Donor Bone Marrow Engraftment Research Articles

Complications of total-body irradiation in allogeneic bone marrow transplantation: A review article

Hematopoietic stem cell transplantation commonly known as bone marrow transplantation (BMT) or allogeneic BMT (using healthy blood stem cells from a donor) is the preferred therapeutic option for numerous blood-related conditions, both malignant and non-malignant. It is often the sole therapy strategy and essential for relapsed and refractory hematologic malignancies. There have studies regarding BMT on regimen containing total body irradiation (TBI) and a regimen without TBI. It is expected that TBI-based conditioning regimens provide better antitumor effects than chemotherapy regimens. The primary objective of TBI is to eradicate the recipient's bone marrow, facilitating the successful engraftment of donor bone marrow. Acute lymphoid leukemia (ALL) is the principal indication for TBI in bone marrow transplantation. Other diseases including Hodgkin’s lymphoma, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), multiple myeloma (MM), etc., may benefit from TBI-based regimens; however, TBI use is associated with many side effects.The main complications of patients who underwent TBI-containing conditioning regimens in bone marrow transplantation are vomiting and nausea, with frequencies of approximately 66% and 35%, respectively. However, these events are easily managed. Acute complications include stomatitis, diarrhea, loss of appetite, temporary loss of taste, rash and asthenia. Moreover, veno-occlusive disease, interstitial pneumonitis, lung side effects, growth hormone deficiency, neurological side effects, cataracts, renal toxicity, endocrine impairments, and infertility are other complications in patients who underwent TBI-containing conditioning regimens in bone marrow transplantation. In review article, a complication of total-body irradiation in allogeneic bone marrow transplantation was assessed.

Resistance of bone marrow stroma to genotoxic preconditioning is determined by p53

Transplantation of bone marrow (BM) is made possible by the differential sensitivity of its stromal and hematopoietic components to preconditioning by radiation and/or chemotherapeutic drugs. These genotoxic treatments eliminate host hematopoietic precursors by inducing p53-mediated apoptosis but keep the stromal niche sufficiently intact for the engraftment of donor hematopoietic cells. We found that p53-null mice cannot be rescued by BM transplantation (BMT) from even the lowest lethal dose of total body irradiation (TBI). We compared structural changes in BM stroma of mice differing in their p53 status to understand why donor BM failed to engraft in the irradiated p53-null mice. Irradiation did not affect the general structural integrity of BM stroma and induced massive expression of alpha-smooth muscle actin in mesenchymal cells followed by increased adiposity in p53 wild-type mice. In contrast, none of these events were found in p53-null mice, whose BM stroma underwent global structural damage following TBI. Similar differences in response to radiation were observed in in vitro-grown bone-adherent mesenchymal cells (BAMC): p53-null cells underwent mitotic catastrophe while p53 wild-type cells stayed arrested but viable. Supplementation with intact BAMC of either genotype enabled donor BM engraftment and significantly extended longevity of irradiated p53-null mice. Thus, successful preconditioning depends on the p53-mediated protection of cells critical for the functionality of BM stroma. Overall, this study reveals a dual positive role of p53 in BMT: it drives apoptotic death of hematopoietic cells and protects BM stromal cells essential for its functionality.

Desensitization using imlifidase and EndoS enables chimerism induction in allosensitized recipient mice.

Mixed hematopoietic chimerism induction as a way to foster tolerance to donor organs in recipients who have been sensitized to donor antigens is challenging. Donor‐specific antibodies (DSA) are a dominant barrier toward successful donor bone marrow engraftment. Although desensitization methods are routinely used in recipients with allosensitization for allogeneic bone marrow transplantation, engraftment is frequently unsuccessful. To overcome the barrier of prior sensitization we tested enzymatic desensitization of donor‐specific IgG using imlifidase and endoglycosidase of Streptococcus pyogenes (EndoS), which both partially block the function of DSA in mice, as a novel approach to improve murine bone marrow engraftment in primed hosts. We found that EndoS was capable of inhibiting antibody‐mediated killing of donor cells in vivo. Furthermore, the effect of EndoS depended on the titer of DSA and the genetic background of the recipients. In combination with imlifidase, EndoS improved the survival of donor bone marrow cells. Together with cyclophosphamide, bortezomib, T cell depletion, and nonlethal irradiation, imlifidase in combination with EndoS allowed allogeneic bone marrow engraftment in sensitized recipients. We conclude that enzymatic inactivation of DSA, using the combination of imlifidase and EndoS, can be used for inducing donor hematopoietic chimerism in allosensitized recipient mice in combination with other desensitization strategies.

A Comparison of Cs-137 γ Rays and 320-kV X-Rays in a Mouse Bone Marrow Transplantation Model.

US homeland security concerns regarding the potential misuse of some radiation sources used in radiobiological research, for example, cesium-137 (137Cs), have resulted in recommendations by the National Research Council to conduct studies into replacing these sources with suitable X-ray instruments. The objective of this research is to compare the effectiveness of an X-RAD 320 irradiator (PXINC 2010) with a 137Cs irradiator (Gammacell-1000 Unit) using an established bone marrow chimeric model. Using measured radiation doses for each instrument, we characterized the dose–response relationships for bone marrow and splenocyte ablation, using a cytotoxicity-hazard model. Our results show that the X-RAD 320 photon energy spectrum was suitable for ablating bone marrow at the 3 exposure levels used, similar to that of 137Cs photons. However, the 320-kV X-rays were not as effective as the much higher energy γ rays at depleting mouse splenocytes. Furthermore, the 3 X-ray levels used were less effective than the higher energy γ rays in allowing the successful engraftment of donor bone marrow, potentially as a result of the incomplete depletion of the spleen cells. More defined studies are warranted for determining whether bone marrow transplantation in mice can be successfully achieved using 320-kV X-rays. A higher X-ray dose then used is likely needed for transplantation success.

Bone marrow-derived cells are recruited by the melanoma tumor with endothelial cells contributing to tumor vasculature.

Tumor expansion is dependent on neovascularization, a process that requires sustained new vessel formation. Although the critical role of angiogenesis by endothelial sprouting in this process, controversy still prevails on whether angiogenesis involving bone marrow-derived endothelial cells, does contribute to this process. This study aims to evaluate the recruitment of bone marrow-derived cells by the melanoma tumor, including endothelial cells, and if they contribute to angiogenesis. A chimeric mouse model of GFP bone marrow was used to induce melanoma tumors derived from murine B16-F10 cell line. These tumors were evaluated for the presence of myeloid cells (CD11b), T lymphocytes (CD3, CD4 and CD8) and endothelial cells (VEGFR2 and CD31) derived from bone marrow. Mice transplanted with GFP+ cells showed significant bone marrow chimerism (90.9±0.87%) when compared to the GFP transgenic mice (90.66±2.1%, p=0.83) demonstrating successful engraftment of donor bone marrow stem/progenitor cells. Analysis of the murine melanoma tumor showed the presence of donor cells in the tumors (3.5±1.7%) and interestingly, these cells represent endothelial cells (CD31+ cells; 11.5±6.85%) and myeloid cells (CD11b+ cells; 80±21%), but also tumor-infiltrating lymphocytes (CD8+ T cells, 13.31±0.2%; CD4+ T-cells, 2.1±1.2%). Examination of the tumor endothelium by confocal microscopy suggests the presence of donor CD31+/GFP+ cells in the wall of some blood vessels. This study demonstrates that bone marrow-derived cells are recruited by the murine melanoma tumor, with myeloid cells and CD4 and CD8 T lymphocytes migrating as antitumor immune response, and endothelial cells participating of the tumor blood vessels formation.

The Ryk Receptor Regulates Hematopoietic Stem and Progenitor Cell Proliferation and Their Sensitivity To Myeloablative Agents

The molecular mechanisms that control the balance between quiescence and proliferation of hematopoietic stem and progenitor cells (HSPCs) are critical for maintaining life-long hematopoiesis. In a recent study (Povinelli, et al. Stem Cells, In Press, 2013) we demonstrated that the Wnt5a ligand inhibits HSPC proliferation through a functional interaction with a non-canonical Wnt ligand receptor termed Related to Receptor Tyrosine Kinase (Ryk). Expression of Ryk on HSPCs in vivo was associated with a decreased rate of proliferation. Following treatment with fluorouracil (5-FU), the percentage of Ryk+ HSPCs increased at the expense of Ryk-/low HSPCs. Based on these data, we hypothesized that one function of the Ryk receptor is to protect HSPCs from the effects of myeloablative agents. To test this hypothesis, we injected 6-8 week old C57BL/6 mice with 150 mg/kg of 5-FU and analyzed bone marrow 48 hours later for the presence of apoptotic HSPCs, defined as lineage negative (Lin-), Sca-1+, CD48- cells positive for active caspase-3. There was a 2.5-fold decrease in the percentage of apoptotic Ryk+ HSPCs (12.9 ± 1.7%) compared to Ryk-/low HSPCs (32.4 ± 5.3%, p < 0.001, n = 3). To test whether this effect was limited to 5-FU, we performed a similar study in which we irradiated C57BL/6 mice with 3 cGy of total body irradiation (TBI) and analyzed bone marrow 72 hours later for apoptotic HSPCs (for this experiment, defined by a Lin-, c-kit+, Sca-1+, CD150+, CD48- immunophenotype or LSK, SLAM). Comparable to the effects of 5-FU, there was a significant 3.0-fold reduction in the percentage of apoptotic Ryk+ HSPCs (3.1 ± 0.2%) compared to Ryk-/low HSPCs (9.2 ± 1.5%, p < 0. 001, n = 3) in mice receiving 3 cGy TBI. These results demonstrated an association between Ryk expression and survival of HSPCs following myeloablative injury. To determine whether in vivo targeting of the Ryk receptor would increase the sensitivity of HSPCs to myeloablative injury, we utilized a neutralizing rabbit anti-Ryk antibody (α-Ryk). We injected C57BL/6 mice with 5 mg/kg α-Ryk or rabbit IgG isotype for 2 consecutive days. Twenty-four hours after the second dose, we determined the frequency and cell cycle status of LSK SLAM cells. Treatment with α-Ryk significantly increased the percentage of LSK SLAM cells in the S/G2/M phases compared to control (α-Ryk: 17.8 ± 2.2%; isotype IgG: 11.6 ± 2.7%, p < 0.05, n = 3). This was associated with a decrease in the percentage of LSK, SLAM cells in G1 following treatment with α-Ryk (α-Ryk: 40.5 ± 3.2%, isotype IgG: 51.3 ± 2.2; p < 0.01, n = 3). The percentage of G0 LSK SLAM cells was unchanged (α-Ryk: 37.9 ± 2.6, isotype IgG: 35.7 ± 3.1% n = 3) indicating that inhibiting Ryk promoted the exit of LSK SLAM cells from G1. Treatment with α-Ryk also increased the percentage of whole bone marrow cells expressing the LSK SLAM phenotype by 1.4-fold compared to controls (p < 0.05, n = 3). To determine if α-Ryk treatment altered HSPC function, we transplanted whole bone marrow cells from C57BL/6 mice treated with two days of α-Ryk or isotype IgG at a 1:1 ratio with whole bone marrow from untreated Ubc-GFP transgenic mice into lethally irradiated B6.SJL mice. Four weeks after transplant, we analyzed peripheral blood cells for the percentage of CD45.2+ GFP- cells. There was no difference in engraftment by transplanted bone marrow cells from mice treated with α-Ryk or isotype IgG (α-Ryk: 61.6 ± 6.1% n = 4, isotype IgG: 52.8 ± 13.6%, n = 5), indicating that the neutralizing antibody does not inhibit short-term HSPC function on its own. We then tested whether blocking Ryk function resulted in greater sensitivity of HSPCs to 5-FU. We treated B6.SJL mice with 5 mg/kg α-Ryk or isotype IgG for 2 consecutive days, followed by 150 mg/kg of 5-FU. Forty-eight hours after 5-FU treatment, we transplanted 2x106 C57BL/6 whole bone marrow cells into treated B6.SJL mice without additional conditioning. Four weeks after transplant, we determined the percentage of donor-derived CD45.2+ peripheral blood cells. Treatment of recipient mice with α-Ryk prior to 5-FU treatment resulted in increased engraftment of donor bone marrow by 3.6-fold compared to isotype (p < 0.05, n = 5), suggesting that inhibition of Ryk resulted in increased elimination of host HSPCs by 5-FU. Collectively, these data suggest a model in which inhibition of the Ryk receptor results in increased proliferation of HSPCs, rendering them more sensitive to the effects of myeloablative agents such as chemotherapy or TBI. Disclosures:No relevant conflicts of interest to declare.

Abstract LB-304: Bone marrow-derived endothelial cells migrate to tumor sites and contribute to functional tumor vasculature

Abstract Tumor expansion is dependent on neovascularization, a process that requires sustained new vessel formation. Although the critical role of angiogenesis by endothelial sprouting in this process, controversy still prevails on the whether vasculogenesis, involving bone marrow (BM)-derived endothelial cells, does contribute to this process. The objective of this study was to investigate the contributions of BM-derived endothelial cells to tumor vasculature, in a melanoma model using chimeric GFP mice. Methodology: First, wild-type C57BL/6 mice were was exposed to a single dose of 10 Gy X-irradition and rescued by transplantation of BM cells from C57BL/6-GFP mice. At d30 post-transplant, the animals received a subcutaneous infusion of melanoma cells stably transfected with the luciferase gene (B16/F10-Luc+). After 20 days, the tumors were harvested, and tumor stroma characterized using flow cytometry, bioluminescence-based image processing and confocal microscopy, to define the specific contribution of BM-derived cells. Results: Mice transplanted with donor GFP+ cells showed significant BM chimerism (90.9 ± 0.87%) demonstrating successful engraftment of donor BM stem/progenitor cells. Analyses of tumor specimens showed the presence of donor cells in the tumor sites (3.5±1.7%) in all animals, showing that BM-derived cells were effectively recruited to the developing melanoma. Interestingly, these cells represent endothelial cells (CD31+ cells; mean=X%) and myeloid-lineage cells (CD11b+ cells; mean=80%), but also tumor-infiltrating lymphocytes (CD8+ T cells, mean = 13.31; CD4+ T-cells, mean=2.1). Examination of the tumor endothelium by confocal microscopy clearly showed the presence of donor CD31/GFP cells in the vessel wall, namely in vessels lacking other mural components (as pericytes). Conclusions: Taken together, this study demonstrates that BM-derived cells are actively recruited to tumor sites, contributing to the tumor stroma and the formation of blood vessels in the developing tumor. We are performing studies to elucidate the molecular mechanisms involved in mobilization of of BM-derived precursor cells to the tumor microenvironment, and whether disruption of this vasculogeneic process significant impairs tumor development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-304. doi:10.1158/1538-7445.AM2011-LB-304

Effective Alloreactive NK Cell Therapy for Breast Cancer In Mice Requires Conditioning with Cyclophosphamide and Total Body Irradiation but Not Bone Marrow Engraftment

AbstractAbstract 2085 Background:Patients with metastasized breast cancer cannot be cured by current standard treatment options. One hypothesis is that slow cycling chemo-resistant tumor stem cells give rise to new tumors after cytoreductive treatment, ultimately leading to chemoresistant tumors. Last year we showed that the 4T1 mouse breast cancer model contains slow-cycling chemo-resistant cells that induce renewed growth of the tumor after chemo- and radiotherapy (abstract 4082). We also showed that haploidentical spleen and bone marrow transplantation (BMT) cures the mice and that donor NK cells are a prerequisite. Our current aim was to study the need of long term BM engraftment and to study the role of the conditioning in the curative process. Methods:The 4T1 breast cancer cell line, originating from a spontaneous Balb/c (H-2d) breast cancer, was cultured under standard conditions. Fifty thousand 4T1 cells were injected s.c. in the flank. For the experiments addressing the need for haploidentical BMT tumor bearing CB6F1 (H-2b/d) recipients were treated with 2x 2Gy total body irradiation and 200 mg/kg cyclophosphamide (CY+TBI) followed by in vitro NK cell enriched haploidentical B6CBAF1 (H-2b/k) spleen cell infusion with or without additional BM cells. Chimerism in tumor-free surviving recipients was measured by flowcytometry of spleens at least 100 days after the treatment. The role of the conditioning in the alloreactive NK cell effect was studied in fully H-2 mismatched B6CBAF1 mice. When indicated, in vivo NK cell depletion was by i.p. injection of anti-AsialoGM1. Results:Figure A shows overall survival of mice with breast cancer after various treatments (10 mice per group). Haploidentical BMT plus spleen cells cured 50% of tumor bearing mice after CY+TBI (♦, dashed line) and survival was at least as good when NK cell enriched spleen cells were co-transplanted (▴, solid line). Transplantation of spleen cells from NK cell depleted mice (•, dotted line) obliterated the beneficial effect of haploidentical transplantation and resulted is similar poor survival as syngeneic BMT plus spleen cells (▪, solid line). The majority of mice that received NK cell enriched spleen cells (10 out of 14 tested) had no bone marrow engraftment and in the other four only 1–5% donor cells were detectable at 150 days. Recipients of unmanipulated haploidentical spleen and BM cells had >90% donor chimerism in 10 out of 14 tested. The cure rate in both groups was nevertheless similarly high. In a subsequent experiment (Figure B, 10 mice per group) we infused haploidentical NK cells only after CY+TBI (▴, solid line); other groups received T cell depleted (x, solid line) or T cell replete (♦, solid line) haploidentical BMT, or syngeneic BMT (▪, solid line). This resulted in a similar superior tumor-free survival (80-90%) than in mice co-transplanted with haploidentical BM (90%), as compared with syngeneic BM and spleen cell transplantation (•, dotted line). [Display omitted] We then planned to study the role of the conditioning in the curative process. For this purpose 4T1 breast cancer cells were injected in fully H-2 mismatched B6CBAF1 mice (H-2b/k). Surprisingly, 4T1 breast cancer is not rejected by B6CBAF1 mice despite the full MHC mismatch. Tumors are only rejected when the mice were treated with CY+TBI. Tumor rejection proved to be NK cell dependant and not a direct result of the conditioning as it was prevented by in vivo NK cell depletion. Conclusions:This report provides the first evidence that chemo resistant tumor cells can be eliminated in vivo by alloreactive NK cells resulting in cure without the need for long term donor bone marrow engraftment. Conditioning with CY+TBI seems essential for this effect. These results set the stage for the exploration of alloreactive NK therapy in patients with metastasized breast cancer. Disclosures:No relevant conflicts of interest to declare.

Dissection of Effector Pathways in the Host-Versus-Graft Response to Bone Marrow Transplantation

We have dissected the role of the primary cytotoxic pathways Fas-FasL and perforin-granzyme in host-versus-graft reactions after allogeneic bone marrow transplantation. Sublethally irradiated female recipient mice deficient for FasL (B6.gld) or perforin (B6.prf-/-) were transplanted with bone marrow from B6 male donors. Donor engraftment in B6.prf-/- recipients was higher when compared with B6.gld, particularly when assessed by in vivo killing, demonstrating the importance of the perforin pathway over the Fas-FasL pathway. In the absence of both pathways, however, donor bone marrow engraftment was not fully restored identifying a role for an additional pathway(s) in the host-versus-graft response.

Mixed Chimerism Achieved by a Nonlethal Conditioning Regimen Induces Donor-Specific Tolerance to Lung Allografts

Graft rejection and toxicity associated with chronic immunosuppressive therapy remain a major problem in lung transplantation (Tx). Mixed hematopoietic chimerism has been shown to produce long-lasting donor-specific transplant tolerance without immunosuppressive drugs in animal models; however, most conditioning regimens required to achieve mixed chimerism are too toxic for clinical use. The aim of this study was to develop a nonlethal conditioning regimen to induce tolerance to lung allografts. Four to 6-wk old ACI (RT1.A(a)) and Wistar Furth (RT1.A(u)) rats were used as organ donors and recipients, respectively. The recipient conditioning regimen included: 10 mg/animal antilymphocyte globulin (on day-5), 1 mg/kg/d tacrolimus (days 1 to 10), total body irradiation (500 cGy; day 0), and donor bone marrow (DBM) Tx (100 x 10(6) T-cell depleted cells on day 0 following irradiation). Six weeks after DBM Tx, chimeric animals received orthotopic left lung Tx. Graft survival was monitored by chest X-ray and histology. Long-term DBM engraftment was observed: hematopoietic chimerism in the peripheral blood was 12.4 +/- 3.4%, 36.7 +/- 14.1%, and 31.9 +/- 14.1% at 30 d, 6 mo, and 16 mo following DBM Tx, respectively. There was no graft versus host disease. Chimeric recipients (RT1.A(u)) permanently accepted (>400 d) donor-specific lungs (RT1.A(a); n = 8), yet rapidly rejected (<8 d) third party hearts (RT1.A(l); n = 5). Graft (lung) tolerant (>150 d) chimeric recipients accepted secondary donor-specific heart grafts (>150 d; n = 4) but rejected third party heart grafts (<7 d; n = 3). Graft tolerant recipients demonstrated reduced (P < 0.05) in vitro donor-specific lymphoproliferative response and cytotoxicity, and no evidence of acute or chronic graft rejection. Mixed chimerism achieved by a nonlethal conditioning regimen induced long-term donor-specific tolerance to lung allografts.

Efficacy and Limitations of Natural Killer Cell Depletion in Cyclophosphamide-Induced Tolerance

We previously developed a cyclophosphamide (CP)-induced tolerance protocol, consisting of an intravenous injection of 1 x 10(8) donor spleen cells (SC) given on day 0 and an intraperitoneal injection of 200 mg/kg CP given on day 2. In the present study, we modified this protocol with natural killer cell (NK) depletion in recipient mice, and evaluated the efficacy of tolerance induction. We used B10.D2 (H-2d; IE+) and B10 (H-2b; IE-) mice as both donors and recipients. The recipient mice were treated with donor SC, CP, and donor bone marrow cells (BMCs) with or without NK depletion. A higher level of mixed chimerism was achieved in the NK-depleted recipients. Survival of both the skin and heart donor grafts was significantly prolonged in the NK-depleted recipients. Donor reactive Vbeta11+ T cells were found at the same level as in untreated control mice. Pretreatment with recipient NK cell depletion was effective in inducing higher levels of donor mixed chimerism; however, permanent engraftment of donor bone marrow was not achieved. Survival of donor grafts was remarkably prolonged in the NK cell-depleted group, but transplantation tolerance could not be induced. Our results suggest that NK cell depletion in CP-induced tolerance conditioning has some effect on the induction of donor-specific tolerance.

Bone marrow transplantation correcting β‐galactosidase activity does not influence neurological outcome in juvenile GM1‐gangliosidosis

Donor bone marrow engraftment, which resulted in complete normalization of white cell beta-galactosidase levels in a patient with presymptomatic juvenile onset GM1-gangliosidosis (McKusick 230600), did not improve long-term clinical outcome.

Depletion of donor-reactive cells as a new concept for improvement of mismatched bone marrow engraftment using reduced-intensity conditioning

New nonmyeloablative strategies to improve acceptance of mismatched bone marrow (BM) may compensate for the inadequate supply of compatible grafts. Recently we proposed to facilitate engraftment of mismatched BM by selective depletion of activated donor-reactive host cells with cyclophosphamide (CY). Here we have compared engraftment of allogeneic BM after depletion of antigen-activated host lymphocytes by CY, with BM engraftment following general immunosuppression by the same CY dose. Naive or mildly irradiated BALB/c mice were primed with C57BL/6 BM cells (day 0), treated with CY in order to deplete activated T cells (day 1), and transplanted with a second C57BL/6 BM inoculum (day 2) in order to achieve BM engraftment. Alternatively, mice received an equal dose of donor BM cells in a single injection one day after the same CY dose. Treated animals were repeatedly tested for persistence of donor cells in the blood. Depletion of alloantigen-primed lymphocytes by 200 mg/kg CY provided stable GVHD-free engraftment of allogeneic BM in nonirradiated mice, while immunosuppressive treatment with the same CY dose alone resulted in BM rejection. Low-dose irradiation before priming with donor BM allowed the tolerance-inducing CY dose to be reduced to 100 mg/kg. Alloantigen-primed lymphocyte depletion (APLD) by a reduced CY dose resulted in engraftment of donor BM after a significantly lower irradiation dose than treatment with irradiation and CY alone. Our results demonstrate that conditioning that focuses on APLD has a definite advantage over general immunosuppression with CY and radiation therapy.

CD154 Blockade for Induction of Mixed Chimerism and Prolonged Renal Allograft Survival in Nonhuman Primates

Costimulatory blockade with anti-CD154 monoclonal antibody (aCD154) prolongs allograft survival in nonhuman primates, but has not reliably induced tolerance when used alone. In the current studies, we evaluated the effect of adding CD154 blockade to a chimerism inducing nonmyeloablative regimen in primates. We observed a significant improvement of donor bone marrow (DBM) engraftment, which has been associated with a lower incidence of acute rejection and long-term survival of renal allografts without the need for previously required splenectomy. Among the long-term survivors, four never showed evidence of rejection, with the longest survival exceeding 1700 days following discontinuation of immunosuppression. Nevertheless, late chronic rejection was observed in three of eight recipients, indicating the necessity of further modifications of the regimen. Control recipients receiving no DBM or donor splenocytes in place of DBM rejected their allografts. Thus, DBM engraftment with, at least, transient mixed chimerism appears essential for induction of allograft tolerance using this conditioning regimen. Modification of the original mixed chimerism approach, by the addition of costimulatory blockade, has been shown to enhance mixed chimerism and induce renal allograft tolerance with less morbidity in nonhuman primates.

Inactivation of macrophage scavenger receptor class B type I promotes atherosclerotic lesion development in apolipoprotein E-deficient mice.

Scavenger receptor class B type I (SR-BI) is expressed in macrophages, where it has been proposed to facilitate cholesterol efflux. However, direct evidence that the expression of macrophage SR-BI is protective against atherosclerosis is lacking. In this study, we examined the in vivo role of macrophage SR-BI in atherosclerotic lesion development in the apolipoprotein (apo) E-deficient mouse model. ApoE-deficient mice with (n=16) or without (n=15) expression of macrophage SR-BI were created by transplanting lethally irradiated apoE-deficient mice with bone marrow cells collected from SR-BI-/- apoE-/- mice or SR-BI+/+ apoE-/- mice. The recipient mice were fed a chow diet for 12 weeks after transplantation for analysis of atherosclerosis. Quantification of macrophage SR-BI mRNA by real-time reverse transcription-polymerase chain reaction indicated successful engraftment of donor bone marrow and inactivation of macrophage SR-BI in recipient mice reconstituted with SR-BI-/- apoE-/- bone marrow. There were no significant differences in plasma lipid levels, lipoprotein distributions, and HDL subpopulations between the 2 groups. Analysis of the proximal aorta demonstrated an 86% increase in mean atherosclerotic lesion area in SR-BI-/- apoE-/- --> apoE-/- mice compared with SR-BI+/+ apoE-/- --> apoE-/- mice (109.50+/-18.08 versus 58.75+/-9.58x10(3) microm2; mean+/-SEM, P=0.017). No difference in cholesterol efflux from SR-BI+/+ apoE-/- or SR-BI-/- apoE-/- macrophages to HDL or apoA-I discs was detected. Expression of macrophage SR-BI protects mice against atherosclerotic lesion development in apoE-deficient mice in vivo without influencing plasma lipids, HDL subpopulations, or cholesterol efflux. Thus, macrophage SR-BI plays an antiatherogenic role in vivo, providing a new therapeutic target for the design of strategies to prevent and treat atherosclerosis.

Murine hematopoietic stem cell distribution and proliferation in ablated and nonablated bone marrow transplantation

The engraftment of donor bone marrow (BM) cells in nonablated mice is inefficient. Niche availability has been thought to be the reason, and cytoablation with irradiation or cytotoxic agents is routinely used with the belief that this frees the preoccupied niches in recipients. In this study, donor cell redistribution and proliferation in ablated and nonablated mice were compared by implanting donor cells directly into the femur cavity of sedated mice. The redistribution of Lin(-) donor cells into BM was similar between ablated and nonablated mice. Poor engraftment in nonablated mice was shown to be the result of inefficient donor cell proliferation rather than because of a lack of space. Competitive repopulation assays demonstrated that the donor hematopoietic stem cells (HSCs) were present in nonirradiated recipients for at least 6 months after transplantation, but that they did not expand as did their counterparts in lethally irradiated mice. This study suggests that efficient bone marrow transplantation in nonablated recipients may be possible as a result of better understanding of HSC proliferative regulation and appropriate in vitro manipulation.

Mechanisms Involved in the Establishment of Tolerance Through Costimulatory Blockade and BMT: Lack of Requirement for CD40L‐Mediated Signaling for Tolerance or Deletion of Donor‐reactive CD4+ Cells

We have previously shown that high levels of multiline-age mixed hematopoietic chimerism and systemic T-cell tolerance can be achieved in mice without myeloablation through the use of anti-CD40L and costimulatory blockade alone (plus CTLA4Ig) or with recipient CD8 depletion and allogeneic bone marrow transplantation. Chimeric mice permanently accept donor skin grafts (> 100 days), and rapidly reject third-party grafts. The mechanisms by which costimulatory blockade facilitates the engraftment of allogeneic hematopoietic cells have not been defined. To further understand the in vivo mechanisms by which the administration of anti-CD40L mAb facilitates the engraftment of donor bone marrow and rapidly tolerizes CD4+ T cells, we analyzed the establishment of chimerism and tolerance in CD40L -/- mice. We demonstrate here that anti-CD40L mAb treatment is required only to prevent CD40L/CD40 interactions, and that no signal to the T cell through CD40L is necessary for the induction of CD4+ tolerance. Peripheral deletion of donor-reactive CD4+ T cells occurs rapidly in CD40L -/- mice receiving bone marrow transplantation (BMT), indicating that this deletion in the presence of anti-CD40L is not due to targeting of activated CD4+ cells by the antibody. Complete CD4+ cell tolerance is observed by both skin graft acceptance and in vitro assays before deletion is complete, indicating that additional mechanisms play a role in inducing CD4+ T-cell tolerance as the result of BMT in the presence of CD40/CD40L blockade.

Delayed cardiac xenograft rejection in a pig-to-baboon model treated with a tolerance-inducing regimen and donor bone marrow infusion

DISCORDANT cardiac xenografts are hyperacutely rejected owing to the binding of natural antibody (NAb) to xenograft endothelial cells and activation of the complement pathway. Depletion of NAb and complement from recipients can prevent hyperacute rejection. However, delayed xenograft rejection (DXR) has become a major barrier to long-term xenograft survival after overcoming hyperacute rejection. The pathogenesis of DXR has been demonstrated to be macrophage-mediated cellular rejection and newly developed NAb. Total lymphoid irradiation (TLI) has been used as a conditioning regimen for bone marrow transplantation, a treatment designed to remove or suppress peripheral T and B cells while leaving bone marrow stem cells intact. TLI has been shown, with some success, to induce specific transplant tolerance to allografts and xenografts. Our previous study has shown alteration of humoral immune response in a rhesus monkey-to-baboon cardiac xenograft model treated with methotrexate and cyclosporine A. In this study, we utilized TIL combined with immunosuppression of humoral immunity by methotrexate to establish donor bone marrow engraftment and immune tolerance, and to prevent DXR in a pig-to-baboon cardiac xenograft model.

Increased expression of Fas (APO-1, CD95) on CD34+ haematopoietic progenitor cells after allogeneic bone marrow transplantation.

Up-regulation of Fas/APO-1 (CD95) on haematopoietic progenitors and Fas-mediated apoptosis have been suggested to occur in a possible pathological mechanism in some bone marrow failure syndromes. We examined the expression of Fas antigen and susceptibility to Fas-mediated suppression of donor-derived haematopoietic cells of allogeneic bone marrow transplantation (BMT) recipients. Cytofluorometric analysis revealed low expression of Fas on CD34+ bone marrow cells from marrow donors or healthy controls. However, significantly higher expression of Fas antigen was observed on CD34+ bone marrow cells of BMT recipients, in whom engraftment of donor bone marrow (BM) cells was confirmed. The addition of an agonistic anti-Fas antibody (Ab) (CH-11) to haematopoietic stem cell culture of BM cells more strongly suppressed colony formation from granulocyte-macrophage colony-forming units (GM-CFU) and erythroid burst-forming units (BFU-E) after BMT. Pretreatment by blocking anti-Fas Ab (ZB4) abrogated the Fas-mediated GM-CFU and BFU-E suppression. Purified marrow CD34+ cells from BMT recipients were also susceptible to the Fas-mediated colony suppression. Thus, donor-derived CD34+ haematopoietic cells increased their expression of Fas antigen and were susceptible to Fas-mediated haematopoietic suppression. These findings provide new insight for understanding the haematological condition after BMT.

Chromosomal aneuploidy in leukemic blast crisis: A potential source of error in interpretation of bone marrow engraftment analysis by VNTR amplification

Polymerase chain reaction (PCR) amplification of polymorphic microsatellite or minisatellite DNA markers has proven to be a fast, sensitive, and specific technique in post-transplantation monitoring of bone marrow engraftment, as well as early detection of residual disease and relapse. Deletion or amplification of chromosomal segments carrying marker loci, as can occur in leukemia and other hematologic malignancies, may result in loss or increased dosage of marker alleles. Examination of these marker alleles by PCR therefore may give aberrant results, which might lead to misinterpretation of bone marrow transplantation (BMT) engraftment studies. We report a case of chronic myelogenous leukemia treated by BMT. PCR amplification of the minisatellite at the apoB locus on chromosome 2 was used to monitor the donor bone marrow engraftment. The patient experienced relapse in blast crisis with a near-haploid karyotype with loss of recipient-specific apoB allele causing an aberrant PCR result for bone marrow engraftment that mimicked full donor engraftment. Loss or gain of polymorphic DNA markers because of chromosomal losses or gains in some hematologic malignancies may affect the interpretation of bone marrow engraftment studies by PCR. When choosing polymorphic markers for such studies, it is important to avoid those that will be affected by expected chromosomal alteration, if possible. In addition, any abberant post-transplantation typing should prompt further investigation to rule out the possibility of chromosomal aberration. Review of all pertinent laboratory studies is important to avoid misinterpretation of results from a single test for engraftment analysis.