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Here we present a mouse model for T-cell targeting of hair follicles, linking the pathogenesis of alopecia to that of depigmentation disorders. Clinically, thymus transplantation has been successfully used to treat T-cell immunodeficiency in congenital athymia, but is associated with autoimmunity. We established a mouse model of thymus transplantation by subcutaneously implanting human thymus tissue into athymic C57BL/6 nude mice. These xenografts supported mouse T-cell development. Surprisingly, we did not detect multiorgan autoimmune disease. However, in all transplanted mice, we noted a striking depigmentation and loss of hair follicles. Transfer of T cells from transplanted nudes to syngeneic black-coated RAG(-/-) recipients caused progressive, persistent coat-hair whitening, which preceded patchy hair loss in depigmented areas. Further transfer experiments revealed that these phenomena could be induced by CD4+ T cells alone. Immunofluorescent analysis suggested that Trp2+ melanocyte-lineage cells were decreased in depigmented hair follicles, and pathogenic T cells upregulated activation markers when exposed to C57BL/6 melanocytes in vitro, suggesting that these T cells are not tolerant to self-melanocyte antigens. Our data raise interesting questions about the mechanisms underlying tissue-specific tolerance to skin antigens.

X-linked lymphoproliferative disease (XLP1) arises from mutations in the gene encoding SLAM-associated protein (SAP) and leads to abnormalities of NKT-cell development, NK-cell cytotoxicity, and T-dependent humoral function. Curative treatment is limited to allogeneic hematopoietic stem cell (HSC) transplantation. We tested whether HSC gene therapy could correct the multilineage defects seen in SAP(-/-) mice. SAP(-/-) murine HSCs were transduced with lentiviral vectors containing either SAP or reporter gene before transplantation into irradiated recipients. NKT-cell development was significantly higher and NK-cell cytotoxicity restored to wild-type levels in mice receiving the SAP vector in comparison to control mice. Baseline immunoglobulin levels were significantly increased and T-dependent humoral responses to NP-CGG, including germinal center formation, were restored in SAP-transduced mice.We demonstrate for the first time that HSC gene transfer corrects the cellular and humoral defects in SAP(-/-) mice providing proof of concept for gene therapy in XLP1.

In vitro exposure of haematopoietic stem and progenitor cells (HSPC) to cytokines in expansion or gene therapy protocols reduces homing and engraftment in vivo. We have previously reported that this is related in part to altered tissue specificity of short-term homing, leading to loss of cells in non-haematopoietic tissues. Here we demonstrate that defective engraftment persists when cultured HSPC are transplanted by intrabone injection. Changes in engraftment function occur within 24 h of cytokine exposure, and are evident when engraftment is analysed solely in the injected bone. A novel ex vivo model of the bone marrow was developed, in which the attachment of infused HSPC in rodent long bones is reduced following culture with cytokines. Finally, cultured HSPC demonstrated reduced adhesion to N-cadherin, osteopontin and vascular cell-adhesion molecule-1, ligands present in bone marrow niches. These changes in adhesive function occur rapidly, and are not related to downregulation of the relevant receptors. Our findings suggest that cytokine exposure of adult human HSPC results in altered adhesion within bone marrow niches, further leading to reduced engraftment potential in vivo.

X-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency caused by mutations in the CYBB gene encoding the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase catalytic subunit gp91(phox). A recent clinical trial for X-CGD using a spleen focus-forming virus (SFFV)-based γ-retroviral vector has demonstrated clear therapeutic benefits in several patients although complicated by enhancer-mediated mutagenesis and diminution of effectiveness over time due to silencing of the viral long terminal repeat (LTR). To improve safety and efficacy, we have designed a lentiviral vector that directs transgene expression primarily in myeloid cells. To this end, we created a synthetic chimeric promoter that contains binding sites for myeloid transcription factors CAAT box enhancer-binding family proteins (C/EBPs) and PU.1, which are highly expressed during granulocytic differentiation. As predicted, the chimeric promoter regulated higher reporter gene expression in myeloid than in nonmyeloid cells, and in human hematopoietic progenitors upon granulocytic differentiation. In a murine model of stem cell gene therapy for X-CGD, the chimeric vector resulted in high levels of gp91(phox) expression in committed myeloid cells and granulocytes, and restored normal NADPH-oxidase activity. These findings were recapitulated in human neutrophils derived from transduced X-CGD CD34(+) cells in vivo, and suggest that the chimeric promoter will have utility for gene therapy of myeloid lineage disorders such as CGD.

Graft versus Host Disease (GvHD) remains one of the main complications after hematopoietic stem cell transplantation (HSCT). Due to their ability to suppress effector cells, regulatory T cells (Tregs) have been proposed as a cellular therapy to prevent GvHD, however they also inhibit the functions of natural killer (NK) cells, key effectors of the Graft versus Leukemia effect. In this study, we have explored whether a Tregs therapy will also impact on NK cell differentiation. Using an in vitro model of hematopoietic stem cell (HSC) differentiation into NK cells, we found that activated Tregs led to a 90% reduction in NK cell numbers when added at the time of commitment to the NK cell lineage. This effect was contact dependent and was reversible upon Tregs depletion. The few NK cells that developed in these cultures were mature and exhibited normal functions. Furthermore, adoptive transfer of activated Tregs in rag-/- γc-/- mice abrogated HSC differentiation into NK cells thus confirming our in vitro findings. Collectively, these results demonstrate for the first time that activated Tregs can inhibit NK cell differentiation from HSC under specific conditions.

The pathophysiology of microthrombocytopenia in the Wiskott-Aldrich syndrome (WAS) and its milder form, X-linked thrombocytopenia (XLT), is unclear. Although quantitative defects are correctable by splenectomy, residual platelet abnormalities are suggestive of intrinsic disturbances of production. In contrast to human patients, murine models of WASp deficiency exhibit only mild thrombocytopenia, and platelets are of normal size. Here, we have identified a critical role for WASp during murine platelet biogenesis. By electron microscopy, WASp-deficient MKs appeared to have shed platelets ectopically within the bone marrow space. WASp-deficient megakaryocytes (MKs) also displayed defects in response to fibrillar collagen I (CI) in vitro, the major matrix component of bone. These included a loss of normal CI receptor (α2β1 integrin)-mediated inhibition of proplatelet formation, a marked abrogation of SDF-1-induced chemotactic migration of CD41+ MKs adherent to CI, and an almost complete lack of actin-rich podosomes, normally induced by interaction between CI and its receptors GPVI or α2β1 integrin. These findings highlight the central and highly specialized role of WASp in MKs during platelet biogenesis, and may provide a mechanism for the mild thrombocytopenia observed in WASp-deficient mice. In addition, they suggest a novel explanation for some of the platelet abnormalities characteristic of patients with WAS. (Blood. 2006;108:134-140)

Regulated migration and spatial localization of dendritic cells (DCs) are critical events during the initiation of physiologic immune responses and maintenance of tolerance. Here we have used cells deficient in the Wiskott-Aldrich syndrome protein (WASp) to demonstrate the importance of dynamic remodeling of the actin cytoskeleton for these trafficking processes to occur in vitro and in vivo. On fibronectin-coated surfaces, WASp-null immature murine DCs exhibited defects both of attachment and detachment, resulting in impaired net translocation compared with normal cells. The chemokinetic response to CCL21, which is critical for normal lymphatic trafficking, was also abrogated in the absence of WASp. In vivo in both fluorescein isothiocyanate (FITC) and oxazolone contact hypersensitivity models, WASp-null Langerhans cell (LC) migration was compromised, as judged by exit from the skin as well as by homing to the draining lymph node (LN). Furthermore, following systemic challenge with lipopolysaccharide (LPS) or toxoplasma-derived antigen, WASp-null DCs showed incomplete redistribution to T-cell areas in the spleen. Instead, they were retained ectopically in the marginal zone. DC trafficking in vivo is therefore dependent on a normally regulated actin cytoskeleton, which performs an essential function during maintenance of physiologic immunity and when disturbed may contribute significantly to the immunopathology of Wiskott-Aldrich Syndrome.

Unmatched allogeneic in utero stem cell transplantation (IUSCT) produces poor engraftment unless the fetus has congenital immunodeficiency, probably because of maternal and fetal immune responses to injected cells. We studied the functional hematopoietic potential of transduced green fluorescent protein (GFP+) sheep amniotic fluid (AF) stem cells, before and after autologous IUSCT. CD34+ cells were selected from first trimester sheep AF, transduced overnight, and injected intravenously into NOD-SCID-gamma (NSG) mice. At 3 months, primary recipient bone marrow (BM) was injected into secondary NSG recipients. GFP+ cells were detected in the hematopoietic organs and peripheral blood of primary and secondary recipients at 3 months. Autologous IUSCT (transduced GFP+CD34+AF) was performed in fetal sheep. Six months postnatally, lamb BM was injected into secondary NSG recipients. GFP+ cells were detected in the peripheral blood of primary and secondary recipients. This confirms the hematopoietic potential of AF stem cells supporting the concept of autologous IUSCT to treat congenital hematopoietic disease. Stem Cells 2015;33:122–132

We have investigated the minimal time required for efficient transduction of human hematopoietic repopulating cells using a surrogate nonobese diabetic (NOD)/severe combined immunodeficient (SCID) xenoengraftment assay. Cord blood CD34+ cells were transduced to high levels over 24-48 hr in the presence of Flt-3 ligand, stem cell factor, interleukin 3, and interleukin 6. Under these conditions, high levels of NOD/SCID repopulating activity were preserved, but the levels of gene marking in engrafting cell populations measured by expression of a reporter transgene were low. Extension of the transduction period by 24 hr (total culture period, 72 hr) under the same cytokine conditions resulted in high levels of gene marking, but on closer analysis expression was limited predominantly to the myeloid population. Efficient transduction of both lymphoid and myeloid lineages could be achieved only if the transduction protocol was extended by a further 24 hr (total culture period, 96 hr), suggesting that myeloid lineage-committed precursors are capable of repopulation, and that over shorter time periods transduction is largely restricted to this population. This adds to the emerging evidence of heterogeneity within the SRC compartment, and has important implications for the interpretation of this assay in stem cell transplantation and gene transfer studies.

Xenotransplantation of human cells into immunodeficient mice has been used to develop models of human haemopoiesis and lymphoid cell function. However, the utility of existing mouse strains can be limited by shortened life-spans, spontaneous production of functional lymphocytes with ageing, and residual innate immunity leading to variable levels of engraftment. Mice with a deletion of the common cytokine receptor gamma chain (gamma c) gene have reduced numbers of peripheral T and B lymphocytes, and absent natural killer cell (NK) activity. A genetic cross with a recombinase activating gene 2 (RAG2)-deficient strain produced mice doubly homozygous for the gamma c and RAG2 null alleles (gamma c-/RAG2-). These mice have a stable phenotype characterized by the absence of all T lymphocyte. B lymphocyte and NK cell function. Injection of human B-lymphoblastoid cells resulted in earlier fatal metastatic lymphoproliferative disease than in NOD/LtSz-scid controls. This was particularly evident in animals injected intravenously, possibly because of residual NK activity in NOD/LtSz-scid mice. Levels of engraftment with peripheral-blood-derived human lymphocytes were also increased and associated with higher CD4/CD8 ratios. These findings demonstrate that this new strain of immunodeficient mice has significant advantages over existing strains for engraftment of human cells, and may be useful for study of adoptive immunotherapy and novel therapies for GvHD and HIV infection.

International audience; The Wiskott-Aldrich syndrome protein is an essential cytoskeleton regulator found in cells of the hematopoietic lineage and controls the motility of leukocytes. The impact of WAS gene deficiency on the mobilization of hematopoietic progenitor/stem cells in circulation has remained unexplored but information would be pertinent in the context of autologous gene therapy of Wiskott-Aldrich syndrome. The response to granulocyte-colony stimulating factor mobilization was investigated in a murine WAS knock-out model of the disease, by measuring hematologic parameters, circulation and engraftment of hematopoietic progenitor/stem cells. In the steady-state, adult WAS knock-out mice have B-cell lymphopenia, marked neutrophilia, increased counts of circulating hematopoietic progenitor cells and splenomegaly, presumably caused by the retention of hematopoietic progenitor cells due to high levels of splenic CXCL12. In spite of these anomalies, the administration of granulocyte-colony-stimulating factor mobilizes progenitor/stem cells in WAS knock-out mice to the same level and with the same kinetics as in wild-type control mice. Mobilized peripheral blood cells from WAS knock-out mice can be transduced and are able to engraft into lethally-irradiated hosts reconstituting multiple lineages of cells and providing more effective radio-protection than mobilized cells from wild-type control mice. Surprisingly, the homing and the peripheral blood recovery of B lymphocytes was influenced by the background of the host. Thus, in the absence of Wiskott-Aldrich syndrome protein, effective mobilization is achieved but partial correction may occur as a result of an abnormal hematopoietic environment.

Megakaryocytes give rise to platelets via extension of proplatelet arms, which are released through the vascular sinusoids into the bloodstream. Megakaryocytes and their precursors undergo varying interactions with the extracellular environment in the bone marrow during their maturation and positioning in the vascular niche. We demonstrate that podosomes are abundant in primary murine megakaryocytes adherent on multiple extracellular matrix substrates, including native basement membrane. Megakaryocyte podosome lifetime and density, but not podosome size, are dependent on the type of matrix, with podosome lifetime dramatically increased on collagen fibers compared with fibrinogen. Podosome stability and dynamics depend on actin cytoskeletal dynamics but not matrix metalloproteases. However, podosomes degrade matrix and appear to be important for megakaryocytes to extend protrusions across a native basement membrane. We thus demonstrate for the first time a fundamental requirement for podosomes in megakaryocyte process extension across a basement membrane, and our results suggest that podosomes may have a role in proplatelet arm extension or penetration of basement membrane.

Introduction Human hematopoietic stem cell transplantation (HSCT) has been used to treat a range of hematological and immunological disorders. As a result, the demand for hematopoietic stem cells (HSC) in clinical applications is increasing. Amniotic fluid stem cells (AFSC) serve as a potential alternative cell source for therapy. Amniotic fluid can be derived by amniocentesis or therapeutic amniodrainage. AFSC are multi-potent, have low risk of tumorigenicity, can be expanded and do not have legal or ethical limitations. The significant hematopoietic activity of murine AFSC led us to explore the potential of human AFSC (CD117/c-Kit+) towards hematopoietic differentiation and to reconstitution in vivo. Methods Human AFSC (2nd and 3rd trimester) and cord blood HSC (CB-HSC; control) were selected for CD117 and CD34 respectively using a MoFlo XDP sorter. Sorted cells (104 in 200μl PBS) were injected intravenously into sub-lethally irradiated NOD-SCID/IL2rγnull (NSG) mice (n=6/group). Hematopoietic engraftment of human cells (% of human CD45+ within total CD45+) and multi-lineage reconstitution (myeloid: CD13, CD14, CD15 and lymphoid: CD3, CD4 and CD8) were assessed at 16 weeks in blood, bone marrow (BM) and spleen by flow cytometry. For subsequent secondary transplants, BM mononuclear cells (MNC) derived from BM harvested from primary recipients of mice were intravenously injected into secondary recipients (1.5x107 MNC in 200μl PBS). Hematopoietic engraftment was assessed at 16 weeks post-transplantation (n=6/group). For further analysis of human donor cell engraftment, Q-PCR was performed on spleen samples harvested from primary and secondary recipients using oligonucleotide primers specific for human ALU repeat sequences; Immunohistochemistry was carried out using anti-human CD45 antibody and detected with a commercially available kit (Dako EnVision Plus, Dako). Results are expressed as mean±SEM, and statistical analysis was performed using 1-way ANOVA with Bonferroni post-hoc tests. Results Human AFSC engrafted the hematopoietic system of NSG mice at levels similar to the ones achieved with CB-HSC (blood: AFSC 7.5±1.3% vs. CB-HSC 6.1±2.2%, p=0.6; BM: AFSC 46.3±7.9% vs. CB-HSC 38.3±8.2%, p=0.6; spleen: AFSC 39.6±9.3% vs. CB-HSC 34.7±10.5%, p=0.7). Similarly, at 16 weeks following secondary transplantation, human donor cell engraftment was comparable between groups in blood (AFSC 11.5 ± 3.9% vs. CB-HSC 16.9 ± 3.9%, p=0.3) and other hematopoietic tissues. Q-PCR and immunohistochemistry confirmed donor cell engraftment in AFSC and CB-HSC groups. Importantly, there were no differences between groups in multi-lineage differentiation at 16 weeks post primary and secondary transplantation. Conclusion Human CD117/c-Kit+ AFSC have functional, multi-lineage hematopoietic potential that is similar to the current "gold-standard" stem cell source for hematopoietic transplantation. The ease of isolation during early gestation, as well as their gene-engineering and expansion potential make human AFSC a novel autologous fetal cell source for pre- and post-natal therapy of inherited hematological disorders. Disclosures No relevant conflicts of interest to declare.

Rearrangement of the cytoskeleton in T cells plays a critical role in the organization of a complex signaling interface referred to as immunologic synapse (IS). Surprisingly, the contribution of antigen presenting cells, in particular dendritic cells (DCs), to the structure and function of the IS has not been investigated in as much detail. We have used a natural model of cytoskeletal dysfunction caused by deficiency of the Wiskott-Aldrich syndrome protein (WASp) to explore the contribution of the DC cytoskeleton to IS formation and to T-cell priming. In an antigen-specific system, T-DC contacts were found to be less stable when DCs alone lacked WASp, and associated with multiple defects of IS structure. As a consequence, DCs were unable to support normal IL-12 secretion, and events downstream of TCR signaling were abrogated, including increased calcium flux, microtubule organizing center (MTOC) polarization, phosphorylation of ZAP-70, and T-cell proliferation. Formation of an effective signaling interface is therefore dependent on active cytoskeletal rearrangements in DCs even when T cells are functionally competent. Deficiency of DC-mediated activities may contribute significantly to the varied immunodysregulation observed in patients with WAS, and also in those with limited myeloid reconstitution after allogeneic hematopoietic stem cell transplantation.

Lentiviral vectors (lentivectors) are effective for stimulation of cell-mediated and humoral immunity following subcutaneous and intramuscular immunization. However, lentivector genome integration carries a risk of perturbation of host gene expression. Here, we demonstrate that lentivectors with multiple mutations that prevent integration are also effective immunogens. First, systemic CD8 + T-cell responses to the model antigen ovalbumin were detected following subcutaneous injection of nonintegrating lentivectors. Transfer of transgenic OT1 T cells demonstrated that antigen presentation persisted for at least 30 days. Furthermore, an enhanced CD8 + T-cell response, peaking at 7 days, was stimulated by coexpression of p38 MAP kinase or an NF-κB activator from the same vector. Second, we demonstrated systemic CD8 + T-cell and antibody responses to the secreted hepatitis B virus (HBV) surface antigen expressed from a nonintegrating lentivector injected intramuscularly. The induction, specificity, and kinetics of antibody production closely mimicked those of natural HBV infection. In this case, both the vector genome and the immune response were maintained for at least 2 months. Together, our data indicate that nonintegrating lentivectors can be employed to generate effective vaccines.

Wiskott–Aldrich syndrome (WAS) is an X-linked hematological disease characterized by immunodeficiency, eczema, and thrombocytopaenia, and shows promise for treatment with hematopoietic stem cell gene therapy. The immunopathology of WAS is attributable at least in part to defects of cell migration and localization as a result of chemotactic, adhesive, and chemokinetic defects. Whereas previous studies using either gammaretroviral or lentiviral vectors have demonstrated variable correction of T-cell proliferation and dendritic cell (DC) cytoarchitecture, we have used a lentiviral vector expressing an eGFP–WASp fusion protein to test the potential for restoration of cell migratory defects. Multilineage expression of the fusion transgene was present for up to 10 months after primary engraftment, and also in secondary recipients analyzed after a further 9 months. Transduced bone marrow–derived dendritic cells (BMDCs) demonstrated recovery of podosome numbers and turnover, while B cells, BMDCs, and Langerhans cells (LCs) exhibited enhanced chemotactic responses to specific stimuli. As an indication of functionality in vivo, splenic marginal zone B cells and a cutaneous contact hypersensitivity (CHS) response to dinitrofluorobenzene (DNFB) were both partially restored. These proof of principle experiments demonstrate that WAS protein (WASp) transgene expression can be successfully maintained long term in primary and secondary recipients, and that it is associated with a significant repair of migratory defects.

The expression of genes specifically in B cells is of great interest in both experimental immunology as well as in future clinical gene therapy. We have constructed a novel enhanced B cell-specific promoter (Igk- E) consisting of an immunoglobulin kappa (Igk) minimal promoter combined with an intronic enhancer sequence and a 30 enhancer sequence from Ig genes. The Igk- E promoter was cloned into a lentiviral vector and used to control expression of enhanced green fluorescent protein (eGFP). Transduction of murine B-cell lymphoma cell lines and activated primary splenic B cells, with IgK-E-eGFP lentivirus, resulted in expression of eGFP, as analysed by flow cytometry, whereas expression in non-B cells was absent. The specificity of the promoter was further examined by transducing Lin bone marrow with Igk-E-eGFP lentivirus and reconstituting lethally irradiated mice. After 16 weeks flow cytometry of lymphoid tissues revealed eGFP expression by CD19(+) cells, but not by CD3(+), CD11b(+), CD11c(+) or Gr-1(+) cells. CD19(+) cells were comprised of both marginal zone B cells and recirculating follicular B cells. Activated human peripheral mononuclear cells were also transduced with Igk-E-eGFP lentivirus under conditions of selective B-cell activation. The Igk-E promoter was able to drive expression of eGFP only in CD19(+) cells, while eGFP was expressed by both spleen focus forming virus and cytomegalovirus constitutive promoters in CD19(+) and CD3(+) lymphocytes. These data demonstrate that in these conditions the Igk-E promoter is cell specific and controls efficient expression of a reporter protein in mouse and human B cells in the context of a lentiviral vector.

Severe congenital neutropenia (SCN) is characterized by neutropenia, recurrent bacterial infections, and maturation arrest in the bone marrow. Although many cases have mutations in the ELA2 gene encoding neutrophil elastase, a significant proportion remain undefined at a molecular level. A mutation (Leu270Pro) in the gene encoding the Wiskott-Aldrich syndrome protein (WASp) resulting in an X-linked SCN kindred has been reported. We therefore screened the WAS gene in 14 young SCN males with wild-type ELA2 and identified 2 with novel mutations, one who presented with myelodysplasia (Ile294Thr) and the other with classic SCN (Ser270Pro). Both patients had defects of immunologic function including a generalized reduction of lymphoid and natural killer cell numbers, reduced lymphocyte proliferation, and abrogated phagocyte activity. In vitro culture of bone marrow progenitors demonstrated a profound reduction in neutrophil production and increased levels of apoptosis, consistent with an intrinsic disturbance of normal myeloid differentiation as the cause of the neutropenia. Both mutations resulted in increased WASp activity and produced marked abnormalities of cytoskeletal structure and dynamics. Furthermore, these results also suggest a novel cause of myelodysplasia and that male children with myelodysplasia and disturbance of immunologic function should be screened for such mutations.

No defects related to deficiency of the Wiskott-Aldrich Syndrome protein (WASp) have been described in osteoclasts. Here we show that there are significant morphologic and functional abnormalities. WASp-null cells spread over a much larger surface area and are highly polykaryotic. In their migratory phase, normal cells assemble clusters of podosomes behind their leading edges, whereas during the bone resorptive phase multiple podosomes are densely aggregated in well-defined actin rings forming the sealing zone. In comparison, WASp-null osteoclasts in either phase are markedly depleted of podosomes. On bone surfaces, this results in a failure to form actin rings at sealing zones. Complementation of WASp-null osteoclasts with an enhanced green fluorescent protein (eGFP)-WASp fusion protein restores normal cytoarchitecture. These structural disturbances translate into abnormal patterns of bone resorption both in vitro on bone slices and in vivo. Although physiologic steady-state levels of bone resorption are maintained, a major impairment is observed when WASp-null animals are exposed to a resorptive challenge. Our results provide clear evidence that WASp is a critical component of podosomes in osteoclasts and indicate a nonredundant role for WASp in the dynamic organization of these actin structures during bone resorption. (Blood. 2004;103:3552-3561)

The reduced engraftment potential of hematopoietic stem/progenitor cells (HSPCs) after exposure to cytokines may be related to the impaired homing ability of actively cycling cells. We tested this hypothesis by quantifying the short-term homing of human adult CD34+ cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) animals. We show that the loss of engraftment ability of cytokine-activated CD34+ cells is associated with a reduction in homing of colony-forming cells (CFCs) to bone marrow (BM) at 24 hours after transplantation (from median 2.8% [range, 1.9%-6.1%] to 0.3% [0.0%-0.7%]; n = 3; P < .01), coincident with an increase in CFC accumulation in the lungs (P < .01). Impaired BM homing of cytokine-activated cells was not restored by using sorted cells in G0G1 or by inducing cell cycle arrest at the G1/S border. Blocking Fas ligation in vivo did not increase the BM homing of cultured cells. Finally, we tested cytokine combinations or culture conditions previously reported to restore the engraftment of cultured cells but did not find that any of these was able to reverse the changes in homing behavior of cytokine-exposed cells. We suggest that these changes in homing and, as a consequence, engraftment result from the increased migratory capacity of infused activated cells, leading to the loss of selectivity of the homing process.

Background Beta thalassaemia is a genetic blood disease that causes life-threatening anemia. Hematopoietic stem cell (HSC) transplantation successfully cures the disease but in only 30% of patients. We hypothesized that in utero gene therapy (IUGT) to the fetal HSC compartment with the corrected beta globin gene might cure the disease before birth. Methods A humanized mouse model of thalassemia (Cooley's anemia; CA) was used in which heterozygous animals are affected by anemia, splenomegaly and extra-medullary hematopoiesis. At E13.5 a “GLOBE” vector (HIV-2 based lentiviral vector that incorporates a mini hemoglobin beta gene, the beta-globin promoter and HS2/3 β-LCR element) was injected into the liver of each fetus (n=12). At 12 weeks of age, recipient blood, liver, spleen and bone marrow were collected for complete blood count, blood film, as well as RNA and DNA isolation. Extra-medullary hematopoiesis was examined in the spleen and liver using flow cytometry (CD71+/Ter119+ cells) and histo-pathological analysis. Gene expression of human and mouse alpha and beta globins, as well as human gamma globin was assessed by quantitative polymerase chain reaction (qPCR). High performance liquid chromatography (HPLC) was used to quantify the presence of human beta globin in the peripheral blood of treated animals. Results are expressed as mean±SEM, and statistical analysis was performed using 1-way ANOVA with Bonferroni post-hoc tests. Experimental protocols were approved by the ethical committee on animal experimentation at University College London. Results Compared to non-injected heterozygous pups (control), IUGT increased hemoglobin levels [11.3±0.4g/dl (n=6) vs. 7.6±0.6g/dl (n=8); p Conclusions IUGT resulted in phenotypic normalization in a heterozygous humanized mouse model of CA. Increased levels of beta globin and associated down-regulation of gamma globin is consistent with a switch from fetal to adult human hemoglobin, confirming successful prenatal correction of the genetic defect. Figure A. Quantification of human beta globin mRNA using qPCR (*p Figure B. HPLC analysis of peripheral blood hemolysates of control versus IUGT treated. Figure B. HPLC analysis of peripheral blood hemolysates of control versus IUGT treated. Disclosures No relevant conflicts of interest to declare.

Background EBV associated lymphoproliferative disease (PTLD) remains a major cause of morbidity and mortality after stem cell (SCT) or solid organ (SOT) transplant. Adoptive transfer of ex vivo-derived EBV-specific cytotoxic T lymphocytes (EBV-CTL) to reconstitute immunity to the oncogenic virus EBV has been highly effective for both the prophylaxis and treatment of PTLD after SCT, where immunosuppression could be withdrawn before transfer of EBV-CTLs. In contrast, the application of this approach for the treatment of PTLD in SOT patients, although feasible, has been challenging with restricted expansion, persistence and efficacy of the adoptive transferred T cells. This difference is likely to reflect the need for the on-going immunosuppression to prevent graft-rejection post SOT which inhibits the virus-specific T cell responses. Our group has previously genetically engineered EBV-CTL to enable them to function in vitro in the presence of the calcineurin inhibitor Tacrolimus (FK506) through retroviral transfer of a calcineurin mutant (CNA12). Aim To examine the ability of genetically engineered EBV-CTLs resistant to FK506 to control EBV+ B cell lymphoma progression in vivoin a xenogeneic mouse model in the presence of FK506. Methods NOD/SCID/IL2rgnull (NSG) mice were inoculated subcutaneously with 5×106 EBV-transformed lymphoblastoid B cell lines (LCL) labelled with F-Luc to develop human EBV+ lymphoma. To evaluate in vivo antitumor activity, 5×106 CTLs retrovirally transduced with CNA12 or eGFP control CTLs were injected intravenously after 7 days in the presence or absence of FK506 (10mg/kg/day). Tumour growth was analysed using the Xenogen-IVIS system. Results Adoptive transfer of autologous CNA12 transduced CTLs induced EBV+ lymphoma regression in the presence of FK506, as assessed both by IVIS and tumour size, whereas FK506 treated mice receiving eGFP control CTLs had tumour progression (P Conclusions Our results demonstrate that CNA12 modified EBV-CTL can induce regression of EBV-associated tumours in vivo in the face of on-going immunosuppression with FK506. Clinical application of this novel approach may enhance the efficacy of adoptive transfer of EBV-CTL in SOT patients developing PTLD without the need for reduction in immunosuppressive therapy. Disclosures: No relevant conflicts of interest to declare.

Background X-linked lymphoproliferative disease (XLP) arises from mutations in the gene encoding SAP, an intracellular adaptor protein expressed in T cells, natural killer (NK) cells, and natural killer T (NKT) cells. SAP deficiency causes abnormalities of NK cell cytotoxicity, NKT cell development, and T-cell-dependent humoral function. Clinical manifestations are characterised by haemophagocytic lymphohistiocytosis (HLH), lymphoma, and dysgammaglobulinaemia. Curative treatment is limited to allogeneic haemopoietic stem-cell transplantion (HSCT). Somatic gene therapy offers a potential cure in XLP. We have developed a lentiviral mediated gene therapy strategy to correct immune defects in XLP. Methods We designed a lentiviral vector incorporating codon-optimised human SAP cDNA and green fluorescent protein (GFP) driven by the elongation factor-1 short (EFS) promoter. Haemopoietic stem-cell progenitors from SAP-deficient mice were transduced with SAP-GFP (n=10) or GFP only (n=9) vectors before transplantation into irradiated SAP-deficient recipients. Animals were sacrificed 3 months later, 10 days post vaccination with NP-CGG to assess functional humoral reconstitution. Findings Both SAP and GFP expression was evident in bone marrow, thymus, spleen, and peripheral blood cells. NK cell cytotoxicity was restored to wild type levels in mice receiving the SAP-GFP vector, and NKT cell development was seen in SAP-GFP transduced mice at levels significantly higher than those seen in SAP-deficient or control mice. Baseline IgG, IgM, IgG1, and IgG3 levels were significantly increased and T-cell-dependent humoral responses were also restored, with SAP-GFP transduced mice having levels of NP-specific immunoglobulin that were significantly higher than SAP-deficient mice or controls. Interpretation We demonstrate correction of cellular and humoral defects in SAP-deficient mice through lentiviral-vector-mediated gene transfer into haemopoietic progenitor cells, providing proof of concept for gene therapy in XLP. Funding Wellcome Trust and Leukaemia Lymphoma Research.

Introduction Mouse amniotic fluid c-Kit(+)/Lin(-) stem (AFS) cells display hematopoietic potential. We explored the haematopoietic potential of sheep and human AFS cells after in utero stem cell transplantation. Methods Human AFS cells (hAFSC) were isolated from women undergoing 3rd trimester amniodrainage. Sheep AFS cells (sAFSC) were collected under ultrasound guidance (59.5±4.5days, term=145days), and isolated using a sheep-specific CD34 antibody. hAFSC were transplanted into the peritoneal cavity of fetal mice from CD1 mothers (14dpc, n=6). The peripheral blood of recipient mice was analysed 4 weeks postnatal for engraftment by flow-cytometry using anti-human beta2-microglobin antibody. Neonatal tissues collected at 6 weeks were analysed by PCR and immuno-staining for anti-human mitochondrial antibody; bone marrow (BM) was assayed for colony-forming cells. sAFSC were transduced overnight using a lentivirus vector (HIV-SFFV-eGFP, MOI=50) and injected either intravenously into NOD-SCID-gamma (NSG) mice (3x10∧5, N=4 per group) or by ultrasound-guided peritoneal injection back into donor sheep fetuses (n=7; 2x10∧4 sAFSC). Results hAFS cells were detectable in the peripheral blood, liver, spleen and BM of neonatal mice at 6 weeks postnatal; harvested BM generated colonies of human origin. GFP+ve cells were detected in the peripheral blood, spleen, liver, and BM of NSG mice 3 months after transplantation of transduced sAFSC. Five lambs injected with autologous transduced GFP+CD34+ cells survived to birth (71.4%); peripheral blood of all lambs contained GFP+ cells (1.9-3.8%) maintained at 6 months postnatal. GFP+ cells were detected in the liver and BM of lambs. Conclusion AFSC have haematopoietic potential and be useful for autologous transplantation.

Abstract 141 Short term cytokine exposure reduces the engraftment potential of haematopoietic stem and progenitor cells (HSPC). We have previously shown, in MPB CD34+ cells, that this occurs in conjunction with reduced short term homing to the BM of irradiated NOD/SCID animals, and is evident by 4 hours of culture (Ahmed at al, Blood 2004; 103:2079). Homing and engraftment of HSPC is dependent on the SDF-1/CXCR4 axis, that is negatively regulated by CD26, a cell-surface peptidase that cleaves SDF-1. Thus, blockade of CD26 with diprotin A improves engraftment of cord blood HSPC. CD26 levels are low on MPB CD34+ cells, but increase on cytokine exposure, an effect that may account for reduced homing function. We tested the effect of diprotin A treatment on the homing and engraftment of MPB CD34+ progenitors in irradiated NOD/SCID mice. Cytokine exposure (SCF, FL, IL3, IL6 for 48–72 hrs) reduced BM homing of progenitors (to 32±7% of uncultured cells, p=0.031). Treatment with Diprotin A significantly improved the homing of cultured progenitors (p=0.0002 cf non-treated cells), to the levels seen in uncultured cells (NS cf homing of uncultured cells with or without Diprotin A). Despite this increase in levels of homing to the BM, long term engraftment of cultured progenitors is not rescued by CD26 blockade, suggesting a defect beyond the initial step of homing to BM. To assess the BM attachment of transplanted cells, we used the intrabone (IB) assay, injecting cells directly into the tibiae of irradiated recipients thus bypassing the need for homing from the systemic circulation. In comparison to intravenously (IV) injected cells, IB delivery improved engraftment of both cultured and uncultured MPB CD34+ cells. At a cell dose of 106, median engraftment of uncultured cells was 13.27% (range 2.43–49.1, n=5) by IB injection compared with 1.5% (0.1–4.6, n=6) by IV delivery (p To study this attachment defect directly, we developed an ex-vivo model where CD34+ cells are incubated in long bones of irradiated, 3-week old Sprague Dawley rats after resident HSPC are removed by vigorous flushing. Irradiation, flushing protocols and cell doses were optimized to ensure sensitivity and reproducibility of the assay. Progenitor adherence was assessed by colony assays on infused and recovered attached cells, correcting for animal weight. In this model, cultured progenitors displayed reduced attachment: median number of attached progenitors, 182.1 (23–384, n=26), compared with 385.4 (49–1124, n=30) for uncultured cells (p Next, we tested the effect of cytokine exposure on ligand-specific adhesion of MPB progenitors. CD34+ cells were incubated on immobilized ligands and progenitor adhesion assessed from the clonogenic output of non-adherent cells. Progenitor adhesion to several putative niche ligands was significantly reduced following cytokine exposure. Specific adhesion of uncultured progenitors to N-cadherin was 31.8±2%, compared to 19.7±3.2% for cultured progenitors (p=0.0058). Cytokine culture also reduced specific adhesion to osteopontin and VCAM-1 (p=0.0025 and p=0.0164 respectively), but not to fibronectin, suggesting that reduced adhesive function of cultured cells is not a global defect. We conclude that whilst short term homing of cultured MPB CD34+ cells to the BM can be improved by CD26 blockade, the resulting long term engraftment defect remains. This defect is at least partly related to altered adhesive interactions of cultured cells to ligands within the BM. Disclosures: No relevant conflicts of interest to declare.

To the Editor: In a recent issue of Blood, Guenechea et al[1][1]reported that ex vivo cytokine-expanded CD34+ cord blood cells retained nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating activity of fresh cells, although there was a significant delay in the time to achieve

The WAS gene is mutated in Wiskott-Aldrich syndrome (WAS) and in X-linked thrombopenia (XLT). These diseases associate platelet defects with variable immune dysfunction, as a result of abnormal signalling and impaired cytoskeletal regulation in hematopoeitic cells. Conceivably, severe forms of WAS could be treated by gene therapy because retroviral or lentiviral-mediated WAS gene transfer restores protein expression and function in several WAS protein-deficient models. The purpose of the present study was to improve the safety and efficacy of such lentiviral vectors. Endogenous WAS promoter elements were used to restrict transgene expression to the target cell population and to provide the possibility of regulated expression in these cells. Sequences 0.5 or 1.6 kb upstream of transcription start, were operational in the transfer vector and restricted transgene expression to hematopoietic cells. Vectors utilizing either one of the WAS promoter sequences or the ubiquitously-active PGK-1, SFFV or EF1-a promoters, were compared. Equivalent levels of WAS protein were induced in lymphocytes and dendritic cells (DC), although slightly inferior mRNA levels were obtained in B cells using WAS promoters. At the functional level, all vectors restored a similar degree of proliferation and IL-2 production in T cells and equivalent numbers of podosome cytoskeletal structures in DC. The 0.5 kb or 1.6 kb-long WAS promoter sequences functioned similarly in WAS B lymphocytes or in a model of WASP-deficient T cells generated by RNA interference. No toxicity was induced by over-expression of these vectors in CD34+ cells. Altogether, the data show that lentiviral vectors with WAS promoters function efficiently in several lineages of patient cells and support further development of a hematopoietic-restricted approach for the gene therapy of WAS.

Summary. Xenotransplantation of human cells into immunodeficient mice has been used to develop models of human haemopoiesis and lymphoid cell function. However, the utility of existing mouse strains can be limited by shortened lifespans, spontaneous production of functional lymphocytes with ageing, and residual innate immunity leading to variable levels of engraftment. Mice with a deletion of the common cytokine receptor g chain (gc) gene have reduced numbers of peripheral T and B lymphocytes, and absent natural killer cell (NK) activity. A genetic cross with a recombinase activating gene 2 (RAG2)-deficient strain produced mice doubly homozygous for the gc and RAG2 null alleles (gc π /RAG2 π ). These mice have a stable phenotype characterized by the absence of all T lymphocyte, B lymphocyte and NK cell function. Injection of human B-lymphoblastoid cells resulted in earlier fatal metastatic lymphoproliferative disease than in NOD/LtSz-scid controls. This was particularly evident in animals injected intravenously, possibly because of residual NK activity in NOD/ LtSz-scid mice. Levels of engraftment with peripheral-bloodderived human lymphocytes were also increased and associated with higher CD4/CD8 ratios. These findings demonstrate that this new strain of immunodeficient mice has significant advantages over existing strains for engraftment of human cells, and may be useful for study of adoptive immunotherapy and novel therapies for GvHD and HIV infection.