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.     

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.     

Characteristics of TCRαβ+CD4-CD8- thymocytes in fetal thymic organ culture

TCRαβ+CD4-CD8- (TCR+ DN) thymocytes at different developmental periods, i.e. after either 9 or 18 days of culture in the fetal thymic organ culture (FTOC) system, were characterized in the properties of phenotype, proliferation, differentiation and apoptosis. The results showed that anti-CD3 mAb significantly promoted proliferation of TCRαβ+ DN cells generated after 18 days of culture in FTOC, whereas the cells generated after 9 days of culture responded to anti-CD3 mAb by proliferation weakly. IL-7 efficiently induced TCRαβ+ DN cells at day 9 of FTOC to differentiate into TCRαβ+CD4+/CD8+ SP cells without detectable transitional stage of TCRαβ+CD4+CD8+ (DP) cells. In contrast, fewer TCRαβ+ DN cells generated after 18 days of FTOC were induced to differentiate into SP cells. The thymic stromal cell line MTEC5 cells synergized with IL-7 to promote the differentiation of TCRαβ+ DN cells. In addition, TCRαβ+ DN cells were shown to be less susceptible to apoptosis compared with the other major thymocyte subsets. Taken together, these data have provided insight into the characteristics of TCRαβ+ DN thymocytes.     

Interleukin-4 mediates CD8 induction on human CD4+ T-cell clones

CD4 and CD8 antigens are simultaneously expressed on most of the cortical thymocytes, that weakly express the T-cell antigen receptor(TCR)/CD3 complex. Mature peripheral T cells, however, strongly express the TCR complex and are positive for either CD4 or CD8. Nevertheless, a small percentage of peripheral CD3+ T cells express CD4 and CD8 simultaneously. These mature, double positive cells could be intermediates between CD4+CD8+ thymocytes and mature, single positive T cells, or they may originate from single positive T cells that acquire either CD4 or CD8. Here we report that activation and culturing of cloned CD4+ T cells in interleukin-4 (IL-4), results in the acquisition of CD8 due to its de novo synthesis. The IL-4-induced co-expression of CD8 on CD4+ T cells is reversible, in that CD8 disappeared from double positive T-cell clones isolated in IL-4, when they were cultured in IL-2. CD8 induced by IL-4 can be functional as a monoclonal antibody to CD8 inhibited anti-CD3-mediated cytotoxicity by a double positive T-cell clone.     

Signal for T-cell differentiation to a CD4 cell lineage is delivered by CD4 transmembrane region and/or cytoplasmic tail.

Mature T cells express either CD4 or CD8 on their surface. Most helper T cells express CD4, which binds to class II major histocompatibility complex (MHC) proteins, and most cytotoxic T cells express CD8, which binds to class I MHC proteins. In the thymus, mature CD4+CD8- and CD4-CD8+ T cells expressing alpha beta T-cell antigen receptors (TCR) develop from immature thymocytes through CD4+CD8+ alpha beta TCR+ intermediates. Experiments using mice transgenic for alpha beta TCR suggest that the specificity of the TCR determines the CD4/CD8 phenotype of mature T cells. These results, however, do not indicate how a T cell differentiates into the CD4 or CD8 lineage. Here we show that the CD4 transmembrane region and/or cytoplasmic tail mediates the delivery of a specific signal that directs differentiation of T cells to a CD4 lineage. We generated transgenic mice expressing a hybrid molecule composed of the CD8 alpha extracellular domains linked to the CD4 transmembrane region and cytoplasmic tail. We predicted that this hybrid molecule would bind to class I MHC proteins through the extracellular domains but deliver the intracellular signals characteristic of CD4. By crossing our transgenic mice with mice expressing a transgenic alpha beta TCR specific for a particular antigen plus class I MHC protein, we were able to express the hybrid molecule in developing thymocytes expressing the class I MHC-restricted TCR. Our results show that the signal transduced by the hybrid molecule results in the differentiation of immature thymocytes expressing a class I-restricted TCR into mature T cells expressing CD4.     

Phenotypic analysis of the medullary-type CD4-CD8+ thymocytes in mice

Phenotypic analysis of the medullary-type CD4-CD8+ (CD8SP) thymocytes has revealed phenotypic heterogeneity within this cell population. The phenotype of mature peripheral CD8+T cells is TCRαβ+CD3+Qa-2+HSA-3G11-6C10-, whereas in the medullary-type CD8SP thymocytes, 20% are Qa-2+; 33%, HAS-; 30%, 3G11-; and 70% are 6C10-. The disparate expression patterns of these four cell surface markers suggest that medullary-type CD8SP thymocytes may undergo phenotypic maturation process. According to the distribution of these four cell surface markers, six subgroups of CD8SP thymocytes have been identified. The precursor-progeny relationship along with developmental pathway is postulated as follows: 6C10+HSA+3G11-Qa-2-(r) 6C10+HSA+ 3G11+Qa-2-(r) 6C10-HSA+3G11+Qa-2-(r) 6C10-HSA-3G11+Qa-2-(r) 6C10-HSA-3G11Qa-2-(r) 6C10-HAS-3G11-Qa-2+, the cells in the last subgroup exit the thymus and home into periphery.     

Recognition of cluster of differentiation 1 antigens by human CD4-CD8-cytolytic T lymphocytes

Human cluster-of-differentiation 1 (CD1) is a family of cell surface glycoproteins of unknown function expressed on immature thymocytes, epidermal Langerhans cells and a subset of B lymphocytes. Three homologous proteins, CD1a, b and c, have been defined serologically, and the CD1 gene locus on human chromosome 1 contains five potential CD1 genes. Analysis of the predicted amino-acid sequences of CD1 molecules reveals a low but significant level of homology to major histocompatibility complex (MHC) class I and class II molecules, and, like MHC class I molecules, CD1 molecules are associated non-covalently with beta 2-microglobulin. These structural similarities to known antigen-presenting molecules, together with the expression of CD1 on cells capable of antigen presentation, suggest a role for CD1 molecules in antigen recognition by T cells. Here we demonstrate the specific recognition of CD1a by a CD4-CD8- alpha beta T-cell receptor (TCR) expressing cytolytic T lymphocyte (CTL) line and the specific recognition of CD1c by a CD4-CD8- gamma delta TCR CTL line. The interaction of CD1-specific CTLs with CD1+ target cells appeared to involve the CD3-TCR complex, and did not show evidence of MHC restriction. These results suggest that for a subset of T cells, CD1 molecules serve a function analogous to that of MHC class I and II molecules.     

CD4+ murine T cells develop from CD8+ precursors in vivo

The adult murine thymus contains four subpopulations of thymocytes defined by the T-cell surface antigens CD4 (L3T4) (a marker of helper T cells) and CD8 (Lyt2) (a marker of cytotoxic/suppressor T cells): CD4+8- and CD4-8+ (single positives), CD4+8+ (double positives) and CD4-8- (double negatives). To understand how T cells develop in the thymus, it is important to determine the lineage relationships among these subpopulations. In particular, the status of double positives, which make up approximately 80% of the total thymocyte population, has long been controversial. Some purpose that double positives are 'dead-end cells' that all die in the thymus, perhaps because they have been rejected by some selection process. Others suggest that, although most double positives die in the thymus, some develop into the more mature single positives that leave the thymus. The experiments presented here show that repeated injections of anti-CD8 monoclonal antibodies block the development of CD4+ cells, demonstrating that these cells develop from CD8+ precursors, probably double positive thymocytes, in vivo.     

Positive selection of CD4-CD8+ T cells in the thymus of normal mice

The diversification of the repertoire of T-cell antigen receptor (TCR) specificities is influenced by at least two selection processes which occur in the thymus. One of these, termed 'negative selection', is required to install a state of tolerance to self-antigens in the T-cell repertoire and is often achieved by clonal deletion. The second type of selection operating in the thymus results in preferential differentiation of T cells that have restriction specificity for thymic major histocompatibility complex glycoproteins, but the mechanisms leading to this selective process are not yet clear. One model used to describe this 'positive selection' proposes that only those T cells with sufficient avidity for the MHC glycoproteins expressed in the thymus are allowed to acquire functional competence. Here we directly investigate the generation of TCR specificities by following the fate of developing V beta 17+ CD4-CD8+ T cells under conditions where one of the main class I-MHC molecules, either H-2K or H-2D, was specifically blocked by in vitro monoclonal antibody treatment. The results show that development of V beta 17+ CD4-CD8+ T cells in the SJL H-2s mouse strain is selectively abrogated by blocking class I-Ks molecules but is unaffected by blocking class I-Ds molecules. These data directly demonstrate that generation of CD4-CD8+ T cells expressing a particular TCR V beta segment can be correlated with the expression of a particular class I-MHC molecule, thereby providing evidence for positive selection.     

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.     

Thymic major histocompatibility complex antigens and the alpha beta T-cell receptor determine the CD4/CD8 phenotype of T cells

T-cell receptors and T-cell subsets were analysed in T-cell receptor transgenic mice expressing alpha and beta T-cell receptor genes isolated from a male-specific, H-2Db-restricted CD4-8+ T-cell clone. The results indicate that the specific interaction of the T-cell receptor on immature thymocytes with thymic major histocompatibility complex antigens determines the differentiation of CD4+8+ thymocytes into either CD4+8- or CD4-8+ mature T cells.     

Mutations in T-cell antigen receptor genes alpha and beta block thymocyte development at different stages.

Analysis of mice carrying mutant T-cell antigen receptor (TCR) genes indicates that TCR-beta gene rearrangement or expression is critical for the differentiation of CD4-CD8- thymocytes to CD4+CD8+ thymocytes, as well as for the expansion of the pool of CD4+CD8+ cells. TCR-alpha is irrelevant in these developmental processes. The development of gamma delta T cells does not depend on either TCR-alpha or TCR-beta.     

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.     

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.     

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.     

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.     

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.     

Selective expression of an antigen receptor on CD8-bearing T lymphocytes in transgenic mice

The major problem in the study of T-cell development is that of tracking thymocytes of a given specificity. Recent studies have exploited natural correlations between the expression of a particular V beta gene segment and T-cell receptor (TCR) specificity. We and others (refs 5, 6 and M. Davis, personal communication) have taken an alternative approach. We have generated transgenic mice expressing the alpha beta antigen receptor from the cytotoxic T-lymphocyte clone 2C (ref. 7). In transgenic mice of the same haplotype as the 2C clone, the 2C TCR was expressed on 20-95% of peripheral T cells. Very few of these T cells carried the CD4 antigen; the vast majority were CD4-CD8+ and were able to lyse targets with the same specificity as the original 2C clone. These results indicate that the alpha beta heterodimer transfers specificity to recipient cells as expected from earlier studies, and that receptor specificity in T-cell repertoire selection is determined by both alpha beta heterodimer and CD4 or CD8 accessory molecules.     

Functional maturation of two murine medullary-type CD8SP thymocytes

TCRαβCD4-CD8+ thymocytes are heterogeneity. They may undergo phenotypic and functional maturation within thymic medulla. Medullary-type CD8SP thymocytes were divided into seven subsets based on phenotypic analysis, and their precursor-progeny relationship along with the differential pathway was also delineated. To further testify the validity of the maturation pathway, we purified 6C10-CD69+ cells representing the early stage and 6C10-Qa-2+ cells representing the later stage among medullary-type CD8SP thymocytes and compared their functional maturation levels. CD8+ T cells of spleen were used as the control. It is shown that there is no obvious difference of proliferation ability among these three subsets; however, intracytoplasmic cytokine assay shows that there is a hier archy of IFN-γ and TNFα secretion among these subsets, strikingly comparable to their phenotypic status among medullary type CD8SP thymocytes. The bioassays of IL-2 and IFN-γ in culture supernatant give the similar results.