Biomedical subjects

Thomas M Schmitt

Publications and source records attributed to Thomas M Schmitt.

11 recordsLinked to original sources

PubMed2006

The basic helix-loop-helix transcription factor HEBAlt is expressed in pro-T cells and enhances the generation of T cell precursors.

The basic helix-loop-helix (bHLH) transcription factors HEB and E2A are critical mediators of gene regulation during lymphocyte development. We have cloned a new transcription factor, called HEBAlt, from a pro-T cell cDNA library. HEBAlt is generated by alternative transcriptional initiation and splicing from the HEB gene locus, which also encodes the previously characterized E box protein HEBCan. HEBAlt contains a unique N-terminal coding exon (the Alt domain) that replaces the first transactivation domain of HEBCan. Downstream of the Alt domain, HEBAlt is identical to HEBCan, including the DNA binding domain. HEBAlt is induced in early thymocyte precursors and down-regulated permanently at the double negative to double positive (DP) transition, whereas HEBCan mRNA expression peaks at the DP stage of thymocyte development. HEBAlt mRNA is up-regulated synergistically by a combination of HEBCan activity and Delta-Notch signaling. Retroviral transduction of HEBAlt or HEBCan into hemopoietic stem cells followed by OP9-DL1 coculture revealed that HEBAlt-transduced precursors generated more early T lineage precursors and more DP pre-T cells than control transduced cells. By contrast, HEBCan-transduced cells that maintained high level expression of the HEBCan transgene were inhibited in expansion and progression through T cell development. HEB(-/-) fetal liver precursors transduced with HEBAlt were rescued from delayed T cell specification, but HEBCan-transduced HEB(-/-) precursors were not. Therefore, HEBAlt and HEBCan are functionally distinct transcription factors, and HEBAlt is specifically required for the efficient generation of early T cell precursors.

Amino Acid Sequence↗

PubMed2006

Notch signaling requires GATA-2 to inhibit myelopoiesis from embryonic stem cells and primary hemopoietic progenitors.

The bone marrow and thymus, although both hemopoietic environments, induce very distinct differentiation outcomes. The former supports hemopoietic stem cell self-renewal and multiple hemopoietic lineages, while the latter supports T lymphopoiesis almost exclusively. This distinction suggests that the thymic environment acts to restrict the hemopoietic fates available to thymic immigrants. In this study, we demonstrate that the addition of the Notch ligand Delta-like-1 (Dll-1) to an in vitro system that otherwise supports myelopoiesis, greatly reduces the myelopoietic potential of stem cells or uncommitted progenitors. In contrast, committed myeloid progenitors mature regardless of the presence of Dll-1. The block in myelopoiesis is the direct result of Notch signaling within the hemopoietic progenitor, and Dll-1-induced signals cause a rapid increase in the expression of the zinc finger transcription factor GATA-2. Importantly, in the absence of GATA-2, Dll-1-induced signals fail to inhibit commitment to the myeloid fate. Taken together, our results support a role for GATA-2 in allowing Dll-1 to restrict non-T cell lineage differentiation outcomes.

Cells, Cultured↗

PubMed2006

T-cell development, doing it in a dish.

The thymus provides a unique environment for the development of T lymphocytes from bone marrow-derived progenitor cells. Several environmental factors have been identified that influence the development of T cells in the thymus. In particular, the Notch pathway has emerged as critical for the induction of T-lineage commitment and differentiation. Until recently, however, the precise nature of the thymus-derived signals that drive T-cell development were unclear, and the only reliable in vitro culture system that supported T-cell differentiation required the use of thymus organ cultures. Here, we discuss recent advances in the identification of critical Notch receptor ligands that have facilitated the development of a simple in vitro model for the differentiation of T cells 'in a dish', providing an alternate approach for studying T lymphopoiesis.

Animals↗

PubMed2006

In vitro generation of T lymphocytes from embryonic stem cells.

Mature hematopoietic cells, like all other terminally differentiated lineages, arise during ontogeny via a series of increasingly restricted intermediates. Hematopoietic progenitors have their origin in the mesoderm, which gives rise to hemangioblasts that can differentiate into endothelial or endocardial precursors or hematopoietic stem cells. These hematopoietic stem cells in turn may either self-renew or differentiate into lineage-restricted progenitors and ultimately mature effector cells. The ability to generate most hematopoietic lineages in a two-dimensional environment in vitro has facilitated our study of this complex process. Until recently, the T-lymphocyte lineage was the exception and appeared to require the specialized three-dimensional microenvironment of the thymus to develop. However, here we describe a protocol for the generation of T lymphocytes from embryonic stem cells in vitro, within a two-dimensional microenvironment, provided by OP9 bone marrow stromal cells. This procedure will facilitate further study of early T lymphopoiesis by providing a simple model system in which the effects of genetic and environmental manipulations of embryonic stem cell-derived progenitors can be examined without requiring other more complex in vivo or in vitro experimental approaches.

Animals↗

PubMed2005

Unraveling the origin of lymphocyte progenitors.

Lymphocytes are ultimately derived from hematopoietic stem cells, however the intervening steps that give rise to lymphocyte progenitors are still under intense investigation. In particular, whether a common lymphocyte progenitor serves exclusively as a lymphoid precursor, or whether this cell retains limited myeloid progenitor potential are addressed by Rolink and colleagues. This issue is highlighted by their identification within the bone marrow of early progenitors with lymphoid and myeloid developmental potential or EPLMs. How these cells fit into our current understanding of lymphocyte development, and how this new subset helps to further refine our view of the common lymphocyte progenitor are discussed.

Animals↗

PubMed2005

Thymus-derived signals regulate early T-cell development.

T cells develop in the thymus from uncommitted progenitors under the influence of multiple soluble and membrane-associated factors that regulate the migration, survival, proliferation, and differentiation of progenitor thymocytes. The role of cytokines such as stem cell factor and IL-7 in this process is well established. However, several recent studies have demonstrated an important role for other soluble factors in T-cell development, including WNTs, bone morphogenetic proteins, and Hedgehog proteins. Other studies have clarified how chemokines regulate the migration of progenitor thymocytes at various stages of development. Furthermore, the Notch pathway has emerged as the critical inducer of T-lineage commitment and differentiation, and Notch ligands expressed by the thymic stroma likely provide essential developmental cues throughout early T-cell development. In this review, we discuss recent advances in our understanding of how thymus-derived signals regulate early T-cell development.

Animals↗

PubMed2004

Maintenance of T cell specification and differentiation requires recurrent notch receptor-ligand interactions.

Notch signaling has been shown to play a pivotal role in inducing T lineage commitment. However, T cell progenitors are known to retain other lineage potential long after the first point at which Notch signaling is required. Thus, additional requirements for Notch signals and the timing of these events relative to intrathymic differentiation remain unknown. Here, we address this issue by culturing subsets of CD4 CD8 double negative (DN) thymocytes on control stromal cells or stromal cells expressing Delta-like 1 (Dll1). All DN subsets were found to require Notch signals to differentiate into CD4+ CD8+ T cells. Using clonal analyses, we show that CD44+ CD25+ (DN2) cells, which appeared committed to the T cell lineage when cultured on Dll1-expressing stromal cells, nonetheless gave rise to natural killer cells with a progenitor frequency similar to that of CD44+ CD25- (DN1) thymocytes when Notch signaling was absent. These data, together with the observation that Dll1 is expressed on stromal cells throughout the thymic cortex, indicates that Notch receptor-ligand interactions are necessary for induction and maintenance of T cell lineage specification at both the DN1 and DN2 stages of T cell development, suggesting that the Notch-induced repression of the B cell fate is temporally separate from Notch-induced commitment to the T lineage.

Animals↗

PubMed2004

Obligatory role for cooperative signaling by pre-TCR and Notch during thymocyte differentiation.

The first checkpoint during T cell development, known as beta selection, requires the successful rearrangement of the TCR-beta gene locus. Notch signaling has been implicated in various stages during T lymphopoiesis. However, it is unclear whether Notch receptor-ligand interactions are necessary during beta selection. Here, we show that pre-TCR signaling concurrent with Notch receptor and Delta-like-1 ligand interactions are required for the survival, proliferation, and differentiation of mouse CD4(-)CD8(-) thymocytes to the CD4(+)CD8(+) stage. Furthermore, we address the minimal signaling requirements underlying beta selection and show a hierarchical positioning of key proximal signaling molecules. Collectively, our results demonstrate an essential role for Notch receptor-ligand interactions in enabling the autonomous signaling capacity of the pre-TCR complex.

Animals↗

PubMed2004

Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro.

Embryonic stem cells (ESCs) have the potential to serve as a renewable source of transplantable tissue-specific stem cells. However, the molecular cues necessary to direct the differentiation of ESCs toward specific cell lineages remain obscure. Here we report the successful induction of ESC differentiation into mature functional T lymphocytes with a simple in vitro coculture system. The directed differentiation of ESCs into T cells required the engagement of Notch receptors by Delta-like 1 ligand (DL1) expressed on the OP9-DL1 stromal cell line. We found a normal program of T cell differentiation in ESC-OP9-DL1 cell cocultures. ESC-derived T cell progenitors effectively reconstituted the T cell compartment of immunodeficient mice, enabling an effective response to a viral infection. These findings provide a powerful tool for the molecular analysis of T cell development and open new avenues for the development of immunotherapeutic approaches using defined sources of stem cells.

Animals↗

PubMed2004

Heterogeneity among DN1 prothymocytes reveals multiple progenitors with different capacities to generate T cell and non-T cell lineages.

The nature of early T lineage progenitors in the thymus or bone marrow remains controversial. Here we assess lineage capacity and proliferative potential among five distinct components of the earliest intrathymic stage (DN1, CD25(-)44(+)). All of these express one or more hemato-lymphoid lineage markers. All can produce T lineage cells, but only two of them display kinetics of differentiation, proliferative capacity, and other traits consistent with being canonical T progenitors. The latter also appeared limited to producing cells of the T or NK lineages, while B lineage potential derived mainly from the other, less typical T progenitors. In addition to precisely defining canonical early progenitors in the thymus, this work reconciles conflicting results from numerous groups by showing that multiple progenitors with a DN1 phenotype home to the thymus and make T cells, but possess different proliferative potentials and lineage capacities.

Animals↗

PubMed2002

Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro.

The molecular interactions provided by the thymic microenvironment that predicate T cell development remain obscure. Here, we show that a bone marrow stromal cell line ectopically expressing the Notch ligand Delta-like-1 loses its ability to support B cell lymphopoiesis, but acquires the capacity to induce the differentiation of hematopoietic progenitors into CD4 CD8 double- and single-positive T cells. Both gammadelta-TCR(+) and alphabeta-TCR(+) T cells are generated, and CD8(+) TCR(hi) cells produce gamma-interferon following CD3/TCR stimulation. These results establish that expression of Delta-like-1 on stromal cells provides key signals for the induction of T cell lineage commitment, stage-specific progenitor expansion, TCR gene rearrangement, and T cell differentiation in the absence of a thymus. Thus, it is likely that Delta-like-1/Notch interactions by the thymus underpin its unique ability to promote lineage commitment and differentiation of T cells.

Animals↗