Functional CD47/signal regulatory protein alpha (SIRP(alpha)) interaction is required for optimal human T- and natural killer- (NK) cell homeostasis in vivo

The homeostatic control mechanisms regulating human leukocyte numbers are poorly understood. Here, we assessed the role of phagocytes in this process using human immune system (HIS) BALB/c Rag2(-/-)IL-2Rγc(-/-) mice in which human leukocytes are generated from transplanted hematopoietic progenitor cells. Interactions between signal regulatory protein alpha (SIRPα; expressed on phagocytes) and CD47 (expressed on hematopoietic cells) negatively regulate phagocyte activity of macrophages and other phagocytic cells. We previously showed that B cells develop and survive robustly in HIS mice, whereas T and natural killer (NK) cells survive poorly. Because human CD47 does not interact with BALB/c mouse SIRPα, we introduced functional CD47/SIRPα interactions in HIS mice by transducing mouse CD47 into human progenitor cells. Here, we show that this procedure resulted in a dramatic and selective improvement of progenitor cell engraftment and human T- and NK-cell homeostasis in HIS mouse peripheral lymphoid organs. The amount of engrafted human B cells also increased but much less than that of T and NK cells, and total plasma IgM and IgG concentrations increased 68- and 35-fold, respectively. Whereas T cells exhibit an activated/memory phenotype in the absence of functional CD47/SIRPα interactions, human T cells accumulated as CD4(+) or CD8(+) single-positive, naive, resting T cells in the presence of functional CD47/SIRPα interactions. Thus, in addition to signals mediated by T cell receptor (TCR)/MHC and/or IL/IL receptor interactions, sensing of cell surface CD47 expression by phagocyte SIRPα is a critical determinant of T- and NK-cell homeostasis under steady-state conditions in vivo.     

Decreased homing of retrovirally transduced human bone marrow CD34+ cells in the NOD/SCID mouse model

OBJECTIVE: Many clinical gene therapy trials have described poor engraftment of retrovirally transduced CD34(+) cells. Because engraftment is dependent upon successful homing of graft cells to the bone marrow (BM), we examined whether retroviral-mediated gene transfer (RMGT) induces a homing defect in CD34(+) cells. METHODS: Homing of fluorescently labeled human BM CD34(+) cells transduced with three separate retroviral vectors (MFG-eGFP, LNC-eGFP, and LXSN) was assessed in nonobese diabetic/severe combined immunodeficient mice. RESULTS: Homing of transduced CD34(+) cells was significantly decreased 20 hours after transplantation compared with freshly isolated control and cultured untransduced control cells. Specifically, homing of GFP(+) cells in the graft was preferentially decreased thus skewing the contribution of transduced cells to engraftment. Transduced cells were not selectively trapped in other organs and BM-homed transduced cells did not undergo apoptosis at a higher rate than untransduced cells. Adhesion molecule expression and binding activity was not altered by RMGT. This homing defect was reversed when transduced cells were cultured over CH-296 for 2 additional days with SCF only. CONCLUSION: These data suggest that RMGT of hematopoietic cells may compromise their homing potential and implicate transduction-induced reduced homing in the observed low engraftment of retrovirally transduced CD34(+) cells. These results may have a direct clinical application in gene therapy protocols.     

Repopulation of adult and neonatal mice with human hepatocytes: a chimeric animal model

We report the successful transplantation of human hepatocytes in immunodeficient, fumarylacetoacetate hydrolase-deficient (fah(-/-)) mice. Engraftment occurs over the entire liver acinus upon transplantation. A few weeks after transplantation, increasing concentrations of human proteins (e.g., human albumin and human C3a) can be measured in the blood of the recipient mouse. No fusion between mouse and human hepatocytes can be detected. Three months after transplantation, up to 20% of the mouse liver is repopulated by human hepatocytes, and sustained expression of lentiviral vector transduced gene can be observed. We further report the development of a hepatocyte transplantation method involving a transcutaneous, intrahepatic injection in neonatal mice. Human hepatocytes engraft over the entire injected lobe with an expansion pattern similar to those observed with intrasplenic transplantation.     

The severe combined immunodeficient-human peripheral blood stem cell (SCID-huPBSC) mouse: a xenotransplant model for huPBSC-initiated hematopoiesis

Mononuclear cells (MNCs) containing peripheral blood stem cells (PBSCs) were obtained from solid-tumor patients undergoing mobilizing chemotherapy followed by granulocyte colony-stimulating factor for PBSC transplantation-supported dose-intensified anticancer chemotherapy and were transplanted into unconditioned "nonleaky" young severe combined immunodeficient mice. Multilineage engraftment was shown by flow cytometry and immunocytochemistry using monoclonal antibodies to various human cell surface antigens as well as identification of human immunoglobulin in murine sera. Within a dose range of MNCs suitable for transplantation (10 to 36 x 10(6) cells/graft) the number of CD34+ cells injected (optimal at > 0.7 x 10(6)/graft) determined the yield of human cells produced in recipient animals. Engraftment of hu PBSC preparations resulted in prolonged generation of physiologic levels of human cytokines including interleukin-3 (IL-3), IL-6, and granulocyte- macrophage colony-stimulating factor, which were detectable in the murine blood over a period of at least 4 months. In vivo survival of immature human progenitor cells was preserved even 9 months after transplantation. Because human IL-3 is known to stimulate early hematopoiesis, a rat fibroblast cell line was stably transfected with a retroviral vector carrying the human IL-3 gene and cotransplanted subcutaneously as additional source of growth factor. Cotransplants of this cell line producing sustained in vivo levels of circulating human IL-3 for at least 12 weeks significantly accelerated the process of engraftment of huPBSC and spurred the spread of mature human cells to the murine spleen, liver, thymus, and peripheral blood. Cotransplants of allogeneic human bone marrow stromal cells derived from long-term cultures resulted in a comparable--though less prominent--support of engraftment.     

The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors

CD47, a "don't eat me" signal for phagocytic cells, is expressed on the surface of all human solid tumor cells. Analysis of patient tumor and matched adjacent normal (nontumor) tissue revealed that CD47 is overexpressed on cancer cells. CD47 mRNA expression levels correlated with a decreased probability of survival for multiple types of cancer. CD47 is a ligand for SIRPα, a protein expressed on macrophages and dendritic cells. In vitro, blockade of CD47 signaling using targeted monoclonal antibodies enabled macrophage phagocytosis of tumor cells that were otherwise protected. Administration of anti-CD47 antibodies inhibited tumor growth in orthotopic immunodeficient mouse xenotransplantation models established with patient tumor cells and increased the survival of the mice over time. Anti-CD47 antibody therapy initiated on larger tumors inhibited tumor growth and prevented or treated metastasis, but initiation of the therapy on smaller tumors was potentially curative. The safety and efficacy of targeting CD47 was further tested and validated in immune competent hosts using an orthotopic mouse breast cancer model. These results suggest all human solid tumor cells require CD47 expression to suppress phagocytic innate immune surveillance and elimination. These data, taken together with similar findings with other human neoplasms, show that CD47 is a commonly expressed molecule on all cancers, its function to block phagocytosis is known, and blockade of its function leads to tumor cell phagocytosis and elimination. CD47 is therefore a validated target for cancer therapies.     

Kinetics and functional assay of liver repopulation after human cord blood transplantation

BACKGROUND AND AIMS: To evaluate donor cell engraftment and the kinetics of cell repopulation in the injured mouse liver following human umbilical cord blood cell transplantation. METHODS: Nonobese diabetic/severe immunodeficient mice were treated with allyl alcohol to induce liver injury. Twenty-four hours later, umbilical cord blood derived mononuclear cells were transplanted by intra-splenic injection. Mice were sacrificed from 1 to 180 days after transplantation. Temporal changes in the ratio of human cells and fluorescence counts of human sex-determining region Y alleles in mouse liver were determined to evaluate the kinetics of cell repopulation. Mouse liver and sera were examined for the presence of human albumin. RESULTS: Human cell repopulation was extremely rapid in the first week following transplantation, with a doubling time of 1.16-1.39 days apparent. Thereafter cell doubling rate slowed significantly. Cells displaying characteristics of human hepatocytes were still evident at 180 days. Human albumin was detected in mouse liver and sera. CONCLUSION: These findings confirm those from previous studies demonstrating that cells derived from human umbilical cord blood have the capacity to differentiate into cells with human hepatocyte characteristics in mouse liver following injury. Moreover, the detailed information collected regarding the kinetics of human cell repopulation in mouse liver will be of relevance to future studies examining the use of umbilical cord blood cells in liver transplantation therapy.     

Recombinant AAV2 transduction of primitive human hematopoietic stem cells capable of serial engraftment in immune-deficient mice

A recombinant AAV2 (rAAV2) vector encoding antisense RNA to HIV-1 transactivating region (TAR) was evaluated for transduction of human cord blood CD34+CD38- hematopoietic stem cells (HSC) capable of serial engraftment in nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mice. Results revealed long-term multilineage marking in primary and secondary recipients, and significantly, an enrichment of transduced cells in secondary hosts, indicating efficient transduction of multipotential self-renewing HSC. These results were confirmed by the persistence of rAAV marking of clonogenic progenitors in serial analyses of recipient marrow. Upon HIV-1 challenge, the macrophage progeny of transduced CD34+ cells expressed antisense RNA and exhibited sustained and significant inhibition of virus replication as compared with controls in every donor tested, without selective pressure. This study represents a clear in vivo demonstration of efficient rAAV2 transduction of human HSC.     

Quantitative assessment of retroviral transfer of the human multidrug resistance 1 gene to human mobilized peripheral blood progenitor cells engrafted in nonobese diabetic/severe combined immunodeficient mice

Mobilized peripheral blood progenitor cells (PBPC) are a potential target for the retrovirus-mediated transfer of cytostatic drug-resistance genes. We analyzed nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse-repopulating CD34+ PBPC from patients with cancer after retroviral transduction in various cytokine combinations with the hybrid vector SF-MDR, which is based on the Friend mink cell focus-forming/murine embryonic stem-cell virus and carries the human multidrug resistance 1 (MDR1) gene. Five to 13 weeks after transplantation of CD34+ PBPC into NOD/SCID mice (n = 84), a cell dose-dependent multilineage engraftment of human leukocytes up to an average of 33% was observed. The SF-MDR provirus was detected in the bone marrow (BM) and in its granulocyte fractions in 96% and 72%, respectively, of chimeric NOD/SCID mice. SF-MDR provirus integration assessed by quantitative real-time polymerase chain reaction (PCR) was optimal in the presence of Flt-3 ligand/thrombopoietin/stem-cell factor, resulting in a 6-fold (24% +/- 5% [mean +/- SE]) higher average proportion of gene-marked human cells in NOD/SCID mice than that achieved with IL-3 alone (P <.01). A population of clearly rhodamine-123(dull) human myeloid progeny cells could be isolated from BM samples from chimeric NOD/SCID mice. On the basis of PCR and rhodamine-123 efflux data, up to 18% +/- 4% of transduced cells were calculated to express the transgene. Our data suggest that the NOD/SCID model provides a valid assay for estimating the gene-transfer efficiency to repopulating human PBPC that may be achievable in clinical autologous transplantation. P-glycoprotein expression sufficient to prevent marrow aplasia in vivo may be obtained with this SF-MDR vector and an optimized transduction protocol. (Blood. 2000;95:1237-1248)     

Engraftment of human CD34+ cells leads to widespread distribution of donor-derived cells and correction of tissue pathology in a novel murine xenotransplantation model of lysosomal storage disease

A novel murine system was developed to study the in vivo localization of xenotransplanted human cells and assess their therapeutic effect in an authentic model of disease. The beta-glucuronidase (GUSB) mutation of the mucopolysaccharidosis type VII (MPSVII) mouse was backcrossed onto the nonobese diabetic/severe combined immunodeficient (NOD/SCID) xenotransplantation strain. The resulting NOD/SCID/MPSVII mice displayed the characteristic features of lysosomal storage disease because of GUSB deficiency and were also capable of engrafting human cells. Human CD34+ hematopoietic progenitor cells from healthy, GUSB+ donors engrafted NOD/SCID/MPSVII mice in a manner similar to that of standard NOD/SCID mice. Six to 12 weeks following transplantation, 1% to 86% of the host bone marrow was positive for human CD45. By using a GUSB-specific histochemical assay, human engraftment was detected with single-cell sensitivity not only in well-characterized hematopoietic tissues like bone marrow, spleen, lymph node, and thymus, but also in other nonhematopoietic organs like liver, kidney, lung, heart, brain, and eye. Quantitative measurements of GUSB activity confirmed this expansive tissue distribution. The GUSB-specific assays were validated for their accuracy in identifying human cells through colocalization of human CD45 expression with GUSB activity in tissues of mice receiving transplants. An analysis of the therapeutic effects of engrafted human cells revealed a reduction of pathologic storage material in host organs, including the bone, spleen, and liver. Such xenotransplantation experiments in the NOD/SCID/MPSVII mouse represent a powerful approach to both study the in vivo biology of human cells and gather preclinical data regarding treatment approaches for a human disease.     

Analysis of optimal conditions for retroviral-mediated transduction of primitive human hematopoietic cells

We sought to define optimal conditions for retroviral-mediated transduction of long-lived human hematopoietic progenitors from bone marrow and peripheral blood. CD34+ cells were transduced by the LN and G2 retroviral vectors in the presence or absence of stromal support and with or without cytokine addition. After transduction, a portion of the cells was plated in methylcellulose colony-forming assay, with or without G418, to assess the extent of gene transfer into committed progenitors. The remaining cells from each experiment were transplanted into immunodeficient mice to allow analysis of transduction of long- lived progenitors. Human colony-forming cells contained within the murine bone marrow were analyzed after engraftment periods of 2 to 11 months. Cells were plated in a human-specific colony-forming assay with and without G418 to assess the extent of transduction of primitive progenitors. Individual human colonies were also analyzed by polymerase chain reaction for the presence of provirus. Bone marrow progenitors were efficiently transduced only when stroma was present, whereas mobilized peripheral blood progenitors were effectively transduced in the presence of either stroma or cytokines. Inclusion of the cytokines interleukin-3, interleukin-6, and stem cell factor did not further augment the extent of gene transfer in the presence of a stromal support layer. Additionally, human CD34+ progenitors from bone marrow or mobilized peripheral blood that had been transduced for 3 days in the absence of stroma failed to produce sustained, long-term engraftment of bnx mice. Mice transplanted with the same pools of human progenitors that had been transduced in the presence of stroma for 3 days had significant levels of human cell engraftment at the same timepoints, 7 to 11 months after transplantation. Our data show loss of long-lived human progenitors during 3-day in vitro transduction periods in the absence of stromal support. Therefore, the presence of bone marrow stroma has dual benefits in that it increases gene transfer efficiency and is essential for survival of long-lived human hematopoietic progenitors.     

Concentrated RD114-pseudotyped MFGS-gp91phox vector achieves high levels of functional correction of the chronic granulomatous disease oxidase defect in NOD/SCID/beta -microglobulin-/- repopulating mobilized human peripheral blood CD34+ cells

In previous studies amphotropic MFGS-gp91phox (murine onco-retrovirus vector) was used in a clinical trial of X-linked chronic granulomatous disease (X-CGD) gene therapy to achieve transient correction of oxidase activity in 0.1% of neutrophils. We later showed that transduced CD34+ peripheral blood stem cells (CD34+ PBSCs) from this trial transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice resulted in correction of only 2.5% of human neutrophils. However, higher rates of transduction into stem cells are required. In the current study we demonstrate that the same vector (MFGS-gp91phox) pseudo-typed with RD114 envelope in a 4-day culture/transduction regimen results in a 7-fold increase in correction of NOD/SCID mouse repopulating X-CGD CD34+ PBSCs (14%-22% corrected human neutrophils; human cell engraftment 13%-67%). This increase may result from high expression of receptor for RD114 that we demonstrate on CD34+CD38- stem cells. Using RD114-MFGS encoding cyan fluorescent protein to allow similar studies of normal CD34+ PBSCs, we show that progressively higher levels of gene marking of human neutrophils (67%-77%) can be achieved by prolongation of culture/transduction to 6 days, but with lower rates of human cell engraftment. Our data demonstrate the highest reported level of functional correction of any inherited metabolic disorder in human cells in vivo with the NOD/SCID mouse system using onco-retrovirus vector.     

Engraftment potential into NOD/SCID mice of CD34+ cells derived from human fetal liver as compared to fetal bone marrow

BACKGROUND AND OBJECTIVES: We hypothesized that qualitative or quantitative differences in hematopoietic stem cells from fetal liver (FL) and fetal bone marrow (FBM) may be the cause of their organ specificity. DESIGN AND METHODS: To analyze possible differences in vivo, we compared the engraftment potential of equal numbers of CD34+ cells isolated from human FL or FBM into immunodeficient NOD/SCID mice. RESULTS: Mice showing engraftment following transplantation of CD34+ cells from FL demonstrated 14% (range 2-76%) CD45+ cells of human origin in the bone marrow compared to significantly lower levels of engraftment (4%, range 2-20%, p < 0.04) of FBM CD34+ cells. Likewise, the percentage of CD34+ CD38- cells in FBM was 4 times lower than the percentage in FL (1.4+/-0.9% and 5.6+/-0.7%, respectively). Similar organ distribution of engrafted human cells was found. Subset analysis of human cells in bone marrow of engrafted mice revealed identical distribution of the lymphoid, myeloid and erythroid lineages after transplantation of CD34+ cells from FL or FBM. INTERPRETATION AND CONCLUSIONS: The FL CD34+ cells showed a four-fold higher content of the CD34+ CD38- subset coinciding with a four-fold higher engraftment of CD34+ cells into NOD/SCID mice. Since the organ distribution and differentiation potential of the cells engrafted were similar, we concluded that CD34+ hematopoietic cells derived from FL and FBM have quantitatively different, but qualitatively the same potential for engraftment into NOD/SCID mice.     

Hepatic parenchymal replacement in mice by transplanted allogeneic hepatocytes is facilitated by bone marrow transplantation and mediated by CD4 cells

The lack of adequate donor organs is a major limitation to the successful widespread use of liver transplantation for numerous human hepatic diseases. A desirable alternative therapeutic option is hepatocyte transplantation (HT), but this approach is similarly restricted by a shortage of donor cells and by immunological barriers. Therefore, in vivo expansion of tolerized transplanted cells is emerging as a novel and clinically relevant potential alternative cellular therapy. Toward this aim, in the present study we established a new mouse model that combines HT with prior bone marrow transplantation (BMT). Donor hepatocytes were derived from human alpha(1)-antitrypsin (hAAT) transgenic mice of the FVB strain. Serial serum enzyme-linked immunosorbent assays for hAAT protein were used to monitor hepatocyte engraftment and expansion. In control recipient mice lacking BMT, we observed long-term yet modest hepatocyte engraftment. In contrast, animals undergoing additional syngeneic BMT prior to HT showed a 3- to 5-fold increase in serum hAAT levels after 24 weeks. Moreover, complete liver repopulation was observed in hepatocyte-transplanted Balb/C mice that had been transplanted with allogeneic FVB-derived bone marrow. These findings were validated by a comparison of hAAT levels between donor and recipient mice and by hAAT-specific immunostaining. Taken together, these findings suggest a synergistic effect of BMT on transplanted hepatocytes for expansion and tolerance induction. Livers of repopulated animals displayed substantial mononuclear infiltrates, consisting predominantly of CD4(+) cells. Blocking the latter prior to HT abrogated proliferation of transplanted hepatocytes, and this implied an essential role played by CD4(+) cells for in vivo hepatocyte selection following allogeneic BMT. CONCLUSION: The present mouse model provides a versatile platform for investigation of the mechanisms governing HT with direct relevance to the development of clinical strategies for the treatment of human hepatic failure.     

Macrophages prevent human red blood cell reconstitution in immunodeficient mice

An animal model supporting human erythropoiesis will be highly valuable for assessing the biologic function of human RBCs under physiologic and disease settings, and for evaluating protocols of in vitro RBC differentiation. Herein, we analyzed human RBC reconstitution in NOD/SCID or NOD/SCID/γc(-/-) mice that were transplanted with human CD34+ fetal liver cells and fetal thymic tissue. Although a large number of human CD45- CD71+ nucleated immature erythroid cells were detected in the bone marrow, human RBCs were undetectable in the blood of these mice. Human RBCs became detectable in blood after macrophage depletion but disappeared again after withdrawal of treatment. Furthermore, treatment with human erythropoietin and IL-3 significantly increased human RBC reconstitution in macrophage-depleted, but not control, humanized mice. Significantly more rapid rejection of human RBCs than CD47-deficient mouse RBCs indicates that mechanisms other than insufficient CD47-SIRPα signaling are involved in human RBC xenorejection in mice. All considered, our data demonstrate that human RBCs are highly susceptible to rejection by macrophages in immunodeficient mice. Thus, strategies for preventing human RBC rejection by macrophages are required for using immunodeficient mice as an in vivo model to study human erythropoiesis and RBC function.     

Hepatocyte transplantation: Studies in preclinical models

Summary Transplantation of allogeneic or genetically modified autologous hepatocytes may be an alternative to whole-liver transplantation for the treatment of hereditary metabolic liver diseases. Human hepatocytes have already been transplanted in patients, demonstrating the safety and feasibility of both approaches. Although a few cases of allogeneic transplantation have resulted in long-term engraftment and function, only a partial and transient correction of the disease was achieved. This may partly result from a lack of proliferation of transplanted cells. In rodents, transplanted hepatocytes do not proliferate in adult quiescent livers and repopulate recipient livers only when they display a proliferative advantage over resident hepatocytes. Most of these models are not transposable to humans, however. Our aim is to develop preclinical approaches to hepatocyte transplantation in nonhuman primates. We have defined a strategy that increases the engraftment efficiency of transplanted hepatocytes by inducing their proliferation together with that of resident hepatocytes. We have also immortalized simian fetal hepatic progenitor cells and shown that these cells do not proliferate in situ after transplantation into the livers of immunodeficient mice. By contrast early human hepatoblasts repopulate mouse livers more efficiently. However, if we consider the number of cells to be transplanted (one to several billion), the means of expanding and differentiating stem or progenitor cells other than hepatocytes will have to be determined prior to envisaging treating patients. Presented at the 42nd Annual Meeting of the SSIEM, Paris, 6–9 September, 2005. Competing interests: None declared     

Enhanced engraftment of human cells in RAG2/gammac double-knockout mice after treatment with CL2MDP liposomes

OBJECTIVE: The ability of human cells to repopulate the bone marrow of nonobese diabetic immunodeficient mice (NOD/SCID) is commonly used as a standard assay to quantify the primitive human hematopoietic stem cell population. We studied the applicability of the immunodeficient RAG2(-/-)gammac(-/-) double-knockout mouse for this purpose. METHODS: RAG2(-/-)gammac(-/-) mice and NOD/SCID mice were injected intravenously (i.v.) with umbilical cord blood-derived CD34(+) cells and engraftment was quantified by determining the human CD45+ cell chimerism in bone marrow at several time points. RAG2(-/-)gammac(-/-) were pretreated with total-body irradiation and depleted of macrophages in liver, spleen, and bone marrow by i.v. injection of clodronate diphosphonate containing liposomes. RESULTS: We demonstrated that the frequency of chimerism and the level of engraftment in macrophage-depleted RAG2(-/-)gammac(-/-) largely resemble that in NOD/SCID mice. Also similar is the multilineage differentiation pattern in the two mouse strains at 7 weeks after transplantation, with a prominent outgrowth in RAG2(-/-)gammac(-/-) of CD19+ cells (88% +/- 10%). Cells of other lineages were clearly less frequent: 9% +/- 2% myeloid cells and 0.1% +/- 0.1% erythroid cells. As for immature progenitors, 6% +/- 1% of the human cells express the CD34 antigen and 0.4% +/- 0.1% have the CD34+,CD33,38,71(-) phenotype. The presence of human committed progenitors (i.e., CFU-GM/BFU-E) was evident. The persistence of human cells at 4 months after transplantation shows that the RAG2(-/-)gammac(-/-) support long-term maintenance of human hematopoiesis. CONCLUSION: Our findings indicate that macrophage-depleted RAG2(-/-)gammac(-/-) are a suitable model for studying human hematopoiesis including multipotential stem cells, and long-term repopulation.     

Stable long-term gene correction with low-dose radiation conditioning in murine X-linked chronic granulomatous disease

We previously demonstrated that low-dose radiation conditioning impairs murine hematopoietic stem cell function, permitting engraftment of syngeneic fresh and transduced marrow cells. In this study, we directly examined the ability of low-dose radiation conditioning to permit engraftment of transduced long-term repopulating cells in murine X-linked chronic granulomatous disease (X-CGD), which closely mimics the human disease. X-CGD mice conditioned with 160 cGy were transplanted with 20 x 10(6) MSCV-m91Neo-transduced syngeneic X-CGD marrow cells. The presence of oxidase-positive neutrophils in two independent cohorts of transplanted 160-cGy-conditioned X-CGD recipients was determined by nitroblue tetrazolium testing. Transplanted X-CGD mice (n = 9 total) displayed 1-17% oxidase-positive neutrophils 6-16 months post-transplant. Retroviral marking and NADPH-oxidase-positive neutrophils persisted through serial transplantation, verifying that stem cells were transduced. These results establish that low-dose radiation conditioning results in durable engraftment of low but potentially clinically relevant numbers of functionally reconstituted blood cells in a murine model of X-CGD.     

Clinical stem-cell sources contain CD8+CD3+ T-cell receptor-negative cells that facilitate bone marrow repopulation with hematopoietic stem cells

Clinical observations in patients undergoing bone marrow transplantation implicate the involvement of CD8(+) cells in promoting the stem-cell engraftment process. These findings are supported by mouse transplant studies, which attributed the engraftment-facilitating function to subpopulations of murine CD8(+) cells, but the analogous cells in humans have not been identified. Here, we report that clinical stem-cell grafts contain a population of CD8alpha(+)CD3epsilon(+) T-cell receptor- negative cells with an engraftment facilitating function, named candidate facilitating cells (cFCs). Purified cFC augmented human hematopoiesis in NOD/SCID mice receiving suboptimal doses of human CD34(+) cells. In vitro, cFCs cocultured with CD34(+) cells increased hematopoietic colony formation, suggesting a direct effect on clonogenic precursors. These results provide evidence for the existence of rare human CD8(+)CD3(+)TCR(-) cells with engraftment facilitating properties, the adoptive transfer of which could improve the therapeutic outcome of stem-cell transplantation.