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.

Acute humoral xenograft rejection: destruction of the microvascular capillary endothelium in pig-to-nonhuman primate renal grafts.

The major cause of xenograft loss beyond hyperacute rejection is a form of injury, traditionally termed delayed xenograft rejection (DXR), whose pathogenesis is unknown. Here we analyze the immunologic and morphologic features of DXR that develops in pig kidney xenografts transplanted into nonhuman primates. Kidneys from miniature swine were transplanted into cynomolgus monkeys (n = 14) or baboons (n = 11) that received regimens aimed to induce mixed chimerism and tolerance. No kidney was rejected hyperacutely. Morphologic and immunohistochemical studies were performed on serial biopsies, and an effort was made to quantify the pathologic features seen. The early phase of DXR (Days 0-12) was characterized by focal deposition of IgM, IgG, C3, and scanty neutrophil and macrophage infiltrates. The first abnormality recognized was glomerular and peritubular capillary endothelial cell death as defined by in situ DNA nick-end labeling (TUNEL). Damaged endothelial cells underwent apoptosis and, later, frank necrosis. The progressive phase developed around Day 6 and was characterized by progressive deposition of IgM, IgG, C3, and prominent infiltration of cytotoxic T cells and macrophages, with a small number of NK cells. Thrombotic microangiopathy developed in the glomeruli and peritubular capillaries with TUNEL+ endothelial cells, platelet aggregation, and destruction of the capillary network. Only rare damaged arterial endothelial cells and tubular epithelial cells were observed, with rare endothelialitis and tubulitis. In the advanced phase of DXR, interstitial hemorrhage and infarction occurred. During the development of DXR, the number of TUNEL+ cells increased, and this correlated with progressive deposition of antibody. The degree of platelet aggregation correlated with the number of TUNEL+ damaged endothelial cells. We conclude that peritubular and glomerular capillary endothelia are the primary targets of renal DXR rather than tubular epithelial cells or arterial endothelium and that the earliest detectable change is endothelial cell death. DXR was characterized by progressive destruction of the microvasculature (glomeruli and peritubular capillaries) and formation of fibrin-platelet thrombi. Both cytotoxic cells and antibodies potentially mediate the endothelial damage in DXR; however, in this model, DXR is largely humorally mediated and is better termed "acute humoral xenograft rejection."

Decreased graft-versus-host disease after haplotype mismatched bone marrow allografts in miniature swine following interleukin-2 treatment.

Graft-versus-host disease (GVHD) is an important complication of bone marrow transplantation after transplants between HLA-mismatched donor/recipient pairs. 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.

Tolerance in a concordant nonhuman primate model.

BACKGROUND: We have previously demonstrated that induction of mixed lymphohematopoietic chimerism resulted in donor specific renal allograft tolerance without the need for chronic immunosuppression in nonhuman primates. Here we have tested whether tolerance can be similarly induced for baboon to cynomolgus renal xenografts. METHODS: After preconditioning with anti-thymocyte globulin (ATG), nonlethal total body irradiation, and thymic irradiation, cynomolgus monkeys underwent splenectomy, native nephrectomies, and baboon marrow and renal transplants. Postoperative cyclosporine was given for 28 days. RESULTS: In Group 1 (n=2, survival= 13, 14 days), both animals developed anti-donor immunoglobulin G, had biopsy findings consistent with humoral rejection, and showed rapidly progressive xenograft failure. In Group 2 (n=5, survival=1, 16, 33, 112, 190 days), 15-deoxyspergualine was added to the regimen (Day 0-13). In one long-term survivor, donor specific hyporesponsiveness was first observed (mixed lymphocyte culture [(MLR]) on Day 48. MLR reactivity returned on Day 64 together with the development of anti-donor antibody and subsequent xenograft failure on Day 112. Donor specific T-cell hyporesponsiveness was detected in the other long-term survivor for the first 133 days, after which a donor-specific skin xenograft was placed, (survival 24 days). Following the skin graft rejection, a rise in the MLR, development of anti-donor antibody and progressive rejection of the renal xenograft were observed. CONCLUSIONS: Antibody-mediated rejection seems to constitute the major difference between concordant xenografts and allografts. Addition of 15-deoxyspergualine for 2 weeks posttransplant extended concordant primate xenograft survival to 6 months without chronic immunosuppression. In contrast to the allogeneic model, renal transplant acceptance in this xenogeneic system was interrupted by placement of a donor-specific skin graft.

Long-term discordant xenogeneic (porcine-to-primate) bone marrow engraftment in a monkey treated with porcine-specific growth factors.

BACKGROUND: Mixed allogeneic hematopoietic chimerism has previously been reliably achieved and shown to induce tolerance to fully MHC-mismatched allografts in mice and monkeys. However, the establishment of hematopoietic chimerism has been difficult to achieve in the discordant pig-to-primate xenogeneic model. METHODS: To address this issue, two cynomolgus monkeys were conditioned by whole body irradiation (total dose 300 cGy) 6 and 5 days before the infusion of pig bone marrow (BM). Monkey anti-pig natural antibodies were immunoadsorbed by extracorporeal perfusion of monkey blood through a pig liver, immediately before the intravenous infusion of porcine BM (day 0). Cyclosporine was administered for 4 weeks and 15-deoxyspergualin for 2 weeks. One monkey received recombinant pig cytokines (stem cell factor and interleukin 3) for 2 weeks, whereas the other received only saline as a control. RESULTS: Both monkeys recovered from pancytopenia within 4 weeks of whole body irradiation. Anti-pig IgM and IgG antibodies were successfully depleted by the liver perfusion but returned to pretreatment levels within 12-14 days. Methylcellulose colony assays at days 180 and 300 revealed that about 2% of the myeloid progenitors in the BM of the cytokine-treated recipient were of pig origin, whereas no chimerism was detected in the BM of the untreated control monkey at similar times. The chimeric animal was less responsive by mixed lymphocyte reaction to pig-specific stimulators than the control monkey and significantly hyporesponsive when compared with a monkey that had rejected a porcine kidney transplant. CONCLUSION: To our knowledge, this is the first report of long-term survival of discordant xenogeneic BM in a primate recipient.

Removal of anti-porcine natural antibodies from human and nonhuman primate plasma in vitro and in vivo by a Galalpha1-3Galbeta1-4betaGlc-X immunoaffinity column.

BACKGROUND: Natural antibodies (NAbs) against a terminal alpha1-3 galactosyl (alphaGal) epitope have been identified as the major human anti-pig NAbs. METHODS AND RESULTS: We used two synthetic alphaGal trisaccharides--type 6 (alphaGal6) and type 2(alphaGal2)--linked to an inert matrix to remove NAbs from human plasma in vitro. Flow cytometry indicated that an average of 85% of the NAb binding activity was depleted by adsorption with alphaGal6. By measuring the binding of NAbs to pig peripheral blood mononuclear cells and bone marrow cells, we demonstrated that alphaGal6 was more effective than alphaGal2 in removing NAbs, and the combination of alphaGal6 + alphaGal2 did not further increase removal of NAbs. The specificity of the removal of NAbs (IgM and IgG) reactive with the alphaGal epitope by alphaGal6 matrix was shown by enzyme-linked immunosorbent assay. In vivo studies in nonhuman primates compared plasma perfusion through a alphaGal6 immunoaffinity column with hemoperfusion through a pig liver for changes in blood pressure, hematocrit, platelets, and NAb adsorption. CONCLUSIONS: Both methods reduced the level of anti-pig IgM and IgG xenoreactive antibodies to nearly background, but column perfusion caused less hypotension and reduction in platelets than liver perfusion. Four pig kidneys transplanted into monkeys after column perfusion did not undergo hyperacute rejection, remaining functional for 2-10 days, with a mean functional period of 7 days, demonstrating that a pig kidney can support renal function in a primate.

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.

Pig to monkey bone marrow and kidney xenotransplantation.

BACKGROUND: The intensity of discordant xenograft cellular rejection makes it unlikely that safe doses of immunosuppressive drugs will alone be sufficient to permit long-term survival. We have therefore concentrated our efforts on establishing tolerance to xenogeneic organs through lymphohematopoietic chimerism and the elimination of preformed natural antibodies (nAbs). METHODS: Here we report the most recent series of 11 technically successful porcine to nonhuman primate transplantation procedures. In eight experimental animals induction therapy consisted of (1) 3 x 100 cGy nonlethal whole body irradiation (day -6 and day -5) to all animals, (2) horse anti-human thymocyte globulin (day -2, day -1, and day 0) to seven of the animals, (3) 700 cGy thymic irradiation (day -1) to five of the animals, and (4) pig bone marrow infused on day 0 (2-9 x 10(8)/cells/kg). On day 0, just before the renal xenograft, the recipient was splenectomized, and antipig nAbs were removed by means of perfusion of the monkey's blood through either a pig liver (n = 6) or a Gal-alpha (1,3)-Gal adsorption column (n = 5). There control animals did not receive this pretransplantation induction therapy but did undergo hemoperfusion and posttransplantation immunosuppression identical to the experimental animals. All 11 recipients were treated after transplantation with cyclosporin A and 15-deoxyspergualin. Recombinant pig-specific growth factors (interleukin-3 and stem cell factor) were given to six experimental animals from day 0 until the termination of the experiment. RESULTS: Analysis of recipients' sera by means of flow cytometry indicated the effective removal of immunoglobulin M and immunoglobulin G nAbs by either liver perfusion or column adsorption. In the eight experimental animals, nAb titers remained low until death (up to 15 days), but in the three control animals nAb titers increased substantially with time. The longest surviving recipient maintained excellent kidney function with creatinine levels at 0.8 to 1.3 mg/dl throughout its course. Death occurred at day 15 from complications caused by a urinary leak and pancytopenia. Histologic examination of the xenograft revealed only focal tubular necrosis and cytoplasmic vacuolization, with trace amounts of fibrin and C3 in peritubular capillaries. In this animal a fraction of the peripheral blood cells (3%) at day 7 were of pig origin as detected by pig-specific monoclonal antibodies. In addition, colony-forming assays performed on a bone marrow biopsy specimen taken at day 14 indicated that approximately 30% of the relatively few myeloid progenitors detected were of swine origin. CONCLUSIONS: We have demonstrated that our protocol is effective in the prevention of hyperacute rejection and in the maintenance of excellent function of the renal xenograft for up to 15 days. These results also indicate that at least short-term engraftment of the xenogeneic donor bone marrow cells is possible to achieve in this discordant large animal combination. Longer survivals will be required to assess the possible effect of this engraftment on induction of tolerance.

A study of tolerance in a concordant xenograft model.

Antibody-mediated rejection appears to constitute the major difference between concordant xenografts and allografts in nonhuman primates. Consistent with its known effect on antibody responses, 5-7 addition of DSG to the conditioning regimen has extended concordant primate xenograft survival for up to 6 months after discontinuation of conventional immunosuppression. In contrast to our observations in recipients of renal allografts, donor-specific skin graft rejection can occur and even in long-term recipients may induce rejection of a previously accepted renal xenograft.

Specific tolerance across a discordant xenogeneic transplantation barrier.

Successful induction of tolerance across disparate (discordant) species barriers could overcome the organ shortage that presently limits clinical transplantation. We demonstrate here that xenogeneic swine thymic transplants can induce tolerance to swine antigens in mice, while positively selecting functional host CD4+ T cells. Immunologically normal C57BL/10 mice were thymectomized and depleted of T and natural killer cells; then they received transplants of fetal pig thymus and liver fragments. Mature mouse CD4+ T cells developed in the pig thymus grafts and migrated to the periphery. Swine grafts grew markedly and no anti-pig IgG response was produced. Mixed lymphocyte reactions confirmed that the new T cells were functional and were tolerant to pig antigens.

In vivo treatment with monoclonal antibodies directed against CD4 and CD8 antigens in miniature swine.

Two monoclonal antibodies (mAb) with specificities for mature T-cell subsets in miniature swine have been characterized previously. Antibody 74-12-4 recognizes the porcine CD4 accessory molecule and 76-2-11 is specific for the CD8 molecule. We have now examined the effects of in vivo administration of 74-12-4 and 76-2-11 on several parameters of transplantation immunity. No prolongation of class I disparate skin or kidney graft survival was observed in animals treated with either mAb alone or with a combination of both. In addition, in vivo treatment with these mAbs, in combination with subtherapeutic total body irradiation, failed to permit engraftment of allogenic bone marrow. These therapeutic failures were thought likely to be a consequence of the fact that 74-12-4 coats, but does not deplete, CD4 cells in vivo. Because there are numerous anti-human mAbs that likewise fail to deplete in vivo, we have used 74-12-4 as a prototype to further manipulations aimed at achieving depletion. We attempted to eliminate 74-12-4-coated cells in two animals by subsequent administration of a hyperimmune pig anti-mouse Ig serum. In both such treated animals, administration of this serum produced surprisingly rapid clearance of 74-12-4 from the circulation and caused uncoating of CD4 cells, but no significant cell elimination was detected by flow cytometry. We have also prepared an anti-porcine-CD4 immunotoxin by conjugating 74-12-4 to the ribosome inhibitory plant toxin, pokeweed antiviral protein. This immunotoxin led to significant but not complete CD4 cell depletion from the peripheral blood in four of four treated animals. Further manipulations and possibly development of new anti-porcine CD4 mAbs may therefore be required to achieve complete depletion and successful mAb-mediated modification of transplantation responses.

The importance of nonimmune factors in reconstitution by discordant xenogeneic hematopoietic cells.

Bone marrow transplantation has been shown to induce donor-specific tolerance in rodent models. This approach could potentially be applied to xenotransplantation across discordant species barriers. To evaluate host factors resisting hematopoietic cell engraftment, we have developed two model systems utilizing the combination of swine into severe combined immunodeficient (SCID) mice. SCID mice lack functional B and T lymphocytes, and can therefore be used to evaluate nonimmune factors resisting marrow engraftment, and for adoptive transfer studies to test the role of immune cells and antibodies. First we transplanted swine bone marrow cells into SCID mice conditioned with whole-body irradiation (4 Gy). For nine weeks following the intravenous administration of 10(8) swine bone marrow cells, up to 3.8% of peripheral blood leukocytes were of swine origin, as determined by flow cytometry (FCM). These cells were all of the myeloid lineage. Swine IgG was also detectable in the serum for up to 14 weeks. The bone marrow of the reconstituted mice contained low percentages of swine myeloid cells, and swine myeloid progenitors could be detected for up to 20 weeks after bone marrow transplantation. In a second model, we grafted thymus and liver tissue from 45-69-day-old swine fetuses under the kidney capsule of 4 Gy-irradiated SCID mice. A suspension containing 10(8) swine fetal liver cells (FLC) was also administered i.p. Long-term repopulation with swine T cells was observed, with up to 1.5% swine T cells detected in the WBC, peritoneum, and spleen for at least 5.5 months postgrafting. These T cells expressed either CD4 or CD8, whereas up to 17.6% of cells in the thymic grafts expressed both CD4 and CD8. The i.p. FLC suspension was required for optimal long-term graft maintenance. Our studies show that (1) low level myeloid and B lymphocyte reconstitution can be achieved by transferring adult swine BMC to irradiated SCID recipients; (2) swine myeloid progenitors were detectable long-term in BMC of these mice, suggesting that stem cell engraftment was achieved; and (3) T cell reconstitution of SCID mice by swine progenitors requires cotransplantation of a swine stromal environment, as is provided by fetal swine thymus/liver grafts. We conclude that nonimmune factors such as those provided by species-specific stromal environments are important for reconstitution of some lineages by discordant hematopoietic stem cells.

Bone marrow culture and transduction of stem cells in a miniature swine model.

Recombinant retroviral vectors, engineered to express the beta-chain gene of swine major histocompatibility complex class II DR, were developed for the genetic modification of swine hematopoietic stem cells (HSC). The expression of these vectors in swine bone marrow has been studied both in culture and after bone marrow transplantation. In addition, myeloid progenitor colony assays were performed on swine umbilical cord blood as part of a study to identify alternative sources of HSC for somatic gene transfer, revealing the presence of both granulocyte macrophage colony forming-units (CFU-GM) and CFU-Mix at frequencies comparable to those found in juvenile swine bone marrow.