Lentiviral-mediated Genetic Correction of Hematopoietic and Mesenchymal Progenitor Cells From Fanconi Anemia Patients

Previous clinical trials based on the genetic correction of purified CD34⁺ cells with γ-retroviral vectors have demonstrated clinical efficacy in different monogenic diseases, including X-linked severe combined immunodeficiency, adenosine deaminase deficient severe combined immunodeficiency and chronic granulomatous disease. Similar protocols, however, failed to engraft Fanconi anemia (FA) patients with genetically corrected cells. In this study, we first aimed to correlate the hematological status of 27 FA patients with CD34⁺ cell values determined in their bone marrow (BM). Strikingly, no correlation between these parameters was observed, although good correlations were obtained when numbers of colony-forming cells (CFCs) were considered. Based on these results, and because purified FA CD34⁺ cells might have suboptimal repopulating properties, we investigated the possibility of genetically correcting unselected BM samples from FA patients. Our data show that the lentiviral transduction of unselected FA BM cells mediates an efficient phenotypic correction of hematopoietic progenitor cells and also of CD34⁻ mesenchymal stromal cells (MSCs), with a reported role in hematopoietic engraftment. Our results suggest that gene therapy protocols appropriate for the treatment of different monogenic diseases may not be adequate for stem cell diseases like FA. We propose a new approach for the gene therapy of FA based on the rapid transduction of unselected hematopoietic grafts with lentiviral vectors (LVs).     

The Chemokine SDF-1 Is a Chemoattractant for Human CD34+ Hematopoietic Progenitor Cells and Provides a New Mechanism to Explain the Mobilization of CD34+ Progenitors to Peripheral Blood

Hematopoietic progenitor cells migrate in vitro and in vivo towards a gradient of the chemotactic factor stromal cell-derived factor-1 (SDF-1) produced by stromal cells. This is the first chemoattractant reported for human CD34⁺ progenitor cells. Concentrations of SDF-1 that elicit chemotaxis also induce a transient elevation of cytoplasmic calcium in CD34⁺ cells. SDF-1-induced chemotaxis is inhibited by pertussis toxin, suggesting that its signaling in CD34⁺ cells is mediated by seven transmembrane receptors coupled to G_i proteins. CD34⁺ cells migrating to SDF-1 include cells with a more primitive (CD34⁺/CD38⁻ or CD34⁺/DR⁻) phenotype as well as CD34⁺ cells phenotypically committed to the erythroid, lymphoid and myeloid lineages, including functional BFU-E, CFU-GM, and CFU-MIX progenitors. Chemotaxis of CD34⁺ cells in response to SDF-1 is increased by IL-3 in vitro and is lower in CD34⁺ progenitors from peripheral blood than in CD34⁺ progenitors from bone marrow, suggesting that an altered response to SDF-1 may be associated with CD34 progenitor mobilization.     

Expression of Stem Cell Markers in the Human Fetal Kidney

In the human fetal kidney (HFK) self-renewing stem cells residing in the metanephric mesenchyme (MM)/blastema are induced to form all cell types of the nephron till 34^th week of gestation. Definition of useful markers is crucial for the identification of HFK stem cells. Because wilms' tumor, a pediatric renal cancer, initiates from retention of renal stem cells, we hypothesized that surface antigens previously up-regulated in microarrays of both HFK and blastema-enriched stem-like wilms' tumor xenografts (NCAM, ACVRIIB, DLK1/PREF, GPR39, FZD7, FZD2, NTRK2) are likely to be relevant markers. Comprehensive profiling of these putative and of additional stem cell markers (CD34, CD133, c-Kit, CD90, CD105, CD24) in mid-gestation HFK was performed using immunostaining and FACS in conjunction with EpCAM, an epithelial surface marker that is absent from the MM and increases along nephron differentiation and hence can be separated into negative, dim or bright fractions. No marker was specifically localized to the MM. Nevertheless, FZD7 and NTRK2 were preferentially localized to the MM and emerging tubules (<10% of HFK cells) and were mostly present within the EpCAM^neg and EpCAM^dim fractions, indicating putative stem/progenitor markers. In contrast, single markers such as CD24 and CD133 as well as double-positive CD24⁺CD133⁺ cells comprise >50% of HFK cells and predominantly co-express EpCAM^bright, indicating they are mostly markers of differentiation. Furthermore, localization of NCAM exclusively in the MM and in its nephron progenitor derivatives but also in stroma and the expression pattern of significantly elevated renal stem/progenitor genes Six2, Wt1, Cited1, and Sall1 in NCAM⁺EpCAM^- and to a lesser extent in NCAM⁺EpCAM⁺ fractions confirmed regional identity of cells and assisted us in pinpointing the presence of subpopulations that are putative MM-derived progenitor cells (NCAM⁺EpCAM⁺FZD7⁺), MM stem cells (NCAM⁺EpCAM^-FZD7⁺) or both (NCAM⁺FZD7⁺). These results and concepts provide a framework for developing cell selection strategies for human renal cell-based therapies.     

Preclinical Safety and Efficacy of Human CD34+ Cells Transduced With Lentiviral Vector for the Treatment of Wiskott-Aldrich Syndrome

Gene therapy with ex vivo-transduced hematopoietic stem/progenitor cells may represent a valid therapeutic option for monogenic immunohematological disorders such as Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency associated with thrombocytopenia. We evaluated the preclinical safety and efficacy of human CD34⁺ cells transduced with lentiviral vectors (LV) encoding WAS protein (WASp). We first set up and validated a transduction protocol for CD34⁺ cells derived from bone marrow (BM) or mobilized peripheral blood (MPB) using a clinical grade, highly purified LV. Robust transduction of progenitor cells was obtained in normal donors and WAS patients'' cells, without evidence of toxicity. To study biodistribution of human cells and exclude vector release in vivo, LV-transduced CD34⁺ cells were transplanted in immunodeficient mice, showing a normal engraftment and differentiation ability towards transduced lymphoid and myeloid cells in hematopoietic tissues. Vector mobilization to host cells and transmission to germline cells of the LV were excluded by different molecular assays. Analysis of vector integrations showed polyclonal integration patterns in vitro and in human engrafted cells in vivo. In summary, this work establishes the preclinical safety and efficacy of human CD34⁺ cells gene therapy for the treatment of WAS.     

Quantitative Analysis Reveals Expansion of Human Hematopoietic Repopulating Cells After Short-term Ex Vivo Culture

Ex vivo culture of human hematopoietic cells is a crucial component of many therapeutic applications. Although current culture conditions have been optimized using quantitative in vitro progenitor assays, knowledge of the conditions that permit maintenance of primitive human repopulating cells is lacking. We report that primitive human cells capable of repopulating nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice (SCID-repopulating cells; SRC) can be maintained and/or modestly increased after culture of CD34⁺CD38⁻ cord blood cells in serum-free conditions. Quantitative analysis demonstrated a 4- and 10-fold increase in the number of CD34⁺CD38⁻ cells and colony-forming cells, respectively, as well as a 2- to 4-fold increase in SRC after 4 d of culture. However, after 9 d of culture, all SRC were lost, despite further increases in total cells, CFC content, and CD34⁺ cells. These studies indicate that caution must be exercised in extending the duration of ex vivo cultures used for transplantation, and demonstrate the importance of the SRC assay in the development of culture conditions that support primitive cells.     

Vascular niche promotes hematopoietic multipotent progenitor formation from pluripotent stem cells

Pluripotent stem cells (PSCs) represent an alternative hematopoietic stem cell (HSC) source for treating hematopoietic disease. The limited engraftment of human PSC–derived (hPSC-derived) multipotent progenitor cells (MPP) has hampered the clinical application of these cells and suggests that MPP require additional cues for definitive hematopoiesis. We hypothesized that the presence of a vascular niche that produces Notch ligands jagged-1 (JAG1) and delta-like ligand-4 (DLL4) drives definitive hematopoiesis. We differentiated hes2 human embryonic stem cells (hESC) and Macaca nemestrina–induced PSC (iPSC) line-7 with cytokines in the presence or absence of endothelial cells (ECs) that express JAG1 and DLL4. Cells cocultured with ECs generated substantially more CD34⁺CD45⁺ hematopoietic progenitors compared with cells cocultured without ECs or with ECs lacking JAG1 or DLL4. EC-induced cells exhibited Notch activation and expressed HSC-specific Notch targets RUNX1 and GATA2. EC-induced PSC-MPP engrafted at a markedly higher level in NOD/SCID/IL-2 receptor γ chain–null (NSG) mice compared with cytokine-induced cells, and low-dose chemotherapy-based selection further increased engraftment. Long-term engraftment and the myeloid-to-lymphoid ratio achieved with vascular niche induction were similar to levels achieved for cord blood–derived MPP and up to 20-fold higher than those achieved with hPSC-derived MPP engraftment. Our findings indicate that endothelial Notch ligands promote PSC-definitive hematopoiesis and production of long-term engrafting CD34⁺ cells, suggesting these ligands are critical for HSC emergence.     

Transduction of Human Primitive Repopulating Hematopoietic Cells With Lentiviral Vectors Pseudotyped With Various Envelope Proteins

Lentiviral vectors are useful for transducing primitive hematopoietic cells. We examined four envelope proteins for their ability to mediate lentiviral transduction of mobilized human CD34⁺ peripheral blood cells. Lentiviral particles encoding green fluorescent protein (GFP) were pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G), the amphotropic (AMPHO) murine leukemia virus envelope protein, the endogenous feline leukemia viral envelope protein or the feline leukemia virus type C envelope protein. Because the relative amount of genome RNA per ml was similar for each pseudotype, we transduced CD34⁺ cells with a fixed volume of each vector preparation. Following an overnight transduction, CD34⁺ cells were transplanted into immunodeficient mice which were sacrificed 12 weeks later. The average percentages of engrafted human CD45⁺ cells in total bone marrow were comparable to that of the control, mock-transduced group (37–45%). Lenti-particles pseudotyped with the VSV-G envelope protein transduced engrafting cells two- to tenfold better than particles pseudotyped with any of the γ-retroviral envelope proteins. There was no correlation between receptor mRNA levels for the γ-retroviral vectors and transduction efficiency of primitive hematopoietic cells. These results support the use of the VSV-G envelope protein for the development of lentiviral producer cell lines for manufacture of clinical-grade vector.     

T cell-independent development and induction of somatic hypermutation in human IgM+ IgD+ CD27+ B cells

IgM⁺IgD⁺CD27⁺ B cells from peripheral blood have been described as circulating marginal zone B cells. It is still unknown when and where these cells develop. These IgM⁺IgD⁺CD27⁺ B cells exhibit somatic hypermutations (SHMs) in their B cell receptors, but the exact nature of the signals leading to induction of these SHMs remains elusive. Here, we show that IgM⁺IgD⁺CD27⁺ B cells carrying SHMs are observed during human fetal development. To examine the role of T cells in human IgM⁺IgD⁺CD27⁺ B cell development we used an in vivo model in which Rag2^−/−γ_C^−/− mice were repopulated with human hematopoietic stem cells. Using Rag2^−/−γ_C^−/− mice on a Nude background, we demonstrated that development and induction of SHMs of human IgM⁺IgD⁺CD27⁺ B cells can occur in a T cell–independent manner.     

PDGFRα and CD51 mark human Nestin+ sphere-forming mesenchymal stem cells capable of hematopoietic progenitor cell expansion

The intermediate filament protein Nestin labels populations of stem/progenitor cells, including self-renewing mesenchymal stem cells (MSCs), a major constituent of the hematopoietic stem cell (HSC) niche. However, the intracellular location of Nestin prevents its use for prospective live cell isolation. Hence it is important to find surface markers specific for Nestin⁺ cells. In this study, we show that the expression of PDGFRα and CD51 among CD45⁻ Ter119⁻ CD31⁻ mouse bone marrow (BM) stromal cells characterizes a large fraction of Nestin⁺ cells, containing most fibroblastic CFUs, mesenspheres, and self-renewal capacity after transplantation. The PDGFRα⁺ CD51⁺ subset of Nestin⁺ cells is also enriched in major HSC maintenance genes, supporting the notion that niche activity co-segregates with MSC activity. Furthermore, we show that PDGFRα⁺ CD51⁺ cells in the human fetal BM represent a small subset of CD146⁺ cells expressing Nestin and enriched for MSC and HSC niche activities. Importantly, cultured human PDGFRα⁺ CD51⁺ nonadherent mesenspheres can significantly expand multipotent hematopoietic progenitors able to engraft immunodeficient mice. These results thus indicate that the HSC niche is conserved between the murine and human species and suggest that highly purified nonadherent cultures of niche cells may represent a useful novel technology to culture human hematopoietic stem and progenitor cells.Hematopoietic stem cells (HSCs) continuously replenish all blood cell lineages throughout their lifetime. Incipient hematopoiesis is first detected extraembryonically in the yolk sac and later in the aorta–gonad–mesonephros region, from where it moves transiently to the placenta and liver before being stabilized in the fetal BM (Wang and Wagers, 2011). In the adult stage, HSCs reside in a highly complex and dynamic microenvironment of the BM commonly referred to as the HSC niche (Schofield, 1978). The interactions between the niche constituents and HSCs ensure hematopoietic homeostasis by regulating HSC self-renewal, differentiation, and migration and by integrating neural and hormonal signals from the periphery (Méndez-Ferrer et al., 2009, 2010; Mercier et al., 2012). However, HSC maintenance and expansion ex vivo still remains challenging mainly because of our limited knowledge on the in vivo HSC niche constituents and the factors that drive HSC self-renewal.Although the cellular constituents of the HSC niche and their role are still poorly understood, in the last decade, several putative cellular components of the murine HSC niche have been proposed, including osteoblastic, endothelial, adipocytic, and perivascular cells (Calvi et al., 2003; Zhang et al., 2003; Arai et al., 2004; Kiel et al., 2005; Sugiyama et al., 2006; Chan et al., 2009; Naveiras et al., 2009; Méndez-Ferrer et al., 2010; Ding et al., 2012). Multipotent BM mesenchymal stem cells (MSCs) have long been suggested to also provide regulatory signals to hematopoietic progenitors, as mixed cultures derived from the adherent fraction of the BM stroma promote the maintenance of HSCs in vitro (Dexter et al., 1977). Although numerous studies explored the ability of mesenchymal stromal cultures to support the ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs), currently these systems are still insufficient to preserve primitive HSCs with long-term multilineage engraftment capacity (Chou et al., 2010; Broxmeyer, 2011). This limitation may in part be associated with the heterogeneous composition of mesenchymal stromal cell cultures. The prospective identification and functional characterization of purified naive populations of mouse and/or human BM stromal MSCs have been mired by the absence of specific cell surface markers allowing prospective isolation. Several MSC-associated antigens have been proposed (such as CD31⁻ CD34⁻ CD45⁻ CD105⁺ CD90⁺ CD73⁺) in cultured cells (Dominici et al., 2006). Nevertheless, these markers are not homogeneously expressed across cultures, varying with isolation protocols and passage and therefore not necessarily representative of MSCs in vivo (Bianco et al., 2013; Frenette et al., 2013). Very few MSC-associated antigens have been validated using rigorous transplantation assays (Sacchetti et al., 2007; Méndez-Ferrer et al., 2010). In the mouse BM, the expression of the intermediate filament protein Nestin characterizes a rare population of multipotent MSCs in close contact with the vasculature and HSCs. Nestin⁺ stromal cells contain all of the fibroblastic CFU (CFU-F) activity within the mouse BM and the exclusive capacity to form clonal nonadherent spheres in culture. The selective ablation of mouse Nestin⁺ cells (Méndez-Ferrer et al., 2010) or CXCL12-abundant reticular (CAR) cells (Omatsu et al., 2010) led to significant alterations in the BM HSC and progenitor maintenance. Serial transplantation analyses revealed that Nestin⁺ cells are able to self-renew and generate hematopoietic activity in heterotopic bone ossicle assays (Méndez-Ferrer et al., 2010). This potential was also associated with a CD45⁻ Tie2⁻ αV⁺ CD105⁺ CD90⁻ subset from the fetal mouse bone (Chan et al., 2009). In the adult mouse BM, PDGFRα⁺ Sca1⁺ CD45⁻ Ter119⁻ cells were also shown capable to give rise to osteoblasts, reticular cells, and adipocytes in vivo upon transplantation into irradiated mice (Morikawa et al., 2009). However, human BM MSCs are still retrospectively isolated based on plastic adherence (Friedenstein et al., 1970; Pittenger et al., 1999). Human CD45⁻ CD146⁺ self-renewing osteoprogenitors isolated from stromal cultures containing all the human BM CFU-F activity were shown capable of generating a heterotopic BM niche in an s.c. transplantation model (Sacchetti et al., 2007). However, a recent study showed that human CD45⁻ CD271⁺ CD146^−/low BM cells also possess these capacities (Tormin et al., 2011).Because Nestin is an intracellular protein, its identification in nontransgenic mice requires cell permeabilization, which precludes prospective isolation of live cells. In this study, we have evaluated putative cell surface MSC markers to identify a stromal population equivalent to Nestin⁺ cells in the mouse and human BM. Our results show that the combination of PDGFRα and CD51 identify a large subset of perivascular Nestin⁺ cells that is highly enriched in MSC and HSC niche activities in both species. Furthermore, we show that PDGFRα⁺ CD51⁺ stromal cells isolated from human BM can also form self-renewing clonal mesenspheres capable of transferring hematopoietic niche activity in vivo and support the ex vivo maintenance and expansion of human HSPCs in a dose-dependent manner.     

Identification of a Novel Developmental Stage Marking Lineage Commitment of Progenitor Thymocytes

Bipotent progenitors for T and natural killer (NK) lymphocytes are thought to exist among early precursor thymocytes. The identification and functional properties of such a progenitor population remain undefined. We report the identification of a novel developmental stage during fetal thymic ontogeny that delineates a population of T/NK-committed progenitors (NK1.1⁺/CD117⁺/CD44⁺/CD25⁻). Thymocytes at this stage in development are phenotypically and functionally distinguishable from the pool of multipotent lymphoid-restricted (B, T, and NK) precursor thymocytes. Exposure of multipotent precursor thymocytes or fetal liver– derived hematopoietic progenitors to thymic stroma induces differentiation to the bipotent developmental stage. Continued exposure to a thymic microenvironment results in predominant commitment to the T cell lineage, whereas coculture with a bone marrow–derived stromal cell line results in the generation of mature NK cells. Thus, the restriction point to T and NK lymphocyte destinies from a multipotent progenitor stage is marked by a thymus-induced differentiation step.     

TGF-β signaling in myeloproliferative neoplasms contributes to myelofibrosis without disrupting the hematopoietic niche

Myeloproliferative neoplasms (MPNs) are associated with significant alterations in the bone marrow microenvironment that include decreased expression of key niche factors and myelofibrosis. Here, we explored the contribution of TGF-β to these alterations by abrogating TGF-β signaling in bone marrow mesenchymal stromal cells. Loss of TGF-β signaling in Osx-Cre–targeted MSCs prevented the development of myelofibrosis in both MPL^W515L and Jak2^V617F models of MPNs. In contrast, despite the absence of myelofibrosis, loss of TGF-β signaling in mesenchymal stromal cells did not rescue the defective hematopoietic niche induced by MPL^W515L, as evidenced by decreased bone marrow cellularity, hematopoietic stem/progenitor cell number, and Cxcl12 and Kitlg expression, and the presence of splenic extramedullary hematopoiesis. Induction of myelofibrosis by MPL^W515L was intact in Osx-Cre Smad4^fl/fl recipients, demonstrating that SMAD4-independent TGF-β signaling mediates the myelofibrosis phenotype. Indeed, treatment with a c-Jun N-terminal kinase (JNK) inhibitor prevented the development of myelofibrosis induced by MPL^W515L. Together, these data show that JNK-dependent TGF-β signaling in mesenchymal stromal cells is responsible for the development of myelofibrosis but not hematopoietic niche disruption in MPNs, suggesting that the signals that regulate niche gene expression in bone marrow mesenchymal stromal cells are distinct from those that induce a fibrogenic program.     

Human interleukin 17-producing cells originate from a CD161+CD4+ T cell precursor

We demonstrate that CD161 is a highly up-regulated gene in human interleukin (IL) 17 T helper cell (Th17) clones and that all IL-17–producing cells are contained in the CD161⁺ fraction of CD4⁺ T cells present in the circulation or in inflamed tissues, although they are not CD1-restricted natural killer T cells. More importantly, we show that all IL-17–producing cells originate from CD161⁺ naive CD4⁺ T cells of umbilical cord blood, as well as of the postnatal thymus, in response to the combined activity of IL-1β and IL-23. These findings implicate CD161 as a novel surface marker for human Th17 cells and demonstrate the exclusive origin of these cells from a CD161⁺CD4⁺ T cell progenitor.     

SCL/TAL1 Regulates Hematopoietic Specification From Human Embryonic Stem Cells

Determining the molecular regulators/pathways responsible for the specification of human embryonic stem cells (hESCs) into hematopoietic precursors has far-reaching implications for potential cell therapies and disease modeling. Mouse models lacking SCL/TAL1 (stem cell leukemia/T-cell acute lymphocytic leukemia 1) do not survive beyond early embryogenesis because of complete absence of hematopoiesis, indicating that SCL is a master early hematopoietic regulator. SCL is commonly found rearranged in human leukemias. However, there is barely information on the role of SCL on human embryonic hematopoietic development. Differentiation and sorting assays show that endogenous SCL expression parallels hematopoietic specification of hESCs and that SCL is specifically expressed in hematoendothelial progenitors (CD45⁻CD31⁺CD34⁺) and, to a lesser extent, on CD45⁺ hematopoietic cells. Enforced expression of SCL in hESCs accelerates the emergence of hematoendothelial progenitors and robustly promotes subsequent differentiation into primitive (CD34⁺CD45⁺) and total (CD45⁺) blood cells with higher clonogenic potential. Short-hairpin RNA–based silencing of endogenous SCL abrogates hematopoietic specification of hESCs, confirming the early hematopoiesis-promoting effect of SCL. Unfortunately, SCL expression on its own is not sufficient to confer in vivo engraftment to hESC-derived hematopoietic cells, suggesting that additional yet undefined master regulators are required to orchestrate the stepwise hematopoietic developmental process leading to the generation of definitive in vivo functional hematopoiesis from hESCs.     

Allelic Exclusion and Peripheral Reconstitution by TCR Transgenic T Cells Arising From Transduced Human Hematopoietic Stem/Progenitor Cells

Transduction and transplantation of human hematopoietic stem/progenitor cells (HSPC) with the genes for a T-cell receptor (TCR) that recognizes a tumor-associated antigen may lead to sustained long-term production of T cells expressing the TCR and confer specific antitumor activity. We evaluated this using a lentiviral vector (CCLc-MND-F5) carrying cDNA for a human TCR specific for an HLA-A*0201-restricted peptide of Melanoma Antigen Recognized by T cells (MART-1). CD34⁺ HSPC were transduced with the F5 TCR lentiviral vector or mock transduced and transplanted into neonatal NSG mice or NSG mice transgenic for human HLA-A*0201 (NSG-A2). Human CD8⁺ and CD4⁺ T cells expressing the human F5 TCR were present in the thymus, spleen, and peripheral blood after 4–5 months. Expression of human HLA-A*0201 in NSG-A2 recipient mice led to significantly increased numbers of human CD8⁺ and CD4⁺ T cells expressing the F5 TCR, compared with control NSG recipients. Transduction of the human CD34⁺ HSPC by the F5 TCR transgene caused a high degree of allelic exclusion, potently suppressing rearrangement of endogenous human TCR-β genes during thymopoiesis. In summary, we demonstrated the feasibility of engineering human HSPC to express a tumor-specific TCR to serve as a long-term source of tumor-targeted mature T cells for immunotherapy of melanoma.     

Toward targeting B cell cancers with CD4+ CTLs: identification of a CD19-encoded minor histocompatibility antigen using a novel genome-wide analysis

Some minor histocompatibility antigens (mHags) are expressed exclusively on patient hematopoietic and malignant cells, and this unique set of antigens enables specific targeting of hematological malignancies after human histocompatability leucocyte antigen (HLA)–matched allogeneic stem cell transplantation (allo-SCT). We report the first hematopoietic mHag presented by HLA class II (HLA-DQA1*05/B1*02) molecules to CD4⁺ T cells. This antigen is encoded by a single-nucleotide polymorphism (SNP) in the B cell lineage-specific CD19 gene, which is an important target antigen for immunotherapy of most B cell malignancies. The CD19^L-encoded antigen was identified using a novel and powerful genetic strategy in which zygosity-genotype correlation scanning was used as the key step for fine mapping the genetic locus defined by pairwise linkage analysis. This strategy was also applicable for genome-wide identification of a wide range of mHags. CD19^L-specific CD4⁺ T cells provided antigen-specific help for maturation of dendritic cells and for expansion of CD8⁺ mHag-specific T cells. They also lysed CD19^L-positive malignant cells, illustrating the potential therapeutic advantages of targeting this novel CD19^L-derived HLA class II–restricted mHag. The currently available immunotherapy strategies enable the exploitation of these therapeutic effects within and beyond allo-SCT settings.Leukemia, lymphoma, and myeloma together account for ∼500,000 deaths per year worldwide (1). HLA-matched allogeneic stem cell transplantation (allo-SCT) is a widely applied immunotherapeutic approach for several of these hematological malignancies. The therapeutic effect of allo-SCT is largely mediated by alloreactive donor T cells directed at polymorphic peptides presented by HLA molecules on the recipient''s malignant cells (2). These polymorphic peptides, also known as minor histocompatibility antigens (mHags), are frequently derived from cellular proteins encoded by allelic genes on autosomal chromosomes. Although several mHags are expressed ubiquitously, some mHags are exclusively expressed on hematopoietic cells and their malignant counterparts (2–4). Hence, targeting donor T cells toward such hematopoietic mHags is considered an ideal strategy to establish specific antitumor effects after allo-SCT (2, 4). Because CD8⁺ T cells are traditionally considered as the effector cells of antitumor responses, over the past years the major focus was to identify hematopoietic mHags presented to CD8⁺ CTLs (5–12). Nonetheless, several reports, including ours, indicate that not only CD8⁺ CTLs but also CD4⁺ T cells may possess immunotherapeutic potential (13–15). Yet no hematopoietic mHag presented by HLA class II has been identified, partly because the available techniques are not well suited for identification of such antigens. More importantly, several of the apparently hematopoietic mHags recognized by CD4⁺ T cells are not derived from genuine hematopoietic antigens. For instance, the recently identified autosomal mHag presented to CD4⁺ T cells is derived from the broadly expressed phosphatidylinositol 4-kinase type II β gene (16).We previously isolated an HLA-DQA1*05/B1*02–restricted mHag-specific CD4⁺ T cell (clone 21) from the PBMC of a multiple myeloma patient after HLA-identical allo-SCT. This clone recognized recipient-derived EBV-transformed B cells (EBV-transformed lymphoblastoid cell lines [EBV-LCLs]) but not the nonhematopoietic fibroblasts and stromal cells, suggesting that its target antigen was encoded by a hematopoietic gene (unpublished data). To identify the mHag recognized by clone 21, we developed a nonlaborious but powerful genetic strategy in which a zygosity-genotype correlation analysis was used for fine mapping of the genomic locus mHag identified by classical pair-wise two-point linkage analysis. The new gene-mapping method was also genomewide applicable for a broad range of mHags. Further investigation on the identified locus revealed that the antigen recognized by clone 21 was encoded by a single-nucleotide polymorphism (SNP) in the B cell lineage-specific CD19 gene, which is a highly important target antigen for immunotherapy of almost all B cell malignancies. The CD19^L-specific CD4⁺ T cells not only mediated antigen-specific help for the induction and expansion of CD8⁺ mHag-specific T cells but also displayed antigen-specific and HLA-restricted lysis of CD19^L-positive malignant cells, illustrating the potential therapeutic advantages of targeting this CD19^L-derived HLA class II–restricted mHag.     

Role of angiogenic growth factors in transplant coronary artery disease

Transplant coronary artery disease (TxCAD) as a manifestation of chronic rejection is a major limitation to long‐term survival of heart transplant recipients. Although the exact molecular and cellular mechanisms contributing to neointimal formation are unknown, it has been generally believed that smooth muscle cells (SMC) of donor origin migrate from the media into the subendothelial layer of the vascular wall, where SMC proliferate and synthesize extracellular matrix resulting in intimal thickening. However, recent observations indicate that hematopoietic and vascular progenitor cells derived from recipient bone marrow may contribute to the arteriosclerotic lesion formation in the coronary arteries of the transplant. On the other hand, studies on postnatal hematopoiesis indicate that angiogenic growth factors such as vascular endothelial growth factor (VEGF) and angiopoietin‐1 (Ang1) may regulate the recruitment of these cells into distant organs. Furthermore, embryonic VEGFR‐2 ⁺ /CD34 ⁺ stem cells may serve as vascular progenitor cells and their differentiation into endothelial cells and SMC may be regulated by VEGF and platelet‐derived growth factor (PDGF), respectively. In this review, we discuss the role of angiogenic growth factors such as VEGF, Ang, and PDGF in the recruitment of hematopoietic and vascular progenitor cells in TxCAD and suggest novel therapies targeted at homing, differentiation and proliferation of these cells in the allograft.     

A human postnatal lymphoid progenitor capable of circulating and seeding the thymus

Identification of a thymus-seeding progenitor originating from human bone marrow (BM) constitutes a key milestone in understanding the mechanisms of T cell development and provides new potential for correcting T cell deficiencies. We report the characterization of a novel lymphoid-restricted subset, which is part of the lineage-negative CD34⁺CD10⁺ progenitor population and which is distinct from B cell–committed precursors (in view of the absence of CD24 expression). We demonstrate that these Lin⁻CD34⁺CD10⁺CD24⁻ progenitors have a very low myeloid potential but can generate B, T, and natural killer lymphocytes and coexpress recombination activating gene 1, terminal deoxynucleotide transferase, PAX5, interleukin 7 receptor α, and CD3ε. These progenitors are present in the cord blood and in the BM but can also be found in the blood throughout life. Moreover, they belong to the most immature thymocyte population. Collectively, these findings unravel the existence of a postnatal lymphoid-polarized population that is capable of migrating from the BM to the thymus.     

不同时相移植人骨髓间充质干细胞对人脐血CD34^＋细胞植入NOD／SCID小鼠的影响

为了观察不同时相移植人骨髓间充质干细胞（MSC）对脐血（UCB）CD34^＋细胞移植的NOD／SCID小鼠造血重建的影响，明确最佳的移植时机，将体外培养扩增的人骨髓MSC分别于UCBCD34^＋细胞移植同时、移植前48小时及移植后48小时输入经^60Coγ射线照射的NOD／SCID小鼠，观察共移植后42天内小鼠外周血白细胞和血小板变化，并于移植后42天处死小鼠，用FACS检测外周血、骨髓和脾脏人源细胞含量。结果表明：（1）MSC和UCBCD34^＋细胞同时输注可明显降低外周血白细胞和血小板下降幅度，缩短白细胞和血小板恢复时间；二者不同时输注均不降低白细胞和血小板下降幅度，且输注UCBCD34^＋细胞后48小时输注MSC时外周血血小板恢复时间明显晚于同时输注者。（2）与单纯UCBCD34^＋细胞移植相比较，不同时相输注MSC均可促进UCBCD34^＋细胞的植入，三个共输注组间促进骨髓各系造血植入效应无明显差异。结论：人骨髓MSC与UCBCD34^＋细胞共移植时，以同时移植效果最佳，此结果为MSC的临床应用提供了实验依据。     

Nanoparticles Deliver Triplex-forming PNAs for Site-specific Genomic Recombination in CD34+ Human Hematopoietic Progenitors

Triplex-forming peptide nucleic acids (PNAs) are powerful gene therapy agents that can enhance recombination of short donor DNAs with genomic DNA, leading to targeted and specific correction of disease-causing genetic mutations. Therapeutic use of PNAs is severely limited, however, by challenges in intracellular delivery, particularly in clinically relevant targets such as hematopoietic stem and progenitor cells. Here, we demonstrate efficient and nontoxic PNA-mediated recombination in human CD34⁺ cells using poly(lactic-co-glycolic acid) (PLGA) nanoparticles for intracellular oligonucleotide delivery. Treatment of progenitor cells with nanoparticles loaded with PNAs and DNAs targeting the β-globin locus led to levels of site-specific modification in the range of 0.5–1% in a single treatment, without detectable loss in cell viability, resulting in a 60-fold increase in modified and viable cells as compared to nucleofection. As well, the differentiation capacity of the progenitor cells treated with nanoparticles did not change relative to untreated progenitor cells, indicating that nanoparticles are safe and minimally disruptive delivery vectors for PNAs and DNAs to mediate gene modification in human primary cells. This is the first demonstration of the use of biodegradable nanoparticles to deliver genome-editing agents to human primary cells, and provides a strong rationale for systemic delivery of complex nucleic acid mixtures designed for gene correction.