The preTCR‐dependent DN3 to DP transition requires Notch signaling, is improved by CXCL12 signaling and is inhibited by IL‐7 signaling

Abstract

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...

Ephrin-B–Dependent Thymic Epithelial Cell–Thymocyte Interactions Are Necessary for Correct T Cell Differentiation and Thymus Histology Organization: Relevance for Thymic Cortex Development

“…During early phases of thymic organogenesis, DLL4 is expressed by the majority of TECs, and its expression diminishes with age, being confined mainly to a fraction of cTECs at the corticomedullary junction in the adult thymus (49,50). Moreover, it has been recently described that DN3 thymocytes require a DLL4-induced Notch signal to differentiate into DP cells (31)(32)(33). In contrast, in RAG 2/2 mice, which have a blockade on T cell development at DN3, most TECs are MHC II hi Ly51 hi , and just 15% of the TECs are MHC II lo Ly51 lo , this latter subpopulation being higher (30%) in TCRa 2/2 mice, in which T cell differentiation is blocked in the DP to SP transition (28).…”

“…1C). The association of the expression of DLL4, specifically to the DN microenvironment, has been reported (31)(32)(33), in that the different levels of expression of Ly51 seem to distinguish between two functionally different cTEC subsets. Mutant mice showed alterations in Ly51 expression and the distribution of these cTEC subpopulations.…”

Developmental gene networks: a triathlon on the course to T cell identity

“…3f) 34 . Despite the downregulation of these growth-supporting systems, the cells shift into very rapid proliferation, as the cells become highly responsive to chemokine signaling through the receptor Cxcr4 139, 140 . Proliferation not only expands the potential TCR repertoire pool, it also helps αβ T-cells confirm their lineage commitment by allowing the cells to dilute out the last vestiges of earlier regulatory molecules and to reset epigenetic marks 37 .…”

Cytokines, Transcription Factors, and the Initiation of T-Cell Development

“…Despite the shutoff of IL-7R- and Notch-dependent growth-supporting systems, the DN4 cells enter a period of very rapid proliferation that is important for full phenotypic differentiation to the next stage (Kreslavsky et al 2012). This is supported in part by chemokine signaling through CXCL12/CXCR4 (Janas et al 2010; Tussiwand et al 2011). The proliferative burst also appears to be supported by very dynamic use of TCF1 and LEF1 in what may be a transient, self-limited canonical Wnt pathway response (Yu et al 2010), although the details are still under debate.…”