Immortalized bone-marrow derived pig endothelial cells.

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 IFNgamma. 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 xenoantigens, 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.

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 (Tg's) in recipient bone marrow cells would allow survival of subsequent Tg-matched renal allografts. Somatic matching between donor kidney class II and the recipient Tg's, 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.

Assessment of transduction rates of porcine bone marrow.

Although drug resistance is commonly used as an indicator of gene transfer in various cellular contexts, the assessment of drug resistance is often imprecise and over-estimated. To measure accurately transduction efficiencies of the retroviral-mediated transfer of genes encoding the neomycine phosphotransferase (Neo(r)) and porcine major histocompatibility (MHC) class II in pig bone marrow cells (BMC), the fraction of targeted progenitors was evaluated by both colony-forming unit granulocytes/macrophages assays (G418r CFU-GM) and by PCR analysis of the transgenes (Tg). Transduced and untransduced BMC were selected at different concentrations of G418 and revealed high individual variability of drug sensitivity. Comparison of the results obtained by estimating the CFU frequency and the PCR assays on drug-resistant colonies demonstrated a marked overestimation of BM transduction rates when determined by G418 resistance alone, because only approximately one-third of individual colonies were positive for both the Neo(r) and the class II Tg. Because this discrepancy is likely to affect the overall assessment of transduction rates using drug resistance markers, our data attest for the need of a combination of molecular assays to determine transduction efficiencies accurately.

Regulated expression of an MHC class II gene from a promoter-inducible retrovirus.

Specific immune tolerance to fully allogeneic kidney grafts can be achieved in a miniature swine transplantation model by retrovirus-mediated transfer of allogeneic MHC class II genes into bone marrow cells (BMCs) of recipient animals. Graft survival correlated with transient expression of the somatic transgene (Tg) in the induction phase of tolerance. With the aim of investigating the effects of timing and threshold levels of Tg expression on induction of hyporesponsiveness to the grafted tissues, two recombinant retrovirus constructs containing the tetracycline binary regulatory system were used to achieve conditional expression of either the green fluorescent protein (tetGFP) as a control, or the porcine MHC class II DRbeta chain (tetDRB). Effective downregulation of GFP gene transcription was demonstrated in transduced murine fibroblasts after doxycycline treatment, leading to a > 90% reduction of GFP fluorescence. Similar diminution of the DRB gene transcription was achieved in transduced pig endothelial cells (ECs). Drug-dependent downregulation of DRBc gene expression in SLAd pig ECs coincided with complete inhibition of allogeneic activation of anti-class IIc-primed SLAd T cells. These in vitro results suggest that the binary tetracycline retrovirus system may also be adequate to regulate MHC class II Tg expression in vivo.

MHC class II alpha/beta heterodimeric cell surface molecules expressed from a single proviral genome.

Transplantation tolerance to renal allografts can be induced in large animal preclinical models if the donor and recipient have identical major histocompatibility complex (MHC) class II loci. Such class II matching is, however, not clinically achievable owing to the extreme diversity of class II sequences. With the ultimate goal of creating a somatic class II match in the bone marrow of an allograft recipient, the aim of the study is to develop a double-copy retrovirus construct to express both chains of the MHC class II DQ glycoprotein on a single transduced cell. Analysis of the expression patterns of the retroviral DQ transgenes in both virus producer and transduced fibroblasts revealed correct transcription and stable surface expression of the DQ heterodimers. In addition, we demonstrate that both the DQA and DQB sequences are functional within the same proviral copy, a prerequisite for efficient induction of transplantation tolerance following transduction of bone marrow precursor cells. The DQ double-copy retrovirus vector showed efficient expression of the transferred class II cDNA in murine colony-forming units for the granulocyte-monocyte lineage (CFU-GM), indicating that it is suitable for gene therapy of multimeric proteins in hematopoietic cells.

Recombinant retrovirus vectors for the expression of MHC class II heterodimers.

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 DQ-retrovirus 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.

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.

Transfer of porcine MHC DRalpha into IEalpha-deficient murine bone marrow results in reduced IE-restricted Vbeta usage.

BACKGROUND: Allogeneic bone marrow transplantation has proven effective for inducing specific tolerance to subsequent solid organ allografts, although the clinical applicability of this approach is limited by the morbidity and mortality associated with this procedure. As an alternative, we are investigating the transfer of allogeneic MHC class II genes into recipient bone marrow cells (BMC), using the miniature swine as a model. METHODS: To understand the mechanism of tolerance induction achieved through class II gene transfer, BMC from C57BL/10 mice, which lack expression of the MHC class II DRalpha equivalent (H-2 IEalpha), were transduced with a retrovirus vector for swine DRalpha. RESULTS: Expression of the DRA-vector in bone marrow-derived cells was demonstrated by Northern analysis of colonies grown in vitro from transduced myeloid progenitors. Taking advantage of the fact that the introduced DRalpha chain was able to form heterodimers with endogenous IEbeta, surface expression of the transgene was demonstrated on splenocytes harvested 1, 17, and 28 weeks after bone marrow transplantation. Transgene expression was confirmed by reverse transcriptase-polymerase chain reaction in the thymus of those animals killed at weeks 17 and 28. Finally, the effects of bone marrow transduction on central tolerance induction was demonstrated by the progressive decrease of IE-reactive T-cell clones bearing Vbeta5 and Vbeta11 T cell receptors in the peripheral blood cells of engineered recipients. CONCLUSIONS: Our results support the notion that transplantation tolerance, induced by class II gene transfer into syngeneic BMC, results in part from durable deletional unresponsiveness of graft-specific alloreactive T cells.

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.

HLA-Cw3 expression on porcine endothelial cells protects against xenogeneic cytotoxicity mediated by a subset of human NK cells.

There is increasing evidence that NK cells make an important contribution to human anti-porcine xenogeneic cytotoxicity. Most allogeneic as well as autologous normal cells are not susceptible to NK cell-mediated cytotoxicity because they express inhibitory molecules encoded within the MHC class I loci. The protective signal is delivered to NK cells through killer cell-inhibitory receptors expressing different MHC class I specificities. It has been proposed that xenogeneic target cells may be susceptible to NK cell-mediated lysis because their MHC class I molecules fail to be recognized by human killer cell-inhibitory receptors. To explore this hypothesis, we examined the effect of human MHC class I expression on porcine target cell lysis by human NK cells. An immortalized porcine bone marrow-derived endothelial cell line (2A2) was transfected with three different human MHC class I allelic genes (HLA-A2, -B27, or -Cw3). The cytotoxic activity of several GL183+ NK clones, which lysed untransfected porcine cells effectively, was substantially blocked by the presence of HLA-Cw3. In contrast, HLA-Cw3-positive cells were not protected against lysis by GL183- EB6+ NK clones. The expression of HLA-B27 or HLA-A2 molecules on pig target cells did not provide substantial protection from lysis by any of the NK clones tested. In addition to confirming the hypothetical basis of NK cell-mediated killing of xenogeneic targets, these results have practical implications as an approach to overcoming NK cell-mediated cytotoxicity, which may be an obstacle to pig-to-human xenotransplantation.

The mechanism of specific prolongation of class I-mismatched skin grafts induced by retroviral gene therapy.

In the present study, we examine the mechanism of specific hyporesponsiveness to major histocompatibility complex (MHC) class I-mismatched skin allografts induced by retrovirus-mediated gene transfer of an allogeneic class I gene into syngeneic bone marrow (BM). Using appropriate congenic recombinant mouse strains, we have mapped MHC determinants that are capable of restoring rapid rejection of Kb-bearing skin grafts. Our results indicate that either a single class I or a single class II alloantigen expressed on skin in association with Kb is able to restore the rapid rejection of Kb-mismatched skin grafts. These data suggest that third-party alloantigens expressed on skin in association with Kb abrogate hyporesponsiveness by providing T cell help. Consistent with this interpretation, spleen cells from mice reconstituted with Kb-transduced BM were unable to elicit a significant anti-Kb cytotoxic T lymphocyte response in vitro unless interleukin-2 was added to the culture medium. Skin graft survival was also analyzed on B10. AKM mice thymectomized 3-4 weeks post-reconstitution with Kb-transduced BM. Thymectomy did not result in significantly prolonged survival of B10. MBR skin grafts compared to euthymic controls, suggesting that even early after reconstitution, intrathymic deletion of Kb-reactive T cells must have been incomplete. Taken together, these data suggest that prolongation of skin allograft survival in this model is controlled at the level of T cell help.

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.

Alloresistance to K locus class I-mismatched bone marrow engraftment is mediated entirely by CD4+ and CD8+ T cells.

Clinical application of approaches to inducing transplantation tolerance that involve bone marrow reconstitution will require achievement of engraftment without major toxicity to the recipient. These requirements are likely to vary according to the type of histoincompatibility between donor and recipient. We have attempted to determine the minimal conditioning required to achieve lasting mixed allogeneic chimerism and tolerance in the presence of a class I MHC disparity by evaluating the host elements that resist alloengraftment. We based our approach on a regimen that was shown to induce mixed chimerism in fully MHC-mismatched strain combinations. Recipient B10.AKM (KkIkDq) mice were treated with 7 Gy thymic irradiation (TI) and 3 Gy whole body irradiation (WBI) and received either anti-CD8 mAb alone or anti-CD4 plus anti-CD8 mAbs before transplantation of K locus-disparate B10.MBR (KbIkDq) marrow. All (27 of 27) animals receiving both mAbs showed lasting multi-lineage mixed chimerism and donor-specific tolerance. In contrast, five of 22 (23%) recipients pre-treated with anti-CD8 mAb alone in the same experiments failed to develop lasting multilineage mixed chimerism, suggesting that the CD4 T cell subset also participates in resistance to class I-mismatched marrow engraftment. We next attempted to determine whether or not host non-T cell elements resist allogeneic engraftment by comparing the minimum number of syngeneic vs allogeneic BMC required to achieve lasting multilineage mixed chimerism. Titrated numbers (10(6) to 10(7)) of B10.MBR (KbIkDq) bone marrow cells were administered to B10.AKM recipients treated with anti-CD4 and -CD8 mAbs, 3 Gy WBI and 7 Gy TI. All recipients of each marrow dose developed lasting multilineage mixed chimerism and showed specific tolerance to B10.MBR skin grafts. The level of donor-type repopulation in recipients of each dose was not lower than that observed in similarly irradiated recipients in an Ly5 congenic, otherwise syngeneic, BMT system. Together, our results suggest that CD4+ T cells contribute to resistance to K locus class I-mismatched marrow allografts and that resistance is mediated only by CD4 and CD8 T cells, with no role for non-T cell host elements.