María L. Toribio

Oncogenic IL7R gain-of-function mutations in childhood T-cell acute lymphoblastic leukemia.

Interleukin 7 (IL-7) and its receptor, formed by IL-7Rα (encoded by IL7R) and γc, are essential for normal T-cell development and homeostasis. Here we show that IL7R is an oncogene mutated in T-cell acute lymphoblastic leukemia (T-ALL). We find that 9% of individuals with T-ALL have somatic gain-of-function IL7R exon 6 mutations. In most cases, these IL7R mutations introduce an unpaired cysteine in the extracellular juxtamembrane-transmembrane region and promote de novo formation of intermolecular disulfide bonds between mutant IL-7Rα subunits, thereby driving constitutive signaling via JAK1 and independently of IL-7, γc or JAK3. IL7R mutations induce a gene expression profile partially resembling that provoked by IL-7 and are enriched in the T-ALL subgroup comprising TLX3 rearranged and HOXA deregulated cases. Notably, IL7R mutations promote cell transformation and tumor formation. Overall, our findings indicate that IL7R mutational activation is involved in human T-cell leukemogenesis, paving the way for therapeutic targeting of IL-7R–mediated signaling in T-ALL.

PHF6 mutations in T-cell acute lymphoblastic leukemia

Tumor suppressor genes on the X chromosome may skew the gender distribution of specific types of cancer. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with an increased incidence in males. In this study, we report the identification of inactivating mutations and deletions in the X-linked plant homeodomain finger 6 (PHF6) gene in 16% of pediatric and 38% of adult primary T-ALL samples. Notably, PHF6 mutations are almost exclusively found in T-ALL samples from male subjects. Mutational loss of PHF6 is importantly associated with leukemias driven by aberrant expression of the homeobox transcription factor oncogenes TLX1 and TLX3. Overall, these results identify PHF6 as a new X-linked tumor suppressor in T-ALL and point to a strong genetic interaction between PHF6 loss and aberrant expression of TLX transcription factors in the pathogenesis of this disease.

Plasmacytoid dendritic cells resident in human thymus drive natural Treg cell development.

The generation of natural regulatory T cells (nTregs) is crucial for the establishment of immunologic self-tolerance and the prevention of autoimmunity. Still, the origin of nTregs and the mechanisms governing their differentiation within the thymus are poorly understood, particularly in humans. It was recently shown that conventional dendritic cells (cDCs) in human thymus were capable of inducing nTreg differentiation. However, the function of plasmacytoid DCs (pDCs), the other major subset of thymic DCs, remains unknown. Here we report that pDCs resident in the human thymus, when activated with CD40 ligand (CD40L) plus interleukin-3, efficiently promoted the generation of CD4(+)CD25(+)Foxp3(+) nTregs from autologous thymocytes. The progenitors of these nTregs were selectively found within CD4(+)CD8(+) thymocytes that had accomplished positive selection, as judged by their CD69(hi)TCR(hi) phenotype. Supporting the involvement of the CD40-CD40L pathway in pDC-induced nTreg generation, we show that positively selected CD4(+)CD8(+) progenitors specifically transcribed CD40L in vivo and up-regulated CD40L expression on T-cell receptor engagement, thereby promoting the activation of pDCs. Finally, evidence is provided that nTregs primed by pDCs displayed reciprocal interleukin-10/transforming growth factor-beta cytokine expression profiles compared with nTregs primed by cDCs. This functional diversity further supports a nonredundant tolerogenic role for thymic pDCs in the human thymus.

CSL–MAML-dependent Notch1 signaling controls T lineage–specific IL-7Rα gene expression in early human thymopoiesis and leukemia

Notch1 activation is essential for T-lineage specification of lymphomyeloid progenitors seeding the thymus. Progression along the T cell lineage further requires cooperative signaling provided by the interleukin 7 receptor (IL-7R), but the molecular mechanisms responsible for the dynamic and lineage-specific regulation of IL-7R during thymopoiesis are unknown. We show that active Notch1 binds to a conserved CSL-binding site in the human IL7R gene promoter and critically regulates IL7R transcription and IL-7R α chain (IL-7Rα) expression via the CSL–MAML complex. Defective Notch1 signaling selectively impaired IL-7Rα expression in T-lineage cells, but not B-lineage cells, and resulted in a compromised expansion of early human developing thymocytes, which was rescued upon ectopic IL-7Rα expression. The pathological implications of these findings are demonstrated by the regulation of IL-7Rα expression downstream of Notch1 in T cell leukemias. Thus, Notch1 controls early T cell development, in part by regulating the stage- and lineage-specific expression of IL-7Rα.

Notch1 and IL-7 Receptor Interplay Maintains Proliferation of Human Thymic Progenitors while Suppressing Non-T Cell Fates

Notch signaling is critical for T cell development of multipotent hemopoietic progenitors. Yet, how Notch regulates T cell fate specification during early thymopoiesis remains unclear. In this study, we have identified an early subset of CD34highc-kit+flt3+IL-7Rα+ cells in the human postnatal thymus, which includes primitive progenitors with combined lymphomyeloid potential. To assess the impact of Notch signaling in early T cell development, we expressed constitutively active Notch1 in such thymic lymphomyeloid precursors (TLMPs), or triggered their endogenous Notch pathway in the OP9-Delta-like1 stroma coculture. Our results show that proliferation vs differentiation is a critical decision influenced by Notch at the TLMP stage. We found that Notch signaling plays a prominent role in inhibiting non-T cell differentiation (i.e., macrophages, dendritic cells, and NK cells) of TLMPs, while sustaining the proliferation of undifferentiated thymocytes with T cell potential in response to unique IL-7 signals. However, Notch activation is not sufficient for inducing T-lineage progression of proliferating progenitors. Rather, stroma-derived signals are concurrently required. Moreover, while ectopic IL-7R expression cannot replace Notch for the maintenance and expansion of undifferentiated thymocytes, Notch signals sustain IL-7R expression in proliferating thymocytes and induce IL-7R up-regulation in a T cell line. Thus, IL-7R and Notch pathways cooperate to synchronize cell proliferation and suppression of non-T lineage choices in primitive intrathymic progenitors, which will be allowed to progress along the T cell pathway only upon interaction with an inductive stromal microenvironment. These data provide insight into a mechanism of Notch-regulated amplification of the intrathymic pool of early human T cell progenitors.

SFRPs act as negative modulators of ADAM10 to regulate retinal neurogenesis

It is well established that retinal neurogenesis in mouse embryos requires the activation of Notch signaling, but is independent of the Wnt signaling pathway. We found that genetic inactivation of Sfrp1 and Sfrp2, two postulated Wnt antagonists, perturbs retinal neurogenesis. In retinas from Sfrp1−/−; Sfrp2−/− embryos, Notch signaling was transiently upregulated because Sfrps bind ADAM10 metalloprotease and downregulate its activity, an important step in Notch activation. The proteolysis of other ADAM10 substrates, including APP, was consistently altered in Sfrp mutants, whereas pharmacological inhibition of ADAM10 partially rescued the Sfrp1−/−; Sfrp2−/− retinal phenotype. Conversely, ectopic Sfrp1 expression in the Drosophila wing imaginal disc prevented the expression of Notch targets, and this was restored by the coexpression of Kuzbanian, the Drosophila ADAM10 homolog. Together, these data indicate that Sfrps inhibit the ADAM10 metalloprotease, which might have important implications in pathological events, including cancer and Alzheimers disease.

Human T‐Cell Precursors: Involvement of the IL‐2 Pathway in the Generation of Mature T Cells

Despite the genetic and structural homoiogy between antigen recognition receptors expressed by both T and B cellular components of the immune system (Kronenberg et al. 1986), T-cell recognition appears to be restricted by the products of the major histocompatibility complex (MHC) (Kindred & ShrefTler 1972, Katz et al. 1973, Zinkernagei & Doherty 1974). Acquisition of MHCrestricted T-ccll function is related to the many, and yet poorly understood, selective and muturational events which take place in the thymus (Fink & Bevan 1978, von Boehmer et ai. 1978, Sprent 1978, Zinkernagel 1978, Moller 1988). Committed hematopoietic T-cell precursors colonize the thymus during ontogeny, undergo a complex developmental process leading to the expression of clonallydistributed T-cell receptors (TCR) and migrate to the periphery as immunocompetent mature T cells (Moller 1984a). Attempts in the past few years to delineate such developmental events led to the characterization of distinct thymus subpopulations by means of the ditTerential expression of various cell surface molecules. Based on phenotypic and functional criteria, further reinforced by molecular genetic approaches, increasing agreement was reached between different laboratories when trying to correlate these subsets with discrete stages of development (reviewed by Moller 1984a). Careful analyses of cell dynamics within the thymus have allowed a better appreciation of the contribution of each thymic subpopulation to the general developmental process (Scollay & Shortman 1984, Scollay et al. 1984). Although the normal rate of stem-cell seeding into the thymus is extremely low, there is an extensive and rapid

The Participation of B Cells and Antibodies in the Selection and Maintenance of T Cell Repertoires

The imtnune system (IS) of vertebrates is endowed with obvious cognitive properties: it learns, remembers, and makes discriminatory inferences about molecular profiles. Typically, properties of this nature cannot be ascribed to individual components in the system (in our case, to the presence or activity of given clones), but they emerge as global behaviors from its general organization. Immune self/ nonself discrimination is, thus, a characteristically dispersed property of the IS (Varela et al. 1987). It follows that, to understand self/nonself discrimination, we have to be concerned at least as much v̂ fith such global behaviors and distributed properties as with individual clonal specificities. Distributed properties require connectivity between the elements of the system, and of these with other self components. Therefore, we believe, the understanding of self/nonself discrimination must start from the consideration of self-directed reactivities, in clear contrast with the classical notion that immune self/nonself discrimination is based upon the elimination of self-reactive clones (Vaz et al. 1984. Coutinho et al. 1984). Clearly, and this is also the case for conventional points of view, the behavior of an IS at any point in life can only be understood in the context of its own history in the ontogenic development of the individual. Snapshots of adult immune systems based on the study of lymphocyte repertoires, while necessary to realize the structure of the systems components and to construct the anatomy of interactions, will always tell us little about history and overall organization. We must therefore make explicit here the limitations of an approach (clonal

The differentiation stage of p53-Rb-deficient bone marrow mesenchymal stem cells imposes the phenotype of in vivo sarcoma development

Increasing evidence suggests that mesenchymal stem/stromal cells (MSCs) carrying specific mutations are at the origin of some sarcomas. We have reported that the deficiency of p53 alone or in combination with Rb (Rb−/− p53−/−) in adipose-derived MSCs (ASCs) promotes leiomyosarcoma-like tumors in vivo. Here, we hypothesized that the source of MSCs and/or the cell differentiation stage could determine the phenotype of sarcoma development. To investigate whether there is a link between the source of MSCs and sarcoma phenotype, we generated p53−/− and Rb−/−p53−/− MSCs from bone marrow (BM-MSCs). Both genotypes of BM-MSCs initiated leiomyosarcoma formation similar to p53−/− and Rb−/−p53−/− ASCs. In addition, gene expression profiling revealed transcriptome similarities between p53- or Rb-p53-deficient BM-MSCs/ASCs and muscle-associated sarcomagenesis. These data suggest that the tissue source of MSC does not seem to determine the development of a particular sarcoma phenotype. To analyze whether the differentiation stage defines the sarcoma phenotype, BM-MSCs and ASCs were induced to differentiate toward the osteogenic lineage, and both p53 and Rb were excised using Cre-expressing adenovectors at different stages along osteogenic differentiation. Regardless the level of osteogenic commitment, the inactivation of Rb and p53 in BM-MSC-derived, but not in ASC-derived, osteogenic progenitors gave rise to osteosarcoma-like tumors, which could be serially transplanted. This indicates that the osteogenic differentiation stage of BM-MSCs imposes the phenotype of in vivo sarcoma development, and that BM-MSC-derived osteogenic progenitors rather than undifferentiated BM-MSCs, undifferentiated ASCs or ASC-derived osteogenic progenitors, represent the cell of origin for osteosarcoma development.

T lymphoid differentiation in human bone marrow.

The unique role of the thymus in the development of T cells was established >4 decades ago. To elucidate how uncommitted lymphoid progenitor cells are instructed to migrate from bone marrow to the thymus to undergo T lymphoid differentiation, we generated and analyzed a genome-wide gene expression profile of CD7+ CD10+ human bone marrow T cell lineage precursors (TLPs) by using the serial analysis of gene expression technique. Unexpectedly, the serial analysis of gene expression profile identified a high number of (pre-) T cell receptor antigen (TCR)-related transcripts in bone marrow TLPs. To determine the configuration of the TCRβ locus in these cells at a quantitative level, we sorted and analyzed bone marrow TLPs from five donors by single-cell PCR. Similar proportions of TLPs harbored TCRβ germ-line alleles, D-J, or V-DJ gene rearrangements. Thus, bone marrow TLPs are heterogenous with respect to TCRβ rearrangement status, suggesting an active recombination machinery that is consistent with the expression of RAG1, RAG2, and TdT in this population. As a hallmark of ongoing TCRβ V-DJ rearrangement, we could amplify broken-ended recombination-signal sequence DNA intermediates from bone marrow TLPs, but not from mature T cells by ligation-mediated PCR. Approximately half of the TCRβ rearrangements were compatible with the expression of a functional pre-TCR, which is in agreement with surface expression of pre-Tα on bone marrow TLPs as shown by confocal laser microscopy and flow cytometry. At a frequency <0.5% of mononucleated cells in human bone marrow, this population is rare, yet it exemplifies T lymphoid differentiation in the human already before immigration into the thymus.