The microRNA biogenesis machinery modulates lineage commitment during αß T cell development.

Differentiation of CD4(+) helper and CD8(+) cytotoxic αß T cells from CD4(+)CD8(+) thymocytes involves upregulation of lineage-specifying transcription factors and transcriptional silencing of CD8 or CD4 coreceptors, respectively, in MHC class II or I (MHCII or I)-restricted thymocytes. In this study, we demonstrate that inactivation of the Dicer RNA endonuclease in murine thymocytes impairs initiation of Cd4 and Cd8 silencing, leading to development of positively selected MHCI- and MHCII-restricted mature CD4(+)CD8(+) thymocytes. Expression of the antiapoptotic BCL2 protein or inactivation of the p53 proapoptotic protein rescues these thymocytes from apoptosis, increasing their frequency and permitting accumulation of CD4(+)CD8(+) αß T cells in the periphery. Dicer-deficient MHCI-restricted αß T cells fail to normally silence Cd4 and display impaired induction of the CD8 lineage-specifying transcription factor Runx3, whereas Dicer-deficient MHCII-restricted αß T cells show impaired Cd8 silencing and impaired induction of the CD4 lineage-specifying transcription factor Thpok. Finally, we show that the Drosha RNA endonuclease, which functions upstream of Dicer in microRNA biogenesis, also regulates Cd4 and Cd8 silencing. Our data demonstrate a previously dismissed function for the microRNA biogenesis machinery in regulating expression of lineage-specifying transcription factors and silencing of Cd4 and Cd8 during αß T cell differentiation.

T cell development requires constraint of the myeloid regulator C/EBP-α by the Notch target and transcriptional repressor Hes1.

Notch signaling induces gene expression of the T cell lineage and discourages alternative fate outcomes. Hematopoietic deficiency in the Notch target Hes1 results in severe T cell lineage defects; however, the underlying mechanism is unknown. We found here that Hes1 constrained myeloid gene-expression programs in T cell progenitor cells, as deletion of the myeloid regulator C/EBP-α restored the development of T cells from Hes1-deficient progenitor cells. Repression of Cebpa by Hes1 required its DNA-binding and Groucho-recruitment domains. Hes1-deficient multipotent progenitor cells showed a developmental bias toward myeloid cells and dendritic cells after Notch signaling, whereas Hes1-deficient lymphoid progenitor cells required additional cytokine signaling for diversion into the myeloid lineage. Our findings establish the importance of constraining developmental programs of the myeloid lineage early in T cell development.

T cell factor 1 is required for group 2 innate lymphoid cell generation.

Group 2 innate lymphoid cells (ILC2) are innate lymphocytes that confer protective type 2 immunity during helminth infection and are also involved in allergic airway inflammation. Here we report that ILC2 development required T cell factor 1 (TCF-1, the product of the Tcf7 gene), a transcription factor also implicated in T cell lineage specification. Tcf7(-/-) mice lack ILC2, and were unable to mount ILC2-mediated innate type 2 immune responses. Forced expression of TCF-1 in bone marrow progenitors partially bypassed the requirement for Notch signaling in the generation of ILC2 in vivo. TCF-1 acted through both GATA-3-dependent and GATA-3-independent pathways to promote the generation of ILC2. These results are reminiscent of the critical roles of TCF-1 in early T cell development. Hence, transcription factors that underlie early steps of T cell development are also implicated in the development of innate lymphoid cells.

Early T-cell progenitors are the major granulocyte precursors in the adult mouse thymus.

The mouse thymus supports T-cell development, but also contains non-T-cell lineages such as dendritic cells, macrophages, and granulocytes that are necessary for T-cell repertoire selection and apoptotic thymocyte clearance. Early thymic progenitors (ETPs) are not committed to the T-cell lineage, as demonstrated by both in vitro and in vivo assays. Whether ETPs realize non-T-cell lineage potentials in vivo is not well understood and indeed is controversial. In the present study, we investigated whether ETPs are the major precursors of any non-T-lineage cells in the thymus. We analyzed the development of these populations under experimental circumstances in which ETPs are nearly absent due to either abrogated thymic settling or inhibition of early thymic development by genetic ablation of IL-7 receptorα or Hes1. Results obtained using multiple in vivo approaches indicate that the majority of thymic granulocytes derive from ETPs. These data indicate that myelolymphoid progenitors settle the thymus and thus clarify the pathways by which stem cells give rise to downstream blood cell lineages.

Erythroid/myeloid progenitors and hematopoietic stem cells originate from distinct populations of endothelial cells.

Hematopoietic stem cells (HSCs) and an earlier wave of definitive erythroid/myeloid progenitors (EMPs) differentiate from hemogenic endothelial cells in the conceptus. EMPs can be generated in vitro from embryonic or induced pluripotent stem cells, but efforts to produce HSCs have largely failed. The formation of both EMPs and HSCs requires the transcription factor Runx1 and its non-DNA binding partner core binding factor ß (CBFß). Here we show that the requirements for CBFß in EMP and HSC formation in the conceptus are temporally and spatially distinct. Panendothelial expression of CBFß in Tek-expressing cells was sufficient for EMP formation, but was not adequate for HSC formation. Expression of CBFß in Ly6a-expressing cells, on the other hand, was sufficient for HSC, but not EMP, formation. The data indicate that EMPs and HSCs differentiate from distinct populations of hemogenic endothelial cells, with Ly6a expression specifically marking the HSC-generating hemogenic endothelium.

Runx1 loss minimally impacts long-term hematopoietic stem cells.

RUNX1 encodes a DNA binding subunit of the core-binding transcription factors and is frequently mutated in acute leukemia, therapy-related leukemia, myelodysplastic syndrome, and chronic myelomonocytic leukemia. Mutations in RUNX1 are thought to confer upon hematopoietic stem cells (HSCs) a pre-leukemic state, but the fundamental properties of Runx1 deficient pre-leukemic HSCs are not well defined. Here we show that Runx1 deficiency decreases both apoptosis and proliferation, but only minimally impacts the frequency of long term repopulating HSCs (LT-HSCs). It has been variously reported that Runx1 loss increases LT-HSC numbers, decreases LT-HSC numbers, or causes age-related HSC exhaustion. We attempt to resolve these discrepancies by showing that Runx1 deficiency alters the expression of several key HSC markers, and that the number of functional LT-HSCs varies depending on the criteria used to score them. Finally, we identify genes and pathways, including the cell cycle and p53 pathways that are dysregulated in Runx1 deficient HSCs.

Delivery of progenitors to the thymus limits T-lineage reconstitution after bone marrow transplantation.

T-cell production depends on the recruitment of hematopoietic progenitors into the thymus. T cells are among the last of the hematopoietic lineages to recover after bone marrow transplantation (BMT), but the reasons for this delay are not well understood. Under normal physiologic conditions, thymic settling is selective and either CCR7 or CCR9 is required for progenitor access into the thymus. The mechanisms of early thymic reconstitution after BMT, however, are unknown. Here we report that thymic settling is briefly CCR7/CCR9-independent after BMT but continues to rely on the selectin ligand PSGL-1. The CCR7/CCR9 independence is transient, and by 3 weeks after BMT these receptors are again strictly required. Despite the normalization of thymic settling signals, the rare bone marrow progenitors that can efficiently repopulate the thymus are poorly reconstituted for at least 4 weeks after BMT. Consistent with reduced progenitor input to the thymus, intrathymic progenitor niches remain unsaturated for at least 10 weeks after BMT. Finally, we show that thymic recovery is limited by the number of progenitors entering the thymus after BMT. Hence, T-lineage reconstitution after BMT is limited by progenitor supply to the thymus.

Eliciting the T cell fate with Notch.

Multipotent progenitors arrive at the thymus via the blood. Constraining the non-T cell fates of these progenitors while promoting the T cell fate is a major task of the thymus. Notch appears to be the initial trigger for a developmental program that eventually results in T cell lineage commitment. Several downstream targets of Notch are known, but the specific roles of each are poorly understood. A greater understanding of how Notch and other thymic signals direct progenitors to a T cell fate could be useful for translational work. For example, such work could eventually allow for the generation of fully competent T cells in vitro that could supplement the waning T cell numbers and function in the elderly and boost T cell-mediated immunity in patients with immunodeficiency and after stem cell transplantation.