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.     

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.     

Antibodies to CD3/T-cell receptor complex induce death by apoptosis in immature T cells in thymic cultures

The receptors found on most T lymphocytes bind to antigen presented on major histocompatibility complex proteins and consist of dimers of alpha- and beta-polypeptides associated with the invariant CD3 complex. A fully competent immune system requires a diverse array of T-cell antigen receptors (TCRs) with different specificities. This diversity is generated by rearrangement of TCR alpha- and beta-chain gene segments within the thymus where the receptors are first expressed. Any cells carrying self-reactive receptors must be eliminated, suppressed or inactivated so that destructive autoimmunity is avoided. Recently, compelling evidence has shown that one process involved in producing such self-tolerance is clonal deletion of autoreactive cells within the thymus by an as-yet-undefined mechanism. Here we show that engaging the CD3/TCR complex of immature mouse thymocytes with anti-CD3 antibodies produces DNA degradation and cell death through the endogenous pathway of apoptosis. Activation of this process in immature T cells by the binding of the TCR to self-antigens may therefore be the mechanism which produces clonal deletion and consequently self-tolerance.     

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.     

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.     

Positive selection of CD4+ T cells mediated by MHC class II-bearing stromal cell in the thymic cortex

T lymphocytes differentiate in the thymus, where functionally immature, CD4+CD8+ (double positive) thymocytes develop into functionally mature CD4+ helper cells and CD8+ cytotoxic (single positive) T cells. The thymus is the site where self-reactive T cells are negatively selected (clonally deleted) and where T cells with the capacity to recognize foreign antigens in association with self-proteins encoded by the major histocompatibility complex (MHC) are positively selected. The net result of these developmental pathways is a T-cell repertoire that is both self-tolerant and self-restricted. One unresolved issue is the identity of the thymic stromal cells that mediate the negative and positive selection of the T-cell repertoire. Previous work has pointed to a bone-marrow-derived macrophage or dendritic cell as the inducer of tolerance, whereas a radiation-resistant, deoxyguanosine-resistant thymic cell seems to mediate the positive selection of self-MHC restricted T cells. Thymic stromal cells in the cortex interact with the T-cell antigen receptor on thymocytes. Using several strains of transgenic mice that express the class II MHC molecule I-E in specific regions of the thymus, we show directly that the positive selection of T cells is mediated by an I-E-bearing cell in the thymic cortex.     

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.     

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.     

Intrathymic deletion of self-reactive cells prevented by neonatal anti-CD4 antibody treatment

T-cell differentiation in the thymus involves the coordinate expression of genes encoding the alpha and beta chains of the major histocompatibility complex-restricted heterodimeric antigen receptor (TCR) complex, as well as other functionally important molecules such as CD4 and CD8. The repertoire of TCR expressed by T cells is generally thought to be influenced by positive and/or negative selection events occurring when TCRs on developing T cells interact with self-antigens and major histocompatibility complex components. Using a model system in which specific antigen-reactive cells can be monitored by virtue of their preferential expression of certain TCR beta-chain variable (V beta) domains, it has been shown that self-reactive T cells are clonally deleted during development. We report here that clonal deletion of V+ beta 6 cells in Mlsa mice can be prevented by in vivo neonatal administration of monoclonal antibodies directed against CD4. Furthermore, as anti-CD4 monoclonal antibody treatment resulted in the reappearance of V+ beta 6 cells in the mature CD8+ T-cell subset, it is likely that clonal deletion acts on the CD4+CD8+ thymocyte subset and that this subset is an intermediate stage in the differentiation pathway of both CD4+ and CD8+ T-cell lineages.     

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.     

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 CDS⁺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→ 6C10⁺HSA⁺ 3G11⁺Qa-2 → 6C10 HSA⁺3G11⁺Qa-2 → 6C10⁻HSA⁻3G11⁺Qa-2 → 6C10⁻HSA⁻3G11 Qa-2 → 6C10⁻HA S 3G11 Qa-2⁺, the cells in the last subgroup exit the thymus and home into periphery.     

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.     

Effect of MTECl on the functional expression of TCRαβ~ + 3G11~- 6C10~-CD4~+CD8~- thymocyte subset

A murine CD4+ thymocyte subset with phenotype of TCRαβ + 3G11- 6C10- CD4 + CD8- CD69 +/- HSAmed/locontains the cells in relatively functional matured status. The functional property of the cells in this subset is characterized by the unique pattern of cytokine production at transitional stage from Th0 to Th2 type with the latter being the dominant type. After being co-cultured with murine thymic medullary epithelial cell line (MTEC1) cells, a murine thymic medullary type epithelial cell line, the TCRαβ(T 3G11 6C10-CD4 + CD8- CD69+/- HSAmed/l? thy-mocytes, has exhibited significantly higher levels of proliferation capability and IL-6 production, whereas the production of IL-4 and IL-10 is suppressed after co-culturing with MTECl. By contrast, MTECl could not induce thymocytes to secrete Th1 type of cytokines. The results suggest that MTECl can regulate functional status of this thymocyte subset and induce them to develop into a specialized Th2 subset.     

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.     

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.     

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.     

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.     

Inefficient positive selection of T cells directed by haematopoietic cells.

Intrathymic differentiation of alpha beta TCR+ T cells depends on positive selection of CD4+CD8+ thymocytes by thymic major histocompatibility complex (MHC) molecules. Positive selection allows the maturation of only those T cells capable of restricted antigen recognition in the context of the hosts' MHC alleles. Studies of normal or T-cell receptor-transgenic mice engrafted with MHC-different bone marrow or thymuses support the conclusion that positive selection is directed by MHC molecules expressed on non-haematopoietic cells, presumably thymic epithelial cells. Here we, present contrary evidence that class I MHC molecules expressed by haematopoietic cell types direct positive selection of CD8+ T cells, though at a reduced rate compared with positive selection directed by thymic epithelial cells. The identity of cell types that direct positive selection bears directly on mechanistic models of the process, including the idea that thymic epithelial cell MHC molecules uniquely present specialized peptides that mediate positive selection, and the notion that thymic epithelial cells express unique differentiation-inducing cell surface molecules.     

Tolerance in T-cell-receptor transgenic mice involves deletion of nonmature CD4+8+ thymocytes

The mechanism of self-tolerance is studied in T-cell-receptor transgenic mice expressing a receptor in many of their T cells for the male (H-Y) antigen in the context of class I H-2Db MHC antigens. Autospecific T cells are deleted in male mice. The deletion affects only transgene-expressing cells with a relatively high surface-density of CD8 molecules, including nonmature CD4+ CD8+ thymocytes, and is not caused by anti-idiotype cells.