig DR. Surface pig DR ± B cells, nevertheless, stimulated autologous human T cells pre-sensitized to pig Ags, a proliferation likely occurring through presentation of class II-derived peptides. Collectively, these data suggest that surface expression of transferred class II molecules is not related to the ability of recipient cells to synthesize xenogeneic class II molecules but rather to their Ag processing capacities.
Bone Marrow Transplantation, 2000
In mice, giving IL-2 post transplant decreases GVHD in this setting. We studied high-dose IL-2 therapy in pigs. Transplants were carried out after conditioning with fractionated total body radiation and cyclophosphamide. Fourteen pigs received a fully mismatched bone marrow transplant (six with IL-2; eight without IL-2), and six received a single haplotype class II mismatched transplant (three with IL-2; three without IL-2). GVHD was evaluated by skin histology. All fully mismatched recipients had severe GVHD (grade 2-3) and died within 13 to 51 days whether or not they received IL-2. Pigs receiving a one haplotype class II mismatched transplant without IL-2 developed severe skin GVHD lasting for 8-45 days; all died within 57 days. Similar pigs receiving IL-2 post transplant had no or only mild skin GVHD for less than 15 days; two are long-term survivors. Bone Marrow Transplantation (2000) 25, 47-52. Keywords: GVHD; interleukin-2; bone marrow transplantation; miniature swine Graft-versus-host disease (GVHD) is caused by T cells in the allograft 1 and can be prevented by removing them. 2,3 However, T cell depletion is associated with less engraftment 4-6 and increased relapse risk. IL-2 is an immunoregulatory protein with a wide range of immune effects. We showed previously that recombinant interleukin-2 (IL-2) therapy in mice decreases severity of GVHD following allogeneic bone marrow transplantation (BMT) 10,11 with no adverse effect on engraftment 12 or relapse.
Journal of Experimental Medicine, 1997
The almost uniform failure in transplant patients of tolerance-inducing regimens that have been found to be effective in rodents, has made it necessary to examine large animal models before testing of new approaches clinically. Miniature swine have been shown to share many relevant immunologic parameters with humans, and because of their reproducible genetics, have proved extremely useful in providing such a large animal model. We have previously shown that indefinite systemic tolerance to renal allografts in miniature swine is induced in 100% of cases across a two-haplotype class I plus minor histocompatibility antigen disparity by a 12-d course of Cyclosporine A (CyA), in contrast to irreversible rejection observed uniformly without CyA treatment. In the present study, we have examined the role of the thymus during the induction of tolerance by performing a complete thymectomy 21 d before renal transplantation. This analysis demonstrated a striking difference between thymectomized and nonthymectomized animals. Thymectomized swine developed acute cellular rejection characterized by a T cell (CD25 ϩ ) infiltrate, tubulitis, endothelialitis and glomerulitis, and anti-donor CTL reactivity in vitro. Nonthymectomized and sham thymectomized animals had a mild T cell infiltrate with few CD25 ϩ cells and no anti-donor CTL response in vitro. These results indicate that the thymus is required for rapid and stable induction of tolerance. M any methods by which transplantation tolerance can be induced in rodents have failed when applied to large animals or to patients (1-4), making testing in large animals a necessary step before applying new techniques clinically. Miniature swine provide the only large animal model in which one can reproducibly study the effects of selective matching within the MHC on parameters of transplantation (5-7). We have therefore used MHC inbred and recombinant lines of miniature swine extensively for preclinical studies of transplantation tolerance (8-12). Previous studies from this laboratory have demonstrated that tolerance to renal allografts in miniature swine occurs spontaneously in about one-third of animals selectively matched for class II antigens and mismatched for a single class I MHC locus plus minor antigens (8, 13). The induction of spontaneous long-term tolerance was associated with a transient antidonor class I humoral response which has been shown to be almost entirely of the IgM class. Rejector animals developed antidonor class I IgG and promptly rejected their allografts. The failure to switch from IgM to IgG in spontaneous acceptors, suggested that the pathway to tolerance involved a deficiency of T cell help. Studies in miniature swine mismatched for two class I haplotypes were consistent with this hypothesis. Such animals reject renal allografts in 100% of cases without immunosuppression, but when T cell help was limited by the administration of a 12-d course of Cyclosporine A (CyA) 1 , 100% of animals developed long-term tolerance . Subsequent studies demonstrated that transplants of second renal allografts, MHC-matched to the original donors, were accepted without further immunosuppression if grafted at the time of the transplant nephrectomy (14). These results indicate that long-term graft acceptance is associated with the induction of systemic tolerance.
Transplantation, 2003
Background. Long-term survival of fully allogeneic cardiac grafts can be induced in mice through transduction of recipient bone marrow cells (BMCs) with a recombinant retroviral vector encoding a single full-length major histocompatibility complex (MHC) class I alloantigen. This study investigated whether cell surface expression of the transduced MHC antigen was necessary for the induction of specific unresponsiveness. Method. The signal sequence for translocation into the endoplasmic reticulum was deleted from H-2K b (SDELK b). Syngeneic BMCs from CBA.Ca (H2 k) recipients were transduced with an MFG retroviral vector encoding either wild-type K b or the mutant SDELK b and reinfused in conjunction with an anti-CD4 therapy. Four weeks later, the recipients underwent transplantation with a fully allogeneic C57BL/10 cardiac graft. Graft survival and the development of transplant arteriosclerosis were assessed. Results. Expression of both the wild-type K b or SDELK b in recipient CBA mice before transplantation resulted in prolonged survival of C57BL/10 grafts. Grafts from recipients pretreated with SDELK b developed 48%؎22% intimal proliferation compared with 61%؎21% in grafts from recipients pretreated with wild-type K b. However, this difference did not reach statistical significance. Conclusion. Cell surface expression, and therefore direct recognition, of an MHC class I alloantigen is not required to induce long-term survival of fully allogeneic cardiac grafts after retroviral transduction of recipient BMCs. MATERIALS AND METHODS Animals CBA.Ca (H2 k) mice were used as recipients and donors of bone marrow and syngeneic heart grafts, and C57BL/10 (H2 b) and NZW (H2 z) mice were used as fully allogeneic heart donors. BM3.6 mice were used as T-cell donors for the cytotoxicity assay. The mice were bred and maintained in the Biomedical Services Unit at the John