Additional supporting information may be found in the online version of this article at the publisher's web-site

The requirement for Notch signaling during T-cell development has been extensively studied. Nevertheless, the developmental stage at which it is required and whether additional signaling pathways are needed are still poorly understood. By using a stromalcell-free culture system, we show that sorted double-negative 3 (DN3) thymocytes only require a Delta-like-4-induced Notch signal to differentiate into double-positive (DP) cells. This differentiation process is preTCR-a dependent. DN3 cells undergo 4-5 proliferation cycles, and the addition of the chemokine CXCL12 improves proliferation. IL-7 blocks the differentiation of DN3 cells to DP cells but not the Notch-induced proliferation of cultured DN3 cells. The impaired differentiation correlates with an inhibition of Rag-2 upregulation. Overall, the in vitro stromal-cell-free culture system presented here also provides a powerful and unique tool for studying the mechanisms involved in the positive and negative selection of T cells.Key words: Delta-like 4 and preTCR . DN3 cells . Double-positive cells . Notch IntroductionTo maintain T lymphopoiesis, progenitor cells from the BM constantly seed the thymus. In transplantation settings, it has been shown that different progenitors can home to the thymus and generate T cells [1][2][3][4][5][6][7][8][9]. However, under physiological conditions the role of each of these progenitors is still unclear. It is generally believed that the so-called thymic seeding precursor (TSP) still has the capability of generating B cells, NK cells, DCs and other myeloid cells [2,5,6,10,11]. Upon receiving the Notch signal, the differentiation potential towards the B-cell lineage of the TSP is rapidly lost [2,10,11], whereas other lineage options are still maintained [2,5,6,10,11]. However, a recent study using an IL-7Ra reporter mouse indicated that the non-T-cell lineage differentiation of these progenitors is not, or only very rarely, occurring under physiologic conditions [12,13].Within the thymus, the earliest thymocytes are characterized by the absence of CD4 and CD8 expression and are therefore called double-negative (DN) cells. Based on the expression of CD25, CD44 and CD117, DN cells can be subdivided into four subpopulations. DN1 cells express high cell surface levels of CD44 and CD117, and are negative for CD25, whereas their DN2 progeny express all three markers [14][15][16]. In vitro, DN1 and DN2 cells still possess the capacity to differentiate into NK cells, DCs and other myeloid cells [5,6,11,17] suggesting that they are not yet restricted to the T-cell lineage. T-cell commitment is achieved at the DN3 stage. However, the mechanisms operating . It is at this stage of development that the rearrangement of the TCR-b chain locus is completed, and the cells are selected for a productive TCR-b chain rearrangement, also known as the b-selection checkpoint [18][19][20]. DN3 cells that have rearranged their TCR-b chain locus unproductively can either differentiate into g/d T cells or will otherwise undergo apoptosis. DN3 cells car...

T cell development is critically dependent on successful rearrangement of antigen-receptor chains. At the β-selection checkpoint, only cells with a functional rearrangement continue in development. However, how nonselected T cells proceed in their dead-end fate is not clear. We identified low CD27 expression to mark pre-T cells that have failed to rearrange their β-chain. Expression profiling and single-cell transcriptome clustering identified a developmental trajectory through β-selection and revealed specific expression of the transcription factor Duxbl at a stage of high recombination activity before β-selection. Conditional transgenic expression of Duxbl resulted in a developmental block at the DN3-to-DN4 transition due to reduced proliferation and enhanced apoptosis, whereas RNA silencing of Duxbl led to a decrease in apoptosis. Transcriptome analysis linked Duxbl to elevated expression of the apoptosis-inducing Oas/RNaseL pathway. RNaseL deficiency or sustained Bcl2 expression led to a partial rescue of cells in Duxbl transgenic mice. These findings identify Duxbl as a regulator of β-selection by inducing apoptosis in cells with a nonfunctional rearrangement.

No abstract

Between 10 and 20% of the peripheral cd T cells express cytoplasmic TCR-b proteins, but whether such TCR-b chains can partake in ab T-cell development has never been systematically investigated. Therefore, we reconstituted the T-cell compartment of CD3e-deficient mice with Pax5-TCR-b deficient proB cells expressing, via a retroviral vector, TCR-b chains from either peripheral cd or ab T cells. Recipient thymi reconstituted with proB cells containing empty vector were small (o15 Â 10 6 cells), contained few cd T but no ab T cells. In contrast, thymi from mice receiving proB cells containing cd or ab T-cellderived TCR-b chains contained 80-130 Â 10 6 cells, and showed a normal CD4, CD8 and ab TCR expression pattern. However, regardless of the source of TCR-b chain, reconstituted mice rapidly showed signs of autoimmunity dying 5-15 wk following reconstitution. Autoimmune disease induction could be prevented by co-transfer of Treg cells thereby allowing the functionality of the generated T cells to be assessed. Results obtained show that TCR-b chains from cd T cells can efficiently take part in ab T-cell development. The implications of these findings for cd T-cell development will be discussed.Key words: ab T cells . gd T cells . TCR-b chains . pre-TCR . Thymus Supporting Information available online Introduction T-cell development takes place in the thymus from progenitors of bone marrow origin. By now it is generally believed that a cell called a thymus settling progenitor (TSP), characterized by the expression of CD44, CD117, CD135 and the chemokine receptor CCR9 [1][2][3][4] is the bone marrow cell that enters the thymus. However, transplantation experiments in T-cell-deficient mice have indicated that other progenitors can enter the thymus and may take part in T-cell development [2,5,6].Early T-cell progenitors in the thymus lack CD4 and CD8 expression and are therefore called double negative (DN) cells. Based on the differential expression of CD44, CD25 and CD117, DN cells can be subdivided into DN1-4 subpopulations. Thus DN1 cells are CD44 + , CD25 À and CD117 High and within this population, TSP are included as a CD135 + subpopulation [2,4,7,8]. DN1 cells differentiate through a CD44 + , CD25 + , CD117 high DN2 stage to become CD44 À , CD25 + , CD117 À/low DN3 cells [8].Ã These authors contributed equally to this work. 3520Both DN1 and DN2 cells still possess multi-lineage developmental potential, including that for NK, dendritic and myeloid cells [9][10][11] whereas B-cell lineage potential seems to be restricted to the TSP population within the DN1 subset [1,2,4,7]. By largely unknown mechanisms, commitment to the T-cell lineage is acquired at the DN2 to DN3 cell transition [9,12]. However, cells at the DN3 stage still have the option to develop into either ab or gd T-cell lineage cells [13][14][15]. A relatively large number of studies have indicated that the expression of the pre-TCR plays a crucial role in the development of ab T cells [14][15][16]. Recent, elegant, single cell studies have indicated ...