HOX11L2/TLX3 is transcriptionally activated through T-cell regulatory elements downstream of BCL11B as a result of the t(5;14)(q35;q32).

International audienceThe t(5;14)(q35;q32) chromosomal translocation is specifically observed in up to 20% of childhood T-cell acute lymphoblastic leukemia (T-ALL). It affects the BCL11B/CTIP2 locus on chromosome 14 and the RANBP17-TLX3/HOX11L2 region on chromosome 5. It leads to ectopic activation of TLX3/HOX11L2. To investigate the reasons of the association between t(5;14) and T-ALL, we isolated the translocation breakpoints in 8 t(5;14) patients. Sequence analyses did not involve recombinase activity in the genesis of the translocation. We used DNAse1 hypersensitive experiments to locate transcriptional regulatory elements downstream of BCL11B. By transient transfection experiments, 2 of the 6 regions demonstrated cis-activation properties in T cells and were also effective on the TLX3 promoter. Our data indicate that the basis of the specific association between t(5;14) and T-ALL lies on the juxtaposition of TLX3 to long-range cis-activating regions active during T-cell differentiation

Hematopoietic Stem Cell Transplant for the Treatment of X-MAID

We report outcomes after hematopoietic stem cell transplant for three patients with X-MAID, including 1 patient from the originally described cohort and two brothers with positive TREC newborn screening for SCID who were found to have a T-B-NK+ SCID phenotype attributable to X-linked moesin associated immunodeficiency (X-MAID). A c.511C>T variant in moesin was identified via exome sequencing in the older of these siblings in the setting of low lymphocyte counts and poor proliferative responses consistent with SCID. He received reduced intensity conditioning due to CMV, and was transplanted with a T-depleted haploidentical (maternal) donor. His post-transplant course was complicated by hemolytic anemia, neutropenia, and sepsis. He had poor engraftment, requiring a 2nd transplant. His younger brother presented with the same clinical phenotype and was treated with umbilical cord blood transplant following myeloablative conditioning, has engrafted and is doing well. The third case also presented with severe lymphopenia in infancy, received a matched related bone marrow transplant following myeloablative conditioning, has engrafted and is doing well. These cases represent a novel manifestation of non-radiosensitive X-linked form of T-B-NK+ SCID that is able to be detected by TREC based newborn screening and effectively treated with HCT

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Génération des progéniteurs lymphoïdes T ex vivo par exposition brève au ligand de Notch DL-4

L allogreffe des cellules souches hématopoïétiques (CSH) dans les situations d incompatibilité HLA partielle représente une option thérapeutique irremplaçable pour des patients nécessitant une greffe de cellules souches hématopoïétiques, en absence d un donneur HLA-identique. Toutefois, le retard de la restauration du système immunitaire en particulier dans du compartiment lymphocytaire après greffe est l'une des complications majeures. Une nouvelle stratégie pour promouvoir la reprise de la thymopoïèse à partir des CSH provenant du donneur et d'accélérer la reconstitution cellulaire T chez des patients après greffe de CSH consiste en le transfert adoptif des progéniteurs T générés in vitro. L identification de Notch1 comme le régulateur-clé du développement lymphocytaire T a permis l établissement de systèmes de culture à base de ligands de Notch, qui permettent la génération efficace de progéniteurs lymphoïdes T in vitro. L'efficacité des progeniteurs T-lymphoïdes murins pour promouvoir la reconstitution des lymphocytes T a été bien démontrée dans des modèles de greffe chez la souris. De même, des progéniteurs T-lymphopoïétiques humains générés in vitro et greffés aux souris humanisées favorisent la reprise de la thymopoïèse. Pourtant, aucune donnée n a encore démontré leur capacité à donner naissance à un compartiment lymphocytaire T périphérique. De plus, les systèmes de co-culture à base de ligand de Notch actuellement utilisés consistent en des lignées stromales murines génétiquement modifiées. Afin d'établir un système cliniquement applicable, il est donc indispensable d établir des systèmes de culture qui soutiennent la génération de progéniteurs T en absence d un support des cellules nourricières. Au cours de mon projet de thèse, j'ai développé un nouveau système de culture pour la génération des progéniteurs T-lymphopoïétiques humains T basé sur l immobilisation du ligand de Notch Delta-like-4 (DL-4) sous sa forme protéique. La culture des progéniteurs hématopoïétiques CD34+ issue de sang en présence de DL-4 immobilisé permet la génération d un grand nombre de cellules ayant un phénotype de progéniteurs thymiques précoces (early thymic progenitor: ETP) et de prothymocytes (proT). Les cellules ETP et ProT ainsi générées expriment à des niveaux élevés des gènes impliqués dans le développement lymphocytaire précoce (i.e. pTa, Rag1, IL7Ra et BCL11b). Elles montrent des signes de réarrangement du récepteur des cellules T (TCR) similaires à leurs homologues thymiques. Par des expériences de dilution limite sur une co-culture OP9/DL-1 secondaire, j ai pu montrer que les progéniteurs générés sur DL-4 possédaient un potentiel lymphoïde T très augmenté, qui pourrait être entièrement attribué aux sous populations ETP et ProT. Suite à leur transfert dans des souris NOD/SCID/gc-/-, les progéniteurs lymphoïde T générés par exposition a DL-4 sont capable de migrer dans le thymus, d y poursuivre des étapes ultérieures de leur développement et d accélérer la différentiation T intra thymique ainsi que l émergence des lymphocytes T mature, polyclonaux et fonctionnels en périphérie. Dans une approche de co-transplantation, qui se rapproche des conditions cliniques envisagées, j ai simultanément injecté dans le même récipient des progéniteurs générées sur DL-4 et des cellules CD34+ non traitées (d un 2èm donneur HLA-incompatible). Cette procédure a permis une reconstitution des lymphocytes T encore plus rapide et plus. Etant donné que les progéniteurs T générées sur DL-4 et les cellules CD34+ non-traitées étaient issue de deux donneurs avec un HLA différent, cette expérience a permis de montrer que les progéniteurs préalablement exposés à DL-4 reconstituaient spécifiquement les compartiments lymphoïdes T alors que les autres lignées hématopoïétiques provenaient des progéniteurs CD34+ non-traités...Human leukocyte antigen (HLA)-mismatched haematopoietic stem cell transplantation (HSCT) represents an important therapeutic option for patients lacking suitable donors. Delayed posttransplant immune recovery constitutes one of its major complications and is most pronounced in the T cellular compartment. A novel strategy to promote de novo thymopoiesis from donor derived HSCs and to accelerate T cellular reconstitution in patients after HSCT consists in the adoptive transfer of in vitro generated T cell progenitor cells. Identification of Notch1 as the key regulator of early T-lineage development has allowed the generation of Notch ligand-based culture systems, which provide a powerful tool to generate T-lymphoid progenitors in vitro. The efficacy of murine T-lymphoid progenitors to promote T cell reconstitution has been well demonstrated in conventional mouse models. In consistency, in vitro-generated human T cell progenitors were demonstrated to promote thymic recovery in humanized mice. Yet, positive effects of in vitro generated human T cell precursors on peripheral T cell reconstitution have not been demonstrated. Moreover currently used Notch-based co-culture systems consist of genetically modified murine cell lines. With view to establishing a clinically applicable system, feeder-cell-free Notch-ligand culture systems for the generation of T-lymphopoietic progenitors are warranted. During my PhD project I developed a new culture system based on the immobilized Notch ligand Delta-like-4 (DL-4). Exposure of human CD34+ cord blood cells to immobilized DL-4 enabled the in vitro generation of high number of T cell progenitors, which harboured the phenotype of immature early thymic progenitor cells (ETP) and prothymocytes (proT). ETP and proT cell generated during DL-4 culture upregulated essential genes involved in early T-lymphoid development (i.e. IL7Ra, PTa, RAG1 and BCL11b) and had undergone stage-specific recombination of the T cell receptor (TCR) locus in a similar way as in native human thymopoiesis. In limiting dilution analysis after secondary OP9/DL-1 co-culture, DL-4 progenitors displayed a highly increased T-lymphoid potential, which could be entirely attributed to the ETP and proT subset. When transferred into NOD/SCID/gc-/- mice, DL-4 primed T cell progenitors migrated to the thymus and accelerated intrathymic T cell differentiation and emergence of functional, mature and polyclonal ab T cells in the periphery. In a co-transplantation approach, which more closely mimics a clinical setting, DL-4 progenitors and untreated CD34+ cells from HLA-disparate donors were simultaneously injected in the same recipient. This procedure allowed even more rapid and more robust T cell reconstitution. HLA-tracking of the distinct graft sources further showed, that DL-4 progenitors specifically reconstituted the T-lymphoid compartments. This work provides further evidence for the ability of in vitro-generated human T cell progenitors to promote de novo thymopoiesis and shows for the first time, that these cells accelerate peripheral T cell reconstitution in humanized mice. The availability of the efficient feeder-cell-free DL-4 culture technique represents an important step towards the future clinical exploitation translation of in vitro generated T-lymphoid progenitor cells to improve posttransplant immune reconstitutionPARIS5-Bibliotheque electronique (751069902) / SudocSudocFranceF

Expansion of Murine Amniotic Fluid c-Kit High Lin- Cells Maintaining Their Hematopoietic Potential.

Abstract Abstract 1475 Poster Board I-498 We have recently described the hematopoietic potential of ckit+ Lin- cells from the murine and human amniotic fluid (1). These cells were able to generate all types of blood cells in vitro and as far as mice are concerned, to generate a complete hematopoietic system once transplanted to immunodeficient recipients. This strong hematopoietic potential was accompanied by a molecular signature measured by unicellular RT-PCR, characteristic of fetal hematopoietic progenitors. Indeed coexpression of Gata2, Lmo2 and Aml1 was found in 28% of ckit+Lin- AF cells. Intriguingly, murine ckit+Lin- AF cells can be subdivided into two fractions depending on the level of ckit expression (low or high). In in vitro assays, we demonstrated that hematopoietic potential was strictly restricted to the ckit high expressing fraction. The expansion of these cells would have great impact even in the clinical field as AF could be seen as a source of transplantable hematopoietic stem cells (HSCs). Many of the early studies that documented some expansion ability of HSCs included fetal calf serum in the protocol. Given the poorly defined combination of factors in serum and the variability between different serum lots, these protocols were often difficult to reproduce. Serum-free media supplied with specific inducers have been shown to bring several advances in driving direct differentiation of embryonic stem cells. Murine AF Lin-ckitlo and ckithi were cultured in serum- and feeder layer-free culture conditions. Lin-ckitlo AF cells died within a few days. Conversely, Lin-ckithi AF cells were maintained for up to 6 weeks, with a proliferation rate of more than 100 during the first three weeks. Their phenotype remained stable, ckithi CD45+ and Lin-. The hematopoietic potential tested in methylcellulose assays showed an increased frequency of mixed CFU-GEMM (from 24% to 84%). In vivo, CFU-S12 composed of erythroid, myeloid and Lin-ckit+Sca1+ progenitor cells were observed after the injection of AF ckithi in lethally irradiated recipients. Gene expression profile analyzed by single cell multiplex RT-PCR analysis correlated with the in vitro and in vivo results of differentiation. LMO2 was coexpressed with Gata2 and Aml1 in 66% of expanded cells, demonstrating the maintenance of an overall pattern of expression. Collectively, our results indicate that the hematopoietic potential of AF resides in the ckithi fraction and that these cells can be expanded in serum-free condition for prolonged periods of time without reduction of their hematopoietic potential. This strongly supports the idea that AF may be an excellent source of cells for therapeutic applications. 1. Ditadi 2009 Disclosures: No relevant conflicts of interest to declare. </jats:sec

In Vitro Generation of Human T-Cell Precursors From Bone Marrow CD34+ Cells by Short Exposure to Immobilized Notch-Ligand Delta-Like 4.

Abstract Abstract 3532 Poster Board III-469 Prolonged posttransplant immune deficiency is a major complication following hematopoietic stem cell transplantation, particularly in the T-lymphoid compartment. Accelerating T-cell development by injecting donor derived T-cell precursors has been proposed as a novel strategy to shorten the immune deficient phase. Several research groups have successfully generated T-cell precursors from murine and human HSC in vitro by transitory exposure to the Notch-ligand presenting murine OP9DL1-cell line. Transfer of the in vitro generated murine T-cell precursors into irradiated NOD/SCID/γcnull-mice accelerated T-cellular reconstitution. However, the clinical application of the OP9DL1-system is limited. Recent studies have demonstrated that short exposure of cord blood CD34+ cells to Notch-ligand Delta-like 4 is sufficient to promote human T-cell differentiation in vitro. Here, we modified this technique to better characterize and ameliorate T-cell development in vitro, with the objective of eventually transferring this method to a clinical phase. Towards this aim, we exposed human CD34+ HSC derived from any available source to immobilized Notch-ligand Delta-like 4 in the presence of different cytokine combinations implicated in human haematopoiesis (IL-7, SCF, Flt3-ligand and TPO). Within 7 days a population of CD34+CD7+ and CD34-CD7++ T-cell precursors emerged in the presence of Delta-like 4, but not under control conditions. After 7 days the CD34+CD7+ population subsequently declined while further amplification of the CD34-CD7++ population was observed. Two distinct progenitor subsets emerged within the CD34-CD7++ population, namely CD34-CD7++CD5+ and CD34-CD7++CD5-. The CD34-CD7++CD5+ subset further acquired CD1a and, thus, adopted a pre-T-cell phenotype. Between days 7 and 14 the CD34-CD7++CD5- acquired a NK-cell phenotype, as indicated by CD16 and CD56 expression. Beyond 14 days no further expansion of the pre-T-cell fraction was observed, while the NK-cell fraction continued proliferating. More advanced stages T-cell development, such as immature single positive CD4+ cells as observable in OP9DL1 co-cultures, did not arise after exposing cells only to immobilized Delta-like4. Intermittent emergence of a CD13+CD14+CD7- myeloid population was observed within the first 14 days of culture on Delta-like 4; however, this population disappeared spontaneously and did not preserve its common myeloid progenitor. Selecting a more immature CD34+CD38- population resulted in a two-fold increase of the frequency of CD34+CD7+ and CD34-CD7++ cells as compared to the whole CD34+ population, while myeloid differentiation was inhibited. A further increase was obtained by replanting cultured cells to freshly coated plates with Delta-like 4 every 3 days of culture. T-cell precursors cells derived after 7 days of culture were injected into NOD/SCID/γcnull mice. The in vivo-experiments are ongoing and results are pending. Our results provide further evidence that human T-cell precursors can be generated in vitro, not only in co-culture with murine OP9DL1-cells but also by short exposure to immobilized Notch-ligand Delta-like 4. These ongoing experiments are an important prerequisite for the potential clinical application of this method. Disclosures: No relevant conflicts of interest to declare. </jats:sec

Gene Therapy of Human Blnk Deficiency in NOD/SCID/Gc Ko Murine Model

Abstract Abstract 4717 Blnk deficiency is an autosomal recessive immune disorder characterized by the absence of B cells in periphery and the absence of any seric immunoglobulins due to an early blocage at the pro-B cell stage in the bone marrow. The very rare patients affected by blnk deficiency develop severe infections. In the murine model of the disease, a similar blocage in B cell development is described as well as susceptibility to infections and to pre-B lymphomas. A homozygote stop mutation in blnk gene was identified in a 8-yr old boy. Bone marrow cells analysis revealed that CD34+CD10+CD24-CD19- lymphoid progenitors were present as well as CD34+CD10+CD24+CD19- early B cells and CD34+CD19+ pro B cells. However, no surface IgM or seric Ig were detected in this patient. To demonstrate the implication of Blnk in the B-cell differentiation process, we transduced CD34+ sorted bone marrow cells from this patient with a lentiviral construct containing human wild type Blnk cDNA. The transduced cells were intravenously injected into irradiated NOD/SCID/IL2rg knock-out mice. Twelve weeks after transplantation, recipients were analysed. Human engraftment was detected in bone marrow and spleen. Among bone marrow human CD45+ cells, more than 80% were CD19+ and 6 to 8% express surface IgM. In the spleen, between 14 and 30% of CD19+ cells were detected. Eight to 42% of CD19+ cells expressed surface IgM. This is the first demonstration that Blnk is absolutely required for the differentiation of pro-B cells toward mature B cells. Disclosures: No relevant conflicts of interest to declare. </jats:sec

In Vitro Exposure to DL-4 Increases the in Vitro and In Vivo T-Lymphopoietic Potential of CB Derived CD34+ Progenitor Cells

Abstract Abstract 2980 Notchligand-based culture systems such as OP9-DL1 cells induce HSC to engage towards the T-cell developmental program and allow generation of T-lymphoid progenitors in vitro. In vitro generated murine T-lymphoid progenitors accelerated T-cell reconstitution in vivo. In consistency, human T-lymphoid progenitors generated in co-culture with OP9-DL1 cells enhanced thymic repopulation when injected into NOD/SCID/gc−/− mice (NSG). However, positive effects of human T-lymphoid progenitors on peripheral T-cell reconstitution have not been reported yet. Besides, Notchligand-based culture systems, consisting of genetically modified murine cells might raise safety concern for clinical use. It has been described that exposure of CD34+ cells to immobilized DL4 induces the T-cell developmental program even in absence of stromal cell support. Recently, we have made use of this system to generate T-lymphoid progenitors in vitro. In the present study we have further characterized their T-lymphoid potential in vitro and in vivo. Exposure of human CB-derived CD34+ cells to immobilized DL4 allowed generation of CD34+CD7+ and CD34−CD7++CD5+ progenitors displaying a similar phenotype as early thymic progenitors (ETP) and the prethymocytes (pre-T). Within the DL-4 derived ETP- and preT-like progenitors we observed subsequent up regulation of genes involved in T-cell development and silencing of genes implied in B-cell and myeloid differentiation. T-cell commitment of DL-4 progenitors could be further confirmed by early and intermediate rearrangement events within the TCR d/g/b genes. The pattern of gene expression profile and TCR-rearrangement events displayed a pattern similar to what we observed in corresponding intrathymic developmental stages. DL4-progenitors obtained after 7 days of culture displayed a 30-fold increased in vitro T-lymphoid potential as compared to untreated CD34+ CB progenitors. DL4 ETP-like and preT-like progenitors further completed T-cell differentiation in vitro (in OP9DL1 co-culture) faster than native CD34+ CB progenitors. When transferred into NSG, DL4 progenitors obtained after 7 days of culture were able to repopulate the recipients' thymus and to give rise to mature, polyclonal intrathymic and peripheral T-cells. Two months after transfer recipients of DL4 progenitors displayed advanced intrathymic T-cell development as compared to recipients of CD34+ CB cells. Furthermore, peripheral T-cells could be observed in a number of DL-4 progenitor recipients but not in control mice. Our experiments provide further evidence that DL4 allows in vitro induction of T-cell development and generation of early T-lymphoid progenitors in a system devoid of stromal cell support. These progenitors feature phenotypical and molecular characteristics of immature thymic developmental stages. Moreover, they are able to accelerate T-cell development in vitro and when transferred into NSG. This work provides further evidence of the ability of in vitro -generated human T-cell progenitors to accelerate T-cell reconstitution and simultaneously introduces a culture technique that could be rapidly transferred into a clinical setting. Disclosures: No relevant conflicts of interest to declare. </jats:sec

Further Characterization of T-Cellular Precursors Generated From CD34+ Progenitors by Exposure to Immobilized Notch Ligand Delta-Like 4 In Vitro.

Abstract Abstract 3712 Injection of donor derived T-cellular precursors has been proposed as a novel strategy to shorten delayed reconstitution of the T-lymphoid compartment following HSCT. In the past years, several research groups have successfully generated murine and human T-cellular precursors in vitro using Notchligand-based coculture systems such as OP9-DL1 or Tst-DL4. Murine T-cellular precursors generated in vitro, promoted reconstitution of the T-cellular compartment when applied in murine HSCT-models. In consistency, transfer of human T-cellular precursors, generated in vitro in coculture with OP9-DL1 or Tst-DL4 resulted in enhanced thymic repopulation in NOD/SCID/gc−/− mice. Yet, positive effects on peripheral T-cell reconstitution have not been reported. Moreover, clinical application of OP9-DL1 or Tst-DL4 coculture systems is limited, since they consist of murine stromal cells transduced with either DL1 or DL4. It has been described that exposure of CD34+ cells to immobilized DL4 induces T-cell differentiation in vitro and allows expansion human T-cellular precursors even in absence of stromal cell support. However, the hypothesis that DL4 alone can drive hematopoietic progenitors towards a T-cell fate in vitro, requires more evidence. Here, we further characterized the in vitro and in vivo potential of T-cellular precursors generated by single exposure to DL4. We exposed human CD34+ progenitors to immobilized DL4 in the presence of different cytokine combinations implicated in human haematopoiesis. Within 7 days, CD34+CD7+ and CD34−CD7++ T-cellular precursors emerged in the presence of DL4, but not under control conditions. After 7 days the CD34+CD7+ population subsequently declined while the CD34−CD7++ population further expanded. Two distinct progenitor subsets, CD5+ and CD5-, emerged within the CD34−CD7++ population. The CD34−CD7++CD5+ subset partially acquired CD1a, corresponding to a developmental stage between the early thymic progenitor (ETP) and the prethymocyte (pre-T) stage. Conversely to what observed in the OP9-DL1 system, T-cell development did not progress beyond the pre-T-stage. Indeed, we neither observed more advanced stages of T-cell development, such as immature single positive CD4+ cells, nor complete TCR-rearrangements. 7-day exposure to immobilized DL4 induced a 90-fold increase of T-precursor frequency in CD34+ progenitors (1/8800 before culture vs. 1/90 after culture) as confirmed by limiting dilution assays on OP9-DL1. All T-cellular precursor activity was restricted to cells expressing CD34, CD7 or both (frequency: 1/9). In particular, elevated T-cellular precursor levels were found in the subsets expressing CD7 (CD34+/CD7+ and CD34−/CD7+), while the T-cellular precursor frequency in the CD34+/CD7− subset was equal to that seen in non-cultured CD34+ progenitors. In consistency the CD34−CD7− population did not contain any detectable T-cellular precursors. After 7 day exposure to DL4, cells phenotypically corresponding to T-cellular precursors were transferred into NOD/SCID/gc−/− mice. Within 2 months following HSCT, cells exposed to DL4 were able to reconstitute the recipients' thymus and partially gave rise to peripheral T-cells. When injecting non-cultured CD34+ progenitors, thymic reconstitution was likewise seen 2 months after HSCT. However, intrathymic T-cell development was less advanced and peripheral T-cells were absent. In contrast, cells cultured in presence of a control peptide did not retain any potential to repopulate the recipients' thymus. Our experiments provide further evidence that exposure DL4 induces early human T-cell development and allows generation of large numbers of T-cellular precursors in vitro. These precursors feature phenotypical and molecular properties corresponding to early precursors found in the human thymus. Furthermore, they have an increased potential to further differentiate into mature T-cells in vitro and when transferred into immunodeficient mice. Our preliminary data suggest, that injection of T-cellular precursors accelerates T-cell reconstitution after HSCT and provides further evidence for the feasibility of this novel strategy of immunotherapy. Disclosures: No relevant conflicts of interest to declare. </jats:sec