Calcineurin sets the bandwidth for discrimination of signals during thymocyte development

At critical times in development, cells are able to convert graded signals into discrete developmental outcomes; however, the mechanisms involved are poorly understood. During thymocyte development, cell fate is determined by signals originating from the alphabeta T-cell receptor. Low-affinity/avidity interactions between the T-cell receptor and peptide-MHC complexes direct differentiation to the single-positive stage (positive selection), whereas high-affinity/avidity interactions induce death by apoptosis (negative selection). Here we show that mice deficient in both calcineurin and nuclear factor of activated T cells (NFAT)c2/c3 lack a population of preselection thymocytes with enhanced ability to activate the mitogen-activated protein kinase (Raf-MEK-ERK) pathway, and fail to undergo positive selection. This defect can be partially rescued with constitutively active Raf, indicating that calcineurin controls MAPK signalling. Analysis of mice deficient in both Bim (which is required for negative selection) and calcineurin revealed that calcineurin-induced ERK (extracellular signal-regulated kinase) sensitization is required for differentiation in response to 'weak' positive selecting signals but not in response to 'strong' negative selecting signals (which normally induce apoptosis). These results indicate that early calcineurin/NFAT signalling produces a developmental period of ERK hypersensitivity, allowing very weak signals to induce positive selection. This mechanism might be generally useful in the discrimination of graded signals that induce different cell fates.     

CD3delta couples T-cell receptor signalling to ERK activation and thymocyte positive selection

Thymocytes from mice lacking the CD3delta chain of the T-cell receptor (TCR), unlike those of other CD3-deficient mice, progress from a CD4- CD8- double-negative to a CD4+ CD8+ double-positive stage. However, CD3delta-/- double-positive cells fail to undergo positive selection, by which double-positive cells differentiate into more mature thymocytes. Positive selection is also impaired in mice expressing inactive components of the Ras/mitogen activated protein (MAP) kinase signalling pathway. Here we show that CD3delta-/- thymocytes are defective in the induction of extracellular signal-regulated protein kinase (ERK) MAP kinases upon TCR engagement, whereas activation of other MAP kinases is unaffected. The requirement for CD3delta maps to its extracellular or transmembrane domains, or both, as expression of a tail-less CD3delta rescues both ERK activation and positive selection in CD3delta-/- mice. Furthermore, the defect correlates with severely impaired tyrosine phosphorylation of the linker protein LAT, and of the CD3zeta chain that is localized to membrane lipid rafts upon TCR engagement. Our data indicate that the blockade of positive selection of CD3delta-/- thymocytes may derive from defective tyrosine phosphorylation of CD3zeta in lipid rafts, resulting in impaired activation of the LAT/Ras/ERK pathway.     

Negative selection of murine medullary-type single positive thymocytes

Negative selection depletes self-reactive T cells, thus ensuring self-tolerance. It is usually considered that negative selection imposed on double-positive (DP) thymocytes that reside at the cortico-medullary junction. Negative selection model was set up by injecting mice with anti-T cell receptor (TCR) monoclonal antibody (mAb) intraperitoneally in this work. As shown in phenotypic analysis of thymocytes, negative selection destroys not only cortical-type DP thymocytes, but also medullary-type CD3+TCRαβ+CD4SP and CD3+TCRαβ+CD8SP thymocytes. Negative selection of medullary-type single positive (SP) are more susceptible to apoptosis, while with development of the cells, their resistance to apoptosis increases. Therefore, negative selection does not operate on functionally mature thymocytes at the late stage. This result is a supplement to the traditional theory of negative selection. Negative selection of medullary-type thymocytes is probably to further deplete self-reactive T cells, thus producing precise TCR repertoire and inducing self-tolerance.     

A motif in the alphabeta T-cell receptor controls positive selection by modulating ERK activity

Positive selection allows thymocytes that recognize an individual's own major histocompatibility complex (self-MHC) molecules to survive and differentiate, whereas negative selection removes overtly self-reactive thymocytes. Although both forms of thymic selection are mediated by the alphabeta T-cell receptor (TCR) and require self-MHC recognition, an important question is whether they are controlled by distinct signalling cascades. We have shown that mutation of an essential motif within the TCR alpha-chain-connecting peptide domain (alpha-CPM) profoundly affects positive but not negative selection. Using transgenic mice expressing a mutant alpha-CPM TCR we examined the contribution of several mitogen-activated protein kinase (MAPK) cascades to thymic selection. Here we show that in thymocytes expressing a mutant alpha-CPM receptor, a positively selecting peptide failed to activate the extracellular signal-regulated kinase (ERK), although other MAPK cascades were induced normally. The defect in ERK activation was associated with impaired recruitment of the activated tyrosine kinases Lck and ZAP-70, phosphorylated forms of the TCR component CD3zeta and the adaptor protein LAT to detergent-insoluble glycolipid-enriched microdomains (DIGs). Therefore, an intact DIG-associated signalosome is essential for sustained ERK activation, which leads to positive selection.     

Intrathymic signalling in immature CD4+CD8+ thymocytes results in tyrosine phosphorylation of the T-cell receptor zeta chain

Thymic selection of the developing T-cell repertoire occurs in immature CD4+CD8+ double-positive thymocytes and is thought to be mediated by signals transduced by T-cell antigen receptor (TCR) molecules and possibly by CD4 and CD8 accessory molecules as well. It is not known, however, which signal-transduction mechanisms function in immature CD4+CD8+ thymocytes on engagement of TCR, CD4 or CD8 molecules. In mature T cells, CD4 and CD8 molecules are each associated with the src-like protein tyrosine kinase p56 lck and signals transduced by TCR and CD4 activate tyrosine kinases that phosphorylate TCR-zeta chains and other intracellular substrates. Consequently, we examined whether tyrosine kinases could be similarly activated in immature CD4+CD8+ thymocytes. Unexpectedly, we found that TCR-zeta chains from CD4+CD8+ thymocytes were already phosphorylated in vivo, and that dephosphorylation of this TCR subunit occurred on removal of CD4+CD8+ cells from their intrathymic environment. Rephosphorylation of TCR-zeta in cultured CD4+CD8+ thymocytes occurred rapidly in vitro, either in response to cross-linking of TCR, CD4 or CD8 by specific monoclonal antibodies, or on cell-cell contact. These observations indicate that tyrosine kinases are activated in vivo in immature CD4+CD8+ thymocytes undergoing thymic differentiation and selection. They also indicate that TCR, CD4 and CD8 molecules can function in CD4+CD8+ thymocytes as signalling molecules to activate tyrosine kinases and that phosphorylated TCR-zeta serves as a marker of these signalling events.     

Major histocompatibility complex-linked specificity of gamma delta receptor-bearing T lymphocytes

Several recent studies have identified a distinct subset of CD3(T3)+CD4-CD8-T lymphocytes that express a CD3-associated heterodimer made up of the protein encoded by the T-cell receptor (TCR) gamma-gene and a second glycoprotein termed TCR delta (refs 1-4). TCR gamma delta is expressed on CD3+ thymocytes during fetal ontogeny before the appearance of TCR alpha-beta (alpha beta) (refs 5-7), on CD3+CD4-CD8- adult thymocytes, and on a subset (1-10%) of CD3+ cells in adult peripheral lymphoid organs and the peripheral blood. TCR gamma delta-expressing T cells probably represent a distinct mature T-cell lineage with the capacity to proliferate in response to receptor-mediated signals, and to display non-major histocompatibility complex (MHC)-restricted cytolysis. Critical to understanding the function of this T-cell subset is the identification of the ligand(s) recognized by TCR gamma delta. Here we describe an alloreactive CD3+CD4-CD8-TCR gamma delta-expressing, TCR alpha beta-negative, T-cell line that manifests MHC-linked recognition specificity for both proliferation and cytotoxicity. Our results suggest that T cells expressing TCR gamma delta are capable of self-non-self MHC discrimination and that they can undergo MHC-influenced selection during differentiation like TCR alpha beta-expressing T cells.     

Distinct sequence of negative or positive selection implied by thymocyte T-cell receptor densities

Recent evidence suggests that positive and negative selection of thymocytes bearing alpha beta T-cell receptors occurs during the predominant double-positive (CD4+CD8+) stage. But the sequence or stage at which positive or negative selection occurs during thymocyte maturation has not been well defined. Here we use transgenic mice to show that the CD4+CD8+ stage might be further subdivided into CD3lo (low) and CD3in (intermediate) stages. The CD3in stage could represent T cells that have been positively selected, as this stage is dependent on the presence of the appropriate major histocompatibility complex restriction element. In addition, we use two different tolerizing antigens to show that negative selection may occur either before or after this CD3in stage.     

Engagement of the CD4 molecule influences cell surface expression of the T-cell receptor on thymocytes

The intrathymic differentiation process by which precursor cells derived from the bone marrow develop into immuno-competent T lymphocytes is poorly understood. Most thymocytes express both CD4 and CD8 accessory molecules, yet little is known about either the function of these molecules or the responsiveness of the CD4+8+ double positive thymocytes that bear them. Here, we address the possibility that CD4 engagement influences T-cell receptor (TCR) expression on developing thymocytes. We engaged CD4 molecules on murine thymocytes by in vivo injection of an anti-CD4 monoclonal antibody, which reduced the surface expression of CD4 on CD4+ thymocytes. More importantly, CD4 engagement also affected TCR expression on CD4+ thymocytes, but the effect on CD4+8+ double positive and CD4+8- single positive thymocytes was very different. CD4+8+ thymocytes responded to CD4 engagement by dramatically increasing surface expression of TCR, whereas CD4+8- thymocytes decreased surface expression of TCR. These results demonstrate that the effect of CD4 engagement on TCR expression is dependent upon the developmental state of the responding thymocyte, and, most interestingly, results in increased TCR expression by double positive thymocytes.     

The generation of mature T cells requires interaction of the alpha beta T-cell receptor with major histocompatibility antigens

THE T-cell repertoire within an individual is biased to recognize antigen in the context of self major histocompatibility complex (MHC) antigens. This is thought to depend on a process of positive selection during development. Support for this notion has recently been obtained in experiments using transgenic mice bearing genes for T-cell receptors (TCR) of defined specificity: T cells expressing the introduced genes form the main part of the mature T-cell population only in mice that express the appropriate MHC product. We have now extended these observations using TCR transgenic mice homozygous for the severe combined immunodeficiency (SCID) mutation which are defective in the rearrangement of both TCR and immunoglobulin genes. In this case mature thymocytes develop only in transgenic mice that express the MHC product which restricts the specificity of the transgenic TCR. This shows that the interaction of the alpha beta TCR with thymic MHC antigen is essential for the development of mature T cells. Furthermore, the peripheral lymph nodes of such mice are underdeveloped, suggesting that the peripheral expansion of mature T cells may require interactions with other lymphocytes expressing a range of receptors.     

Differential expression of two distinct T-cell receptors during thymocyte development

The product of the T-cell receptor (TCR) gamma-gene has recently been found to be expressed on a subset of both peripheral cells and thymocytes. As an initial approach to understanding the role of this gamma-chain of TCR (TCR gamma) in T-cell development, we have studied the ontogeny of TCR expression at the protein level in the developing murine thymus. We show here that the first T3-associated TCR to be expressed in the developing thymus is a disulphide-linked heterodimer composed of a gamma-chain of relative molecular mass 35,000 (Mr 35K) and a 45K partner (termed TCR delta). This TCR gamma delta is first detected approximately two days before the appearance of cell-surface TCR alpha beta heterodimers. We report that N-glycosidase digestions reveal that all of the gamma-protein expressed on fetal thymocytes, as in adult CD4-8-(L3T4-, Lyt2-) thymocytes, bear N-linked carbohydrate side chains. The major gamma-gene transcribed in mature, alpha beta-bearing T cells (V gamma 1.2C gamma 2)encodes no N-linked glycosylation site so these results suggest that the fetal gamma delta receptor defines a distinct T-cell lineage whose development in the thymus precedes classical alpha beta-bearing cells.     

Positive selection of antigen-specific T cells in thymus by restricting MHC molecules

Thymus-derived lymphocytes (T cells) recognize antigen in the context of class I or class II molecules encoded by the major histocompatibility complex (MHC) by virtue of the heterodimeric alpha beta T-cell receptor (TCR). CD4 and CD8 molecules expressed on the surface of T cells bind to nonpolymorphic portions of class II and class I MHC molecules and assist the TCR in binding and possibly in signalling. The analysis of T-cell development in TCR transgenic mice has shown that the CD4/CD8 phenotype of T cells is determined by the interaction of the alpha beta TCR expressed on immature CD4+8+ thymocytes with polymorphic domains of thymic MHC molecules in the absence of nominal antigen. Here we provide direct evidence that positive selection of antigen-specific, class I MHC-restricted CD4-8+ T cells in the thymus requires the specific interaction of the alpha beta TCR with the restricting class I MHC molecule.     

Thymic selection process induced by hybrid antibodies

Thymus-derived (T) lymphocytes using the alpha beta T-cell antigen receptor (TCR) recognize fragmented antigen in conjunction with surface molecules encoded by genes of the major histocompatibility complex (MHC). Peripheral T lymphocytes preferentially see antigen presented by self rather than by foreign MHC molecules, and autoreactive T lymphocytes are deleted. Thus, the peripheral T-lymphocyte repertoire is skewed towards recognition of antigen in the context of self-MHC and towards tolerance to self-antigens. During T-lymphocyte development in the thymus, this repertoire is formed by the interaction of TCR with MHC molecules resulting in positive and negative selection phenomena. Hybrid antibodies (HAbs) that carry binding sites to the TCR and to a surface marker on another cell can engage all T lymphocytes regardless of their specificity. It should be possible to mimic selection processes in normal animals with HAb that specifically link members of a TCR family to MHC molecules on the thymic stroma. We have probed T-lymphocyte development with HAbs linking V beta 8-positive TCR to either class I or class II MHC products in thymic organ culture. Thymocytes exposed to either HAb in an early stage of maturation respond with a significant increase in the frequency of V beta 8-carrying cells. At a later stage of development V beta 8-positive thymocytes are depleted. These results illustrate the succession of positive and negative selection in the developing thymus of normal mice.     

Self-tolerance to transgenic gamma delta T cells by intrathymic inactivation

During their intrathymic differentiation, T lymphocytes expressing alpha beta T-cell receptors (TCR) are negatively and positively selected. This selection contributes to the establishment of self-tolerance and ensures that mature CD4+ and CD8+ cell populations are restricted by the self major histocompatibility complex. Little is known, however, about gamma delta T-cell development. To investigate whether selection operates in the establishment of the gamma delta T-cell class, we have generated transgenic mice using gamma- and delta-transgenes encoding a TCR that is specific for a product of a gene in the TL-region of the TLb haplotype. Similar numbers of thymocytes expressing the transgenic TCR were generated in mice of TLb and TLd haplotypes. But gamma delta thymocytes from TLb and TLd transgenic mice differed in cell size, TCR density and in their capacity to respond to TLb stimulator cells or interleukin-2 (IL-2). In contrast to gamma delta T cells from TLd transgenic mice, gamma delta T cells from TLb transgenic mice did not produce IL-2 and did not proliferate in response to TLb stimulator cells, but they did proliferate in the presence of exogenous IL-2. These results indicate that functional inactivation of self-antigen-specific T cells could contribute to the establishment of self-tolerance to thymic determinants.     

A novel population of T-cell receptor alpha beta-bearing thymocytes which predominantly expresses a single V beta gene family

Recent studies have demonstrated that CD3 is expressed on a subset of thymocytes with a CD4-CD8- (double negative) phenotype. At least some of these cells bear the CD3-associated gamma delta T-cell receptor (TCR gamma delta). Here we describe a second subset of double negative thymocytes which expresses CD3-associated alpha beta receptors (TCR alpha beta). Surprisingly, these cells express predominantly the products of a single V beta gene family (V beta 8). These CD4-CD8-, TCR alpha beta+ cells appear relatively late in ontogeny (between birth and day 5 of life) and thus are unlikely to be the precursors to the TCR alpha beta-bearing cells (CD4+CD8- and CD4-CD8+) already present at birth. They can be selectively expanded in vitro by stimulation with a monoclonal antibody to V beta 8 (F23.1) in the presence of interleukin I (IL-1). We propose that this cell type is a unique T-cell population distinguishable from typical TCR alpha beta+ T cells by its CD4-CD8- phenotype and a restricted TCR V beta repertoire. Analysis of the unique phenotype of these cells suggests that they may represent the normal counterpart of the defective CD4-CD8- T cells found in the lpr autoimmune mouse.     

Development of CD4-CD8+ cytotoxic T cells requires interactions with class I MHC determinants

Differentiation of bone marrow derived precursors into mature T cells takes place in the thymus. During differentiation, T cells develop the receptor repertoire which allows them to recognize antigen in the context of self major histocompatibility complex (MHC) molecules. Mature T helper cells (mostly CD4+ CD8-) recognize antigen in the context of class II MHC molecules, whereas cytotoxic T cells (mostly CD4-CD8+) recognize antigen in the context of class I MHC determinants. Thymic MHC-encoded determinants greatly influence the selection of the T-cell receptor repertoire. In addition to positive selection, a negative selection to eliminate self-reactive T-cell clones is thought to occur in the thymus, but how this 'education' occurs is not well understood. It has been suggested that during differentiation an interaction between the T-cell receptor (TCR) and MHC-encoded determinants occurs, leading to the selection of an MHC-restricted receptor repertoire. In support of this hypothesis, class-II-specific, CD4+ CD8- helper T cells fail to develop in mice neonatally treated with anti-class II monoclonal antibody (mAb). As CD4-CD8+ cells differ from the CD4+ CD8- lineage (in function, MHC-restriction specificity and perhaps site of education) we examined whether interactions with MHC determinants are also necessary for the development of class-I-specific T cells. Here we show that mice chronically treated with anti-class I mAb from birth lack CD4-CD8+ cells and cytotoxic T-cell precursors, indicating that most CD4-CD8+ T cells need interaction with class I MHC molecules during differentiation.     

Thymic selection threshold defined by compartmentalization of Ras/MAPK signalling

A healthy individual can mount an immune response to exogenous pathogens while avoiding an autoimmune attack on normal tissues. The ability to distinguish between self and non-self is called 'immunological tolerance' and, for T lymphocytes, involves the generation of a diverse pool of functional T cells through positive selection and the removal of overtly self-reactive thymocytes by negative selection during T-cell ontogeny. To elucidate how thymocytes arrive at these cell fate decisions, here we have identified ligands that define an extremely narrow gap spanning the threshold that distinguishes positive from negative selection. We show that, at the selection threshold, a small increase in ligand affinity for the T-cell antigen receptor leads to a marked change in the activation and subcellular localization of Ras and mitogen-activated protein kinase (MAPK) signalling intermediates and the induction of negative selection. The ability to compartmentalize signalling molecules differentially in the cell endows the thymocyte with the ability to convert a small change in analogue input (affinity) into a digital output (positive versus negative selection) and provides the basis for establishing central tolerance.     

Selective development of CD4+ T cells in transgenic mice expressing a class II MHC-restricted antigen receptor

T lymphocytes are predisposed to recognition of foreign protein fragments bound to cell-surface molecules encoded by the major histocompatibility complex (MHC). There is now compelling evidence that this specificity is a consequence of a selection process operating on developing T lymphocytes in the thymus. As a result of this positive selection, thymocytes that express antigen receptors with a threshold affinity for self MHC-encoded glycoproteins preferentially emigrate from the thymus and seed peripheral lymphoid organs. The specificity for both foreign antigen and MHC molecules is imparted by the alpha and beta chains of the T-cell antigen receptor (TCR). Two other T-cell surface proteins, CD4 and CD8, which bind non-polymorphic regions of class II and class I MHC molecules respectively, are also involved in these recognition events and play an integral role in thymic selection. In order to elucidate the developmental pathways of class II MHC-restricted T cells in relation to these essential accessory molecules, we have produced TCR-transgenic mice expressing a receptor specific for a fragment of pigeon cytochrome c and the Ek (class II MHC) molecule. The transgenic TCR is expressed on virtually all T cells in mice expressing Ek. The thymuses of these mice contain an abnormally high percentage of mature CD4+CD8- cells. In addition, the peripheral T-cell population is almost exclusively CD4+, demonstrating that the MHC specificity of the TCR determines the phenotype of T cells during selection in the thymus.     

Programmed death of autoreactive thymocytes

T lymphocytes bearing high-affinity T-cell receptors (TCR) for self-antigens are clonally deleted during thymus development. Several recent studies have identified variable domains of the beta-chain of the TCR that are specifically deleted in vivo in mouse strains that express major histocompatibility complex class II molecules in addition to poorly defined self-antigens, including those encoded by the Mls-1a and Mls-2a loci. Deletion of autoreactive cells in these systems occurs in the thymus, and antibody blocking experiments in vivo have implicated the phenotypically immature CD4+CD8+ 'cortical' subset as the target population for clonal deletion. Similarly, studies with transgenic mice bearing autoreactive TCR have provided independent evidence that clonal deletion occurs at the CD4+CD8+ stage of development. But none of these studies directly identified dying autoreactive cells, and the circumstances leading to deletion remain unclear. Here we report that neonatal thymus contains a significant population of phenotypically mature CD4+CD8- cells bearing autoreactive TCR. When placed in short-term culture, a large proportion (60%) of these autoreactive cells die selectively. Furthermore, their death can be prevented by inhibitors of macromolecule (RNA and protein) synthesis, as is the case for glucocorticoid-induced death of thymocytes. These data indicate that physiological clonal deletion of autoreactive cells involves 'programmed' cell death, and that it can occur in cells with a mature (CD4+CD8-) surface phenotype.     

Participation of CD4 coreceptor molecules in T-cell repertoire selection.

During thymocyte development, progenitor cells bearing both CD4 and CD8 coreceptor molecules mature into functional T lymphocytes that express these proteins in a mutually exclusive way. Although T-cell specificity is determined primarily by the structure of the T-cell antigen receptor (TCR) heterodimer, a developmentally regulated process acts to ensure that cells bearing class II-restricted TCRs are CD4+ and those bearing class I-restricted TCRs express only CD8. To investigate this maturation process, we have engineered transgenic mice in which CD4 is expressed in all thymocyte subsets and in all peripheral T cells. Peripheral CD4+8+ T lymphocytes from these mice react with both class I and class II alloantigens. Moreover, expression of the CD4 transgene disrupts the positive selection of doubly transgenic thymocytes bearing a class I-restricted TCR specific for the male (H-Y) antigen. Hence the CD4 coreceptor participates directly in T-cell repertoire selection.     

The structural basis for autonomous dimerization of the pre-T-cell antigen receptor

The pre-T-cell antigen receptor (pre-TCR), expressed by immature thymocytes, has a pivotal role in early T-cell development, including TCR β-selection, survival and proliferation of CD4(-)CD8(-) double-negative thymocytes, and subsequent αβ T-cell lineage differentiation. Whereas αβTCR ligation by the peptide-loaded major histocompatibility complex initiates T-cell signalling, pre-TCR-induced signalling occurs by means of a ligand-independent dimerization event. The pre-TCR comprises an invariant α-chain (pre-Tα) that pairs with any TCR β-chain (TCRβ) following successful TCR β-gene rearrangement. Here we provide the basis of pre-Tα-TCRβ assembly and pre-TCR dimerization. The pre-Tα chain comprised a single immunoglobulin-like domain that is structurally distinct from the constant (C) domain of the TCR α-chain; nevertheless, the mode of association between pre-Tα and TCRβ mirrored that mediated by the Cα-Cβ domains of the αβTCR. The pre-TCR had a propensity to dimerize in solution, and the molecular envelope of the pre-TCR dimer correlated well with the observed head-to-tail pre-TCR dimer. This mode of pre-TCR dimerization enabled the pre-Tα domain to interact with the variable (V) β domain through residues that are highly conserved across the Vβ and joining (J) β gene families, thus mimicking the interactions at the core of the αβTCR's Vα-Vβ interface. Disruption of this pre-Tα-Vβ dimer interface abrogated pre-TCR dimerization in solution and impaired pre-TCR expression on the cell surface. Accordingly, we provide a mechanism of pre-TCR self-association that allows the pre-Tα chain to simultaneously 'sample' the correct folding of both the V and C domains of any TCR β-chain, regardless of its ultimate specificity, which represents a critical checkpoint in T-cell development. This unusual dual-chaperone-like sensing function of pre-Tα represents a unique mechanism in nature whereby developmental quality control regulates the expression and signalling of an integral membrane receptor complex.