Infusion Of Donor Bone Marrow Cells Research Articles

A Prospective Controlled Trial to Evaluate Safety and Efficacy of in vitro Expanded Recipient Regulatory T Cell Therapy and Tocilizumab Together With Donor Bone Marrow Infusion in HLA-Mismatched Living Donor Kidney Transplant Recipients (Trex001).

Background: The induction of donor-specific immunological tolerance could improve outcome after kidney transplantation. However, no tolerance protocol is available for routine clinical use. Chimerism-based regimens hold promise, but their widespread application is impeded in part by unresolved safety issues. This study tests the hypothesis that therapy with polyclonal recipient regulatory T cells (Tregs) and anti-IL6R (tocilizumab) leads to transient chimerism and achieves pro-tolerogenic immunomodulation in kidney transplant recipients also receiving donor bone marrow (BM) without myelosuppressive conditioning of the recipient.Methods/design: A prospective, open-label, controlled, single-center, phase I/IIa academic study is performed in HLA-mismatched living donor kidney transplant recipients.Study group: Recipients of the study group receive in vitro expanded recipient Tregs and a donor bone marrow cell infusion within 3 days after transplantation and tocilizumab for the first 3 weeks post-transplant. In addition they are treated with thymoglobulin, belatacept, sirolimus, and steroids as immunosuppression. Starting 6 months post-transplant, sirolimus and steroids are withdrawn in a step-wise manner in stable patients.Control group: Recipients of the control group are treated with thymoglobulin, belatacept, sirolimus, and steroids as immunosuppression. Co-primary endpoints of safety (impaired graft function [eGFR <35 mL/min/1.73 m2], graft-vs.-host disease or patient death by 12 months) and efficacy (total leukocyte donor chimerism within 28 days post-transplant) are assessed. Secondary endpoints include frequency of biopsy-proven acute rejection episodes and subclinical rejection episodes on surveillance biopsies, assessment of kidney graft function, and the evaluation whether the study protocol leads to detectable changes in the immune system indicative of pro-tolerogenic immune modulation.Discussion: The results of this trial will provide evidence whether treatment with recipient Tregs and donor BM is feasible, safe and efficacious in leading to transient chimerism. If successful, this combination cell therapy has the potential to become a novel treatment option for immunomodulation in organ transplantation without the toxicities associated with myelosuppressive recipient conditioning.Trial registration: European Clinical Trials Database EudraCT Nr 2018-003142-16 and clinicaltrials.gov NCT03867617.

Mechanisms of Vascularized Bone Marrow Graft Protection of Vascularized Composite Allografts

Background Co-transplantation of vascularized bone marrow (VBM) promotes vascularized composite allograft (VCA) survival with standard immunosuppression in a non-human primate (NHP) model. We investigated mechanisms of immunomodulation by comparing VCA survival following donor bone marrow cell (BMC) infusion, donor and recipient conditioning strategies, and heterotopic VBM allografts to historical groups. Methods Groups of 3–4 NHP received facial subunit VCA and immunosuppression of tacrolimus (Tac) and mycophenolate mofetil (MMF). Comparative groups, VCA+VBM (n=4) and VCA (no VBM, n=3) were from historical cohorts (Group 1 and 2, respectively). Group 3 received VCA without VBM and transfused donor BMC at 20x 106 cells/kg on d 1. Group 4 received VCA+VBM following donor mandible irradiation (1.5 Gy) on d −1, while Group 5 received irradiated donor mandible followed by BMC transfusion on d 1. Group 6 underwent co-transplantation of heterotopic VBM and VCA without VBM. Group 7 received VCA+VBM after donor T cell depletion with anti-thymocyte globulin (ATGAM, 50 mg/kg) on days −2, and −1. An additional dose of ATGAM was given on d 0 if CD3+ cells remained >50/mm3. Group 8 recipients received ATGAM depletion as above, VCA+VBM, and a 21-d steroid taper. Banff classification was determined by histopathologic analysis. Chimerism was assessed by flow cytometry from peripheral blood and sampled tissues. Results VCA + VBM treated with Tac/MMF demonstrated prolonged graft survival and rejected only after immunosuppression was withdrawn compared to Groups 2–8 (mean survival 348d vs 36d). This effect was statistically significant (p=0.05). Endpoints were met secondary to Banff IV rejection and malignancy. Macrochimerism was present only in Group 1; remaining groups demonstrated only transient microchimerism. Discussion The immunologic advantage of VBM prolonging VCA graft survival is contingent on proximity of the VBM to the VCA allograft. Donor conditioning nullifies this benefit and it is not reconstituted by transfusion of donor BMC. Recipient T cell depletion mirrors the early graft loss observed with donor T cell depletion. Conclusion Our data elucidate a mechanism for VCA graft survival that is mediated by locally acting, radiosensitive T cell populations in both donor and recipient and is dependent on the unique immunologic and structural niche of VBM. Astellas Pharma, Inc.

Combined Co-Stimulatory Blockade and Donor Bone Marrow Cells Induce Robust Immune Tolerance in a Fully MHC-Mismatched Swine Hind Limb Transplant Model

INTRODUCTION: Vascularized Composite Allografts (VCA), such as hand and upper extremity transplants, contain vascularized bone marrow (BM) and a BM niche representing a constant source of donor-derived stem cells and hence can favor chimerism and tolerance induction.1,2 This study investigates the immunological effects of vascularized BM within VCA under co-stimulation blockade-based regimen and its impact on allograft survival and tolerance induction. METHODS: Fully MHC- and gender mismatched MGH miniature swine (n=3-5 per group) underwent heterotopic hind-limb transplantation containing intact vascularized BM component (Figure 1).Figure 1: Schematic diagram of swine heterotopic hind limb transplant. Osteomyocutaneous flap is transplanted to a subcutaneous pocket with externalized skin component.Recipient animals received a short course (30 days) of tacrolimus monotherapy with or without donor BM infusion (60x106 cells/kg), and CTLA4Ig. Short course tacrolimus only and untreated animals served as controls. Chimerism was assessed by SRY PCR analysis. Sequential skin and muscle biopsies were performed for histology. Alloreactivity against donor antigens was assessed in vitro using mixed lymphocyte reaction (MLR) assays. Challenge with secondary skin grafts was utilized to demonstrate robust immune tolerance in vivo. RESULTS: The co-stimulation blockade based immunomodulatory protocol resulted in indefinite graft survival (>150 days) in 3 out of 5 animals whereas control and tacrolimus only groups rejected allografts at days 7+/-1 and 29+/-2 respectively (Figure 2).Figure 2: Kaplan-Meier survival curve demonstrating rejection free survival of skin component of hind limb allograft.Combined costimulation blockade with augmented donor BM infusion resulted in indefinite graft survival in 2 out of 3 animals (>150 days). Long-term survivors demonstrated stable micro-chimerism in various graft and recipient tissues including skin, lymph node, bone marrow, and spleen. MLR data showed unresponsiveness to donor but not to third party allogeneic controls. Secondary skin grafting demonstrated advanced rejection of third party grafts on day 7 while donor-matched grafts were accepted indicating donor-specific immune tolerance. There was no evidence of donor specific antibody formation in long-term survivors. Donor unresponsiveness in MLR was lost five weeks post-graftectomy, which demonstrated that persistent antigenic stimulation was required for operational tolerance. CONCLUSION: Combined costimulation blockade and donor BM cell infusion can induce robust immune tolerance in a fully MHC mismatched hind limb transplant model. Such targeted immunomodulatory protocols might eliminate the need for long-term multi-drug immunosuppression after reconstructive transplantation.

Immune tolerance across a full MHC barrier in a swine hind limb transplantation model using a combined co-stimulatory blockade and donor bone marrow cell infusion

Immune tolerance across a full MHC barrier in a swine hind limb transplantation model using a combined co-stimulatory blockade and donor bone marrow cell infusion

Five-year clinical effects of donor bone marrow cells infusions in kidney allograft recipients

Augmentation of microchimerism in solid organ transplant recipients by donor bone marrow cells (DBMC) infusion may promote immune hyporesponsiveness and consequently improve long-term allograft survival. Between March 2005 and July 2007, outcomes for 20 living unrelated donor (LURD) primary kidney recipients with concurrent DBMC infusion (an average of 2.19 ± 1.13 x 10⁹ donor cells consisting of 2.66 ± 1.70 x 10⁷ CD34⁺ cells) were prospectively compared with 20 non-infused control allograft recipients given similar conventional immunosuppressive regimens. With five years of clinical follow up, a total of 11 cases experienced rejection episodes (3 DBMI patients vs. 8 controls, p = 0.15). One DBMC-infused patient experienced chronic rejection vs. two episodes (1 biopsy-confirmed) in the control patients. Actuarial and death-censored 5-y graft survival was significantly higher in infused patients compared with controls (p = 0.01 and p = 0.03, respectively). Long-term graft survival was significantly associated with pre-transplant anti-HLA antibodies (p = 0.01), slightly with peripheral microchimerism (p = 0.09) and CD4⁺CD25⁺FoxP3⁺ T cells (p = 0.09). Immunosuppressant dosing was lower in infused patients than controls, particularly for mycophenolate mofetil (p = 0.001). The current findings as well as our previous reports on these patients indicates clinical improvement in long-term graft survival of renal transplant patients resulting from low-dose DBMC infusion given without induction therapy.

Upper-Extremity Transplantation Using a Cell-Based Protocol to Minimize Immunosuppression

To minimize maintenance immunosuppression in upper-extremity transplantation to favor the risk-benefit balance of this procedure. Despite favorable outcomes, broad clinical application of reconstructive transplantation is limited by the risks and side effects of multidrug immunosuppression. We present our experience with upper-extremity transplantation under a novel, donor bone marrow (BM) cell-based treatment protocol ("Pittsburgh protocol"). Between March 2009 and September 2010, 5 patients received a bilateral hand (n = 2), a bilateral hand/forearm (n = 1), or a unilateral (n = 2) hand transplant. Patients were treated with alemtuzumab and methylprednisolone for induction, followed by tacrolimus monotherapy. On day 14, patients received an infusion of donor BM cells isolated from 9 vertebral bodies. Comprehensive follow-up included functional evaluation, imaging, and immunomonitoring. All patients are maintained on tacrolimus monotherapy with trough levels ranging between 4 and 12 ng/mL. Skin rejections were infrequent and reversible. Patients demonstrated sustained improvements in motor function and sensory return correlating with time after transplantation and level of amputation. Side effects included transient increase in serum creatinine, hyperglycemia managed with oral hypoglycemics, minor wound infection, and hyperuricemia but no infections. Immunomonitoring revealed transient moderate levels of donor-specific antibodies, adequate immunocompetence, and no peripheral blood chimerism. Imaging demonstrated patent vessels with only mild luminal narrowing/occlusion in 1 case. Protocol skin biopsies showed absent or minimal perivascular cellular infiltrates. Our data suggest that this BM cell-based treatment protocol is safe, is well tolerated, and allows upper-extremity transplantation using low-dose tacrolimus monotherapy.

Co-Stimulatory Blockade and Donor Bone Marrow Cell Infusion for Immunomodulation and Tolerance Induction in Vascularized Composite Allografts - A Large Animal Translational Trial

Introduction: Particular Vascular Composite Allografts (VCA), such as hand or arm transplants, contain vascularized long bones that instantly and continuously produce bone marrow (BM) cells after engraftment and thus can be considered to be both a VCA and vascularized BM graft. The creation of hematopoietic chimerism through BM transplantation remains the most reliable method for inducing transplantation tolerance. However, the contribution of a vascularized bone graft in VCA towards such effects is largely unknown. Therefore, this study investigates the long-term effects of vascularized BM within VCA under costimulation blockade-based immunosuppression. Materials and methods: Complete MHC-mismatched miniature swine (n=12) underwent total body and thymic irradiation for cytodepletion. Animal groups received 15, 30 or 60 × 106 cells/kg of whole, unmodified BM. The optimal dose of BM cell infusion was then applied to subsequent experiments evaluating the addition of CTLA4Ig. MGH miniature swine (n=24) underwent heterotopic hind limb transplantation and received a short course of 30 days of Tacrolimus monotherapy. Group 1 (control) received no treatment. Group 2 received FK506 only; Group 3 received irradiation and FK506; Group 4 received irradiation, BM infusion and FK506; Group 5 received FK506 and CTLA4-lg; Group 6 received irradiation, BM infusion, FK506 and CTLA4Ig; and Group 7 received BM infusion, FK 506 and CTLA4Ig fusion protein. Results: Microchimerism was established in all animals after BM cell infusion; at postoperative day 9, it was significantly increased for 60 ×106 cells/kg (P = .0001). Transplanted animals in Group 1 rejected the allograft 5 to 8 days post-transplant. Group 2 rejected the allograft (skin and muscle) 30-32 days post- transplant. Group 3 rejected the allograft (skin and muscle) 35-37 days post-transplant. Group 4 rejected the skin portion of the allograft at 50-53 days post-transplant. The remaining allograft components (muscle, bone, nerve, and vessel) survived indefinitely. Group 5 animals demonstrated significantly prolonged muscle survival beyond 150 days post-transplant; the skin component survived past 150 days in 3 out of 5 animals. Skin and muscle histology in all long-term surviving animals was rejection-free. Two animals in Group 5 developed parasitic diarrhea and were euthanized at post-operative day 45. Both skin and muscle components were rejection free at the time of euthanasia. All animals in Group 6 had unexpected weight loss leading to sudden death (n=1) or euthanasia (n=2). Group 7 is currently in progress with rejection-free survival beyond 52 days post-transplant. Conclusion: High-dose BM cell infusion results in stable levels of micro-chimerism. The addition of co-stimulatory blockade (CTLA4Ig) enabled us to optimize induction therapy, further reduce maintenance immunosuppression, and indefinitely prolong graft survival. Such targeted immunomodulatory protocols that combine BM cell-based strategies and biologics might facilitate immune tolerance and eliminate the need for multi-drug immunosuppression to maintain graft survival after vascularized composite allotransplants.

Infused Bone Marrow Fails to Prolong Vascularized Composite Allograft Survival in Nonhuman Primates

Introduction: Clinical protocols for hand and face vascularized composite allografts (VCA) have included varying amounts donor bone marrow cell (BMC) infusion after lymphocyte depletion as an immunomodulatory strategy. We previously demonstrated that vascularized bone marrow (VBM) protects facial VCA from rejection in unconditioned nonhuman primates. To investigate the potential immunologic benefit of infused BMC, we substituted infused BMC for VBM in the transplanted facial segment. Methods: Three cynomolgus macaques underwent heterotopic facial segment transplantation with mandibular segment removed, followed by central venous infusion of BMC from the same MHC-mismatched donor on postoperative day 1. Bone marrow cells were collected from the donor lumbar vertebrae following euthanasia. Cells counts and viability were confirmed using trypan blue and automated complete blood count. Donor BMC were infused at 200 million cells per kg of recipient weight, a standard for bone marrow transplantation. CD34+ hematopoeitic stem and progenitor cells were identified using a modified ISHAGE gating strategy. Immunosuppression was maintained with tacrolimus and mycophenolate mofetil. Rejection episodes were not treated. Chimerism was measured in blood samples using flow cytometry. Results: Mean cell content of the mandibular segments set aside before transplantation was 48.2 million cells/kg of donor weight. Hematopoeitic stem and progenitor cells comprised 5.63% of vertebral bone marrow BMC and 7.60% of mandibular BMC. Animals in the previous VCA+VBM treatment group had no graft loss while on immunosuppression. Additionally, graft survival was longer vs. VCA alone (205-430 vs. 9-42 days), as was rejection-free survival (44-358 vs. 7-15 days). In the VCA + BMC group all animals developed acute rejection (Banff grade I or higher) by day 14. One progressed to grade IV rejection and graft loss by day 28, while another resolved but progressed to chronic rejection by day 50 and endpoint at day 115 for post-transplant lymphoproliferative disease with chronic rejection. Only one animal receiving BMC demonstrated transient macrochimerism days 85-98. Chimerism was similarly observed transiently in only one animal receiving VCA without either VBM or BMC. In contrast, 3 of 4 animals with VBM demonstrated macrochimerism.Figure: [Graft Survival]Conclusion: Despite larger cell counts, BMC infusion failed to prolong facial VCA survival compared to VBM. These data support that VBM is a more effective immunomodulatory strategy than infusion of donor BMC without depletional pre-conditioning. The addition of irradiation and T cell depletion therapies to VBM may further enhance the amount and durability of chimerism, and potentially allow for the development tolerance.

Regulatory T-cell subset analysis and profile of interleukin (IL)-10, IL-17 and interferon-gamma cytokine-producing cells in kidney allograft recipients with donor cells infusion

This pilot study aimed to assess whether the perioperative infusion of donor bone marrow cells (DBMC) in renal allograft recipients can affect the appearance of peripheral regulatory T-cell subsets and the profile of cytokine-producing cells [interferon-gamma (IFN-γ), interleukin (IL)-17 and IL-10] 2 years after transplantation. Fresh blood samples were collected from 14 kidney recipients who received infusion and from 13 kidney recipients without infusion who served as controls at the end of the second post-transplantation year. Initially the percentages of CD4(+)CD25(+)FoxP3(+) T cells and CD3(+)CD8(+)CD28(-) T cells were quantified using flowcytometry. Thereafter, the frequencies of IL-10-, IL-17- and IFN-γ-producing cells were determined separately using the ELISPOT technique with peptides corresponding to mismatched donor HLA-DR molecules and phytohemagglutinin (PHA). The mean numbers of IFN-γ- and IL-17-producing cells in response to PHA were lower in infused patients than in controls (P = 0.02 and P = 0.18, respectively); however, an increased frequency of IL-10-producing cells was observed compared to controls (P = 0.07). Furthermore, the ratio of IL-10/IFN-γ-producing cells was significantly higher in the DBMC-infused group versus controls (P = 0.01). There was a negative correlation between the percentage of CD3(+)CD8(+)CD28(-)T cells and IL-17-producing cells in the infused group (r = -0.539, P = 0.04). The mean levels and the frequency of microchimerism within the first post-transplantation year were also significantly higher in infused patients than in controls (P = 0.007 and P = 0.001, respectively). Our findings suggest that DBMC infusion could partially stimulate the regulatory mechanisms against alloimmune responses in kidney allograft recipients

MP-06.18 Augmentation of Chimerism by Donor Bone Marrow Cells Infusion in Kidney Allograft Recipients

MP-06.18 Augmentation of Chimerism by Donor Bone Marrow Cells Infusion in Kidney Allograft Recipients

Serum Levels of Interleukin (IL)-10, IL-17, Transforming Growth Factor (TGF)-β1, and Interferon-γ Cytokines and Expression Levels of IL-10 and TGF-β1 Genes in Renal Allograft Recipients After Donor Bone Marrow Cell Infusion

Background Cytokine storm generated by an alloimmune response after transplantation can lead to either graft survival or rejection. The aim of this study was to evaluate the serum levels of interleukin (IL)-10, IL-17, transforming growth factor (TGF)-β1, and interferon (IFN)-γ and expression levels of IL-10 and TGF-β1 in renal allograft recipients with or without donor bone marrow cell infusion (DBMI). Methods We retrospectively followed 28 living unrelated kidney recipients, including 14 with and 14 without DBMI infusion for 2 years. Also, 14 healthy subjects were included as a normal control group. PBMC gene expression analysis for mRNA levels of IL-10 and TGF-β1 cytokines relative to β-actin as a reference gene was performed using quantitative fluorescence real-time polymerase chain reaction at the end of 2 years posttransplantation. Also, serum levels of IL-10, TGF-β1, IFN-γ, and IL-17 in the 3 groups were measured by enzyme-linked immunosorbent assay at the same time. Results Both patient groups showed increased gene expression and serum content of IL-10 compared with normal controls. The expression levels were only significant between control patients and normal subjects ( P = .02). Serum levels of IFN-γ and IL-17 were higher in untreated patients compared with normal controls ( P = .03 and P = .07, respectively). DBMI patients showed significantly lower levels of serum TGF-β1 and IL-17 compared with normal subjects ( P = .05 and P = .06, respectively). Also, infused patients showed a positive correlation between circulating levels of IL-17 and IL-10 ( r = 0.692; P = .006), and an inverse correlation between serum creatinine and TGF-β1 levels ( r = −0.580; P = .03). Conclusion The decreased levels of inflammatory cytokines besides IL-10 with increased TGF-β1 levels and better allograft function with improved clinical outcomes were observed among infused patients, possibly indicating immunomodulatory effects of this approach in kidney allograft patients.

Rational Targeting of the Signaling GTPase Cdc42 Establishes a Non-Myeloid Ablative Regimen for Hematopoietic Stem Cell Engraftment In Mouse Models

AbstractAbstract 73Hematopoietic stem cell (HSC) transplantation has become a standard of care for the treatment of many hematological disorders such as Fanconi anemia. However, current myeloid ablative or chemotherapy conditioning regimens are associated with significant morbidity and mortality. In addition, outcomes of transplantation can be limited by low infused stem cell numbers, especially in the setting of umbilical cord blood or autologous transplantation. Thus, availability of agents that could optimize entry of stem cells into bone marrow (BM) niche would be of great value in improving HSC transplant outcome. Recent studies have shown that HSC mobilization before transplantation might vacate BM niche and thus allow improved engraftment of donor HSCs (Blood 113, 4856). Previously we reported that conditional knockout of the Rho GTPase Cdc42 from BM cells led to massive egress of hematopoietic stem/progenitor cells (HSPCs) from BM to peripheral blood (PB) in mice (PNAS 104, 5091). We have also shown that a novel Cdc42 activity-specific inhibitor, CASIN, is able to transiently mimic Cdc42 knockout phenotype in mice in inducing HSPC mobilization from mouse BM by suppressing actin polymerization, adhesion, and directional migration (Blood 112, 68). In the present studies, we hypothesize Cdc42 targeting can open up BM niche to facilitate subsequent HSC engraftment. First, in a conditional Cdc42 knockout mouse transplant model (CD45.2+ MxCre;Cdc42flox/flox BM engrafted in congenic recipients) we showed that poly I:C induction followed by transplant of congenic donor BM cells resulted in significant engraftment of donor blood accompanied by a loss of Cdc42−/− cells. The donor-derived cells in both BM and PB made up of ∼70% chimerism in multiple blood lineages including Lin−Sca+c-Kit+ primitive progenitors (74.62±5.5%). The multi-lineage donor chimerism was maintained in secondary transplant, indicating effective long-term HSC engraftment. In contrast, no significant engraftment was observed in mice of MxCre;Cdc42wt/wt BM genotype after similar polyI:C induction. These experiments indicate that genetic deletion of Cdc42 in host HSCs can allow efficient donor engraftment without irradiation or myeloid ablative conditioning. Second, we found CASIN treatment of recipient mice transiently suppressed low density BM cell Cdc42 activity and mimicked the Cdc42 knockout effect in mobilizing long-term HSCs to allow congenic donor HSC engraftment. Three i.p. injections of CASIN to recipient mice prior to infusion of donor BM cells resulted in 8.66±2.3% chimerism of multi-lineage donor derived cells 4 months post-transplant, while the vehicle treated mice showed less than 1% chimerism. Third, similar CASIN preconditioning allowed the engraftment of 5.53±2.68% CD34+ human cord blood cells into immunodeficient NSG mice compared with less than 1% by vehicle. Fourth, CASIN preconditioning alone was able to achieve 13.08±2.94% and 11.17±2.86% chimerisms of WT and Fanca gene-corrected Fanca−/− donor BM cells, respectively, in Fanca−/− mouse recipients. This effect in engraftment was particularly evident when CASIN was administrated in combination with the immunosuppressant, Fludarabine, as donor chimerism reached 22±3.05% compared with 3.63±0.87% by Fludarabine alone or 11.3±1.08% by CASIN alone. Throughout these studies, no toxicity was detected in CASIN-treated recipient mice based on examinations of CASIN treated blood, BM, spleen, liver, and other organs at one or seven days. Together, our studies demonstrate that Cdc42 is a useful target for enhancing HSC engraftment in transplantation by opening BM niche. The results present a novel preclinical regimen of pharmacological targeting Cdc42 to facilitate the engraftment of murine HSCs and human cord blood HSPCs and to improve Fanconi anemia HSC transplantation therapy, clinical settings in which stem cell numbers critically limit success. Disclosures:No relevant conflicts of interest to declare.

Immune “tolerance profiles” in donor bone marrow infused kidney transplant patients using multiple ex vivo functional assays

Ex vivo identification of donor-specific unresponsiveness in organ transplant recipients is important for immunosuppression (IS) minimization. We tested three groups of stable living, related-donor kidney transplant patients up to 11 years postoperatively, i.e., 20 haploidenticals with donor bone marrow cell (DBMC) infusions, eight noninfused haploidentical controls (haplo controls), and 11 HLA-identical controls (HLA-id), using multiple ex vivo immune assays. We observed that no patients developed donor-specific antibodies. The majority showed donor-specific CTL unresponsiveness from year 1 onward. Thirteen of 20 DBMC recipients became specifically donor MLR nonreactive. Depletion of donor cells in DBMC recipients still MLR reactive increased donor-specific reactivity by 75% ± 36% ( p = 0.04). Adding them back in low concentration caused antigen specific inhibition. The frequencies of ELISPOT granzyme-B and interferon-γ–producing cells somewhat paralleled the CTL and MLR responses. In the trans vivo DTH, 14 of 19 DBMC recipients demonstrated donor-specific unresponsiveness and 16 of 19 showed “linked suppression,” vs none of eight and one of eight haplo controls and vs six of 10 and one of 10 HLA-ids, respectively. Most importantly, when all six assays were performed simultaneously, 10 of 18 DBMC, five of 10 HLA-ids, and no haplo controls were specifically donor unresponsive long term. We propose that a cluster analysis combining these assays will reveal tolerant recipients in whom IS minimization may safely be tested. This appears to have occurred in many DBMC-infused recipients.

Enhancement of limb transplantation tolerance induced by infusion of donor bone marrow cell in rats

Objective To investigate the effects and mechanism of hmb transplantation tolerance induced by cyclophosphamide (CP),donor specific spleen cell transfusion (DST) and infusion of donor bone marrow cells (DBMC) in rats.Methods Twenty-five male Wistar rats and 25 female SD rats were used respectively as donors and recipients of limb transplantation.The recipient rats were divided randomly into 5 groups: blank control group,DST group,CP group,DST +CP group and DST +CP + DBMC (experimental) group.After the allogeneic limb transplantation models were established,immune tolerance was induced and the general condition,rejection and survival time of limb grafts were observed.The mixed lymphocytes (MLR) were used to determine the state of tolerance and the pelymerase chain reaction (PCR) was employed for detection of macro-chimerism.Results The survival time of limb allograft was significantly prolonged in the experimental group[(27.6±1.1) d],as compared with the blank control group [(6.8±0.4) d],the DST group[(7.2±0.8) d],the CP group[(7.8±1.3) d],and the DST + CP group [(17.8±0.8) d](P＜0.01).The MLR demonstrated that the donor specific inhibition of lymphocyte proliferation in the experimental group[(88.00±1.06)%]was significantly higher than in the DST group[(36.90±1.08)%],the CP group[(37.90±0.95)%],the DST+CP group[(67.20±1.12)%](P ＜ 0.01).Macrochimerism developed in the experimental group.Conclusions DST and CP combined with infusion of DBMC can induce donor specific transplantation tolerance and prolong murine limb graft survival.The macrochimerism may be related to the generation and maintenance of immune tolerance. Key words: Transplantation,homologous; Bone marrow cells; Chimerism; Immune tolerance

MP-03.08: Effect of Donor Bone Marrow Cells Infusion on Alloimmunization in Kidney Allograft Patients

MP-03.08: Effect of Donor Bone Marrow Cells Infusion on Alloimmunization in Kidney Allograft Patients

Th1/Th2 Cytokines and Soluble CD30 Levels in Kidney Allograft Patients With Donor Bone Marrow Cell Infusion

We investigated the relevance of donor bone marrow cell infusion (DBMI) and serum levels of interferon-gamma (IFN-gamma), interleukin-10 (IL-10), and soluble CD30 (sCD30) in kidney recipients. We analyzed the allograft outcomes correlated with sCD30, IFN-gamma, and IL-10 levels using pre- and posttransplantation sera from 40 live donor renal transplants (20 patients with DBMI [2.1 x 10(9) +/- 1.3 x 10(9) mononuclear cells/body] and 20 controls). Patients with acute rejection episodes (ARE)-3/20 DBMI and 6/20 controls-showed increased sCD30 and IFN-gamma as well as decreased IL-10 posttransplantation compared with nonrejectors. Significant differences were observed for sCD30 and IFN-gamma levels: 59.54 vs 30.92 ng/mL (P = .02) and 11.91 vs 3.01 pg/mL (P = .01), respectively. Comparison of pre- and posttransplant levels of IFN-gamma, IL-10, and sCD30 in ARE patients showed higher levels in posttransplant sera except for IFN-gamma in controls (6.37 vs 11.93; P = .01). Increased IFN-gamma and IL-10 were correlated with rejection (r = .93; P = .008). sCD30 correlated with serum creatinine among ARE patients in control and DBMI groups (r = .89; P = .019; and r = 1.00; P < .0001, respectively). Higher levels of sCD30, IFN-gamma, and IL-10 posttransplantation in rejecting patients provided evidence for coexistence of cellular and humoral responses in ARE. There appeared to be a down-regulatory effect of infusion on alloresponses.

Allogeneic donor bone marrow cells recovery and infusion after allogeneic face transplantation from the same donor

Allogeneic donor bone marrow cells recovery and infusion after allogeneic face transplantation from the same donor

Addition of Cyclophosphamide to T-cell Depletion–Based Nonmyeloablative Conditioning Allows Donor T-cell Engraftment and Clonal Deletion of Alloreactive Host T-cells After Bone Marrow Transplantation

Bone marrow (BM) chimerism has been shown to have a beneficial effect on allograft survival. We recently found that production of donor T-cells was highly correlated with induction of tolerance in minimally conditioned chimeras. In the present studies, we demonstrate that nonmyeloablative conditioning and BM cell infusion modulate innate and adaptive host immune responses. Chimeras were generated by bone marrow transplantation (B10.BR to B10). Recipients were preconditioned with T-cell depleting antibodies and total body irradiation with or without cyclophosphamide. Donor-specific tolerance was tested by skin grafting. Transfer of tolerant splenocytes to immunocompetent secondary recipients did not transfer tolerance, nor did infusion of tolerant CD4+/CD25+ T-cells into chimeras without donor T-cell production, demonstrating that linked suppression is an unlikely mechanism in tolerance induction in the context of BM cell infusion. The addition of a single dose of cyclophosphamide to the conditioning enhanced engraftment and tolerance. This was associated with production of donor T-cells and effective clonal deletion, and a significant reduction in activated recipient plasmacytoid dendritic cells (pDC) and natural killer (NK) cells. Chimeras without donor T-cell production that eventually lost their chimerism did not generate an antidonor humoral response, whereas unconditioned controls infused with similar numbers of BM cells did, indicating that infusion of donor BM cells into conditioned recipients induced immune deviation for adaptive B-cell immunity, preventing sensitization to major histocompatibility complex (MHC) alloantigens. These results demonstrate that recipient T-cells, pDC, and NK cells contribute to the host barrier for establishing chimerism, implicate deletional tolerance as the mechanism for total body irradiation-based nonmyeloablative conditioning for BM transplantation, and show a beneficial effect of BM cells in preventing sensitization to MHC alloantigens.

Campath-1H Does Not Alter Bone Marrow Cell Regulatory Function

We have previously reported in laboratory volunteers ( in vitro) and renal transplant recipients ( ex vivo) that bone marrow cells (BMC) are potent downregulators of the immune response. Also, the use of alemtuzumab (Campath-1H, C1H) for immunodepletion is associated with the most potent lasting effects yet seen on T-cell immunity in renal transplantation. We questioned whether the administration of C1H to kidney allograft recipients of donor bone marrow cell (DBMC) infusions would lead to stronger or weaker immunoregulatory effects. Human BMC depleted of T cells (nT-BMC) were either untreated or treated with C1H and rabbit complement and compared for their ability to downregulate autologous or allogeneic T-cell responses and to generate T regulatory (T reg) cells. The proliferative responses to anti-CD3 monoclonal antibody of T cells derived from cocultures with C1H-treated or untreated autologous nT-BMC were equally suppressed, i.e., an equivalent alteration in CD3 complex signaling, not regained by the addition of interleukin 2. Adenosine triphosphate levels were also markedly reduced in T cells both from C1H-treated and untreated nT-BMC cocultures. The ability of C1H-treated or untreated nT-BMC to suppress autologous T-cell cytotoxic function was also equivalent, with a marked, but equivalent, capacity to induce CD4/CD25 high T regs from CD3 + cells, which effectively downregulated cytotoxic T cells. To mimic the clinical infusion of DBMC into (allogeneic) recipients, peripheral blood mononuclear cells were also cultured with allogeneic C1H-treated and untreated nT-BMC. T cells derived from these cultures secondarily stimulated with the same-donor mature antigen-presenting cells exhibited suppressed cytotoxicity by 85% and 54%, respectively. These in vitro studies suggest that C1H does not abrogate BMC immunoregulation and thus may allow its lympho-depleting effect to be synergistic.

The Role of Donor Bone Marrow Infusions in Withdrawal of Immunosuppression in Adult Liver Allotransplantation

We investigated the role of donor bone marrow cell (DBMC) infusions in immunosuppression withdrawal in adult liver transplantation. Patients enrolled were at least 3 years post-transplantation, with stable graft function. Forty-five (study group: G1) received DBMC, and 59 (control group: G2) did not. Immunosuppression was reduced by one third upon enrollment, by another third the second year of the study and was completely withdrawn the third year. Patient and graft survival were similar between the two groups. Although rejection episodes were significantly less in G1 the first 2 years of the study (35% vs. 57%, p = 0.016), there was no significant difference overall (74% vs. 81%, p = 0.14). Until February 2004, 20 patients, 10 in each group, were immunosuppression free for 1-3 years. Approximately 20% of long-term survivors of liver transplantation can successfully discontinue their immunosuppression. DBMC infusions, do not increase this likelihood.