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Cyclin C was cloned as a growth-promoting G1 cyclin, and was also shown to regulate gene transcription. Here we report that in vivo cyclin C acts as a haploinsufficient tumour suppressor, by controlling Notch1 oncogene levels. Cyclin C activates an 'orphan' CDK19 kinase, as well as CDK8 and CDK3. These cyclin-C-CDK complexes phosphorylate the Notch1 intracellular domain (ICN1) and promote ICN1 degradation. Genetic ablation of cyclin C blocks ICN1 phosphorylation in vivo, thereby elevating ICN1 levels in cyclin-C-knockout mice. Cyclin C ablation or heterozygosity collaborates with other oncogenic lesions and accelerates development of T-cell acute lymphoblastic leukaemia (T-ALL). Furthermore, the cyclin C encoding gene CCNC is heterozygously deleted in a significant fraction of human T-ALLs, and these tumours express reduced cyclin C levels. We also describe point mutations in human T-ALL that render cyclin-C-CDK unable to phosphorylate ICN1. Hence, tumour cells may develop different strategies to evade inhibition by cyclin C.

SummaryD-cyclins represent components of cell cycle machinery. To test the efficacy of targeting D-cyclins in cancer treatment, we engineered mouse strains that allow acute and global ablation of individual D-cyclins in a living animal. Ubiquitous shutdown of cyclin D1 or inhibition of cyclin D-associated kinase activity in mice bearing ErbB2-driven mammary carcinomas triggered tumor cell senescence, without compromising the animals’ health. Ablation of cyclin D3 in mice bearing Notch1-driven T cell acute lymphoblastic leukemias (T-ALL) triggered tumor cell apoptosis. Such selective killing of leukemic cells can also be achieved by inhibiting cyclin D associated kinase activity in mouse and human T-ALL models. Inhibition of cyclin D-kinase activity represents a highly-selective anticancer strategy that specifically targets cancer cells without significantly affecting normal tissues.

Binding of Smad3 to the foxp3 enhancer is not required for thymic T reg cell development, but thymic involution with aging reveals the contribution of TGF-β–Smad2 signaling in gut T reg cell development., Regulatory T cells (T reg cells) are essential for the prevention of autoimmunity throughout life. T reg cell development occurs intrathymically but a subset of T reg cells can also differentiate from naive T cells in the periphery. In vitro, Smad signaling facilitates conversion of naive T cells into T reg cells but results in unstable Foxp3 expression. The TGF-β–Smad response element in the foxp3 locus is located in the CNS1 region in close proximity to binding sites for transcription factors implicated in TCR and retinoic acid signaling. From in vitro experiments it was previously postulated that foxp3 transcription represents a hierarchical process of transcription factor binding in which Smad3 would play a central role in transcription initiation. However, in vitro conditions generate T reg cells that differ from T reg cells encountered in vivo. To address the relevance of Smad3 binding to the CNS1 enhancer in vivo, we generated mice that exclusively lack the Smad binding site (foxp3CNS1mut). We show that binding of Smad3 to the foxp3 enhancer is dispensable for T reg cell development in newborn and adult mice with the exception of the gut.

Fopx3(+) Treg safeguard against autoimmune diseases and immune pathology. The extrathymic conversion of naïve T cells into Foxp3(+) regulatory T cells can be achieved in vivo by the delivery of strong-agonist ligands under subimmunogenic conditions. Tolerogenic vaccination with strong-agonist mimetopes of self-antigen to promote self-antigen specific tolerance may represent the most specific and safest means of preventing autoimmunity. This review discusses the requirements for induction of dominant tolerance exerted by Foxp3(+) Tregs in autoimmunity with special emphasis on their impact to interfere with T1D. The future goals are the understanding of self-non-self discrimination at the cellular and molecular level, which should then enable investigators to develop clinical vaccination protocols that specifically interfere with unwanted immune responses.

The Notch signaling pathway is an evolutionarily conserved cell signaling system present in most multicellular organisms, as it controls cell fate specification by regulating cell proliferation, differentiation, apoptosis, and survival. Regulation of the Notch signaling pathway can be achieved at multiple levels. Notch proteins are involved in lineage fate decisions in a variety of tissues in various species. Notch is essential for T lineage cell differentiation including T versus B and αβ versus γδ lineage specification. In this paper, we discuss Notch signaling in normal T-cell maturation and differentiation as well as in T-cell acute lymphoblastic lymphoma/leukemia.

Both αβ and γδ T cells develop in the thymus from a common progenitor. Historically distinguished by their T-cell receptor (TCR), these lineages are now defined on the basis of distinct molecular programs. Intriguingly, in many transgenic and knockout systems these programs are mismatched with the TCR type, leading to the development of γδ lineage cells driven by αβTCR and vice versa. These puzzling observations were recently explained by the demonstration that TCR signal strength, rather than TCR type per se, instructs lineage fate, with stronger TCR signal favoring γδ and weaker signal favoring αβ lineage fates. These studies also highlighted the ERK (extracellular signal regulated kinase)-Egr (early growth response)-Id3 (inhibitor of differentiation 3) axis as a potential molecular switch downstream of TCR that determines lineage choice. Indeed, removal of Id3 was sufficient to redirect TCRγδ transgenic cells to the αβ lineage, even in the presence of strong TCR signal. However, in TCR non-transgenic Id3 knockout mice the overall number of γδ lineage cells was increased due to an outgrowth of a Vγ1Vδ6.3 subset, suggesting that not all γδ T cells depend on this molecular switch for lineage commitment. Thus, the γδ lineage may in fact be a collection of two or more lineages not sharing a common molecular program and thus equipollent to the αβ lineage. TCR signaling is not the only factor that is required for development of αβ and γδ lineage cells; other pathways, such as signaling from Notch and CXCR4 receptors, cooperate with the TCR in this process.

In T cell-mediated autoimmune diseases, self-reactive T cells with known antigen specificity appear to be particularly promising targets for antigen-specific induction of tolerance without compromising desired protective host immune responses. Several lines of evidence suggest that delivery of antigens to antigen-presenting dendritic cells (DCs) in the steady state (i.e., to immature DCs) may represent a suitable approach to induce antigen-specific T-cell tolerance peripherally. Here, we report that anti-DEC205–mediated delivery of the self-peptide proteolipid protein (PLP)139–151 to DCs ameliorated clinical symptoms in the PLP-induced SJL model of experimental autoimmune encephalomyelitis. Splenocytes from treated mice were anergized to PLP139–151, and IL-17 secretion was markedly reduced. Moreover, we show directly, using transgenic CD4 + Vβ6 + TCR T cells specific for PLP139–151, that, under the conditions of the present experiments, these cells also became anergic. In addition, evidence for a CD4 + T cell-mediated suppressor mechanism was obtained.

Two major T lymphocyte lineages--alphabeta and gammadelta T cells--develop in the thymus from common precursors. Differentiation of both lineages requires signals coming from TCRs. Development of alphabeta T cells is driven at early stages by signaling from the pre-TCR, most likely in a ligand-independent fashion, and later--by signals delivered by alphabetaTCRs binding to their ligands--classical or non-classical MHC molecules. gammadelta lineage cells likewise require TCR signaling for their differentiation. Recent work from several groups suggests that TCR signaling not only ensures the developmental progression towards alphabeta and gammadelta lineages but that signal strength instructs lineage fate: weaker TCR signal results in alphabeta and stronger--in gammadelta lineage commitment. However, as most gammadeltaTCRs remain orphan receptors, it is still debated whether strong signals from gammadeltaTCRs in development are generated in a ligand-dependent manner (as in the case of alphabetaTCRs), ligand-independent manner (as for pre-TCR) or both. Here we summarize evidence supporting a possible role for ligands in gammadelta T cell lineage commitment and the generation of gammadelta sublineages.

αβ and γδ T cells develop in the thymus from a common precursor. Although lineages initially were defined by the type of TCR they express, it soon became clear that the TCR type per se does not play a deterministic role in the lineage decision, since in various transgenic and knockout models, as well as in a small fraction of cells in wt mice, the TCRγδ can drive the differentiation of αβ lineage cells and the TCRαβ can drive differentiation of γδ lineage cells. Thus until recently it was unclear what determines lineage choice and at which stage the two lineages diverge. Recent observations suggest that TCR signal strength determines lineage fate and that lineage choice is made at or shortly after the first TCR-controlled checkpoint. While it is clear that the decision between αβ and γδ lineages is made at the first TCR-controlled checkpoint and the αβ sublineages split off later, it is less clear whether γδ sublineages divert already at the first TCR-controlled checkpoint or later. Recent experiments support the former view.

Regulatory T (Treg) cells that express the Foxp3 transcription factor are essential for lymphoid homeostasis and immune tolerance to self. Other nonimmunological functions of Treg cells, such as controlling metabolic function in adipose tissue, are also emerging. Treg cells originate primarily in the thymus, but can also be elicited from conventional T cells by in vivo exposure to low-dose antigen or homeostatic expansion or by activation in the presence of TGFβ in vitro. Treg cells are characterized by a distinct transcriptional signature controlled in part, but not solely, by Foxp3. For a better perspective on transcriptional control in Treg cells, we compared gene expression profiles of a broad panel of Treg cells from various origins or anatomical locations. Treg cells generated by different means form different subphenotypes and were identifiable by particular combinations of transcripts, none of which fully encompassed the entire Treg signature. Molecules involved in Treg cell effector function, chemokine receptors, and the transcription factors that control them were differentially represented in these subphenotypes. Treg cells from the gut proved dissimilar to cells elicited by exposure to TGFβ in vitro, but instead they resembled a CD103 + Klrg1 + subphenotype preferentially generated in response to lymphopenia.

To identify dysregulated pathways in distinct phases of NOTCH1-mediated T-cell leukemogenesis, as well as small-molecule inhibitors that could synergize with or substitute for γ-secretase inhibitors (GSIs) in T-cell acute lymphoblastic leukemia (T-ALL) therapy, we compared gene expression profiles in a Notch1-induced mouse model of T-ALL with those in human T-ALL. The overall patterns of NOTCH1-mediated gene expression in human and mouse T-ALLs were remarkably similar, as defined early in transformation in the mouse by the regulation of MYC and its target genes and activation of nuclear factor-κB and PI3K/AKT pathways. Later events in murine Notch1-mediated leukemogenesis included down-regulation of genes encoding tumor suppressors and negative cell cycle regulators. Gene set enrichment analysis and connectivity map algorithm predicted that small-molecule inhibitors, including heat-shock protein 90, histone deacetylase, PI3K/AKT, and proteasome inhibitors, could reverse the gene expression changes induced by NOTCH1. When tested in vitro, histone deacetylase, PI3K and proteasome inhibitors synergized with GSI in suppressing T-ALL cell growth in GSI-sensitive cells. Interestingly, alvespimycin, a potent inhibitor of the heat-shock protein 90 molecular chaperone, markedly inhibited the growth of both GSI-sensitive and -resistant T-ALL cells, suggesting that its loss disrupts signal transduction pathways crucial for the growth and survival of T-ALL cells.

International audience; Some gammadelta and alphabeta T lymphocytes exhibit an "innate" phenotype associated with rapid cytokine responses. The PLZF transcription factor is essential for the innate phenotype of NKT cells. This report shows that PLZF is likewise responsible for the innate, NKT-like phenotype of Vgamma1+Vdelta6.3/Vdelta6.4+ cells. TCR cross-linking induced PLZF expression in all polyclonal immature gammadelta thymocytes, suggesting that agonist selection might be required for PLZF induction. Transgenic expression of Vgamma1Vdelta6.4 TCR was sufficient to support the development of large numbers of PLZF+ T cells, further supporting the importance of the TCR for PLZF induction. Interestingly, expression of this TCR transgene led to the development of spontaneous dermatitis.

It has been reported that retinoic acid (RA) enhances regulatory T (T reg) cell conversion by inhibiting the secretion of cytokines that interfere with conversion. This report shows that these conclusions provide a partial explanation at best. First, RA not only interfered with cytokine secretion but also with the ability of these cytokines to inhibit T reg cell conversion of naive T cells. Furthermore, RA enhanced conversion even in the absence of inhibitory cytokines. The latter effect depended on the RA receptor alpha (RAR alpha) but did not require Smad3, despite the fact that RA enhanced Smad3 expression. The RAR alpha 1 isoform was not essential for RA-dependent enhancement of transforming growth factor beta-driven conversion, suggesting that conversion can also be mediated by RAR alpha 2. Interleukin (IL)-6 strongly reduced RAR alpha expression levels such that a deficiency of the predominant RAR alpha 1 isoform leaves too little RAR alpha 2 for RA to inhibit the generation of Th17 cells in the presence of IL-6.

αβ and γδ T cell lineages develop in the thymus from a common precursor. It is unclear at which stage of development commitment to these lineages takes place and in which way T cell receptor signaling contributes to the process. Recently, it was demonstrated that strong TCR signals favor γδ lineage development, whereas weaker TCR signals promote αβ lineage fate. Two models have been proposed to explain these results. The first model suggests that commitment occurs after TCR expression and TCR signaling directly instructs lymphocytes to adopt one or the other lineage fate. The second model suggests that commitment occurs before TCR expression and that TCR signaling merely confirms the lineage choice. By tracing the fate of single T cell precursors, this study shows that there is no commitment to either the αβ or γδ lineage before TCR expression and that modulation of TCR signaling in progeny of a single TCR-expressing cell changes lineage commitment.

Background & Aims: Disruption of the enteric glial cell (EGC) network is an early pathologic feature in Crohn's disease. To determine the contribution of antigen-specific CD8 and CD4 T cells to the breakdown of the EGC network, we studied specific autoimmune targeting of an ectopic antigen expressed by EGCs. Methods: Transgenic mice (GFAP-HA), which express the influenza hemagglutinin (HA) in EGCs, were either crossed with mice transgenic for a T-cell receptor (TCR) specific for a major histocompatibility complex (MHC) class I epitope of HA (CL4-TCR) or were adoptively transferred with conventional CL4 T cells. These were compared with GFAP-HA mice transferred with conventional T cells specific for an MHC class II epitope of HA (6.5). Results: Both CD8 and CD4 T-cell subtypes were activated in vivo in an antigen-specific manner; however, they differed substantially in their ability to expand in the mesenteric lymph nodes, trigger proinflammatory cytokines, and induce autoimmune damage in the intestine. Direct presentation of antigen, provided by lymph node stromal cells, caused the activation and deletion of CD8 T cells. This mechanism of T-cell tolerance did not affect CD4 T cells, which produced antigen-specific lethal autoimmunity. Conclusions: Our observations support a recently identified mechanism of peripheral T-cell tolerance that specifically protects against autoimmunity mediated by conventional CD8 T cells. Furthermore, we show that conventional CD4 T cells are not affected by this mechanism of tolerance, and their targeting of EGCs produces lethal intestinal autoimmunity.

Mutations resulting in overexpression of intracellular Notch1 (ICN1) are frequently observed in human T cell acute lymphoblastic leukemia (T-ALL). We have determined the consequences of ICN1 overexpression from retroviral vectors introduced into bone marrow cells. Early consequences are the generation of polyclonal nontumorigenic CD4(+)8(+) T cell receptor (TCR)-alphabeta(+) cells that do not qualify as tumor precursors despite the observation that they overexpress Notch 1 and c-Myc and degrade the tumor suppressor E2A by posttranslational modification. The first tumorigenic cells are detected among more immature CD4(-)8(+)TCR-alphabeta(-) cells that give rise to monoclonal tumors with a single, unique TCR-beta chain and diverse TCR-alpha chains, pinpointing malignant transformation to a stage after pre-TCR signaling and before completion of TCR-alpha rearrangement. In T-ALL, E2A deficiency is accompanied by further transcriptional up-regulation of c-Myc and concomitant dysregulation of the c-Myc-p53 axis at the transcriptional level. Even though the tumors consist of phenotypically heterogeneous cells, no evidence for tumor stem cells was found. As judged by array-based comparative genomic hybridization (array CGH) and spectral karyotype (SKY) analysis, none of the tumors arise because of genomic instability.

Inactivation of the CDKN2 genes that encode the p16(INK4A) and p14(ARF) proteins occurs in the majority of human T-cell acute lymphoblastic leukemias (T-ALLs). Ectopic expression of TAL1 and LMO1 genes is linked to the development of T-ALL in humans. In TAL1xLMO1 mice, leukemia develops in 100% of mice at 5 months. To identify the molecular events crucial to leukemic transformation, we produced several mouse models. We report here that expression of P16(INK4A) in developing TAL1xLMO1 thymocytes blocks leukemogenesis in the majority of the mice, and the leukemias that eventually develop show P16(INK4A) loss of expression. Events related to the T-cell receptor beta selection process are thought to be important for leukemic transformation. We show here that the absence of the pTalpha chain only slightly delays the appearance of TAL1xLMO1-induced T-ALL, which indicates a minor role of the pTalpha chain. We also show that the CD3epsilon-mediated signal transduction pathway is essential for this transformation process, since the TAL1xLMO1xCD3epsilon-deficient mice do not develop T-ALL for up to 1 year.

Activation of β-catenin has been causatively linked to the etiology of colon cancer. Conditional stabilization of this molecule in pro-T cells promotes thymocyte development without the requirement for pre-TCR signaling. We show here that activated β-catenin stalls the developmental transition from the double-positive (DP) to the single-positive (SP) thymocyte stage and predisposes DP thymocytes to transformation. β-Catenin–induced thymic lymphomas have a leukemic arrest at the early DP stage. Lymphomagenesis requires Rag activity, which peaks at this developmental stage, as well as additional secondary genetic events. A consistent secondary event is the transcriptional up-regulation of c-Myc, whose activity is required for transformation because its conditional ablation abrogates lymphomagenesis. In contrast, the expression of Notch receptors as well as targets is reduced in DP thymocytes with stabilized β-catenin and remains low in the lymphomas, indicating that Notch activation is not required or selected for in β-catenin–induced lymphomas. Thus, β-catenin activation may provide a mechanism for the induction of T-cell–acute lymphoblastic leukemia (T-ALL) that does not depend on Notch activation.

T cells developing in the thymus are derived from hematopoietic stem cells (HSCs) in the bone marrow (BM). Understanding the developmental steps linking multipotent HSCs to intrathymic T lineage-committed progenitors is important for understanding cancer in T lineage cells, improving T cell reconstitution after BM transplantation, and designing gene-therapy approaches to treat defective T cell development or function. Such an understanding may also help ameliorate immunological defects in aging. This review covers the differentiation steps between HSCs and committed T cell progenitors within the thymus.

Foxp3+CD4+CD25+ regulatory T (Treg) cells are essential for the prevention of autoimmunity1,2. Treg cells have an attenuated cytokine response to T-cell receptor stimulation, and can suppress the proliferation and effector function of neighbouring T cells3,4. The forkhead transcription factor Foxp3 (forkhead box P3) is selectively expressed in Treg cells, is required for Treg development and function, and is sufficient to induce a Treg phenotype in conventional CD4+CD25- T cells5,6,7,8. Mutations in Foxp3 cause severe, multi-organ autoimmunity in both human and mouse9,10,11. FOXP3 can cooperate in a DNA-binding complex with NFAT (nuclear factor of activated T cells) to regulate the transcription of several known target genes12. However, the global set of genes regulated directly by Foxp3 is not known and consequently, how this transcription factor controls the gene expression programme for Treg function is not understood. Here we identify Foxp3 target genes and report that many of these are key modulators of T-cell activation and function. Remarkably, the predominant, although not exclusive, effect of Foxp3 occupancy is to suppress the activation of target genes on T-cell stimulation. Foxp3 suppression of its targets appears to be crucial for the normal function of Treg cells, because overactive variants of some target genes are known to be associated with autoimmune disease.

Summary Mechanisms of dominant tolerance have evolved within the mammalian immune system to prevent inappropriate immune responses. CD4 + CD25 + regulatory T (T reg ) cells have emerged as central constituents of this suppressive activity. By using multiphoton intravital microscopy in lymph nodes (LNs) of anesthetized mice, we have analyzed how cytotoxic T lymphocytes (CTLs) interact with antigen-presenting target cells in the presence or absence of activated T reg cells. Nonregulated CTLs killed their targets at a 6.6-fold faster rate than regulated CTLs. In spite of this compromised effector activity, regulated CTLs exhibited no defect in proliferation, induction of cytotoxic effector molecules and secretory granules, in situ motility, or ability to form antigen-dependent conjugates with target cells. Only granule exocytosis by CTLs was markedly impaired in the presence of T reg cells. This selective form of regulation did not require prolonged contact between CTLs and T reg cells but depended on CTL responsiveness to transforming growth factor-β. CTLs quickly regained full killing capacity in LNs upon selective removal of T reg cells. Thus, T reg cells reversibly suppress CTL-mediated immunity by allowing acquisition of full effector potential but withholding the license to kill.

Signals transduced by Notch receptors are indispensable for T cell specification and differentiation of alphabeta T lineage cells. However, the role of Notch signals during alphabeta versus gammadelta T lineage decision remains controversial. Here, we addressed this question by employing a clonal analysis of CD4(-)CD8(-) (DN) progenitor potential to position the divergence of alphabeta and gammadelta T cell lineages to the late DN2 to DN3 developmental stages. Accordingly, alphabeta and gammadelta precursor frequencies within these T cell progenitor subsets were determined, both in the presence and absence of Notch signaling through Delta-like 1. Notch signals were found to be critical for the DN to CD4(+)CD8(+) (DP) transition, irrespective of the identity (pTalphabeta or gammadelta) of the inducing T cell receptor complex, whereas gammadelta T cells developed from gammadeltaTCR-expressing T cell progenitors in the absence of further Notch ligand interaction. Collectively, our findings demonstrate a differential, stage-specific requirement for Notch receptor-ligand interactions in the differentiation of alphabeta and gammadelta T cells from T cell progenitors.

It is well established that the pre-T cell receptor for antigen (TCR) is responsible for efficient expansion and differentiation of thymocytes with productive TCRbeta rearrangements. However, Ptcra- as well as Tcra-targeting experiments have suggested that the early expression of Tcra in CD4- CD8- cells can partially rescue the development of alphabeta CD4+ CD8+ cells in Ptcra-deficient mice. In this study, we show that the TCR E delta but not E alpha enhancer function is required for the cell surface expression of alphabetaTCR on immature CD4- CD8- T cell precursors, which play a crucial role in promoting alphabeta T cell development in the absence of pre-TCR. Thus, alphabetaTCR expression by CD4- CD8- thymocytes not only represents a transgenic artifact but occurs under physiological conditions.

Thymic precursors expressing the pre–T cell receptor (TCR), the γδTCR, or the αβTCR can all enter the CD4+8+ αβ lineage, albeit with different efficacy. Here it is shown that proliferation and differentiation of precursors with the different TCRs into αβ lineage cells require Notch signaling at the DN3 stage of thymic development. At the DN4 stage, Notch signaling still significantly contributes to the generation of αβ T cells. In particular, in αβ lineage commitment, the pre-TCR synergizes more efficiently with Notch signals than the other two TCRs, whereas γδTCR-expressing cells can survive and expand in the absence of Notch signals, even though Notch signaling enhances their proliferation. These observations suggest a new model of αβ versus γδ lineage choice in which lineage fate is determined by the extent of synergy between TCR and Notch signaling and in which the evolutionarily recent advent of the cell-autonomously signaling pre-TCR increased the efficacy of αβ T cell generation.

Homing of bone marrow (BM)-derived progenitors to the thymus is essential for T cell development. We have previously reported that two subpopulations of common lymphoid progenitors, CLP-1 and CLP-2, coexist in the BM and give rise to lymphocytes. We demonstrate that CLP-2 migrate to the thymus more efficiently than any other BM-derived progenitors. Short-term adoptive transfer experiments revealed that CLP-2 homing involves P-selectin/P-selectin glycoprotein ligand 1 interactions, pertussis toxin-sensitive chemoattractant signaling by CC chemokine ligand 25 through CC chemokine receptor 9, and binding of the integrins α4β1 and αLβ2 to their respective ligands, vascular cell adhesion molecule 1 and intercellular adhesion molecule 1. Preferential thymus-tropism of CLP-2 correlated with higher chemokine receptor 9 expression than on other BM progenitors. Thus, CLP access to the thymus is controlled by a tissue-specific and subset-selective multistep adhesion cascade.

Despite many efforts, the nature of thymic immigrants that give rise to T cells has remained obscure, especially since it became known that extrathymic lineage-negative, Sca-1–positive, c-kit high progenitor cells differ from intrathymic early T cell progenitors (ETPs) by functional potential and dependence on Notch signaling. After our observation that intrathymic T cell precursors expressing a human CD25 reporter under control of pre-TCRα regulatory elements almost exclusively have the ETP phenotype, we have analyzed the phenotypic changes of reporter-expressing common lymphoid progenitor (CLP) cells in the bone marrow when cultured on Delta-like 1–expressing stromal cells. We note that these quickly adopt the phenotype of double negative (DN)2 thymocytes with little display of the ETP phenotype. Our data suggest that common lymphoid progenitor (CLP) cells could be responsible for the rapid reconstitution of thymus function after bone marrow transplantation since CLP cells in the blood have the capacity to rapidly enter the thymus and become DN2 thymocytes.

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.

Cancer patients can harbor significant numbers of CD8 and CD4 T cells with specificities to tumor antigens (Ags). Yet, in most cases, such T cells fail to eradicate the tumorin vivo.Here, we investigated the interference of Ag-specific CD4+CD25+regulatory T cells (Treg) with the tumor-specific CD8 T cell immune responsein vivo, by monitoring the homing, expansion, and effector function of both subsets in draining and nondraining lymph nodes. The results show that CD8 cells expand to the same extent and produce similar levels of IFN-γ in the presence or absence of Ag-specific Treg. Nevertheless, these Treg abrogate CD8 T cell-mediated tumor rejection by specifically suppressing the cytotoxicity of expanded CD8 cells. The molecular mechanism of suppression involves TGF-β because expression of a dominant-negative TGF-β receptor by tumor-specific CD8 cells renders them resistant to suppression and is associated with tumor rejection and unimpaired cytotoxicity.

In contrast with the alphabeta T cell receptor (TCR), the pre-TCR spontaneously segregates to membrane rafts from where it signals in a cell-autonomous fashion. The disparate behaviors of these two receptors may stem either from differences inherent to the distinct developmental stages during which they are expressed, or from features intrinsic and unique to the receptor components themselves. Here, we express TCRalpha precisely at the pre-TCR checkpoint, at levels resembling those of endogenous pre-TCRalpha (pTalpha), and in the absence of endogenous pTalpha. Both in isolation and more dramatically when in competition with pTalpha, TCRalpha induced defective proliferation, survival, and differentiation of alphabeta T lymphocyte precursors, as well as impaired commitment to the alphabeta T lymphocyte lineage. Substitution of TCRalpha transmembrane and cytoplasmic domains with those of pTalpha generated a hybrid molecule possessing enhanced competitive abilities. We conclude that features intrinsic to the pre-TCR, which are absent in TCRalpha, are essential for its unique function.

Notch and basic helix–loop–helix E2A pathways specify cell fate and regulate neoplastic transformation in a variety of cell types. Whereas Notch enhances tumorigenesis, E2A suppresses it. However, whether and how Notch and E2A interact functionally in an integrative mechanism for regulating neoplastic transformation remains to be understood. It has been shown that Notch3-induced T-cell leukaemia is abrogated by the inactivation of pTα/pre-T-cell antigen receptor (pre-TCR). We report here that Notch3-induced transcriptional activation of pTα/pre-TCR is responsible for the downregulation of E2A DNA binding and transcriptional activity. Further, the E2A messenger RNA and protein levels remain unaltered but the E2A inhibitor Id1 expression is augmented in thymocytes and T lymphoma cells derived from Notch3 transgenic mice. The increase in Id1 expression is achieved by pre-TCR-induced extracellular-signalling-regulated kinase 1/2. These observations support a model in which the upregulation of pre-TCR signalling seems to be the prerequi-site for Notch3-induced inhibition of E2A, thus leading to the development of lymphoma in Notch3 transgenic mice.

Immunity in the absence of autoimmunity reflects “self–nonself” discrimination by the immune system. The search for mechanisms preventing autoimmunity or enabling “self” tolerance has been at the root of immunology as evidenced by ample conceptual framework, rhetoric, sophism, and

Adoptive transfer of antigen-specific CD25+CD4+regulatory T cells was used to analyze the stability of their phenotype, their behavior after immunization, and their mode of suppressing cotransferred naive T cellsin vivo. We found that regulatory T cells maintained their phenotype in the absence of antigen, were not anergicin vivo, and proliferated as extensively as naive CD4+T cells after immunization without losing their suppressive functionin vivoandin vitro. In vivo, the expansion of cotransferred naive T cells was suppressed relatively late in the response such that regulatory T cells expressing mostly IL-10 but not IL-2 or IFN-γ represented the dominant subset of cells. Our results reveal properties of regulatory T cells that were not predicted fromin vitrostudies.

Differentiation of hematopoietic cells from multipotential progenitors is regulated by multiple growth factors and cytokines. A prominent feature of these soluble factors is promotion of cell survival, in part mediated by expression of either of the anti-apoptotic proteins, BCL-2 and BCL-XL. The complex expression pattern of these frequently redundant survival factors during hematopoiesis may indicate a role in lineage determination. To investigate the latter possibility, we analyzed factor-dependent cell-Patersen (FDCP)-Mix multipotent progenitor cells in which we stably expressed BCL-2 or BCL-XL. Each factor maintained complete survival of interleukin-3 (IL-3)-deprived FDCP-Mix cells but, unexpectedly, directed FDCP-Mix cells along restricted and divergent differentiation pathways. Thus, IL-3-deprived FDCP-Mix BCL-2 cells differentiated exclusively to granulocytes and monocytes/macrophages, whereas FDCP-Mix BCL-XL cells became erythroid. FDCP-Mix BCL-2 cells grown in IL-3 were distinguished from FDCP-Mix and FDCP-Mix BCL-XL cells by a striking reduction in cellular levels of Raf-1 protein. Replacement of the BCL-2 BH4 domain with the related BCL-XL BH4 sequence resulted in a switch of FDCP-Mix BCL-2 cells to erythroid fate accompanied by persistence of Raf-1 protein expression. Moreover, enforced expression of Raf-1 redirected FDCP-Mix BCL-2 cells to an erythroid fate, and prohibited generation of myeloid cells. These results identify novel roles for BCL-2 and BCL-XL in cell fate decisions beyond cell survival. These effects are associated with differential regulation of Raf-1 expression, perhaps involving the previously identified interaction between BCL-2-BH4 and the catalytic domain of Raf-1.

In type 1 diabetes, autoimmune T cells cause destruction of pancreatic β cells by largely unknown mechanism. Previous analyses have shown that β cell destruction is delayed but can occur in perforin-deficient nonobese diabetic (NOD) mice and that Fas-deficient NOD mice do not develop diabetes. However, because of possible pleiotropic functions of Fas, it was not clear whether the Fas receptor was an essential mediator of β cell death in type 1 diabetes. To directly test this hypothesis, we have generated a β cell–specific knockout of the Fas gene in a transgenic model of type 1 autoimmune diabetes in which CD4+ T cells with a transgenic TCR specific for influenza hemagglutinin (HA) are causing diabetes in mice that express HA under control of the rat insulin promoter. Here we show that the Fas-deficient mice develop autoimmune diabetes with slightly accelerated kinetics indicating that Fas-dependent apoptosis of β cells is a dispensable mode of cell death in this disease.

It has been of much interest whether there is functional redundancy between the constitutively signaling pre-Talpha/TCRbeta (pre-TCR) and ligated TCRalphabeta complexes, which independently operate the two distinct checkpoints during thymocyte development, i.e., the pre-TCR involved in beta-selection at the CD4(-)CD8(-) double-negative stage and the TCRalphabeta being crucial for positive/negative selection at the CD4(+)CD8(+) double-positive stage. We found that the pre-TCR expressed on double-positive cells in TCRalpha-deficient (TCRalpha(-/-)) mice produced a small number of mature CD8(+) T cells. Surprisingly, when pre-Talpha was overexpressed, resulting in augmentation of pre-TCR expression, there was a striking increase of the CD8(+) T cells. In addition, even in the absence of up-regulation of pre-TCR expression, a similar increase of CD8(+) T cells was also observed in TCRalpha(-/-) mice overexpressing Egr-1, which lowers the threshold of signal strength required for positive selection. In sharp contrast, the CD8(+) T cells drastically decreased in the absence of pre-Talpha on a TCRalpha(-/-) background. Thus, the pre-TCR appears to functionally promote positive selection of CD8(+) T cells. The biased production of CD8(+) T cells via the pre-TCR might also support the potential involvement of signal strength in CD4/CD8 lineage commitment.

Notch receptors are conserved regulators of cell fate and have been implicated in the regulation of T cell differentiation and lymphomagenesis. However, neither the generality of Notch involvement in leukemia, nor the molecules with which Notch may interact have been clarified. Recently, we showed that transgenic mice expressing the constitutively active intracellular domain of Notch3 in thymocytes and T cells developed early and aggressive T cell neoplasias. Although primarily splenic, the tumors sustained features of immature thymocytes, including expression of pTalpha, a defining component of the pre T cell receptor, known to be a potent signaling complex provoking thymocyte survival, proliferation, and activation. Thus, enforced expression of Notch3, which is ordinarily down-regulated as thymocytes mature, may sustain pre T cell receptor expression, causing dysregulated hyperplasia. This hypothesis has been successfully tested in this article by the observation that deletion of pTalpha in Notch3 transgenic mice abrogates tumor development, indicating a crucial role for pTalpha in T cell leukemogenesis. Parallel observations were made in humans, in that all T cell acute lymphoblastic leukemias examined showed expression of Notch3 and of the Notch target gene HES-1, as well as of pTalpha a and b transcripts, whereas the expression of all these genes was dramatically reduced or absent in remission. Together, these results suggest that the combined expression of Notch3 and pTalpha sustains T cell leukemogenesis and may represent pathognomonic molecular features of human T-ALL.

In 1979, some of us were surprised by the (1) conclusion of Doherty and Zinkernagel on MHC-restricted antigen recognition following the lead of Katz, Hamaoka, and Benacerraf (2) describing the same for the interaction of T helper cells with B cells as well as Rosenthal and Shevach (3) describing it for the interaction of T cells with macrophages. Since Doherty and Zinkernagel offered the least complicated system, they got most of the credit. We wanted to know whether a single effector cell was involved and therefore analyzed a clone of cells, specific for the HY transplantation antigen. If it could be shown that the progeny of a single cell was MHC restricted, we had something to explain, which was not obvious. The cloning in Basel worked fine, with the competent help of Hans Hengartner before his departure to the lab of Zinkernagel in Zurich. The clone was in fact MHC restricted, telling us that MHC restriction was the property of a single cell. The same conclusion was derived from experiments with Matthias Wabl, who observed the killing of targets by single killer cell (4). The clone was also alloreactive, which was observed prior to the realization that a significant portion of T cells carried two receptors and thus it is unclear to date whether a second receptor was involved or not. This was just the first example of a clone, which was MHC restricted and HY specific as well as H-2Dd specific and this overlap in specificity was subsequently observed in other clones such that the high frequency of alloreactive T cells is not really an issue. Follow up experiments with the Michael Steinmetz lab transferring TCR alfa and beta genes from one T cell clone to another allowed us to unequivocally conclude that the MHC-restricted specificity was encoded by a single receptor long before crystallographic studies reached the same conclusion (5). This surprised some molecular biologists somewhat who thought that the cloning of the TCR put an end to the mysteries of the immune system. This lead automatically to the next step, the construction of TCR transgenic mice, to analyze the selection of T cells according to their specificity. Initially, we were interested to test the ideas of Burnet and Lederberg that autoaggressive cells were eliminated in primary lymphoid organs. For this reason, we used again the genes of HY specific clones since then we could easily compare female and male mice. Here, I have to tell a little tale that characterizes (some?) scientists: it was Michael Steinmetz, who had previously spoken to Fritz Melchers, who asked at the Reisensburg in the South of Germany whether there would be any interest in generating TCR transgenic mice. I answered with a clear yes saying that this would allow to test Burnets and Lederbergs ideas. So, it was concluded to go ahead and initially Georges Koehler was singled out as the scientist residing by now in Freiburg to help with the construction of mice since he had succeeded to generate immunoglobulin transgenic mice. I was therefore mildly surprised when one day Georges entered my lab and asked me whether I could give him an HY specific clone since he had the idea of testing Burnets and Lederbergs ideas. I told him that this sounded familiar, he blushed only a little and then asked Hans Georg Rammensee who was in the same office a related question. I leave it to the audience to imagine what Michael Steinmetz told Georges Koehler or better what he did not tell him, even though one cannot be completely sure of it. So Georges did not produce the mice but Anton Berns in Amsterdam cooperated and very nicely mapped what was required to express TCR beta genes in transgenic mice. Finally, the co-injection of alfa and beta genes from an HY specific clone was done by Horst Bluethmann at Hoffmann La Roche in Basel where Michael Steinmetz had moved. When the mice had grown up, we tested them with a variety of reagents prepared for this task and could report on the deletion of CD4+8+ cells in male mice even though these mice came with an anomaly, the too early expression of the transgenic TCR, which made proper quantitation difficult (6). Only recently could we address this problem and reported deletion of CD4+8+ thymocytes in the absence of TCR editing (7). This ended a long story on the deletion of autoaggressive cells at a certain stage of development, something that had not been addressed in mice expressing superantigen specific receptors, which somewhat compromised our transgenic approach since they were conducted later and yielded results earlier albeit with the limitation that the conclusions had to be restricted to superantigens (8) whereas we dealt with conventional antigens for T cells. The next step was related to positive selection and the matching of specificity and function. Here, the first realization was that a receptor derived from a CD8+ cell would only be expressed on CD8+ cells in the transgenic mice (9, 10). The second was that there was in fact positive selection as mice with inappropriate MHC antigens not restricting the specificity of the cell from which receptor genes were obtained, failed to generate single positive cells and thus development was arrested at the CD4+8+ stage where cells died (9). This was then named death from neglect as opposed to death by negative selection which eliminated likewise CD4+8+ cells, at least when the receptor was derived from CD8+ cells (7). It was then clear that it was the MHC molecules expressed in the thymus and the TCR specificity, which determined positive selection, which also led to the matching of specificity and function (10), such that CD8+ killer cells were generated from immature cells expressing a class I restricted TCR (11) and as shown later CD4+ helper cells were generated from immature cells expressing a class II restricted TCR and thus in other words helper cells recognized as a rule peptides entering the target cell from the outside whereas killer cells recognized peptides produced in the target cell itself. This relates to the different modes of peptide loading by class I and class II MHC antigens (12–16). In the meantime, the molecular details of this matching process have been worked out mostly by the work of Dietmar Kappes (17) as well as Dan Littman (18) who identified transcription factors guiding this process in dependence of the signaling by the receptor expressed by immature cells. Thus, at present we have a fairly complete picture of positive selection as far as the selectable T cells are concerned while still we know relatively little about the TCR ligands that are responsible for positive selection. Here, one wonders whether thymus-specific proteasome subunits play an essential role (19). Thus, there are still some secrets in T cell development even after decades of the identification of the TCR (20). The curiosity in T cell development is still very much alive even after retirement but I trust that the remaining issues are in good hands of younger scientific colleagues who identify the outstanding questions and think of clever experiments to address them.

beta selection is a major checkpoint in early thymocyte differentiation, mediated by successful expression of the pre-T cell receptor (TCR) comprising the TCRbeta chain, CD3 proteins, and a surrogate TCRalpha chain, pTalpha. The mechanism of action of the pre-TCR is unresolved. In humans and mice, the pTalpha gene encodes two RNAs, pTalpha(a), and a substantially truncated form, pTalpha(b). This study shows that both are biologically active in their capacity to rescue multiple thymocyte defects in pTalpha(-/-) mice. Further active alleles of pTalpha include one that lacks both the major ectodomain and much of the long cytoplasmic tail (which is unique among antigen receptor chains), and another in which the cytoplasmic tail is substituted with the short tail of TCR Calpha. Thus, very little of the pTalpha chain is required for function. These data support a hypothesis that the primary role of pTalpha is to stabilize the pre-TCR, and that much of the conserved structure of pTalpha probably plays a critical regulatory role.

Peripheral T cell tolerance may result from activation-induced cell death [1], anergy [1], and/or immune response modulation by regulatory T cells [2] . In mice that express a transgenic receptor specific for peptide 111–119 of influenza hemagglutinin presented by E d class II MHC molecules as well as hemagglutinin under control of the immunoglobulin-κ promoter, we have found that anergic T cells [3] can also have immunoregulatory function and secrete IL-10 [4]. In order to obtain information on molecular mechanisms involved in anergy and immunoregulation, we have compared expression levels of 1176 genes in anergic, naive, and recently activated CD4 + T cells of the same specificity by gene array analysis. The results provide a plausible explanation for the anergic phenotype in terms of proliferation, provide new information on the surface phenotype of in vivo-generated anergic CD4 + T cells, and yield clues with regard to new candidate genes that may be responsible for the restricted cytokine production of in vivo-anergized CD4 + T cells. The molecular fingerprints of such T cells should enable the tracking of this small population in the normal organism and the study of their role in immunoregulation.

Summary The development and function of lymphocytes depend upon their precise migration in response to chemoattractant cytokines, or chemokines. Two recent reports suggest that, during thymic β-selection, the binding of the chemokine CXCL12 to the receptor CXCR4 on thymocytes provides not only directional but also developmental cues.

Autoimmune diabetes results from destruction of pancreatic beta-cells by islet-infiltrating leukocytes. Different molecular mechanisms seem to be involved in this destruction but the results from many studies have not provided a clear picture so far. Therefore, we have developed a multiplex single-cell reverse transcription polymerase chain reaction to analyze the expression of genes of the tumor necrosis factor receptor (TNFR) family in pancreatic beta-cells during the development of autoimmune diabetes in a TCR-HA x INS-HA double transgenic as well as a non-obese diabetic (NOD) animal model. To this end we have followed the expression of cell surface receptors of the TNFR family in NOD mice as well as in double transgenic mice that express in their T cells class II MHC-restricted TCR specific for peptide 111 - 119 from influenza hemagglutinin (TCR-HA) as well as hemagglutinin under the control of the rat insulin promoter (INS-HA). Both types of mice develop insulitis and diabetes spontaneously. The data show a significant increase in the expression of Fas and TNFR2 (p75) during the development of insulitis, whereas TNFR1 (p55) is already expressed in beta-cells before the onset of insulitis. As ligands for these receptors are already expressed at high levels during the phase of insulitis, it is possible that beta-cell death is regulated by intracellular inhibitors of apoptosis pathways.

Signaling via the pre-T cell receptor (TCR) is required for the proliferative expansion and maturation of CD4(-)CD8(-) double-negative (DN) thymocytes into CD4(+)CD8(+) double-positive (DP) cells and for TCR-beta allelic exclusion. The adaptor protein SH2 domain-containing leukocyte protein (SLP)-76 has been shown to play a crucial role in thymic development, because thymocytes of SLP-76(-/-) mice are arrested at the CD25(+)CD44(-) DN stage. Here we show that SLP-76(-/-) DN thymocytes express the pre-TCR on their surfaces and that introduction of a TCR-alpha/beta transgene into the SLP-76(-/-) background fails to cause expansion of DN thymocytes or developmental progression to the DP stage. Moreover, analysis of TCR-beta rearrangement in SLP-76(-/-) TCR-transgenic mice or in single CD25(+)CD44(-) DN cells from SLP-76(-/-) mice indicates an essential role of SLP-76 in TCR-beta allelic exclusion.

Autoimmune diabetes develops following recognition of organ-specific antigens by T cells. The disease begins with peri-islet infiltration by mononuclear cells, proceeds with insulitis and becomes manifest with destruction of insulin-producing islet beta-cells. T cells are necessary to induce insulitis and diabetes, but it is not clear by what mechanisms they can do so, i. e. whether the T cells need to make antigen-specific contact with the beta-cell or whether other interactions are sufficient to induce beta-cell death. In the present study we have constructed chimeric mice in which the bone marrow-derived antigen-presenting cells, but not the islet beta-cells, are capable of presenting antigen to monospecific T cells. We show that both insulitis as well as beta-cell destruction can proceed in the absence of islet beta-cell surface antigen recognition by T cells. Our results support the notion that diabetes can be caused by distinct effector mechanisms.

Intracellular signals emanating from cytokine and antigen receptors are integrated during the process of intrathymic development. Still, the relative contributions of cytokine receptor signaling to pre-T cell receptor (TCR) and TCR-mediated differentiation remain undefined. Interleukin (IL)-7 interactions with its cognate receptor complex (IL-7Ralpha coupled to the common cytokine receptor gamma chain, gammac) play a dominant role in early thymopoiesis. However, alpha/beta T cell development in IL-7-, IL-7Ralpha-, and gammac-deficient mice is only partially compromised, suggesting that additional pathways can rescue alpha/beta T lineage cells in these mice. We have investigated the potential interdependence of gammac- and pre-TCR-dependent pathways during intrathymic alpha/beta T cell differentiation. We demonstrate that gammac-dependent cytokines do not appear to be required for normal pre-TCR function, and that the rate-limiting step in alpha/beta T cell development in gammac- mice does not involve TCR-beta chain rearrangements, but rather results from poor maintenance of early thymocytes. Moreover, mice double mutant for both gammac and pre-Talpha show vastly reduced thymic cellularity and a complete arrest of thymocyte differentiation at the CD44(+)CD25(+) cell stage. These observations demonstrate that the pre-TCR provides the gammac-independent signal which allows alpha/beta T cell development in gammac- mice. Thus, a series of overlapping signals derived from cytokine and T cell receptors guide the process of alpha/beta thymocyte development.

We have examined the question of whether there is an additional checkpoint in T cell development that regulates T cell receptor (TCR)-β expression in CD25+44− thymocytes by mechanisms that are independent of the pre-TCR. Our analysis in various mutant mice indicates that all changes in cytoplasmic TCR-β expression can be accounted for by pre-TCR–dependent signal mediation, putting into question the function of a putative pro-TCR.

The pre-TCR, which is minimally composed of the TCRbeta chain, the pre-Talpha chain, and the CD3 complex, regulates early T cell development. The pre-Talpha chain is a 33-kDa type I transmembrane glycoprotein with an extracellular part similar to the constant domain of the immunoglobulin supergene family. We have sequenced (11 kb) the human pTalpha gene, which like the murine pTalpha gene consists of four exons: exon 1 encodes the 5' untranslated region, the leader peptide and the first three amino acids of the mature protein, exon 2 the extracellular immunoglobulin (Ig)-like domain, exon 3 a 15-amino acid peptide including a cysteine required for heterodimerization with TCRbeta, exon 4 the transmembrane region, the cytoplasmic tail and the 3' untranslated sequence. The human pTalpha gene is located on chromosome 6p21.3, close to the HLA-A locus. Reverse transcription-PCR studies with human thymus and leukemic cells showed that alternative splicing produces a shorter pTalpha isoform, which lacks the Ig-like domain but contains the transmembrane elements and the extracytoplasmic cystein and which could thus permit pairing with TCRbeta chain and association with CD3 molecules. The conserved splice sites suggest a yet ill-defined biological function of the short pTalpha protein.

Organ-specific autoimmune disease can be caused by αβ T cells that have escaped self-tolerance induction. Here we show that one of the causes of escape from self-tolerance is the coexpression of two different T cell receptors by the same cell, which can occur in up to 30% of all T cells in normal mice and can lead to low-level surface expression of an autospecific TCR. We found that double receptor–expressing T cells can escape tolerance even to ubiquitously expressed antigens but can nevertheless induce autoimmune diabetes when the relevant protein is expressed in pancreatic tissue. Such diabetogenic T cells are absent, however, among T cells expressing the autospecific TCR as the sole receptor.

In the thymus, T cells are selected according to their T cell receptor (TCR) specificity. After positive selection, mature cells are exported from primary lymphoid organs to seed the secondary lymphoid tissue. An important question is whether survival of mature T cells is an intrinsic property or requires continuous survival signals, i.e., engagement of the TCR by major histocompatibility complex (MHC) molecules in the periphery, perhaps in a similar way as occurring during thymic positive selection. To address this issue we used recombination-activating gene (Rag)-deficient H-2b mice expressing a transgenic TCR restricted by I-Ed class II MHC molecules. After engraftment with Rag−/− H-2d fetal thymi, CD4+8− peripheral T cells emerged. These cells were isolated and transferred into immunodeficient hosts of H-2b or H-2d haplotype, some of the latter being common cytokine receptor γ chain deficient to exclude rejection of H-2b donor cells by host natural killer cells. Our results show that in the absence, but not in the presence, of selecting MHC molecules, peripheral mature T cells are short lived and disappear within 7 wk, indicating that continuous contact of the TCR with selecting MHC molecules is required for survival of T cells.

Although individual T lymphocytes have the potential to generate two distinct T cell receptor (TCR)-beta chains, they usually express only one allele, a phenomenon termed allelic exclusion. Expression of a functional TCR-beta chain during early T cell development leads to the formation of a pre-T cell receptor (pre-TCR) complex and, at the same developmental stage, arrest of further TCR-beta rearrangements, suggesting a role of the pre-TCR in mediating allelic exclusion. To investigate the potential link between pre-TCR formation and inhibition of further TCR-beta rearrangements, we have studied the efficiency of allelic exclusion in mice lacking the pre-TCR-alpha (pTalpha) chain, a core component of the pre-TCR. Staining of CD3+ thymocytes and lymph node cells with antibodies specific for Vbeta6 or Vbeta8 and a pool of antibodies specific for most other Vbeta elements, did not reveal any violation of allelic exclusion at the level of cell surface expression. This was also true for pTalpha-deficient mice expressing a functionally rearranged TCR-beta transgene. Interestingly, although the transgenic TCR-beta chain significantly influenced thymocyte development even in the absence of pTalpha, it was not able to inhibit fully endogeneous TCR-beta rearrangements either in total thymocytes or in sorted CD25+ pre-T cells of pTalpha-/- mice, clearly indicating an involvement of the pre-TCR in allelic exclusion.