Notch/Delta signaling constrains reengineering of pro-T cells by PU.1

PU.1 is essential for early stages of mouse T cell development but antagonizes it if expressed constitutively. Two separable mechanisms are involved: attenuation and diversion. Dysregulated PU.1 expression inhibits pro-T cell survival, proliferation, and passage through β-selection by blocking essential T cell transcription factors, signaling molecules, and Rag gene expression, which expression of a rearranged T cell antigen receptor transgene cannot rescue. However, Bcl2 transgenic cells are protected from this attenuation and may even undergo β-selection, as shown by PU.1 transduction of defined subsets of Bcl2 transgenic fetal thymocytes with differentiation in OP9-DL1 and OP9 control cultures. The outcome of PU.1 expression in these cells depends on Notch/Delta signaling. PU.1 can efficiently divert thymocytes toward a myeloid-like state with multigene regulatory changes, but Notch/Delta signaling vetoes diversion. Gene expression analysis distinguishes sets of critical T lineage regulatory genes with different combinatorial responses to PU.1 and Notch/Delta signals, suggesting particular importance for inhibition of E proteins, Myb, and/or Gfi1 (growth factor independence 1) in diversion. However, Notch signaling only protects against diversion of cells that have undergone T lineage specification after Thy-1 and CD25 up-regulation. The results imply that in T cell precursors, Notch/Delta signaling normally acts to modulate and channel PU.1 transcriptional activities during the stages from T lineage specification until commitment

Transcription factor expression dynamics of early T-lymphocyte specification and commitment

Mammalian T lymphocytes are a prototype for development from adult pluripotent stem cells. While T-cell specification is driven by Notch signaling, T-lineage commitment is only finalized after prolonged Notch activation. However, no T-lineage specific regulatory factor has been reported that mediates commitment. We used a gene-discovery approach to identify additional candidate T-lineage transcription factors and characterized expression of > 100 regulatory genes in early T-cell precursors using realtime RT-PCR. These regulatory genes were also monitored in multilineage precursors as they entered T-cell or non-T-cell pathways in vitro; in non-T cells ex vivo; and in later T-cell developmental stages after lineage commitment. At least three major expression patterns were observed. Transcription factors in the largest group are expressed at relatively stable levels throughout T-lineage specification as a legacy from prethymic precursors, with some continuing while others are downregulated after commitment. Another group is highly expressed in the earliest stages only, and is downregulated before or during commitment. Genes in a third group undergo upregulation at one of three distinct transitions, suggesting a positive regulatory cascade. However, the transcription factors induced during commitment are not T-lineage specific. Different members of the same transcription factor family can follow opposite trajectories during specification and commitment, while factors co-expressed early can be expressed in divergent patterns in later T-cell development. Some factors reveal new regulatory distinctions between αβ and γδ T-lineage differentiation. These results show that T-cell identity has an essentially complex regulatory basis and provide a detailed framework for regulatory network modeling of T-cell specification

Developmental and Molecular Characterization of Emerging β- and γδ-Selected Pre-T Cells in the Adult Mouse Thymus

The first checkpoint in T cell development, β selection, has remained incompletely characterized for lack of specific surface markers. We show that CD27 is upregulated in DN3 thymocytes initiating β selection, concomitant with intracellular TCR-β expression. Clonal analysis determined that CD27^(high) DN3 cells generate CD4^+CD8^+ progeny with more than 90% efficiency, faster and more efficiently than the CD27^(low) majority. CD27 upregulation also occurs in γδ-selected DN3 thymocytes in TCR-β−/− mice and in IL2-GFP transgenic reporter mice where GFP marks the earliest emerging TCR-γδ cells from DN3 thymocytes. With CD27 to distinguish pre- and postselection DN3 cells, a detailed gene expression analysis defined regulatory changes associated with checkpoint arrest, with β selection, and with γδ selection. γδ selection induces higher CD5, Egr, and Runx3 expression as compared to β selection, but it triggers less proliferation. Our results also reveal differences in Notch/Delta dependence at the earliest stages of divergence between developing αβ and γδ T-lineage cells

Progression of regulatory gene expression states in fetal and adult pro-T-cell development

Precursors entering the T-cell developmental pathway traverse a progression of states characterized by distinctive patterns of gene expression. Of particular interest are regulatory genes, which ultimately control the dwell time of cells in each state and establish the mechanisms that propel them forward to subsequent states. Under particular genetic and developmental circumstances, the transitions between these states occur with different timing, and environmental feedbacks may shift the steady-state accumulations of cells in each state. The fetal transit through pro-T-cell stages is faster than in the adult and subject to somewhat different genetic requirements. To explore causes of such variation, this review presents previously unpublished data on differentiation gene activation in pro-T cells of pre-T-cell receptor-deficient mutant mice and a quantitative comparison of the profiles of transcription factor gene expression in pro-T-cell subsets of fetal and adult wildtype mice. Against a background of consistent gene expression, several regulatory genes show marked differences between fetal and adult expression profiles, including those encoding two basic helix-loop-helix antagonist Id factors, the Ets family factor SpiB and the Notch target gene Deltex1. The results also reveal global differences in regulatory alterations triggered by the first T-cell receptor-dependent selection events in fetal and adult thymopoiesis

Developmental and Molecular Characterization of Emerging β- and γδ-Selected Pre-T Cells in the Adult Mouse Thymus

The first checkpoint in T cell development, β selection, has remained incompletely characterized for lack of specific surface markers. We show that CD27 is upregulated in DN3 thymocytes initiating β selection, concomitant with intracellular TCR-β expression. Clonal analysis determined that CD27^(high) DN3 cells generate CD4^+CD8^+ progeny with more than 90% efficiency, faster and more efficiently than the CD27^(low) majority. CD27 upregulation also occurs in γδ-selected DN3 thymocytes in TCR-β−/− mice and in IL2-GFP transgenic reporter mice where GFP marks the earliest emerging TCR-γδ cells from DN3 thymocytes. With CD27 to distinguish pre- and postselection DN3 cells, a detailed gene expression analysis defined regulatory changes associated with checkpoint arrest, with β selection, and with γδ selection. γδ selection induces higher CD5, Egr, and Runx3 expression as compared to β selection, but it triggers less proliferation. Our results also reveal differences in Notch/Delta dependence at the earliest stages of divergence between developing αβ and γδ T-lineage cells

Costimulation by interleukin-1 of multiple activation responses in a developmentally restricted subset of immature thymocytes

An intriguing feature of thymocyte differentiation is that the competence to express both interleukin-(IL)2 and CD25 is acquired even prior to T cell recept or (TcR) expression. When T cell receptor-independent stimuli are used, immature cells can express IL-2 at levels comparable to mature cells, but unlike the mature cells, immature cells require IL-1 as a costimulus. Here we present evidence that IL-1 affects a variety of responses by members of the CD25^+ subset of immature thymocytes. Cells in this population are IL-1 dependent not only for induction of IL-2 expression, but also for high-level maintenance of CD25 expression. CD^(25+) expression is amplified by IL-1 through a mechanism highly sensitive to changes in Ca^(2+) ionophore concentration. The effects of IL-1 on CD25 maintenance are not mediated by IL-2, because of the divergent effects of cAMP on IL-2 and CD^(25+) expression. IL-1 costimulation also increases RNA accumulation in the cell cycle, and this effect too seems to be separable from the effects on IL-2 and CD^(25+) expression. All these effects of IL-1 are developmentally stage-specific, manifest in the CD25^+ subset of immature thymocytes but not in later-stage thymocytes or splenic T cells. Multiparameter cell sorting experiments that dissect the transitional stages between immature and TcR^+ thymocytes imply that all immature cells pass through an IL-1 responsive state. Responsiveness to IL-1 costimulation is then lost by these cells, apparently irreversibly, at a stage just prior to detectable cell-surface TcR expression. These results indicate that IL-1 responsiveness is a defining characteristic of the activation physiology of cells in a particularly important developmental stage

Activation of T cell antigen receptor alpha- and beta-chain genes in the thymus: implications for the lineages of developing cortical thymocytes

Mammalian T lymphocytes mature in the thymus through a series of differentiation events that involve both rapid proliferation and extensive cell death. The mechanisms that govern these processes are currently unknown; however, both mitogenesis and death affect particular subpopulations of cells, suggesting the selective amplification and destruction of specific T cell clones. In mature peripheral T cells, proliferation is most commonly triggered by the recognition of antigen through the T cell antigen receptor complex. If antigen recognition also controls proliferation in the thymus, the differential expression of antigen receptor genes during maturation could play some role in determining the fate of developing T cells. In this study, we examined the expression of the alpha- and beta-chain genes of the T cell antigen receptor in different subpopulations of adult thymocytes. We compared two postmitotic populations--one that appears committed to die and one that appears mature--and several blast cell populations that are enriched for precursors of one or another presumptive lineage. We have found that Lyt-2-, L3T4- precursor thymocytes express much lower levels of both alpha- and beta-chain mRNA than the cells likely to be their immediate descendents. Furthermore, our results show that the cells of the major cortical lineage, which have at least a 95% probability of death, nevertheless express high levels of mature mRNA encoding both the alpha- and the beta-chains of the T cell antigen receptor. These results have important implications for the mechanisms involved in the overproduction and elimination of this major class of T lymphocyte

Functional and phenotypic analysis of thymocytes in SCID mice. Evidence for functional response transitions before and after the SCID arrest point

Thymocytes from severe combined immune deficient (SCID) mice undergo developmental arrest at an early stage, before most TCR gene rearrangement. They therefore represent a natural test case to assess those aspects of T cell development that are TCR independent. Multiparameter flow cytometry was used to analyze the array of immature phenotypes present in the SCID thymus at steady state, as defined by the markers CD4, CD5, Sca-1, NK1.1, CD44, heat-stable antigen (HSA), and IL-2R alpha. The results suggest a simple developmental block in SCID mice rather than a program of aberrant differentiation. SCID thymocytes displayed efficient, developmentally regulated functional responses. Approximately 20-25% of the cells, mostly within the IL-2R alpha +HSA+CD44low fraction, could be induced to express IL-2. This IL-2 inducibility was highly dependent on IL-1 costimulation, in agreement with the behavior of normal immature thymocytes. These results formally demonstrate that competence to express IL-2 is developed independently of TCR expression or gene rearrangement. Comparison of the response properties of various SCID thymocyte subsets indicated that IL-2 inducibility is first likely to be acquired at an early (Sca-1++CD44++HSAlow) stage. A later functional transition was revealed by comparing patterns of IL-2R alpha regulation in normal and SCID IL-2R alpha +HSA+CD44low thymocytes. The SCID thymocytes uniformly maintained IL-2R alpha expression on in vitro stimulation, whereas only a minority of the normal cells in the corresponding subset could do so unless IL-1 was also added. The SCID arrest point thus appears to separate the IL-2R alpha +HSA+CD44low stage into distinct early (TCR independent) and late phases. Normal cells that progress beyond the SCID arrest point appear to lose, rather than gain, competence to make various responses, even before they leave the IL-2R alpha +HSA+CD44low stage. A model is proposed in which discrete changes in functional competence define novel transitions in early thymocyte development, at least some of which may be linked to TCR-beta gene rearrangement before positive or negative selection

Cross-lineage expression of Ig-β (B29) in thymocytes: Positive and negative gene regulation to establish T cell identity

Developmental commitment involves activation of lineage-specific genes, stabilization of a lineage-specific gene expression program, and permanent inhibition of inappropriate characteristics. To determine how these processes are coordinated in early T cell development, the expression of T and B lineage-specific genes was assessed in staged subsets of immature thymocytes. T lineage characteristics are acquired sequentially, with germ-line T cell antigen receptor-β transcripts detected very early, followed by CD3ɛ and terminal deoxynucleotidyl transferase, then pTα, and finally RAG1. Only RAG1 expression coincides with commitment. Thus, much T lineage gene expression precedes commitment and does not depend on it. Early in the course of commitment to the T lineage, thymocytes lose the ability to develop into B cells. To understand how this occurs, we also examined expression of well defined B lineage-specific genes. Although λ5 and Ig-α are not expressed, the μ(0) and Iμ transcripts from the unrearranged IgH locus are expressed early, in distinct patterns, then repressed just before RAG1 expression. By contrast, RNA encoding the B cell receptor component Ig-β was found to be transcribed in all immature thymocyte subpopulations and throughout most thymocyte differentiation. Ig-β expression is down-regulated only during positive selection of CD4(+)CD8(–) cells. Thus several key participants in the B cell developmental program are expressed in non-B lineage-committed cells, and one is maintained even through commitment to an alternative lineage, and repressed only after extensive T lineage differentiation. The results show that transcriptional activation of “lymphocyte-specific” genes can occur in uncommitted precursors, and that T lineage commitment is a composite of distinct positive and negative regulatory events