Chimerism induction in vascularized bone marrow transplants augmented with bone marrow cells

Composite tissue allografts (CTAs) are currently accepted in the clinic; however, long-term immunosuppression is still needed for allograft survival. The presence of donor-specific chimerism may induce tolerance. Thirty-six vascularized bone marrow transplantation (VBMT) allotransplantation were performed across MHC barrier under short-term protocol of 7-day alphabeta-TCRmAb and Cyclosporin A therapy to determine the efficacy of VBMT alone and VBMT augmented with donor bone marrow transplantation (BMT) in chimerism induction. Flow cytometry analysis revealed that VBMT supported with donor BMT directly into the bone resulted in chimerism augmentation and maintenance compared to VBMT. In vivo and in vitro tolerance testing showed prolonged survival of donor skin graft up to 35 days and moderate reactivity in MLR assay that suggests only tolerance induction. Transplantation of vascularized bone without chronic immunosuppression provides a substantial source of bone marrow cells, leading to the development of stable donor-specific chimerism.     

In utero bone marrow transplantation induces kidney allograft tolerance across a full major histocompatibility complex barrier in Swine

BACKGROUND: In utero hematopoietic stem-cell transplantation has been shown to induce donor-specific tolerance in small-animal models. However, tolerance has been difficult to achieve in large-animal studies. METHODS: Outbred swine underwent in utero transplantation of fully major histocompatibility complex (MHC)-mismatched CD3-depleted bone marrow mixed with fresh bone marrow to achieve a final CD3 content of 1.5%. Transplantation was performed at 50 to 55 days' gestation and two animals survived long term and demonstrated multilineage peripheral blood hematopoietic chimerism. These two long-term survivors were analyzed for in vitro evidence of donor-specific tolerance by mixed leukocyte reaction (MLR), cell-mediated lysis (CML), and antibody testing and in vivo by kidney transplantation. RESULTS: Both animals demonstrated in vitro donor-specific unresponsiveness by MLR and CML and did not demonstrate anti-donor antibody production. Donor matched kidney transplants were performed without immunosuppression and functioned for more than 100 days, with no evidence for rejection. CONCLUSIONS: The authors demonstrate conclusively that in utero transplantation of fully MHC-mismatched bone marrow in swine can lead to engraftment and stable multilineage hematopoietic chimerism and tolerance to postnatal donor MHC-matched kidney transplantation without the need for immunosuppression.     

Mesenchymal Stem Cells Facilitate the Induction of Mixed Hematopoietic Chimerism and Islet Allograft Tolerance without GVHD in the Rat

Induction of hematopoietic chimerism and subsequent donor-specific immune tolerance via bone marrow transplantation is an ideal approach for islet transplantation to treat type-1 diabetes. We examined the potential of mesenchymal stem cells (MSCs) in the induction of chimerism and islet allograft tolerance without the incidence of graft-versus-host disease (GVHD). Streptozotocin-diabetic rats received a conditioning regimen consisting of antilymphocyte serum and 5 Gy total body irradiation, followed by an intraportal co-infusion of allogeneic MSCs, bone marrow cells (BMCs) and islets. Although all the recipients rejected the islets initially, half of them developed stable mixed chimerism and donor-specific immune tolerance, shown by the engraftment of donor skin and second-set islet transplants and acute rejection of a third-party skin. The engraftment of the primary islet allografts with stable chimerism was achieved by the addition of a 2-week peritransplant administration of 15-deoxyspergualin (DSG). Without MSCs, none of the recipients treated with DSG developed chimerism or reversal of diabetes. GVHD was not observed in any of the recipients infused with MSCs (0/15), whereas it occurred in 4/11 recipients without MSCs. These results indicate a potential use of MSCs for induction of hematopoietic chimerism and subsequent immune tolerance in clinical islet transplantation.     

Induction of stable chimerism and elimination of graft-versus-host disease by depletion of T lymphocytes from bone marrow using immunomagnetic beads

The goal of transplantation is the induction of immunologic tolerance. At present, nonspecific immunosuppression is used to prevent graft rejection and, commonly, graft-versus-host disease (GVHD). Nevertheless, nonspecific immunosuppressive therapy is frequently complicated by infection, malignant tumors, and drug toxicity. In order to examine whether hematopoietic chimerism can be used to induce specific allograft tolerance, we have reconstituted lethally irradiated Lewis rats with ACI bone marrow that has been depleted of T cells with use of immunomagnetic beads. This technique consists of binding OX-19, a mouse anti-rat pan-T lymphocyte monoclonal antibody, to magnetic polymer beads. Mixing of bone marrow or splenocytes with the bead/OX-19 complexes, followed by magnetic separation, results in significant depletion of T cells with minimal nonspecific cell loss. Immunomagnetic T-cell depletion of bone marrow, followed by reconstitution of a lethally irradiated host, allows for the development of stable, mixed hematopoietic chimerae without evidence of GVHD. These hosts are immunocompetent by clinical criteria. Recipients of untreated donor bone marrow that did or did not receive nonspecific immunosuppression demonstrated varying degrees of GVHD and reduced survival. The ability to rapidly and simply deplete T lymphocytes from bone marrow and produce stable, immunocompetent hematopoietic chimerae without GVHD may be an important method for tolerance induction to vascularized allografts.     

Allogeneic hematopoietic stem-cell transplantation, mixed chimerism, and tolerance in living related donor renal allograft recipients

OBJECTIVE: We designed a prospective, randomized, and controlled clinical trial to evaluate the efficacy and safety of achieving a mixed chimerism-associated tolerance protocol for recipients of living related donor (LRD) renal allografts. PATIENTS AND METHODS: Sixty-six consecutive patients were divided into two equal groups of 33 patients with end-stage renal disease. They were enrolled for transplantation after negative lymphocytotoxicity cross-matching (LCM). Both groups (treated [Tn] and control [Cn]) showed similar clinical and laboratory parameters and donor HLA match profiles. The Tn group underwent thymic transplantation of donor renal tissue, two donor-specific transfusions, low-intensity conditioning, and high-dose hematopoietic stem-cell transplantation (HSCT) before renal transplantation. The conditioning regimen included low-dose, target-specific irradiation (to abdominal and inguinal lymph nodes, bone marrow [BM] from thoracolumbar vertebrae and part of the pelvis on alternate days, 100 rad x 4), anti-T-cell antibodies (1.5 mg/kg body weight [BW]), cyclophosphamide (10 mg/kg BW x 2 consecutive days), and cyclosporine (CyA; >3 mg/kg BW/d). Unfractionated HSCT procured from the donor marrow was administered into the BM, portal and peripheral circulations, within 24 hours of achieving CD 4+/CD 8+ T-cell count less than 10% of normal. This infusion was supplemented with a dose of peripherally mobilized stem cells (mean total dose of 20 x 10(8) cells/kg recipient BW) administered peripherally. Renal transplantation was performed after negative LCM. Donor-specific cytotoxic antibodies were eliminated with intravenous immunoglobulins and plasmapheresis before renal transplantation. Mixed chimerism was evaluated before and after transplantation at monthly intervals in patients with donors of opposite gender by the FISH technique. Both groups received CyA and prednisolone for immunosuppression; Cn subjects also received mycophenolate mofetil/azathioprine. Rejection was treated with standard treatment. Immunosuppression was withdrawn 6 months after renal transplantation for patients with consistently positive chimerism. Clinical tolerance was defined as stable allograft function for more than 100 days without immunosuppression and confirmed by allograft biopsy. RESULTS: Over a mean follow-up of 210 days, all Tn patients showed stable allograft function with mean serum creatinines (SCr) of 1.20 mg/dL, no rejection/CMV infections/graft or patient loss. A low-level donor-specific cytotoxic antibody was observed in all Tn patients. The CyA toxicity was noted in 10 (30.3%) patients. Persistent mixed hematopoietic chimerism was seen in all 21 patients irrespective of donor-recipient HLA matching (mean 0.5% before and 1 +/- 0.3% after transplantation). All four patients on drug withdrawal have shown donor-specific tolerance at a mean follow-up of 129.8 days. Other Tn patients are in the process of being weaned off immunosuppression. Mean SCr of controls was 1.45 mg/dL over a mean follow-up of 216 days. Acute rejection was observed in 17 (51.5%) patients; no CMV infection/patient loss was noted and one (3.03%) graft was lost in controls. No patient was lost in controls. No graft-versus-host disease was observed in Tn patients. CONCLUSION: We have achieved mixed hematopoietic chimerism-associated tolerance with high-dose HSCT, intrathymic donor renal tissue transplantation, and minimal conditioning without any adverse effects.     

Tolerance to musculoskeletal allografts with transient lymphocyte chimerism in miniature swine

BACKGROUND: Although transplantation of musculoskeletal allografts in humans is technically feasible, the adverse effects of long-term immunosuppression subject the patient to high risks for correcting a non-life-threatening condition. Achieving immunologic tolerance to musculoskeletal allografts, without the need for chronic immunosuppression, could expand the clinical application of limb tissue allografting. Tolerance to musculoskeletal allografts has been accomplished previously in miniature swine in our laboratory. Although stable, mixed chimerism has been suggested as the mechanism underlying long-term tolerance in a rat limb model, the mechanism of this tolerance induction has not been established. This report explores the possible relationship between hematopoietic chimerism and tolerance to musculoskeletal allografts in swine. METHODS: Twelve miniature swine underwent vascularized musculoskeletal allograft transplantation from histocompatibility complex (MHC) matched, minor antigen-mismatched donors. Eight animals received a 12-day coprse of cyclosporine, one of which was excluded due to subtherapeutic levels. Four recipients were not immunosuppressed. Serial biopsies to assess graft viability and flow cytometry to assess chimerism were performed. Donor and third-party skin grafts were placed on recipients with surviving allografts greater than 100 days to validate tolerance. RESULTS: Both groups developed early peripheral chimerism, but this chimerism became undetectable by postoperative day 19 in the cyclosporine group and by day 13 in the control group. Animals receiving cyclosporine developed permanent tolerance to their allografts, whereas those not receiving cyclosporine rejected their allografts in 6-9 weeks. Animals demonstrating tolerance to their bone allografts also demonstrated prolonged donor skin graft survival. CONCLUSIONS: Induction of tolerance to musculoskeletal allografts can be achieved in the MHC matched swine. Although hematopoietic chimerism is present in the immediate postoperative period, persistent, long-term chimerism does not seem to be necessary for maintenance of such tolerance.     

Tolerance to composite tissue allografts across a major histocompatibility barrier in miniature swine

BACKGROUND: Tolerance to composite tissue allografts might allow the widespread clinical use of reconstructive allotransplantation if protocols to achieve this could be rendered sufficiently nontoxic. The authors investigated whether tolerance could be generated in miniature swine to composite tissue allografts across a major histocompatibility (MHC) barrier. A clinically relevant tolerance protocol involving hematopoietic cell transplantation without the need for irradiation or myelosuppressive drugs was tested. METHODS: Seven recipient animals were transiently T-cell depleted and a short course of cyclosporine was initiated. Twenty-four hours later, a donor hematopoietic cell transplant consisting of cytokine-mobilized peripheral blood mononuclear cells or bone marrow cells and a heterotopic limb transplant were performed. In vitro anti-donor responsiveness was assessed by mixed-lymphocyte reaction and cell-mediated lympholysis assays. Acceptance of the limb allografts was determined by gross and histologic appearance. Chimerism in the peripheral blood and lymphohematopoietic organs was assessed by flow cytometry. RESULTS: All seven experimental animals accepted the musculoskeletal elements but rejected the skin of the allografts. All but one of the animals displayed donor-specific unresponsiveness in vitro. The animals that received cytokine mobilized-peripheral blood mononuclear cells showed chimerism but had clinical evidence of graft-versus-host disease (GVHD). None of the animals that received bone marrow cells showed stable chimerism and none developed GVHD. CONCLUSIONS: This protocol can achieve tolerance to the musculoskeletal elements of composite tissue allografts across an MHC barrier in miniature swine. Stable chimerism does not appear to be necessary for tolerance and may not be desirable because of the risk of GVHD.     

Targeted bone marrow radioablation with 153Samarium-lexidronam promotes allogeneic hematopoietic chimerism and donor-specific immunologic hyporesponsiveness

BACKGROUND: Transplantation tolerance, defined as acceptance of a graft by an otherwise fully immunocompetent host, has been an elusive goal. Although robust tolerance has been achieved by the induction of stable hematopoietic chimerism after bone marrow transplantation, lethal or sublethal radiation conditioning used to induce long-term chimerism precludes its clinical use. We studied whether targeted delivery of radiation to bone marrow could allow for bone marrow cell (BMC) engraftment, chimerism, and donor-specific tolerance in the absence of the side effects associated with external irradiation. METHODS: We administered a radioactive bone-seeking compound (Samarium-Lexidronam, Quadramet, Berlex Laboratories, Wayne, NJ) together with transient T-cell costimulatory blockade to recipient mice. Allogeneic BMCs were given 7 or 14 days after preconditioning. Costimulatory blockade was obtained by the use of an anti-CD154 antibody for 4 weeks. Chimerism was assessed by flow cytometry. Mice then received donor-specific and third-party skin grafts. Graft survival was analyzed with mechanisms of donor-specific hyporesponsiveness. RESULTS: High levels of stable chimerism across an allogeneic barrier were achieved in mice by a single administration of Samarium-Lexidronam, transient T-cell costimulatory blockade, and BMC transplantation. A large percentage of chimeric animals retained donor-derived skin grafts for more than 120 days without requiring additional immunosuppression, suggesting that harsh cytotoxic preconditioning is not necessary to achieve stable chimerism and donor specific hyporesponsiveness. Analysis of the T-cell repertoire in chimeras indicates T-cell deletional mechanisms. CONCLUSIONS: These data broaden the potential use of BMC transplantation for tolerance induction and argue for its potential in treating autoimmune diseases.     

Efficacy of donor splenocytes mixed with bone marrow cells for induction of tolerance in sublethally irradiated mice

BACKGROUND: High dose of bone marrow cells (BMCs) has been reported to be essential to establish donor-specific tolerance. In clinical settings, a large quantity of BMCs is very difficult to be obtained. Our previous report demonstrated that even a low dose of BMCs could establish donor-specific tolerance if mixed with splenocytes (SPLCs). In the present study, various components of SPLCs were purified or removed and were investigated their contribution for enhancement of bone marrow engraftment leading to donor-specific tolerance in sublethally irradiated mice. METHODS: Sublethally irradiated C57BL/6 recipient mice were intravenously injected 3 x 10(6) BMCs mixed with various components and various numbers of SPLCs harvested from BALB/c donor mice. One week after injection, skin grafting was performed. The degree of chimerism in peripheral blood lymphocytes (PBLs) and in SPLCs was analyzed by FACS 3 months after transplantation. RESULTS: Recipients receiving 3 X 106 BMCs mixed with 10 x 10(6) T cell-enriched SPLCs established chimerism. Recipients receiving BMCs mixed with macrophage-depleted SPLCs also showed chimeirism and donor-specific tolerance. B cell-enriched SPLCs did not help small dose of BMCs to establish chimerism. Irradiated SPLCs were not effective to induce tolerance even with additional infusion to recipients. CONCLUSIONS: Active effects of splenic T cells were more important to help engraftment of small dose of BMCs than B cells, but the interaction between T and B cells might play some roles to enhance BMC engraftment. Splenic macrophages or dendritic cells might have some adverse effects against tolerance induction. Fatal graft-versus-host disease (GVHD) might be avoided by depleting adherent cells from SPLCs, so macrophages or dendritic cells were also considered as key components to induce donor-specific tolerance and prevent GVHD in this model.     

Transplantation of the bone marrow microenvironment leads to hematopoietic chimerism without cytoreductive conditioning

BACKGROUND: It has been hypothesized that regimens to induce transplantation tolerance and long-term hematopoietic chimerism require recipient conditioning with whole body irradiation or a cytoablative regimen to create space within the marrow microenvironment to permit pluripotent stem cell engraftment. The purpose of this study was to determine if transplantation of an intact bone marrow microenvironment in the form of a bone graft would permit stable hematopoietic stem cell engraftment, shape the repertoire of developing T cells, and induce donor-specific unresponsiveness in the absence of a conditioning regimen. METHODS: Fragments of femur were transplanted under the kidney capsule of recipient mice. At defined time points after bone graft transplantation recipients were assayed for chimerism, bone graft viability, and responses to donor and third party alloantigens in vitro and in vivo. RESULTS: In the absence of an immunological barrier, bone graft transplantation resulted in long-term multi-lineage hematopoietic chimerism in the peripheral blood. Nude bone graft transplantation into SCID recipients resulted in development of donor- derived T cells that underwent negative selection on bone graft derived I-E+ cells within the thymus. Across a fully allogeneic barrier in immunocompetent recipients treated with combined blockade of the CD40 and CD28 pathways bone graft transplantation resulted in long-term donor-specific hyporesponsiveness in vitro and acceptance of donor specific skin grafts. CONCLUSIONS: Transplantation of bone marrow in the form of a bone graft may facilitate the production of hematopoietic chimerism and lead to long-term donor-specific hyporesponsiveness in the absence of a cytoreductive conditioning regimen.     

Composite tissue allotransplantation in chimeric hosts: part I. Prevention of graft-versus-host disease

BACKGROUND: Mixed allogeneic chimerism (MAC) has been shown to induce tolerance to composite tissue allografts (CTA). However, transplantation of unmanipulated donor-specific limbs results in severe graft-versus-host disease (GVHD). This suggests that nontolerant mature donor-derived cells in the CTA may affect the stability of chimerism, potentially resulting in GVHD. The aim of this study was to develop an approach to study and prevent GVHD in a mixed chimeric-rat hind-limb transplantation model. METHODS: [ACI-->WF] chimeras received a limb from Wistar Furth (WF) (syngeneic), Fisher (third-party), or ACI (irradiated [1,050 cGy] or nonirradiated) rats. In vitro tolerance was assessed using mixed lymphocyte reactivity (MLR) assays at the time the animals were killed. RESULTS:[ACI-->WF] chimeras with greater than 85% chimerism exhibited rejection-free survival of donor-specific hind limbs. However, 100% of these animals developed lethal GVHD 22.4+/-2.8 days after limb transplantation. [ACI-->WF] chimeras that underwent transplantation with irradiated ACI or syngeneic WF limbs showed no signs of rejection or GVHD at 5 months. Nonchimeric and third-party controls rejected limbs within 10 days. CONCLUSIONS: Conditioning of the host WF rats with 950 cGy of irradiation (sublethal, myeloablative) led to high levels of MAC without GVHD. The mature T-cell content of nonirradiated donor (ACI) limbs was sufficient to induce lethal GVHD in 100% of tolerant mixed chimeric [ACI-->WF] hosts. Irradiation of donor limbs before transplantation resulted in long-term donor-specific tolerance and prevented GVHD. These data demonstrate that (1) established chimeras could be susceptible to GVHD caused by immunocompetent donor cells transferred with the hind limb, and (2) inactivating these cells with irradiation prevents GVHD and destabilization of chimerism, and permits rejection-free graft acceptance.     

Murine Mobilized Peripheral Blood Stem Cells Have a Lower Capacity than Bone Marrow to Induce Mixed Chimerism and Tolerance

Allogeneic bone marrow transplantation (BMT) under costimulation blockade allows induction of mixed chimerism and tolerance without global T‐cell depletion (TCD). The mildest such protocols without recipient cytoreduction, however, require clinically impracticable bone marrow (BM) doses. The successful use of mobilized peripheral blood stem cells (PBSC) instead of BM in such regimens would provide a substantial advance, allowing transplantation of higher doses of hematopoietic donor cells. We thus transplanted fully allogeneic murine granulocyte colony‐stimulating factor (G‐CSF) mobilized PBSC under costimulation blockade (anti‐CD40L and CTLA4Ig). Unexpectedly, PBSC did not engraft, even when very high cell doses together with nonmyeloablative total body irradiation (TBI) were used. We show that, paradoxically, T cells contained in the donor PBSC triggered rejection of the transplanted donor cells. Rejection of donor BM was also triggered by the cotransplantation of unmanipulated donor T cells isolated from naïve (nonmobilized) donors. Donor‐specific transfusion and transient immunosuppression prevented PBSC‐triggered rejection and mixed chimerism and tolerance were achieved, but graft‐versus‐host disease (GVHD) occurred. The combination of in vivo TCD with costimulation blockade prevented rejection and GVHD. Thus, if allogeneic PBSC are transplanted instead of BM, costimulation blockade alone does not induce chimerism and tolerance without unacceptable GVHD‐toxicity, and the addition of TCD is required for success.     

Immunological unresponsiveness in chimeric miniature swine following MHC-mismatched spleen transplantation

BACKGROUND: In rodents, spleen allotransplantation (SpTx) induces tolerance. We investigated the induction of chimerism and donor-specific unresponsiveness following pig SpTx. METHODS: Thirteen pigs underwent splenectomy (day 0); all received a blood transfusion. In 11/13 pigs, SpTx was performed across a MHC class I (n=1) or full (n=10) barrier; two control pigs received no SpTx. All pigs were monitored for chimerism, and anti-donor immune responses, including suppressor assays. Four pigs (two asplenic controls and two with SpTx) underwent delayed donor-matched kidney transplantation without immunosuppression. RESULTS: Six of the 11 spleen grafts were lost from rejection (n=5) or splenic vein thrombosis (n=1), and five remained viable. All 11 SpTx recipients developed multilineage chimerism, but chimerism was rapidly lost if the graft failed. Two control pigs showed <6% blood chimerism for 4 and 11 days only. Pigs with functioning spleen grafts had multilineage chimerism in blood, thymus and bone marrow for at least 2-6 months, without graft-versus-host disease. These pigs developed in vitro donor-specific hyporesponsiveness and suppression. In 2 pigs tolerant to the spleen graft, donor MHC-matched kidney grafts survived for >4 and >7 months in the absence of exogenous immunosuppression; in two asplenic pigs, kidney grafts were rejected on days 4 and 15. CONCLUSIONS: Successful SpTx can result in hematopoietic cell engraftment and in vitro donor-specific unresponsiveness, enabling prolonged survival of subsequent donor-matched kidney grafts without immunosuppression.     

A substantial level of donor hematopoietic chimerism is required to protect donor-specific islet grafts in diabetic NOD mice

BACKGROUND: Mixed chimerism can induce tolerance to alloantigens and restore self-tolerance to autoantigens, thereby permitting islet transplantation. However, the minimal level of donor chimerism that is required to prevent islet allograft rejection and recurrence of autoimmune diabetes has not been established. METHODS: We investigated whether allogeneic Balb/c donor chimerism can be induced in C57BL/6 mice, in prediabetic NOD mice, and in diabetic NOD mice after transplantation of a modest dose of bone marrow by using purine nucleoside analogue, fludarabine phosphate and cyclophosphamide conditioning therapy, followed by short-term anti-CD40 ligand monoclonal antibody and rapamycin posttransplant treatment. We also investigated whether the induced donor chimerism is sufficient to prevent the onset of diabetes in prediabetic NOD mice and protect donor islet grafts in diabetic NOD mice. RESULTS: Allogeneic donor chimerism could be induced under the authors' approach that is nonmyeloablative and radiation-free. Diabetes onset was prevented in chimeric prediabetic NOD mice. The induction of mixed chimerism protected donor-specific islet grafts in diabetic NOD mice. At 60 days after islet transplantation, all donor Balb/c islet grafts survived in diabetic NOD mice whose level of donor-derived lymphocytes was higher than 30% at the time of islet transplantation (n=8). In contrast, Balb/c islet grafts were rejected in five of seven diabetic NOD mice whose level was lower than 30%. CONCLUSIONS: Our data demonstrate that a donor lymphocyte chimerism (>30%) at the time of islet transplantation is required to protect donor-specific islet grafts, and indicate that a strictly non-irradiation-based protocol can be used to achieve this goal.     

Injection of allogeneic bone marrow cells into the portal vein of swine in utero

The ability to safely manipulate the immune system of the developing fetus carries the hope of effective treatment strategies for certain congenital disorders that can be diagnosed during gestation. One possible intervention is the induction of specific transplantation tolerance to an adult donor who could provide tissue after birth without the need for immunosuppression. Although the introduction of allogeneic stem cells to a developing immune system has been shown to result in hematopoietic chimerism, donor-specific transplantation tolerance has not been demonstrated in a large animal model. In previous reports of in utero stem-cell transplantation, the cells were injected into the fetus by an intraperitoneal route. We sought to improve upon this technique of cell transplantation by developing a method for the safe delivery of allogeneic stem cells directly into the hepatic circulation of fetal swine. In the second phase of our study, we determined if adult allogeneic bone marrow cells delivered to the fetus by this intravascular route could result in result in hematopoietic chimerism and donor-specific transplantation tolerance. A method of successful intravascular injection was designed in which a laparotomy was performed on a sow at midgestation (50-55 days) to administer 1 cc of inoculum into the portal vein of each fetus using transuterine ultrasound guidance and a 25-gauge spinal needle. In one sow, 10 piglets were injected with saline to test safety, and 8 piglets were born. For transplantation of stem cells to the fetuses, donor bone marrow was harvested from a genetically defined miniature swine. In one sow the marrow was injected without T-cell depletion resulting in abortion. In the third sow, the marrow was depleted of T-cells to less than 0.01% using magnetic beads conjugated to anti-CD3 monoclonal antibodies. No chimerism was detected in these offspring. Only in the fourth sow where the T-cell depletion was reduced to about 1% of the cells in the inoculum did one animal demonstrate chimerism. This piglet showed reproducible blood chimerism (0.95% donor cells) detected by flow cytometry measurement of monoclonal antibodies to the donor MHC. In addition, this animal demonstrated hyporesponsiveness to donor lymphocytes in an MLR assay while reacting strongly to third-party stimulator cells. A split-thickness skin graft from the donor was accepted, and a third-party graft was rapidly rejected.     

Permanent acceptance of both cardiac and skin allografts using a mild conditioning regimen for the induction of stable mixed chimerism in mice

Patients who are receiving an organ transplant nowadays are sentenced to the life-long administration of immunosuppressive drugs, which have serious side effects. The reliable induction of donor-specific tolerance therefore remains a major goal in organ transplantation. Previously, we have developed a sublethal, non-myeloablative murine model in which permanent mixed, multilineage chimerism and donor-specific tolerance are established. Our model involves engraftment of fully allogeneic T cell depleted donor bone marrow cells in low dose irradiated and anti-CD3 treated major histocompatibility complex (MHC)-disparate recipient mice. To investigate whether vascularized organ grafts are accepted in our model, we performed heterotopic heart transplantations in our mixed chimeric mice. Chimeric mice permanently accepted hearts from the bone marrow donor (>130 days) and rapidly rejected third party-type allografts (median survival time 9 days). Untreated control recipient mice rejected both donor- and third party-type allografts. In addition, mice that accepted their cardiac grafts, donor-specific acceptance of skin grafts was observed. In conclusion, the establishment of stable mixed chimerism with this low-toxicity regimen resulted in permanent donor-specific acceptance of vascularized organ as well as skin grafts across a full MHC barrier.     

Renal allograft tolerance in DLA-identical and haploidentical dogs after nonmyeloablative conditioning and transient immunosuppression with cyclosporine and mycophenolate mofetil

BACKGROUND: Canine models of bone marrow and renal transplantation have provided important preclinical data relevant to developing novel therapeutic protocols for hematopoietic and solid organ transplantation in human beings. Nonmyeloablative transplantation has been shown to induce stable mixed hematopoietic chimerism in normal dogs and correct the phenotype of canine pyruvate kinase deficiency and Glanzman's thrombasthenia. In this study, we investigated the potential for inducing renal allograft tolerance using a nonmyeloablative bone marrow transplantation strategy that induces mixed chimerism in DLA-identical dogs. METHODS: Reciprocal renal allografts were performed in 4 DLA-identical and 4 DLA-haploidentical dogs with nonmyeloablative conditioning (200 cGy total body irradiation [TBI]) and transient immunosuppression with cyclosporine (CSP) and mycophenolate mofetil (MMF) with and without simultaneous bone marrow transplantation. Two DLA-identical control dogs received reciprocal renal allografts without TBI or immunosuppression with CSP and MMF. Serum creatinine (Cr) concentration was monitored to assess renal allograft function. RESULTS: The renal allografts were acutely rejected in the 2 DLA-identical dogs without TBI or immunosuppression. There was long-term (>1 year) renal allograft survival as evidenced by a normal (<2.0 mg/dL) serum Cr concentration in both the DLA-identical and DLA-haploidentical dogs that underwent 200 cGy TBI and transient immunosuppression with CSP and MMF either with or without simultaneous bone marrow transplantation. CONCLUSIONS: Nonmyeloablative conditioning (200 cGy TBI) and transient immunosuppression with CSP and MMF induce renal allograft tolerance in DLA-identical and DLA-haploidentical dogs without donor/host mixed hematopoietic chimerism. These findings suggest it may be possible to induce tolerance to solid organ transplants without the need for chronic immunosuppressive therapy or stable hematopoietic chimerism in the setting of both DLA-matched and haploidentical transplants.     

Flt3-L augments the engraftment of donor-derived bone marrow cells when combined with sublethal irradiation and costimulatory (CD28/B7 and CD40/CD40L) blockade

T-cell costimulatory blockade as a constituent for recipient conditioning prior to bone marrow transplantation has led to the development of less toxic protocols for the establishment of donor cell chimerism. We therefore hypothesized that the addition of the hematopoietic growth factor, Flt3-ligand (Flt3-L), to the perioperative inhibition of the CD28/B7 and CD40/CD40 ligand costimulatory pathways would enhance the engraftment of allogeneic bone marrow. Recipient BALB/c ByJ (H-2(d), Mls(c), Vbeta6+/Vbeta8+ TCR) received a single sublethal dose of total body irradiation (300 rad) 6 h prior to transplantation IV with unfractionated donor CBA/J (H-2(k), Mls(d), Vbeta6-/Vbeta8+ TCR) bone marrow cells. CTLA4-Ig and/or MRI were administered at 500 microg IP on days 0, 2, 4, and 6 posttransplantation. Flt3-L was administered at 10 microg IP on days 0-6. Donor cell chimerism was determined on days 30-90 by flow cytometric analysis. Donor-specific tolerance was assessed by skin grafting. In vitro TCR cross-linking assays and flow cytometry were utilized to explore the deletion of donor-reactive T cells. Recipients receiving CTLA4-Ig and MRI engrafted allogeneic bone marrow cells in the peripheral blood (3/6; 50%) with chimerism being detected at 2-31%. Addition of Flt3-L to this preconditioning regimen enhanced the incidence of engraftment of donor bone marrow cells (10/13; 3-70%). Long-term survival of donor but not third-party-specific skin grafts demonstrated that donor-specific tolerance had been achieved in the chimeric recipients. Deletion of the donor-reactive T cells within the chimeric recipients was also observed. The addition of hematopoietic growth factors and cytokines to the nonmyeloablative regimen of sublethal irradiation and T-cell costimulatory blockade provides a novel strategy for the establishment of donor cell chimerism and for the induction of stable and robust donor-specific tolerance. The deletion of donor-reactive T cells using this protocol suggests the reliability and feasibility of this protocol for clinical transplantation.     

Recipient bone marrow engraftment in donor tissue after long-term tolerance to a composite tissue allograft

BACKGROUND: An important component of a composite tissue limb allograft (CTA) is the vascularized bone marrow and bone marrow stroma, which when transplanted could create immediate marrow space and engraftment. We have previously demonstrated that tolerance to musculoskeletal allografts can be achieved with a 12-day course of cyclosporine without the presence of long-term peripheral donor cell chimerism. The objective of this study was to determine the fate of the donor bone marrow after transplantation of a limb allograft in a miniature swine model. METHODS: CTAs from donor swine were heterotopically transplanted into six MHC-matched, minor-antigen-mismatched recipients, and a 12-day course of cyclosporine was given. Previous animals transplanted without cyclosporine rejected their grafts in less than 42 days. A non-MHC-linked marker, pig allelic antigen (PAA), was used to distinguish host and donor cells. Three PAA- animals received PAA+ CTAs, and three PAA+ animals received PAA- CTAs. Bone marrow was harvested from the donor limb grafts and the recipient and analyzed by flow cytometry and histology. Thymus, spleen, and mesenteric lymph nodes were also harvested from the recipient swine and evaluated for the presence of donor cells by flow cytometry. RESULTS: All animals receiving cyclosporine demonstrated permanent tolerance to their allografts. Donor bone marrow cells were present in all grafts at the time of transplantation and during the immediate postoperative period. By 48 weeks, donor cells were no longer detectable within the marrow space of the allograft. In long-term animals host bone marrow cells replaced donor cells in the graft marrow space. No evidence of donor cell engraftment was found in recipient animals. CONCLUSION: This study demonstrates that in long-term tolerant recipients of musculoskeletal allografts there is no evidence of persistent donor bone marrow cells in the hematopoietic tissues of the graft or the host. Rather, the recipient's bone marrow cells and lymphocytes repopulate the donor marrow space of the graft.     

Development of stable mixed T cell chimerism and transplantation tolerance without immune modulation in recipients of vascularized bone marrow allografts

A consistent majority (62.5%) of immunologically unmodified rat recipients transplanted with vascularized hind-limb bone marrow allografts across a semiallogeneic transplant barrier developed tolerance with absence of graft-versus-host disease. A minority of recipients (37.5%) demonstrated lethal GVHD. Transplantation tolerance in the majority was associated with the induction of stable low-level mixed T cell chimerism, including donor CD5+, CD4+, and CD8+ lymphocytes. Chimeras were specifically immune nonresponsive to host alloantigenic determinants. These results emphasized a potentially important mechanism for low-level stable mixed lymphoid chimerism (SMLC) in tolerance induction, independent of immune suppressive effects due to irradiation or immunopharmacologic intervention. These vascularized bone marrow transplantation (VBMT) results may establish the experimental foundation for a novel approach to stem cell transfer and bone marrow transplantation.