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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.

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.

Foxp3-expressing regulatory T cells (Treg) have an essential function of preventing autoimmune disease in man and mouse. Foxp3 binds to forkhead motifs of about 1,100 genes and the strength of binding increases upon phorbol 12-myristate 13-acetate/ionomycin stimulation. In Foxp3-expressing T cell hybridomas, Foxp3 promoter binding does not lead to activation or suppression of genes which becomes only visible after T cell activation. These findings are in line with observations by others that Foxp3 exerts important functions in collaboration with T cell receptor (TCR)-dependent transcription factors in a DNA-binding complex. Tregs can be generated when developing T cells encounter TCR agonist ligands in the thymus. This process apparently depends on costimulatory signals. In contrast, extrathymic conversion of naïve T cells into Tregs appears to depend on transforming growth factor (TGF)-beta and is inhibited by costimulation. In fact, dendritic cell-derived retinoic acid helps the conversion process by counteracting the negative impact of costimulation. Tregs induced by subimmunogenic antigen delivery in vivo are much more stable than Tregs induced by antigenic stimulation in the presence of TGF-beta in vitro which correlates with the extent of demethylation of the Foxp3 locus. Tregs can be induced by conversion of antigen-specific T cells that occur with a very low frequency in wt mice. Conversion of naïve cluster of differentiation (CD)4 T cells into Tregs by a single peptide of HY antigens results in complete antigen-specific tolerance to an entire set of HY epitopes recognized by CD4 as well as CD8 T cells when presented with male skin or hemopoietic grafts.

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.

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.

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.

At two checkpoints, T cell development is controlled by T cell receptor (TCR) signaling, which determines survival and lineage commitment. At the first of these checkpoints, signaling by the pre-TCR, the γδTCR or the αβTCR has a major but nonexclusive impact on whether cells will become CD4 − CD8 − γδ or CD4 + CD8 + αβ lineage cells. Pre-TCR signals synergize with moderate Notch signals to generate αβ lineage cells. Relatively strong signals by the γδTCR (or early expressed αβTCR) in the absence of Notch signaling are sufficient to yield γδ lineage cells. However, relatively weak signals of the latter two receptors combined with strong Notch signaling result in the formation of αβ lineage cells that generate a diverse αβTCR repertoire in pre-TCR-deficient mice. It remains to be determined whether TCR and/or Notch signals instruct or confirm predetermined lineage fate.

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.

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.

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.

Intrathymic T cell development represents one of the best studied paradigms of mammalian development. Lymphoid committed precursors enter the thymus and the Notch1 receptor plays an essential role in committing them to the T cell lineages. The pre-T cell receptor (TCR), as an autonomous cell signaling receptor, commits cells to the alphabeta lineage while its rival, the gammadeltaTCR, is involved in generating the gammadelta lineage of T cells. Positive and negative selection of immature alphabetaTCR-expressing cells are essential mechanisms for generating mature T cells, committing them to the CD4 and CD8 lineages and avoiding autoimmunity. Additional lineages of alphabetaT cells, such as the natural killer T cell lineage and the CD25+ regulatory T cell lineage, are formed when the alphabetaTCR encounters specific ligands in suitable microenvironments. Thus, positive selection and receptor-instructed lineage commitment represent a hallmark of the thymus. Ectopically expressed organ-specific antigens contribute to thymic self-nonself discrimination, which represents an essential feature for the evolutionary fitness of mammalian species.

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.

Mutation or ablation of T cell factor 1 and lymphocyte enhancer factor 1 indicated involvement of the Wnt pathway in thymocyte development. The central effector of the Wnt pathway is beta-catenin, which undergoes stabilization upon binding of Wnt ligands to frizzled receptors. We report here that conditional stabilization of beta-catenin in immature thymocytes resulted in the generation of single positive T cells that lacked the alpha beta TCR and developed in the absence of pre-TCR signaling and TCR selection. Although active beta-catenin induced differentiation in the absence of TCRs, its action was associated with reduced proliferation and survival when compared to developmental changes induced by the pre-TCR or the alpha beta TCR.

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.

To examine the role of cognate peptide in establishing CD4(+) T cell tolerance, we have mated transgenic mice that express the major I-E(d)-restricted determinant (S1) from the influenza virus PR8 hemagglutinin (HA28 mice) with mice expressing a S1-specific T cell receptor (TS1 mice). Surprisingly, S1-specific CD4(+) T cells were not substantially deleted in TS1xHA28 mice; indeed, lymph node cells expressing the S1-specific TCR were as abundant in TS1xHA28 mice as in TS1 mice. The S1-specific T cells in TS1xHA28 mice were, however, impaired in their ability to respond to S1 peptide both in vitro and in vivo, and contained two distinct populations. Approximately half expressed a unique cell surface phenotype (CD25(hi)/CD45RB(int)) and had been anergized by the neo-self S1 peptide. The remainder responded normally to the S1 peptide if purified away from the anergic T cells, but their proliferation was suppressed when the anergic T cells were also present in unfractionated lymphnode cells or in mixed cultures. These findings establish that anergy and suppression are coordinated mechanisms by which autoreactive CD4(+) T cells are regulated and that anergic/suppressor CD4(+) T cells can develop in response to self peptides.

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.

International audience; Most natural killer T (NKT) cells express a highly skewed alphabeta TCR repertoire, consisting of an invariant V alpha14-J alpha281 chain paired preferentially with a polyclonal Vbeta8.2 chain. This repertoire is positively selected by the monomorphic CD1d molecule expressed on cells of hematopoietic origin. The origin of NKT cells and their lineage relationship to conventional T cells is controversial. We show here that the development of NKT cells is absolutely dependent on expression of the pre-TCRalpha chain, in marked contrast to conventional T cells which arise in significant numbers even in the absence of a functional pre-TCR. Distinct developmental requirements for pre-TCR expression in the NKT and T cell lineages may reflect differences in the ability of the TCRalphabeta to substitute functionally for the pre-TCR in immature precursor cells.

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 analysis of T-cell receptor (TCR) beta selection, TCR beta allelic exclusion and TCR beta rearrangement in gamma delta T cells from normal and pre-TCR-deficient mice has shown that the pre-TCR has a crucial role in T-lymphocyte development: The pre-TCR is by far the most effective receptor that generates large numbers of CD4+8+ T cells with productive TCR beta rearrangements. In the absence of the pre-TCR, TCR beta rearrangement proceeds in developing cells irrespective of whether they already contain a productive TCR beta gene. The pre-TCR directs developing T cells to the alpha beta lineage because gamma delta T cells from pT alpha-/- mice proceed much further in TCR beta rearrangement than gamma delta T cells from wild-type mice. It is argued that the pre-TCR commits developing T cells to the alpha beta lineage by an instructive mechanism, which has largely replaced an evolutionarily more ancient mechanism that involves stochastic alpha beta lineage commitment.

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.

The development of pre-T-cells with productive T-cell receptor beta (TCR beta) rearrangements can be furthered by each of the pre-T-cell receptors (pre-TCR), the alpha beta TCR as well as the gamma delta TCR, albeit by distinct mechanisms. While the gamma delta TCR affects CD4-8- precursor cells irrespective of their TCR beta rearrangement status both the pre-TCR and the alpha beta TCR select only cells with productive TCR beta genes for expansion and maturation. The alpha beta TCR is much less effective than the pre-TCR because of the paucity of TCR alpha proteins in TCR beta positive precursors.

The vast majority of T lymphocytes, with the exception of gut-associated, intraepithelial lymphocytes, differentiate and mature inside the thymus. Early T cell development is characterized by expansion and differentiation of thymocytes which do not yet express mature TCRs on their cell surface. Important events in early thymocyte development are controlled by a pre-TCR complex consisting of a conventional TCR beta chain and a novel transmembrane protein termed pre-TCR alpha (p T alpha chain) which are noncovalently associated with components of CD3. Recent studies of pre-TCR function have led to a better understanding of the molecular events in early thymocyte development.

In peripheral lymphoid tissues of TCR transgenic mice that express the nominal antigen (HY peptide plus H-2Db MHC) recognized by the transgenic TCR, there exist unusual CD4-CD8- and CD4-CD8low cells bearing the transgenic TCR. Here we show that, unlike TCR alpha beta T cells that are generated in the absence of nominal antigen, these unusual cells do not express endogenous TCR alpha genes, have maintained the TCR delta locus on both chromosomes, and can coexpress TCR alpha beta and TCR gamma delta chains on the cell surface. The latter is also true for CD4-CD8-, HSA+ TCR alpha beta + thymocytes in male and female TCR transgenic mice. The number of TCR alpha beta and TCR gamma delta coexpressing cells is increased in pre-TCR-deficient mice. The data indicate that the TCR alpha beta can replace the TCR gamma delta in the development of gamma delta lineage cells and that the pre-TCR interferes with the generation of gamma delta-expressing cells.

We have analyzed pre-T cell receptor alpha (pT alpha) gene expression in cells from various anatomical sites to investigate the lineage specificity of pT alpha RNA as well as its presence in pro-T cells and in sites of extrathymic T cell development. pT alpha RNA is found in precursors of alpha beta T cells but is absent from mature alpha beta T cells as well as T cells that express the gamma delta T cell receptor on the cell surface. pT alpha expression is exquisitely T lineage specific in that mature and immature B cells, myeloid cells, NK cells and pluripotent stem cells are pT alpha negative. On the other hand, pT alpha expression is found in pro-T cells outside the thymus as well as in intra- and extra-thymic sites of T cell development. The latter finding is consistent with the notion that early steps of T cell development within and outside the thymus may be similar.

We have studied memory in T cell receptor (TCR) transgenic mice expressing a Db-restricted TCR specific for the male peptide (H-Y). CD8+ T cells from female TCR transgenic C57BL/6 (B6) mice were activated by transferring them Into X-irradiated male (B6 x bm12)F1 hybrid recipients. Subsequently, they were highly purified by cell sorting and transferred for various lengths of time into female B6 nu/nu recipient mice. Other nu/nu recipient mice received highly purified naive T cells expressing the transgenic TCR. The functional potential of naive and “memory” T cells was analyzed by stimulation with male cells In vivo. The results show that memory cells can be derived from activated T cells and persist in the absence of antigen for at least 13 weeks. Naive and memory T cells differ in that memory T cells give a more vigorous and sustained response than naive T cells.

The differentiation of αβT cells from thymic precursors is a complex process essential for adaptive immunity. Here we exploited the breadth of expression data sets from the Immunological Genome Project to analyze how the differentiation of thymic precursors gives rise to mature T cell transcriptomes. We found that early T cell commitment was driven by unexpectedly gradual changes. In contrast, transit through the CD4[superscript +]CD8[superscript +] stage involved a global shutdown of housekeeping genes that is rare among cells of the immune system and correlated tightly with expression of the transcription factor c-Myc. Selection driven by major histocompatibility complex (MHC) molecules promoted a large-scale transcriptional reactivation. We identified distinct signatures that marked cells destined for positive selection versus apoptotic deletion. Differences in the expression of unexpectedly few genes accompanied commitment to the CD4[superscript +] or CD8[superscript +] lineage, a similarity that carried through to peripheral T cells and their activation, demonstrated by mass cytometry phosphoproteomics. The transcripts newly identified as encoding candidate mediators of key transitions help define the 'known unknowns' of thymocyte differentiation., National Institutes of Health (U.S.) (AI072073)

The oncogenic transcription factor TAL1/SCL is aberrantly expressed in 60% of cases of human T cell acute lymphoblastic leukemia (T-ALL) and initiates T-ALL in mouse models. By performing global microRNA (miRNA) expression profiling after depletion of TAL1, together with genome-wide analysis of TAL1 occupancy by chromatin immunoprecipitation coupled to massively parallel DNA sequencing, we identified the miRNA genes directly controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3, and RUNX1. The most dynamically regulated miRNA was miR-223, which is bound at its promoter and up-regulated by the TAL1 complex. miR-223 expression mirrors TAL1 levels during thymic development, with high expression in early thymocytes and marked down-regulation after the double-negative-2 stage of maturation. We demonstrate that aberrant miR-223 up-regulation by TAL1 is important for optimal growth of TAL1-positive T-ALL cells and that sustained expression of miR-223 partially rescues T-ALL cells after TAL1 knockdown. Overexpression of miR-223 also leads to marked down-regulation of FBXW7 protein expression, whereas knockdown of TAL1 leads to up-regulation of FBXW7 protein levels, with a marked reduction of its substrates MYC, MYB, NOTCH1, and CYCLIN E. We conclude that TAL1-mediated up-regulation of miR-223 promotes the malignant phenotype in T-ALL through repression of the FBXW7 tumor suppressor., National Cancer Institute (U.S.) (5P01CA109901), National Cancer Institute (U.S.) (5P01CA68484), National Cancer Institute (U.S.) (1K99CA157951), National Institutes of Health (U.S.). Intramural Research Program, Center for Cancer Research (National Cancer Institute (U.S.))

SummaryProliferation and differentiation are tightly coordinated to produce an appropriate number of differentiated cells and often exhibit an antagonistic relationship. Developing T cells, which arise in the thymus from a minute number of bone-marrow-derived progenitors, undergo a major expansion upon pre-T cell receptor (TCR) expression. The burst of proliferation coincides with differentiation toward the αβ T cell lineage—but the two processes were previously thought to be independent from one another, although both were driven by signaling from pre-TCR and Notch receptors. Here we report that proliferation at this step was not only absolutely required for differentiation but also that its ectopic activation was sufficient to substantially rescue differentiation in the absence of Notch signaling. Consistently, pharmacological inhibition of the cell cycle machinery also blocked differentiation in vivo. Thus the proliferation step is strictly required prior to differentiation of immature thymocytes.

Subimmunogenic vaccination with an agonist mimetope of insulin converts naive T cells into regulatory T cells and prevents type 1 diabetes in NOD mice., Type 1 diabetes (T1D) results from the destruction of insulin-secreting pancreatic β cells by autoreactive T cells. Insulin is an essential target of the autoimmune attack. Insulin epitopes recognized by diabetogenic T cell clones bind poorly to the class II I-Ag7 molecules of nonobese diabetic (NOD) mice, which results in weak agonistic activity of the peptide MHC complex. Here, we describe a strongly agonistic insulin mimetope that effectively converts naive T cells into Foxp3+ regulatory T cells in vivo, thereby completely preventing T1D in NOD mice. In contrast, natural insulin epitopes are ineffective. Subimmunogenic vaccination with strongly agonistic insulin mimetopes might represent a novel strategy to prevent T1D in humans at risk for the disease.

Expression of a productive T-cell receptor (TCR) beta gene has profound consequences for T-cell development, preceding an increase in thymocyte number, appearance of CD4 and CD8 coreceptors and suppression of further TCR beta-gene rearrangement. Here Marcus Groettrup and Harald von Boehmer discuss the data obtained in various experimental models and describe a novel signal transducing receptor complex on pre-T cells, which may regulate the TCR beta-induced developmental changes.

We describe a novel signal-transducing protein complex, which consists of the T cell receptor (TCR) beta chain that is disulfide linked to a 33 kd glycoprotein and noncovalently associated with proteins of the CD3 complex on the surface of the pre-T cell line SCB.29. This 33 kd glycoprotein, provisionally designated gp33, represents neither of the known TCR chains and has escaped previous detection because it labels poorly by surface iodination. This glycoprotein is absent from the surface of mature T cell lines. A TCR beta complex with identical molecular masses before and after reduction can be immunoprecipitated from surface-iodinated large thymocytes of TCR alpha-deficient mice. The novel gp33-TCR beta complex may be entirely or partly responsible for control of early T cell development exerted by the TCR beta protein.

The thymus produces many more cells than it releases into the periphery. According to generally accepted models of T cell development most of this loss occurs in the thymic cortex, among CD4+8+ thymocytes. An interesting situation arises in the case of T cell receptor (TcR) transgenic mice in which all cells can potentially be positively selected, leading to a theoretical increase of about 30-fold in the survival rate of CD4+8+ cells and in their transition to mature CD4+8- or CD4-8+ thymocytes. This in turn should lead to a 30-fold increase in the size of the thymic medulla, in the emigration rate and in the size of the peripheral T cell pool. Increases in medullary or peripheral pool sizes of this magnitude are not seen in TcR transgenic mice. The question was therefore asked whether some form of homeostatic process regulated the size of the mature T cell pool and at what level it might operate. In this report we demonstrate that the increased rate of double-positive to single-positive transition in the TcR transgenic mice is directly reflected in an increased emigration rate, and that the medulla seems to be relatively efficient regardless of the number of cells passing through it. However, the potential increases in emigrant numbers in TcR transgenic mice are offset by the reduced size of the CD4+8+ thymocyte pool. It would appear then that regulation of T cell production, if it occurs, probably does so through regulation of the size of the CD4+8+ thymocyte pool. Mechanisms for regulation of this kind are not yet known.

Exclusion and inclusion of T cell receptor (TCR) genes were analyzed in alpha beta TCR transgenic mice. Both transgenes are expressed unusually early on the surface of CD4-8-, HSA+, IL-2R- thymocytes. These progenitor cells give rise to progeny, which at the single-cell level contains endogenous alpha but not beta TCR-RNA as well as protein, in addition to products encoded by the transgenes. Thus, the surface expression of an alpha beta TCR does not prevent further alpha TCR rearrangement in immature thymocytes that still transcribe RAG-1 and RAG-2 genes. Reduced levels of RAG-1 and RAG-2 RNA are detectable only in CD4+8+ TCR high cells, which result from positive selection in the thymus. The results suggest that a developing T cell may try different alpha beta TCRs for binding to thymic MHC ligands, and that recombination at the alpha locus ceases only after positive selection.

Antigen receptor-controlled checkpoints in B lymphocyte development are crucial for the prevention of autoimmune diseases such as systemic lupus erythematosus. Checkpoints at the stage of pre-B cell receptor (pre-BCR) and BCR expression can eliminate certain autoreactive BCRs either by deletion of or anergy induction in cells expressing autoreactive BCRs or by receptor editing. For T cells, the picture is more complex because there are regulatory T (T(reg)) cells that mediate dominant tolerance, which differs from the recessive tolerance mediated by deletion and anergy. Negative selection of thymocytes may be as essential as T(reg) cell generation in preventing autoimmune diseases such as type 1 diabetes, but supporting evidence is scarce. Here we discuss several scenarios in which failures at developmental checkpoints result in autoimmunity.

CD4(+)CD25(+) regulatory T cells (T regs) play a major role in the maintenance of self-tolerance and immune suppression, although the mechanisms controlling T reg development and suppressor function remain incompletely understood. Herein, we provide evidence that Kruppel-like factor 10 (KLF10/TIEG1) constitutes an important regulator of T regulatory cell suppressor function and CD4(+)CD25(-) T cell activation through distinct mechanisms involving transforming growth factor (TGF)-beta1 and Foxp3. KLF10 overexpressing CD4(+)CD25(-) T cells induced both TGF-beta1 and Foxp3 expression, an effect associated with reduced T-Bet (Th1 marker) and Gata3 (Th2 marker) mRNA expression. Consistently, KLF10(-/-) CD4(+)CD25(-) T cells have enhanced differentiation along both Th1 and Th2 pathways and elaborate higher levels of Th1 and Th2 cytokines. Furthermore, KLF10(-/-) CD4(+)CD25(-) T cell effectors cannot be appropriately suppressed by wild-type T regs. Surprisingly, KLF10(-/-) T reg cells have reduced suppressor function, independent of Foxp3 expression, with decreased expression and elaboration of TGF-beta1, an effect completely rescued by exogenous treatment with TGF-beta1. Mechanistic studies demonstrate that in response to TGF-beta1, KLF10 can transactivate both TGF-beta1 and Foxp3 promoters, implicating KLF10 in a positive feedback loop that may promote cell-intrinsic control of T cell activation. Finally, KLF10(-/-) CD4(+)CD25(-) T cells promoted atherosclerosis by approximately 2-fold in ApoE(-/-)/scid/scid mice with increased leukocyte accumulation and peripheral pro-inflammatory cytokines. Thus, KLF10 is a critical regulator in the transcriptional network controlling TGF-beta1 in both CD4(+)CD25(-) T cells and T regs and plays an important role in regulating atherosclerotic lesion formation in mice.

During their development, T cells are rescued from apoptotic cell death to follow distinct lineage fates. Recent data concerned with the role of the Notch transmembrane receptor in these events are interpreted to show that Notch promotes survival, but contrary to earlier reports has no function in lineage commitment.

Compelling evidence suggests that Foxp3-expressing CD25(+)CD4(+) regulatory T cells (Treg) are generated within the thymus as a separate lineage. However, Foxp3(+)CD4(+) Treg can also be generated de novo in a TGF-beta-dependent process from naive T cells by TCR triggering. Recently, we have shown that naturally occurring, but not in vitro TGF-beta-induced Foxp3(+) Treg display stable Foxp3 expression that was associated with selective demethylation of an evolutionarily conserved element within the Foxp3 locus named TSDR (Treg-specific demethylated region). Here, we report that inhibition of DNA methylation by azacytidine, even in absence of exogenous TGF-beta, not only promoted de novo induction of Foxp3 expression during priming, but also conferred stability of Foxp3 expression upon restimulation. Most notably, such stable Foxp3 expression was found only for cells displaying enhanced TSDR demethylation. In contrast, in vitro TSDR methylation diminished its transcriptional activity. Foxp3(+) Treg generated in vivo by DEC-205-mediated targeting of agonist ligands to dendritic cells showed long-term survival in the absence of the inducing antigen and exhibited efficient TSDR demethylation. Together, our data suggest that TSDR is an important methylation-sensitive element regulating Foxp3 expression and demonstrate that epigenetic imprinting in this region is critical for establishment of a stable Treg lineage.

Harald von Boehmer describes how he used mice transgenic for T cell receptor αβ to identify T cell receptor–dependent cellular selection and lineage commitment as mechanisms responsible for generating an effective and self-tolerant adaptive immune system.