Notch1 regulates maturation of CD4+ and CD8+ thymocytes by modulating TCR signal strength

Notch signaling regulates cell fate decisions in multiple lineages. We demonstrate in this report that retroviral expression of activated Notch1 in mouse thymocytes abrogates differentiation of immature CD4+CD8+ thymocytes into both CD4 and CD8 mature single-positive T cells. The ability of Notch1 to inhibit T cell development was observed in vitro and in vivo with both normal and TCR transgenic thymocytes. Notch1-mediated developmental arrest was dose dependent and was associated with impaired thymocyte responses to TCR stimulation. Notch1 also inhibited TCR-mediated signaling in Jurkat T cells. These data indicate that constitutively active Notch1 abrogates CD4+ and CD8+ maturation by interfering with TCR signal strength and provide an explanation for the physiological regulation of Notch expression during thymocyte development.     

Inactivation of Notch 1 in immature thymocytes does not perturb CD4 or CD8T cell development

Notch proteins influence cell-fate decisions in many developing systems. Several gain-of-function studies have suggested a critical role for Notch 1 signaling in CD4-CD8 lineage commitment, maturation and survival in the thymus. However, we show here that tissue-specific inactivation of the gene encoding Notch 1 in immature (CD25+CD44-)T cell precursors does not affect subsequent thymocyte development. Neither steady-state numbers nor the rate of production of CD4+ and CD8+ mature thymocytes is perturbed in the absence of Notch 1. In addition, Notch 1-deficient thymocytes are normally sensitive to spontaneous or glucocorticoid-induced apoptosis. In contrast to earlier reports, these data formally exclude an essential role for Notch 1 in CD4-CD8 lineage commitment, maturation or survival.     

Ontogeny of T cell maturation in LEC mutant rats which bear a congenital arrest of maturation from CD4+CD8+ to CD4+CD8- thymocytes.

LEC rats bear a congenital deficiency in CD4+CD8- thymocytes and peripheral CD4+ T cells, and consequently a deficiency in Th cell functions. Ontogeny of T cell maturation in normal and LEC mutant rats was, therefore, investigated. Prenatal development of thymocytes in normal rat strains, with respect to the expression of CD4/CD8 and TcR antigens, was similar to that of mice except that its kinetics was delayed by approximately 24 h. The kinetics of T cell maturation in LEC rats was comparable with that of normal rats up to day 19 of gestation, at which stage double-negative thymocytes (CD4-CD8-) developed into double positives (CD4+CD8+) through immature CD4-CD8+ subset. At day 19 of gestation in LEC as well as normal rats, double positives occupied approximately 80% of the total thymocytes, half of which were TcR-dull positive, indicating that TcR was normally rearranged and then expressed in LEC rat thymocytes. These data indicate that double negatives normally mature into at least double positives in LEC rats. Both single positives appeared after day 19 of gestation in normal rats, while in LEC rats CD4+CD8- cells did not appear, suggesting that the deficiency in CD4+CD8- cells is due to a congenital arrest of maturation from CD4+CD8+ to CD4+CD8- cells, but not due to a postnatal deletion.     

Bone marrow-derived progenitor T cells convey the origin of maturational arrest from CD4+CD8+ to CD4-CD8+ thymocytes in LEC mutant rats.

A mutant strain of rats, LEC, shows a novel arrest of T cell maturation from CD4+CD8+ to CD4+CD8- but not to CD4-CD8+ cells in the thymus. Transplantation of LEC rat fetal thymuses into the subcapsule of the kidney of athymic nude rats resulted in a normal maturation of thymocytes in the thymus graft. Furthermore, both single-positive thymocytes and peripheral lymph node T cells expressed T cell receptor alpha/beta antigen, and lymph node T cells acquired the ability to produce interleukin 2 upon mitogen stimulation. Transplantation of fetal thymuses from LEA rats, which express the same major histocompatibility complex haplotype as LEC rats, into LEC rat kidney subcapsule resulted in the maturational arrest from CD4+CD8+ to CD4+CD8- cells in the thymus graft. These data strongly suggest that bone marrow-derived progenitor T cells carry the cause of maturational arrest and that the thymic stroma of LEC rats has a normal potential to nurse thymocytes.     

p53 regulates thymic Notch1 activation

Notch is crucial for multiple stages of T cell development, including the CD4+CD8+ double positive (DP)/CD8+ single positive (SP) transition, but regulation of Notchactivation is not well understood. p53 regulates Presenilin1 (PS1) expression, and PS1 cleaves Notch, releasing its intracellular domain (NIC), leading to the expression of downstream targets, e.g. the HES1 gene. We hypothesize that p53 regulates Notch activity during T cell development. We found that Notch1 expression and activation were negatively regulated by p53in several thymoma lines. Additionally, NIC was elevated in Trp53(-/-) thymocytes as compared to Trp53(+/+) thymocytes. To determine if elevated Notch1 activation in Trp53(-/-) thymocytes had an effect on T cell development, CD4 and CD8 expression were analyzed. The CD4+ SP/CD8+ SP T cell ratio was decreased in Trp53(-/-) splenocytes and thymocytes. This alteration in T cell development correlated with the increased Notch1 activation observed in the absence of p53. These data indicate that p53 negatively regulates Notch1 activation during T cell development. Skewing of T cell development toward CD8+SP T cells in Trp53(-/-) mice is reminiscent of the phenotype of NIC-overexpressing mice. Thus, we suggest that p53 plays a role in T cell development, in part by regulating Notch1 activation.     

Thymic Nurse Cells Support CD4^-CD8^＋ Thymocytes to Differentiate into CD4^＋CD8^＋ Cells

Thymic nurse cells （TNCs） represent a unique microenvironment in the thymus for T cell maturation. In order to investigate the role of thymic nurse cells during T cell differentiation, a TNC clone, RWTE-1, which formed a typical complex with fetal thymocytes in vitro was established from normal Wistar rat. Hanging drop culture method was applied to reveal the interaction between TNCs and thymocytes. Our result revealed that eighty percent of immature CD4^-CD8^＋ cells differentiated into CD4^＋CD8^＋ cells after a 12-hour hanging drop culture with RWTE-1. However, in a 12-hour culture of immature CD4^-CD8^＋ cells with or without RWTE-1 supernatant, only 30% of the cells differentiated into CD4^＋CD8^＋ cells spontaneously. This observation led to the conclusion that RWTE-1 cell has the capacity to facilitate immature CD4^-CD8^＋ thymocytes to differentiate into CD4^＋CD8^＋ T cells by direct interaction.     

TCR and Notch signaling in CD4 and CD8 T-cell development

Summary:  The generation of CD4 and CD8 αβ T-cell lineages from CD4⁺CD8⁺ double-positive (DP) thymocyte precursors is a complex process initiated by engagement of major histocompatibility complex (MHC) by T-cell receptor (TCR) and coreceptor. Quantitative differences in TCR signaling induced by this interaction impose an instructional bias on CD4/CD8 lineage commitment that must be reinforced by MHC recognition and TCR signaling over subsequent selection steps in order for the thymocyte to progress and mature in the adopted lineage. Our studies show that the transmembrane receptor Notch plays a role in this process by modifying TCR signal transduction in DP thymocytes. In this review, we consider the functional relationship of TCR and Notch signaling pathways in the selection and specification of CD4 and CD8 T-cell lineages.     

Notch signalling regulates cytokine production by CD8+ and CD4+ T cells

The Notch signalling pathway regulates several aspects of cellular differentiation such as T lineage commitment and effector functions on peripheral T cells; however, there is limited information regarding Notch receptor expression on different T cell subsets and the putative role of the different receptors on T cell effector function. Here, we studied the protein expression of Notch receptors on murine T cells in vitro and in vivo and analysed the role of the Notch pathway in cytokine production by CD4+ and CD8+ T cells. We found that resting CD4+ and CD8+ T cells do not express Notch receptors, but they upregulate Notch 1 and Notch 2 shortly after in vitro and in vivo activation. Using a γ-secretase inhibitor, which blocks Notch signalling through all Notch receptors, we demonstrated that the Notch pathway regulates IL-10 production by CD4+ T cells and IFN-γ and IL-17 production by CD8+ T cells. These results suggest that Notch 1 and 2 are expressed by CD4+ and CD8+ T cells and represent the putative Notch receptors that regulate effector functions and cytokine production by these cells.     

Generation of alphabeta T-cell receptor+ CD4- CD8+ cells in major histocompatibility complex class I-deficient mice upon activation of the aryl hydrocarbon receptor by 2,3,7,8-tetrachlorodibenzo-p-dioxin

Gene-targeted mice lacking the beta2 microglobulin gene (beta2m-/- mice), and hence functional major histocompatibility complex (MHC) class I molecules, do not develop CD4- CD8+ cells. We show here that both in vitro and in vivo treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a trans-activating ligand of the endogenous aryl hydrocarbon receptor (Ah-R), bypasses the need for MHC class I molecules for selection into the CD4- CD8+ cell pool. When beta2m-/- dams were given a single dose of 50 microg of TCDD, approximately 13% of CD4- CD8+ thymocytes could be detected in their newborn pups. In TCDD-exposed fetal thymus organ cultures of beta2m-/- mice, approximately 35% CD4- CD8+ thymocytes were detectable. About 16% of these CD4- CD8+ cells bore the alpha beta T-cell receptor (TCR) and approximately 33% bore CD3. Only a minority of the CD8+ cells were heat-shock antigen positive. The cells possessed killing activity as shown using the 51Cr-release assay comprising gamma delta TCR- CD4- CD8+ thymocytes from 3 to 4-day-old b2m-/- mice. Thus, TCDD leads to a significant increase of mature CD4- CD8+ thymocytes in relative and absolute numbers. High numbers of CD4- CD8+ thymocytes developed also in organ cultures from thymi, lacking both MHC class I and class II molecules, exposed to TCDD. A 10-fold transient increase of Notch1 mRNA in thymocytes from fetal thymus organ culture, exposed for 4 days to TCDD, was detected in CD4+ CD8+ cells compared with controls. We suggest that TCDD affects thymic selection and directs the lineage commitment of CD4+ CD8+ thymocytes towards CD4- CD8+ cells, possibly via up-regulation of the Notch1 gene.     

Delineation of human thymocytes with or without functional potential by CD1-specific antibodies

Hematopoietic precursors lacking T cell antigen receptors (TCR-CD3-) and CD4 and CD8 surface markers (i.e. double-negative thymocytes) give rise to functionally mature T lymphocytes. Yet their major progeny are immunologically unresponsive thymocytes in spite of having acquired TCR-CD3 and CD4-CD8. Because only mature thymocytes migrate to peripheral lymphoid organs and most thymocytes die in situ, the knowledge of the events associated with functional maturation in the double-negative thymocyte progeny is a fundamental question in T cell development. We reasoned that a clue to trace the fate of early human thymocytes may perhaps come from the study of the developmental acquisition of CD1 antigen, currently used to define better the functionally inert CD4+8+ (double-positive) stage and absent in mature, medullary thymocytes and peripheral T cells. By using antibodies specific for CD1 (HTA 1/T6) we show here that a large fraction of double-negative thymocytes also express CD1. CD1+3-, CD1+3+, CD1-3+, and CD1-3- subsets all exist. The CD1+3- subset generates CD1+3-4-8+ precursors of CD1+ double-positive cells. A large portion of the CD1+3+ subset bears TCR gamma delta-CD3 complexes. The CD1- subsets are responsive in assays of function, in which they can be stimulated to use the interleukin 2 pathway of proliferation and to mediate cytotoxicity. In contrast, all CD1+ thymocytes behave as functionally inert cells. Thus, the CD1 surface marker delineates human thymocyte precursors and their products which lack, or possess, functional potential in vitro, on both alpha beta and gamma delta lineages.     

T cell receptor-negative thymocytes from SCID mice can be induced to enter the CD4/CD8 differentiation pathway

In order to investigate the role of T cell receptor (TcR) expression in thymocyte maturation, we have analyzed thymocytes from C.B-17/SCID mice, which are unable to productively rearrange their antigen receptor genes and fail to express TcR. Despite this defect, SCID thymocytes are functional as they produce lymphokines and proliferate in response to a variety of stimuli. Phenotypic analysis revealed that thymocyte populations from young adult SCID mice resemble thymocyte populations from normal embryonic mice in that they are large, Thy-1.2+, CD4-, CD8-, TcR- and enriched in CD5lo, IL2R+ and Pgp1+ cells. However, other TcR- populations normally present in adult mice (i.e., CD4-CD8+ cells and CD4+CD8+ cells) are absent from the thymus of TcR- adult SCID mice. To understand the basis of the developmental arrest of TcR- SCID thymocytes at the CD4-CD8- stage of differentiation, we analyzed thymi from the occasional "leaky" SCID mouse which possesses small numbers of TcR+ thymocytes. We found that the presence of TcR+ cells within a SCID thymus was invariably associated with the presence of CD4+ and/or CD8+ SCID thymocytes. Interestingly, however, the CD4+/CD8+ SCID thymocytes were not themselves necessarily TcR+. That is, emergence of SCID thymocytes expressing CD4/CD8 was tightly linked to the presence of TcR+ cells within that SCID thymus, but the SCID thymocytes that expressed CD4/CD8 were not necessarily the same cells that expressed TcR. Finally, we found that the introduction into TcR- SCID mice of normal bone marrow cells that give rise to TcR+ cells within the SCID thymus promoted the differentiation of SCID thymocytes into CD4-CD8+ and CD4+CD8+ TcR- cells. These data indicate that TcR+ cells within the thymic milieu provide critical signals which promote entry of CD4-CD8-TcR- precursor T cells into the CD4/CD8 differentiation pathway. When applied to differentiation of normal thymocytes, these findings may imply a critical role for early appearing CD4-CD8- TcR (gamma/delta)+ cells in initiating normal thymic ontogeny.     

TOX: an HMG box protein implicated in the regulation of thymocyte selection

In the thymus, pre-T cell receptor (pre-TCR)--mediated signaling and then TCR-mediated signaling initiate changes in gene expression that result in the maturation of CD4 and CD8 lineage T cells from common precursors. Using gene chip technology, we isolated a murine gene, designated Tox, that encodes a member of the HMG (high-mobility group) box family of DNA-binding proteins. TOX expression is up-regulated by both pre-TCR and TCR activation of immature thymocytes but not by TCR activation of mature na?ve T cells. Transgenic mice that express TOX show expanded CD8+ and reduced CD4+ single positive thymocyte subpopulations. We present evidence here that this phenotype results from a perturbation in lineage commitment due to reduced sensitivity to TCR-mediated signaling. This molecular marker of thymic selection events may therefore play a role in establishing the activation threshold of developing T cells and patterning changes in gene expression.     

Strength of signaling by CD4 and CD8 coreceptor tails determines the number but not the lineage direction of positively selected thymocytes

The present study has assessed the impact of the intracellular domains of CD4 and CD8 on positive selection and lineage direction of MHC class I-restricted thymocytes. Contrary to current presumption, we found that the CD4 tail promotes the generation of both CD4+ and CD8+ T cells without preference for the CD4+ T cell lineage. We also found that the identity of the coreceptor tail and hence the strength of coreceptor signaling determine the number of thymocytes undergoing positive selection but not their ultimate CD4/CD8 phenotype. These findings demonstrate that the strength of coreceptor signaling has a significant quantitative but not qualitative impact on positive selection and provide a simple explanation for the greater numbers of CD4+ than CD8+ T cells selected in the normal thymus.     

Maturation of CD4-8- alpha/beta TCR+ T cells induced by CD2-stimulation in vivo and in vitro.

Newly generated ('virgin') rat thymocytes of the immature CD4+8+ double positive (DP) subset were treated in suspension culture for 2 days with the stimulatory pair of anti-CD2 monoclonal antibodies OX-54 and OX-55. Approximately 50% of the recovered cells had downregulated CD4 and CD8 and upregulated the T cell antigen receptor (TCR). CD2-stimulated, but not control thymocytes proliferated in response to TCR plus IL-2 stimulation. In vivo, postnatal injection of OX-54/55 led to a dramatic and selective increase in functionally mature CD4-CD8- double negative (DN) alpha/beta--TCR(high) thymocytes and peripheral T cells. These findings show that CD2 stimulation can promote T cell differentiation and suggest that DN TCR(high) thymocytes can be generated from DP thymocytes via alternative pathways of T cell maturation.     

Thymic selection of CD4+CD25+ regulatory T cells induced by an agonist self-peptide

Despite accumulating evidence that regulatory T cells play a crucial role in preventing autoimmunity, the processes underlying their generation during immune repertoire formation are unknown. We show here that interactions with a single self-peptide can induce thymocytes that bear an autoreactive T cell receptor (TCR) to undergo selection to become CD4+CD25+ regulatory T cells. Selection of CD4+CD25+ thymocytes appears to require a TCR with high affinity for a self peptide because thymocytes that bear TCRs with low affinity do not undergo selection into this pathway. Our findings indicate that specificity for self-peptides directs the selection of CD4+CD25+ regulatory thymocytes by a process that is distinct from positive selection and deletion.     

Subpopulations of CD4-, CD8- thymocytes

A population of highly purified CD4-,CD8- thymocytes was analyzed by both flow microfluorometry and in situ hybridization in an attempt to further elucidate thymocyte subpopulations. The double-negative cells had a marked increase in expression of the c-myb and T cell gamma genes relative to unseparated thymocytes. Approximately 27% were Ly-24+, 8% Ly-6C+ and 6% 6B2+. All of the Ly-6C+ cells were also Ly-24+. A small population (6%) of the CD4-,CD8- thymocytes had surface expression of the T cell receptor for antigen (F23.1); all were bright Ly-1+ and half were Ly-24+. These studies demonstrate that there are further subdivisions of the CD4-, CD8- thymocytes based upon cell surface expression of markers previously found on bone marrow cells and their non-T cell progeny. Studies are in progress to determine whether these represent different stages of activation-maturation or different lineages of cells.