Our laboratory has previously reported a nonmyelosuppressive preparative regimen for hematopoietic cell transplantation that leads to mixed chimerism and allograft tolerance in miniature swine across minor and major histocompatibility disparities. Stable chimerism persisted in most of these animals but was restricted to T cells and confined to peripheral blood. Because of the importance of myeloid and erythroid progenitors for the treatment of hematologic disorders, the objective of this study was to assess whether such cells existed in the bone marrow of these lymphoid chimeras as an indication of functional engraftment. Colony-formation assays were performed on donor inocula before infusion and on bone marrow cells harvested from the transplant recipients. Donor-origin myeloid/erythroid progenitor colonies were detected in bone marrow from 6 of 7 lymphoid chimeric recipients. A delayed donor leukocyte infusion successfully converted a stable lymphoid chimera to full multilineage chimerism within 2 weeks. Donor-origin myeloid/erythroid progenitors could be detected in the bone marrow of a host-matched recipient after myeloablation and adoptive transfer of mobilized cells from one of the engrafted lymphoid chimeras. These data suggest that even when only lymphoid chimerism is readily detected by flow cytometry, dormant myeloid/erythroid progenitors can exist and subsequent conversion to full donor chimerism can be achieved. The ability to establish multilineage engraftment and chimerism without significant toxicity may have important clinical implications for the management of nonmalignant hematopoietic disorders and hematologic malignancies.

BACKGROUND In an attempt to induce mixed hematopoietic chimerism and transplantation tolerance in the pig-to-primate model, we have infused high-dose porcine peripheral blood progenitor cells (PBPC) into baboons pretreated with a nonmyeloablative regimen and anti-CD154 monoclonal antibody (mAb). METHODS Group 1 baboons (n=2) received a nonmyeloablative regimen including whole body irradiation, pharmacological immunosuppression, porcine hematopoietic growth factors, and immunoadsorption of anti-Galalpha1,3Gal (Gal) antibody before infusion of high doses of PBPC (2.7-4.6x10(10) cells/kg). In group 2 (n=5), cyclosporine was replaced by anti-CD154 mAb. Group 3 (n=3) received the group 1 regimen plus anti-CD154 mAb. RESULTS In group 1, pig chimerism was detected in the blood by flow cytometry (FACS) for 5 days (with a maximum of 14%), and continuously up to 13 days by polymerase chain reaction (PCR). In group 2, pig chimerism was detectable for 5 days by FACS (maximum 33%) and continuously up to 28 days by PCR. In group 3, initial pig chimerism was detectable for 5 days by FACS (maximum 73%). Two of three baboons showed reappearance of pig cells on days 11 and 16, respectively. In one, in which no anti-Gal IgG could be detected for 30 days, pig cells were documented in the blood by FACS on days 16-22 (maximum 6% on day 19) and pig colony-forming cells were present in the blood on days 19-33, which we interpreted as evidence of engraftment. Microchimerism was continuous by PCR up to 33 days. CONCLUSIONS These results suggest that there is no absolute barrier to pig hematopoietic cell engraftment in primates, and that this may be facilitated if the return of anti-Gal IgG can be prevented.

The aim of the present study was to determine whether certain components of nonmyeloablative regimens for hematopoietic cell transplantation might compromise the growth of hematopoietic progenitors.

Thrombotic microangiopathy (TM) is a serious complication of bone marrow transplantation (BMT) that resembles thrombotic thrombocytopenic purpura (TTP). In attempting to achieve hematopoietic cell chimerism in the pig-to-baboon model, we have observed TM following infusion of high doses (>1010 cells/kg) of porcine peripheral blood mobilized progenitor cells (PBPC) into baboons. We performed investigations to analyze the pathobiology of this TM and to test therapeutic interventions to ameliorate it. PBPC were obtained by leukapheresis of cytokine-stimulated swine. The initial observations were made in two baboons that underwent a non-myeloablative regimen (NMR) prior to PBPC transplantation (TX) (group 1). We then studied three experimental groups. Group 2 (n = 2) received NMR without PBPC TX. Group 3 (n = 2) received PBPC TX alone. Group 4 (n = 6) received NMR + PBPC TX combined with prostacyclin, low-dose heparin, methylprednisolone, and cyclosporine was replaced by anti-CD40L mAb in five cases. Baboons in groups 1 and 3 developed severe thrombocytopenia (<10 000/mm3), intravascular hemolysis with schistocytosis (>10/high powered field (hpf)), increase in plasma lactate dehydrogenase (LDH) (2500–9000 U/l), transient neurologic changes, renal insufficiency, and purpura. Autopsy on two baboons confirmed extensive platelet thrombi in the microcirculation, and, similar to clinical BMT-associated TM/TTP, no unusually large vWF multimers or changes in vWF protease activity were observed in the plasma of baboons with TM. In group 2, self-limited thrombocytopenia occurred for 10–15 days following NMR. Group 4 baboons developed thrombocytopenia (<20 000/mm3) rarely requiring platelet transfusion, minimal schistocytosis (<3/hpf), minor increase in LDH (<1000 U/l), with no clinical sequelae. We conclude that high-dose porcine PBPC infusion into baboons induces a microangiopathic state with vWF biochemical parameters resembling clinical BMT-associated TM/TTP and that administration of antithrombotic and anti-inflammatory agents can ameliorate this complication. Bone Marrow Transplantation (2001) 27, 1227–1236.

BACKGROUND:In pig-to-primate organ transplantation, hyperacute rejection can be prevented, but the organ is rejected within days by acute vascular rejection, in which induced high-affinity anti-Gal alpha1-3Gal (alphaGal) IgG and possibly antibodies directed against new porcine (non-alphaGal) antigenic determinants are considered to play a major role. We have explored the role of an anti-CD40L monoclonal antibody in modifying the humoral response to porcine hematopoietic cells in baboons pretreated with a nonmyeloablative regimen.METHODS:Porcine peripheral blood mobilized progenitor cells obtained by leukapheresis from both major histocompatibility complex-inbred miniature swine (n=7) and human decay-accelerating factor pigs (n=3) were transplanted into baboons. Group 1 baboons (n=3) underwent whole body (300 cGy) and thymic (700 cGy) irradiation, T cell depletion with ATG, complement depletion with cobra venom factor, short courses of cyclosporine, mycophenolate mofetil, porcine hematopoietic growth factors, and anti-alphaGal antibody depletion by immunoadsorption before transplantation of high doses (2-4 x 10(10)/cells/kg) of peripheral blood mobilized progenitor cells. In group 2 (n=5), cyclosporine was replaced by eight doses of anti-CD40L monoclonal antibodies over 14 days. The group 3 baboons (n=2) received the group 1 regimen plus 2 doses of anti-CD40L monoclonal antibodies (on days 0 and 2).RESULTS:In group 1, sensitization to alphaGal (with increases in IgM and IgG of 3- to 6-fold and 100-fold, respectively) and the development of antibodies to new non-alphaGal porcine antigens occurred within 20 days. In group 2, no sensitization to alphaGal or non-alphaGal determinants was seen, but alphaGal-reactive antibodies did return to their pre- peripheral blood mobilized progenitor cells transplant levels. In group 3, attenuated sensitization to alphaGal antigens was seen after cessation of cyclosporine and mycophenolate mofetil therapy at 30 days (IgM 4-fold, IgG 8-30-fold), but no antibodies developed against new porcine determinants. In no baboon did anti-CD40L monoclonal antibodies prevent sensitization to its own murine antigens.CONCLUSIONS:We believe these studies are the first to consistently demonstrate prevention of a secondary humoral response after cell or organ transplantation in a pig-to-primate model. The development of sensitization to the murine elements of the anti-CD40L monoclonal antibodies suggests that nonresponsiveness to cell membrane-bound antigen (e.g., alphaGal) is a specific phenomenon and not a general manifestation of immunological unresponsiveness. T cell costimulatory blockade may facilitate induction of mixed hematopoietic chimerism and, consequently, of tolerance to pig organs and tissues.

BACKGROUND:Because of the relative ease of acquisition, increased yield, and improved engraftment characteristics, mobilized peripheral blood progenitor (stem) cells (PBSCs) have recently become the preferred source for hematopoietic stem cell transplantation. In our laboratory, procurement of a megadose of PBSCs is necessary for on-going studies evaluating non-myelosuppressive transplant regimens for the induction of mixed chimerism and allograft tolerance. To exploit hematopoietic growth factor synergy, we have sought to combine growth factors with proven utility to improve PBSC mobilization and maximize our PBSC procurement through an automated collection procedure.METHODS:Mobilization characteristics of PBSCs were determined in 2-5-month-old miniature swine. Animals received either swine recombinant stem cell factor (pSCF, 100 microg/kg) and swine recombinant interleukin 3 (pIL-3, 100 microg/kg), administered intramuscularly for 8 days, or pSCF, pIL-3, and human recombinant granulocyte-colony stimulating factor (hG-CSF), at 10 microg/kg. Leukapheresis was performed beginning on day 5 of cytokine treatment and continued daily for 3 days.RESULTS:Collection of PBSCs from cytokine-mobilized animals via an automated leukapheresis procedure demonstrated a 10-fold increase in the number of total nucleated cells (TNC) (20-30 x 10(10) TNC) compared to bone marrow harvesting (2-3 x 10(10) total TNC). A more rapid rise in white blood cells (WBCs) was seen after administration of all three cytokines compared to pSCF and pIL-3 alone. An increase in colony-forming unit granulocyte-macrophage frequency measured daily from peripheral blood during cytokine treatment, was seen with the addition of hG-CSF to pSCF/pIL-3 correlating well with the rise in WBCs. Similarly, the addition of hG-CSF demonstrated a notable increase in the median progenitor cell yield from the 3-day leukapheresis procedure. Cytokine-mobilized PBSCs were capable of hematopoietic reconstitution. PBSCs mobilized with pSCF/pIL-3 were infused into an SLA-matched recipient conditioned with cyclophosphamide (50 mg/kg) and total body irradiation 1150 cGy. Neutrophil and platelet engraftment occurred on days 5 and 7, respectively, with minimal evidence of graft-versus-host disease. Complete donor chimerism has been demonstrated 331 days after transplant.CONCLUSIONS:Our preliminary results show that in this well-defined miniature swine model, recombinant swine cytokine combinations (pSCF, pIL-3 with or without hG-CSF) successfully mobilize a high yield of progenitor cells for allogeneic transplantation. Furthermore, these cytokine-mobilized PBSCs demonstrate the potential to reconstitute hematopoiesis and provide long-term engraftment in miniature swine.

512 In allograft models, the induction of mixed hematopoietc cell chimerism by bone marrow (BM) or peripheral blood stem cells (PBSC) transplantation (Tx) leads to donor-specific tolerance of subsequently transplanted organs. We are investigating the induction of mixed chimerism in the discordant pig-to-baboon model. METHODS. Leukopheresis of cytokine-stimulated (pIL3, pSCF, hGCSF) mobilised PBSC in pigs results in the collection of 30-90×10⁁10 cells. Pig leukocytes (15-35×10⁁10) were transplanted into 3 groups of splenectomized baboons. Group 1 (n=2) received no preparative therapy. Group 2 (n=2) received whole body (300cGy) and thymic (700cGy) irradiation, ATG, cyclosporine, mycophenolate mofetil, cobra venom factor, pig-specific cytokines, and extracorporeal immunoadsorption to remove anti-Gal antibodies before PBSC Tx. Group 3 (n=6) received the above regimen combined with prostacyclin, low-dose heparin and methylprednisolone (PHM) +/− antiCD40L mAb (20mg/kg ×2 or ×8 doses). RESULTS. Baboons in Groups 1 and 2 developed severe thrombocytopenia (<10,000cu.mm) requiring multiple platelet transfusions, marked schistocytosis (>12hpf), increase in plasma LDH (<25,000U/L), loss of high molecular von Willebrand factor (vWF), and mild anemia, transient neurologic changes, renal insufficiency and clinical purpura. Two baboons died of these complications; autopsy confirmed extensive platelet thrombi in the microcirculation. (The Group 2 conditioning regimen alone - without PBSC Tx - does not induce these changes.) Group 3 baboons developed moderate thrombocytopenia (not requiring transfusion), mild schistocytosis (<3hpf), mild increase in LDH (<1000U/L), with no other sequelae, despite loss of vWF. CONCLUSIONS. These data are in keeping with a thrombotic thrombocytopenic purpura-like state induced in baboons by porcine PBSC Tx, which can be fatal. Prophylactic therapy with PHM, particularly when combined with anti-CD40L mAb, reduces the sequelae of endothelial activation, markedly reduces microangiopathic hemolysis, and facilitates the induction of mixed chimerism.

284 The induction of mixed hematopoietic chimerism is a means of achieving specific immunological tolerance. Chimerism and skin allograft tolerance have been induced in mice by costimulatory blockade combined with a nonmyeloablative regimen. We have therefore incorporated the anti-CD40L mAb in our pig-to-primate nonmyeloablative hematopoietic cell transplantation protocol. We have previously employed a regimen that includes whole body (300cGy) and thymic (700 cGy) irradiation, complement depletion with cobra venom factor, short courses of ATG, cyclosporine (CYA), mycophenolate mofetil (MMF), porcine hematopoietic growth factors, and preformed anti-Gal antibody depletion by immunoadsorption, in attempts to induce chimerism in baboons transplanted with high doses (2-3 × 10⁁10 cells/kg) of porcine mobilized peripheral blood stem cells (PBSC) obtained by leukapheresis. In the present study, Group 1 baboons (n=3) received the above regimen +/− therapy aimed at reducing a thrombotic microangiopathic state that follows PBSC infusion. Sensitization to Gal (IgM × 4-fold, IgG × 100-fold) and nonGal porcine antigens developed within 7-10 days. Group 2 baboons (n=4) received the same regimen except for the replacement of CYA by a 14 day course (8 doses of 20mg/kg) of anti-CD40L mAb. No sensitization to Gal or nonGal determinants was seen, with follow-up for 100days. Group 3 baboons (n=2) received the Group 1 regimen and, in addition, 2 doses (days 0 and 2) of anti-CD40L mAb. In the 1 baboon in which follow-up to date extends beyond 30 days (and in which pig cell engraftment occurred with pig cells detected by FACS for >3 weeks), late and attenuated sensitization was seen after cessation of CYA and MMF therapy (anti-Gal IgM × 3-fold, IgG × 20-fold). In Groups 2 and 3 baboons, anti-CD40L mAb did not prevent sensitization to its own murine antigens. We conclude that, in this model, (1) high-dose PBSC transplantation led to humoral sensitization; (2) sensitization was prevented by the incorporation of an 8-dose course of anti-CD40L mAb; (3) the combination of 2 doses of mAb and CYA did not prevent sensitization in 1 of 2 baboons; (4) the development of sensitization to the murine elements of the mAb indicates that nonresponsiveness to Gal was not a general manifestation of immunological hyporesponsiveness. These studies demonstrate prevention of a humoral response following cell or organ transplantation in a pig-to-primate model, and suggest that T cell costimulatory blockade may play a valuable role in achieving successful discordant xenotransplantation.

112 Miniature swine provide a large animal model with well-defined genetics for studying allogeneic transplantation. We report here that stable mixed chimerism tolerance can be established in miniature swine using peripheral blood progenitor cells (PBPC) and a non-myelosuppressive conditioning protocol. Methods: Donor swine were mobilized with pig cytokines (pSCF and pIL-3, with or without hG-CSF). Apheresis was initiated on day 5 and continued on a daily basis until sufficient numbers of cells were collected. Recipients were conditioned for transplantation by in vivo depletion of T cells with anti-CD3 immunotoxin and thymic irradiation (TI). PBPC were infused in multiple doses over 3-6 days. Cyclosporine A (CyA) coverage was maintained for 30-60 days post-PBPC transplant. Results: Engraftment of PBPC across a minor histocompatibility barrier was successful in animals undergoing T cell depletion, 700 cGy TI and 30 days of CYA. Stable multilineage chimerism could be detected for more than 300 days with no evidence of clinically significant GvHD. Immunocompetence was demonstrated by prompt rejection of third party minor antigen mismatched skin grafts. Donor specific tolerance was demonstrated by prolongation of donor skin grafts without immunosuppression. Successful engraftment was achieved across a single haplotype MHC barrier when thymic irradiation was increased to 1000 cGy. Grade 2 GvHD was observed in two animals following CYA cessation at 30 days. In one animal, skin and intestinal GvHD was self limited and completely resolved by 90 days. A donor kidney graft placed in this animal without immunosuppression has not shown any sign of rejection (>60d). The second animal was sacrificed at day 73 due to persistent intestinal GvHD. GvHD was completely avoided in an animal given single haplotype mismatched PBPC when the dose of CYA was extended and tapered by day 60. Stable multilineage chimerism was demonstrated in this animal along with immunocompetence and tolerance to donor kidney graft (>30d). Conclusions: Immunocompetence and specific immune tolerance have been demonstrated in a miniature swine allogeneic transplantation model across major histocompatibility barriers using a non-myelosuppressive conditioning regimen. This is an attractive protocol for potential clinical application.

BACKGROUND AND PURPOSE:The pig is being investigated as an organ donor for humans. Induction of immunologic tolerance to pig tissues in primates would overcome the major immunologic barriers to xenotransplantation. A proven method of inducing tolerance to allografts is by the induction of mixed hematopoietic chimerism by bone marrow transplantation. We are therefore investigating induction of mixed hematopoietic chimerism in the pig-to-baboon model.METHODS:To obtain large numbers of pig hematopoietic cells, leukapheresis was used to collect blood cell products in miniature swine (n = 5) after progenitor cell mobilization by use of a course of hematopoietic growth factors (cytokines), consisting of porcine interleukin 3, porcine stem cell factor, and human granulocyte colony-stimulating factor.RESULTS:Cytokine therapy and leukapheresis were well tolerated. Cytokine therapy increased the total white blood cell count and allowed large numbers of leukocytes (60 x 10(10)) to be obtained by apheresis, of which approximately 0.1% were granulocyte-erythrocyte-monocyte-megakaryocyte colony-forming units (CFU-GEMMs), which are considered to be representative of hematopoietic progenitors with multi-lineage potential.CONCLUSIONS:The combination of cytokine therapy and leukapheresis enables hematopoietic progenitor cells to be obtained safely from miniature swine.