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.     

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.     

Clonal deletion of immature CD4+8+ thymocytes in suspension culture by extrathymic antigen-presenting cells

One mechanism ensuring self tolerance of T cells is the clonal deletion of thymocytes bearing alpha beta T-cell receptors. The stage of thymocyte development at which the interaction with antigen-presenting cells (APCs) leads to deletion, however, has not been determined directly. Indirect evidence suggests that intrathymic APCs induce deletion of CD4+8+ thymocytes (which die by apoptosis) but deletion at less and more mature developmental stages has also been implied. It is also not clear if clonal elimination of thymocytes can be triggered by peripheral antigens carried on extrathymic APCs migrating through the thymus. Here we show antigen-specific induction of apoptosis in CD4+8+ thymocytes cultured in suspension, by thymic as well as splenic APCs. Thus the recognition of antigen by CD4+8+ thymocytes may lead to deletion, suggesting that this is the central mechanism of tolerance induction, which is not limited by the antigen-presenting ability of the thymic stroma.     

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.     

CD4+ and CD8+ T cells acquire specific lymphokine secretion potentials during thymic maturation.

Peripheral CD4+ and CD8+ T lymphocytes carry out different functions during immune reactions, partly as a result of the distinct patterns of lymphokines that they secrete upon stimulation. Using thymic cells from adult and newborn mice as well as from fetal organ cultures, we show here that this functional differentiation occurs inside the thymus and is completed during the single positive stage by the time the T-cell receptor becomes fully coupled to the intracellular activation pathways leading to lymphokine secretion. Surprisingly, CD4+8- thymocytes differ from their immediate progeny, naive peripheral CD4+ cells, in that they secrete a broader range of lymphokines, including interleukins 4, 5 and 10 and gamma-interferon, and more closely resemble immunologically experienced (activated or memory) CD4+ lymphocytes.     

Deletion of self-reactive thymocytes occurs at a CD4+8+ precursor stage

As T cells develop in the thymus, they become tolerant of self-antigens. A major advance in the understanding of how this process occurs was the direct demonstration that cells bearing autoreactive T-cell receptors (TCRs) are physically eliminated from the population of functionally mature T cells present in both the thymus and periphery. We have sought to determine the developmental stage at which autoreactive T cells are eliminated by examining the expression of V beta 17a anti-I-E TCRs under various experimental conditions. In vivo antibody blockage of the CD4 molecule on developing thymocytes of I-E+ C57BR mice was found to inhibit the deletion of V beta 17a-bearing cells from the CD4-8+ single positive thymocyte subset. This result provides strong evidence that deletion of potentially autoreactive T cells occurs at a CD4+8+ precursor stage, that the non-clonally distributed accessory molecules (CD4, CD8) are significant participants in the self-recognition process that leads to clonal elimination, and that thymic class II major histocompatibility complex (MHC) molecules can influence the repertoire of CD4-8+ cells.     

Expression of interleukin-2 receptors as a differentiation marker on intrathymic stem cells

The thymus is regarded as the primary site for T-cell lymphopoiesis, but very little is known about the lineage inter-relationships of cells within that organ. At least four subpopulations of mouse thymocytes can be defined on the basis of staining with monoclonal antibodies directed against the T-cell differentiation antigens Lyt-2 and L3T4 (ref. 2). Thus immunocompetent (medullary) thymocytes, like peripheral T cells, express either Lyt-2 (cytotoxic phenotype) or L3T4 (helper phenotype) but not both, whereas non-functional (cortical) thymocytes express both markers. In addition, a small subpopulation comprising 2-3% of cells in the thymus and expressing neither Lyt-2 nor L3T4 has recently been described. The latter cells have the properties of intrathymic 'stem cells' in that they are the first to appear in the embryonic thymus and at least some can be shown to give rise, both in vivo (ref. 4. and our unpublished data) and in vitro, to other thymocyte subpopulations. We show here that 50% of Lyt-2-/L3T4- cells in the adult thymus express receptors for the polypeptide growth hormone interleukin-2 (IL-2) whereas other cells in the thymus do not. Furthermore, immunohistochemical localization studies on frozen sections indicate a disperse distribution of IL-2 receptor-positive cells in both the cortex and medulla. These novel findings have potential implications in the context of current models of differentiation pathways within the thymus.     

Differentiation potential of subsets of CD4-8- thymocytes

Precursor T cells in the thymus are contained within a subpopulation of thymocytes that lack the markers CD4 and CD8. We have examined the heterogeneity of these cells by flow cytometric analysis, and defined four subpopulations using the cell surface markers Thy-1, J11d and the IL-2 receptor (IL-2R). The J11d+ subset of CD4-8- cells all bear the antigen Thy-1, and some express the IL-2R. Staining and RNA analysis of J11d+ cells suggest that some express receptors of the CD3 gamma delta type, but none express CD3 alpha beta receptors. In fetal thymus organ culture, the J11d+ cells diversify to form 'cortical type' CD4+8+ cells and 'medullary type' cells expressing either CD4 or CD8; in vivo they repopulate the thymus of an irradiated host and seed the periphery with T cells. In contrast, the J11d- subset of CD4-8- thymocytes do not all bear Thy-1 and none express the IL-2R, but some express antigen receptors of the CD3 alpha beta type. They have more limited diversification potential in organ culture, and in vivo fail to recolonize the irradiated host in a homing-independent assay. We conclude that they are not precursor T cells, but rather a side-branch from the main line of T cell differentiation.     

Normal development and function of CD8+ cells but markedly decreased helper cell activity in mice lacking CD4

T cells express T-cell antigen receptors (TCR) for the recognition of antigen in conjunction with the products of the major histocompatibility complex. They also express two key surface coreceptors, CD4 and CD8, which are involved in the interaction with their ligands. As CD4 is expressed on the early haemopoietic progenitor as well as the early thymic precursor cells, a role for CD4 in haemopoiesis and T-cell development is implicated. Thymocytes undergo a series of differentiation and selection steps to become mature CD4+8- or CD4-8+ (single positive) T cells. Studies of the role of CD4+ T cells in vivo have been based on adoptive transfer of selected or depleted lymphocytes, or in vivo treatment of thymectomized mice with monoclonal antibodies causing depletion of CD4+ T cells. In order to study the role of the CD4 molecule in the development and function of lymphocytes, we have disrupted the CD4 gene in embryonic stem cells by homologous recombination. Germ-line transmission of the mutation produces mutant mouse strains that do not express CD4 on the cell surface. In these mice, the development of CD8+ T cells and myeloid components is unaltered, indicating that expression of CD4 on progenitor cells and CD4+ CD8+ (double positive) thymocytes is not obligatory. Here we report that these mice have markedly decreased helper cell activity for antibody responses, although cytotoxic T-cell activity against viruses is in the normal range. This differential requirement for CD4+ helper T cells is important to our understanding of immune disorders including AIDS, in which CD4+ cells are reduced or absent.     

Transient expression of IL-2 receptor precedes the differentiation of immature thymocytes

The growth of mature T lymphocytes after activation by antigen is regulated by the binding and endocytosis of interleukin-2 (IL-2). In the thymus, approximately 50% of adult thymocytes that carry neither the CD4 nor the CD8 antigen and day 14-15 fetal CD4-8- thymocytes express receptors for IL-2(IL-2R). The CD4-8- (double-negative) subpopulation of thymocytes contains the precursors of cells that can differentiate along an unknown pathway into thymocytes bearing either CD8 or CD4, with the characteristics of mature T lymphocytes. The basis for IL-2R expression by double-negative thymocytes is unclear as they appear to lack a functional T-cell receptor/CD3 complex through which activation of peripheral T cells is mediated. The argument for a role for IL-2 in thymocyte differentiation has also been complicated by conflicting reports on the inability or capability of double-negative thymocytes to respond to IL-2 in vitro. At present, both the nature of the stimuli within the thymic micro-environment which induce IL-2R expression and its relevance to thymocyte differentiation are not known. We show here that the IL-2R-bearing subset has a greater potential to differentiate into phenotypically mature T lymphocytes than do IL-2R-negative thymocytes. In addition, progeny of IL-2R-negative donor cells transiently express IL-2R in the thymuses of adoptive hosts before generating CD8 and/or CD4-positive thymocytes. These results identify the IL-2R-positive cells as a more differentiated double-negative thymocyte subset on the pathway to mature T lymphocytes.     

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.     

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.     

Goblet cells deliver luminal antigen to CD103+ dendritic cells in the small intestine

The intestinal immune system is exposed to a mixture of foreign antigens from diet, commensal flora and potential pathogens. Understanding how pathogen-specific immunity is elicited while avoiding inappropriate responses to the background of innocuous antigens is essential for understanding and treating intestinal infections and inflammatory diseases. The ingestion of protein antigen can induce oral tolerance, which is mediated in part by a subset of intestinal dendritic cells (DCs) that promote the development of regulatory T cells. The lamina propria (LP) underlies the expansive single-cell absorptive villous epithelium and contains a large population of DCs (CD11c(+) CD11b(+) MHCII(+) cells) comprised of two predominant subsets: CD103(+) CX(3)CR1(-) DCs, which promote IgA production, imprint gut homing on lymphocytes and induce the development of regulatory T cells, and CD103(-) CX(3)CR1(+) DCs (with features of macrophages), which promote tumour necrosis factor-α (TNF-α) production, colitis, and the development of T(H)17 T cells. However, the mechanisms by which different intestinal LP-DC subsets capture luminal antigens in vivo remains largely unexplored. Using a minimally disruptive in vivo imaging approach we show that in the steady state, small intestine goblet cells (GCs) function as passages delivering low molecular weight soluble antigens from the intestinal lumen to underlying CD103(+) LP-DCs. The preferential delivery of antigens to DCs with tolerogenic properties implies a key role for this GC function in intestinal immune homeostasis.     

Tolerance induction in double specific T-cell receptor transgenic mice varies with antigen

The crucial role of the thymus in immunological tolerance has been demonstrated by establishing that T cells are positively selected to express a specificity for self major histocompatibility complex (MHC), and that those T cells bearing receptors potentially reactive to self antigen fragments, presumably presented by thymic MHC, are selected against. The precise mechanism by which tolerance is induced and the stage of T-cell development at which it occurs are not known. We have now studied T-cell tolerance in transgenic mice expressing a T-cell receptor with double specificities for lymphocytic choriomeningitis virus (LCMV)-H-2Db and for the mixed-lymphocyte stimulatory (MIsa) antigen. We report that alpha beta TCR transgenic mice tolerant to LCMV have drastically reduced numbers of CD4+CD8+ thymocytes and of peripheral T cells carrying the CD8 antigen. By contrast, tolerance to MIsa antigen in the same alpha beta TCR transgenic MIsa mice leads to deletion of only mature thymocytes and peripheral T cells and does not affect CD4+CD8+ thymocytes. Thus the same transgenic TCR-expressing T cells may be tolerized at different stages of their maturation and at different locations in the thymus depending on the antigen involved.     

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.     

Expression of the gamma-delta T-cell receptor on intestinal CD8+ intraepithelial lymphocytes

The vast majority of mature T lymphocytes in the peripheral blood and lymphoid organs use the CD3-associated alpha, beta T-cell receptor (TCR) heterodimer for antigen recognition. A second class of TCRs consists of disulphide-linked gamma and delta proteins that are also CD3-associated. A subset of early CD3+ fetal and adult CD4- 8- thymocytes express gamma, delta TCRs before alpha, beta TCRs are detectable. In addition, a minor (1-5%) subpopulation of peripheral T lymphocytes, and some spleen cells from nude mice express gamma, delta TCRs. Notably, dendritic epidermal cells have also been shown to express gamma, delta TCRs. All of these populations lack CD4 and CD8 molecules. We now report that most mature T cells residing in the murine intestinal epithelium express CD3-associated TCRs composed of gamma-chains disulphide-linked to a protein resembling the delta-chain. The striking feature of these intraepithelial lymphocytes (IEL) was that they were exclusively CD4-8+. In addition, approximately half of CD3-bearing IEL lacked detectable Thy-1 on the cell surface, which is unprecedented for murine T cells. In contrast to other CD8+ peripheral T cells, freshly isolated IEL could be induced to display cytolytic activity by engaging the CD3 molecule, indicating that activation had occurred in vivo. Thus, CD8+ IEL are a phenotypically diverse and anatomically restricted population of lymphocytes that use gamma-chain containing heterodimers for antigen recognition.     

CD4 expressed on earliest T-lineage precursor cells in the adult murine thymus.

A continuous but low input of stem cells or 'prothymocytes' is necessary to maintain T-cell development in the adult thymus, but the colonizing cell has not been characterized. Precursors of T cells have been found in the minor CD4-8- population of thymocytes, but even the earliest cells of this population already have partially rearranged T-cell antigen receptor (TCR) genes. We now demonstrate that the thymus contains a minute population of lymphoid cells similar in some but not all respects to bone marrow-derived haemopoietic stem cells. This population has TCR genes in a germline state. It gives a slow but extensive reconstitution of both alpha beta and gamma delta lineages on transfer into an irradiated thymus, with kinetics indicating that it includes the earliest intrathymic precursor cells so far isolated. Surprisingly, these cells express low surface levels of the mature T-cell marker CD4.     

Beta 2-microglobulin deficient mice lack CD4-8+ cytolytic T cells

Mice homozygous for a beta 2-microglobulin gene disruption do not express any detectable beta 2-m protein. They express little if any functional major histocompatibility complex (MHC) class I antigen on the cell surface yet are fertile and apparently healthy. They show a normal distribution of gamma delta, CD4+8+ and CD4+8- T cells, but have no mature CD4-8+ T cells and are defective in CD4-8+ T cell-mediated cytotoxicity. Our results strongly support earlier evidence that MHC class I molecules are crucial for positive selection of T cell antigen receptor alpha beta+ CD4-8+ T cells in the thymus and call into question the non-immune functions that have been ascribed to MHC class I molecules.     

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.