The BCL2A1 gene as a pre–T cell receptor–induced regulator of thymocyte survival

The pre–T cell receptor (TCR) is expressed early during T cell development and imposes a tight selection for differentiating T cell progenitors. Pre-TCR–expressing cells are selected to survive and differentiate further, whereas pre-TCR− cells are “negatively” selected to die. The mechanisms of pre-TCR–mediated survival are poorly understood. Here, we describe the induction of the antiapoptotic gene BCL2A1 (A1) as a potential mechanism regulating inhibition of pre–T cell death. We characterize in detail the signaling pathway involved in A1 induction and show that A1 expression can induce pre–T cell survival by inhibiting activation of caspase-3. Moreover, we show that in vitro “knockdown” of A1 expression can compromise survival even in the presence of a functional pre-TCR. Finally, we suggest that pre-TCR–induced A1 overexpression can contribute to T cell leukemia in both mice and humans

Negative and positive selection of antigen-specific cytotoxic T lymphocytes affected by the α3 domain of MHC I molecules

THE α1 and α2 domains of major histocompatibility complex (MHC) class I molecules function in the binding and presentation of foreign peptides to the T-cell antigen receptor and control both negative and positive selection of the T-cell repertoire. Although the α3 domain of class I is not involved in peptide binding, it does interact with the T-cell accessory molecule, CDS. CDS is important in the selection of T cells as anti-CDS antibody injected into perinatal mice interfers with this process. We previously used a hybrid class I molecule with the α1/α2 domains from L^d and the α3 domain from Q7^b and showed that this molecule binds an L^d-restricted peptide but does not interact with CD8-dependent cytotoxic T lymphocytes. Expression of this molecule in transgenic mice fails to negatively select a subpopulation of anti-L^d cytotoxic T lymphocytes. In addition, positive selection of virus-specific L^d-restricted cytotoxic T lymphocytes does not occur. We conclude that besides the α1/α2 domains of class I, the α3 domain plays an important part in both positive and negative selection of antigen-specific cells

The RhoA transcriptional program in pre-T cells

The GTPase RhoA is essential for the development of pre-T cells in the thymus. To investigate the mechanisms used by RhoA to control thymocyte development we have used Affymetrix gene profiling to identify RhoA regulated genes in T cell progenitors. The data show that RhoA plays a specific and essential role in pre-T cells because it is required for the expression of transcription factors of the Egr-1 and AP-1 families that have critical functions in thymocyte development. Loss of RhoA function in T cell progenitors causes a developmental block that pheno-copies the consequence of losing pre-TCR expression in Recombinase gene 2 (Rag2) null mice. Transcriptional profiling reveals both common and unique gene targets for RhoA and the pre-TCR indicating that RhoA participates in the pre-TCR induced transcriptional program but also mediates pre-TCR independent gene transcription

A genome wide transcriptional model of the complex response to pre-TCR signalling during thymocyte differentiation

Developing thymocytes require pre-TCR signalling to differentiate from CD4−CD8− double negative to CD4+CD8+ double positive cell. Here we followed the transcriptional response to pre-TCR signalling in a synchronised population of differentiating double negative thymocytes. This time series analysis revealed a complex transcriptional response, in which thousands of genes were up and down-regulated before changes in cell surface phenotype were detected. Genome-wide measurement of RNA degradation of individual genes showed great heterogeneity in the rate of degradation between different genes. We therefore used time course expression and degradation data and a genome wide transcriptional modelling (GWTM) strategy to model the transcriptional response of genes up-regulated on pre-TCR signal transduction. This analysis revealed five major temporally distinct transcriptional activities that up regulate transcription through time, whereas down-regulation of expression occurred in three waves. Our model thus placed known regulators in a temporal perspective, and in addition identified novel candidate regulators of thymocyte differentiation

Characterization of Developmental Pathway of Natural Killer Cells from Embryonic Stem Cells In Vitro

In vitro differentiation of embryonic stem (ES) cells is often used to study hematopoiesis. However, the differentiation pathway of lymphocytes, in particular natural killer (NK) cells, from ES cells is still unclear. Here, we used a multi-step in vitro ES cell differentiation system to study lymphocyte development from ES cells, and to characterize NK developmental intermediates. We generated embryoid bodies (EBs) from ES cells, isolated CD34(+) EB cells and cultured them on OP9 stroma with a cocktail of cytokines to generate cells we termed ES-derived hematopoietic progenitors (ES-HPs). EB cell subsets, as well as ES-HPs derived from EBs, were tested for NK, T, B and myeloid lineage potentials using lineage specific cultures. ES-HPs derived from CD34(+) EBs differentiated into NK cells when cultured on OP9 stroma with IL-2 and IL-15, and into T cells on Delta-like 1-transduced OP9 (OP9-DL1) with IL-7 and Flt3-L. Among CD34(+) EB cells, NK and T cell potentials were detected in a CD45(−) subset, whereas CD45(+) EB cells had myeloid but not lymphoid potentials. Limiting dilution analysis of ES-HPs generated from CD34(+)CD45(−) EB cells showed that CD45(+)Mac-1(−)Ter119(−) ES-HPs are highly enriched for NK progenitors, but they also have T, B and myeloid potentials. We concluded that CD45(−)CD34(+) EB cells have lymphoid potential, and they differentiate into more mature CD45(+)Lin(−) hematopoietic progenitors that have lymphoid and myeloid potential. NK progenitors among ES-HPs are CD122(−) and they rapidly acquire CD122 as they differentiate along the NK lineage

The kinesin motor protein Kif7 is required for T-cell development and normal MHC expression on thymic epithelial cells (TEC) in the thymus

Kif7 is a ciliary kinesin motor protein that regulates mammalian Hedgehog pathway activation through influencing structure of the primary cilium. Here we show that Kif7 is required for normal T-cell development, despite the fact that T-cells lack primary cilia. Analysis of Kif7-deficient thymus showed that Kif7-deficiency increases the early CD44+CD25+CD4-CD8- thymocyte progenitor population but reduces differentiation to CD4+CD8+ double positive (DP) cell. At the transition from DP to mature T-cell, Kif7-deficiency selectively delayed maturation to the CD8 lineage. Expression of CD5, which correlates with TCR signal strength, was reduced on DP and mature CD4 and CD8 cells, as a result of thymocyte-intrinsic Kif7-deficiency, and Kif7-deficient T-cells from radiation chimeras activated less efficiently when stimulated with anti-CD3 and anti-CD28 in vitro. Kif7-deficient thymocytes showed higher expression of the Hedgehog target gene Ptch1 than WT, but were less sensitive to treatment with recombinant Shh, and Kif7-deficient T-cell development was refractory to neutralisation of endogenous Hh proteins, indicating that Kif7-deficient thymocytes were unable to interpret changes in the Hedgehog signal. In addition, Kif7-deficiency reduced cell-surface MHCII expression on thymic epithelial cells

Down-regulation of Tumor Necrosis Factor α Expression by Activating Transcription Factor 2 Increases UVC-Induced Apoptosis of Late-stage Melanoma Cells

To identify mechanisms whereby activating transcription factor 2 (ATF2) alters the radiation resistance of human melanoma cells, we examined the possible role of ATF2 in UVC-induced apoptosis. Forced expression of full-length or truncated (Δ1–195 amino acids) forms of ATF2 in LU1205, a late-stage human melanoma cell line, elevated the levels of UVC-induced apoptosis. At the same time, either truncated or full-length forms of ATF2 reduced UVC-induced activation of the tumor necrosis factor-α (TNFα) promoter and decreased expression of TNFα. Forced expression of c-Jun in ATF2-expressing melanoma cells restored TNFα expression, suggesting that both forms of ATF2 sequestered transcription factors that positively regulate TNFα expression in response to UV irradiation. Antagonistic antibodies to Fas, but not to TNFR1, efficiently suppressed UVC-induced apoptosis, suggesting that the Fas pathway mediates the primary apoptotic signal in melanoma cells whereas the TNFR1 pathway elicits a survival signal. Indeed, treatment of melanoma cells with TNFα before UVC irradiation partially suppressed UVC-induced apoptosis, further supporting the protective role of TNFα in UVC-treated melanoma cells. Taken together, our findings suggest that ATF2 contributes to UVC-induced apoptosis through transcriptional silencing of TNFα, which balances Fas-mediated cell death in melanoma