Enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck.

Lymphocyte-specific tyrosine protein kinase p56lck is physically associated with CD4 and CD8 T-cell surface molecules, suggesting that it may transduce CD4/CD8-triggered tyrosine phosphorylation signals during antigen stimulation. Indeed, antibody-mediated aggregation of CD4 (to mimic interaction with its ligand, major histocompatibility complex (MHC) class II molecules), rapidly elevates the kinase activity of p56lck and is associated with marked changes in tyrosine protein phosphorylation. Genetic analyses suggest that the interaction of CD4/CD8 with p56lck results in a positive signal during antigen-induced T-cell activation. To evaluate directly the role of p56lck in T-cell activation, we introduced a constitutively activated form of Lck protein (tyrosine 505 to phenylalanine 505 mutant); in a CD4-negative, MHC-class II restricted mouse T-cell hybridoma. We report here that, as for transfection of CD4, expression of the Lck mutant enhanced T-lymphocyte responsiveness. This finding provides direct evidence that p56lck can positively regulate T-cell functions and that it mediates at least some of the effects of CD4 and CD8 on T-cell activation.     

No T-cell tyrosine protein kinase signalling or calcium mobilization after CD4 association with HIV-1 or HIV-1 gp120.

The T lymphocyte surface protein CD4 is an integral membrane glycoprotein noncovalently associated with the tyrosine protein kinase p56lck. In normal T cells, surface association of CD4 molecules with other CD4 molecules or other T-cell surface proteins, such as the T-cell antigen receptor, stimulates the activity of the p56lck tyrosine kinase, resulting in the phosphorylation of various cellular proteins at tyrosine residues. Thus, the signal transduction in T cells generated through the surface engagement of CD4 is similar to that observed for the class of growth factor receptors possessing endogenous tyrosine kinase activity. As CD4 is also the cellular receptor for the human immunodeficiency virus (HIV), binding of the virus or gp120 (the virus surface protein responsible for specific CD4+ T-cell association) could mimic the types of immunological interactions that have previously been found to stimulate p56lck and trigger T-cell activation pathways. We have evaluated this possibility and report here that binding of HIV-1 or the virus glycoprotein gp120 to CD4+ human T cells fails to elicit detectable p56lck-dependent tyrosine kinase activation and signalling, alterations in the composition of cellular phosphotyrosine-containing proteins, or changes in intracellular Ca2+ concentration.     

Signal transduction through the CD4 receptor involves the activation of the internal membrane tyrosine-protein kinase p56lck

The CD4 T-cell surface antigen is an integral membrane glycoprotein of relative molecular mass 55,000 which binds class II major histocompatibility complex (MHC) molecules expressed on antigen presenting cells (APCs). It is thought to stabilize physical interactions between T cells and APCs (for a review, see ref. 1). Evidence is accumulating that suggests that CD4 can transduce an independent signal during T-cell activation. It has recently been shown that CD4 expressed on human and murine T cells is physically associated with the Src-related tyrosine protein kinase p56lck (refs 7, 8). These results indicate that CD4 can function as a signal transducer and suggest that tyrosine phosphorylation events may be important in CD4-mediated signalling. Here, we present evidence that cross-linking of the CD4 receptor induces a rapid increase in the tyrosine-specific protein kinase activity of p56lck and is associated with the rapid phosphorylation of one of the subunits (zeta) of the T-cell receptor complex on tyrosine residues. These data provide direct evidence for a specific CD4 signal transduction pathway that is mediated through p56lck and suggest that some of the tyrosine phosphorylation events detected during antigen-mediated T-cell activation may result from signalling through this surface molecule.     

Inability of CD8 alpha' polypeptides to associate with p56lck correlates with impaired function in vitro and lack of expression in vivo

T-cell accessory molecules, particularly CD4 and CD8, seem to be involved in the control of T-cell activation by antigen. Precisely how such molecules operate is not fully understood, but evidence to date suggests a dual role, as receptors binding ligands on stimulator cells and by direct or indirect involvement in intracellular signalling events. In mouse, truncated 'tailless' CD8 molecules occur naturally (CD8 alpha' polypeptides) and although they are expressed on the surface of thymocytes, they are not expressed on the surface of mature T cells. In this study, we show that truncated CD8 molecules are impaired in their ability to interact with the protein tyrosine kinase, p56lck, and have decreased ability to restore immune responsiveness in vitro. Our data support a dual function for CD8 molecules correlated with expression of external domains and cytoplasmic domains, respectively. Both functions appear to be critical for a competent immune system in vivo.     

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.     

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.     

Association of tyrosine kinase p56lck with CD4 inhibits the induction of growth through the alpha beta T-cell receptor.

The membrane glycoprotein CD4 enhances antigen-mediated activation of T cells restricted by class II molecules of the major histocompatibility complex (MHC). This positive function has been attributed to the protein tyrosine kinase p56lck (ref. 4), which is noncovalently associated with the cytoplasmic portion of CD4, and is activated on CD4 aggregation. Antigen presentation by MHC class II molecules coaggregates CD4 and the T-cell antigen receptor (TCR alpha beta-CD3). Thus, the mutual specificity of CD4 and TCR alpha beta for the MHC-antigen complex results in the juxtaposition of p56lck and TCR alpha beta-CD3. In contrast, anti-CD4 antibodies can abrogate antigen-induced, as well as anti-TCR-induced T-cell activation, indicating that CD4 might also transduce negative signals. The molecular basis for this opposing function remains unclear. Here we show that the CD4-p56lck complex prohibits the induction of activation signals through the TCR-CD3 complex when not specifically included in the signalling process. This negative effect does not require anti-CD4 treatment, indicating that the induction of distinct negative signals is probably not involved. Rather, the results demonstrate that the CD4-p56lck complex provides prerequisite signals for antigen-receptor-induced T-cell growth and thus characterize a molecular mechanism for functional constraints imposed on T-cell activation by the MHC.     

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.     

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.     

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.     

Phenotypic differences between alpha beta versus beta T-cell receptor transgenic mice undergoing negative selection

T-cell differentiation in the thymus is thought to involve a progression from the CD4-CD8- phenotype through CD4+CD8+ intermediates to mature CD4+ or CD8+ cells. There is evidence that during this process T cells bearing receptors potentially reactive to 'self' are deleted by a process termed 'negative selection' One example of this process occurs in mice carrying polymorphic Mls antigens, against which a detectable proportion of T cells are autoreactive. These mice show clonal deletion of thymic and peripheral T-cell subsets that express the autoreactive V beta 3 segment of the T-cell antigen receptor, but at most a two-fold depletion of thymic cells at the CD4+CD8+ stage. By contrast, transgenic mice bearing both alpha and beta chain genes encoding autoreactive receptors recognizing other ligands, show severe depletion of CD4+CD8+ thymocytes as well, suggesting that negative selection occurs much earlier. We report here the Mls 2a/3a mediated elimination of T cells expressing a transgene encoded V beta 3-segment, in T-cell receptor alpha/beta and beta-transgenic mice. Severe depletion of CD4+CD8+ thymocytes is seen only in the alpha/beta chain transgenic mice, whereas both strains delete mature V beta 3 bearing CD4+ and CD8+ T cells efficiently. We conclude that severe CD4+CD8+ thymocyte deletion in alpha/beta transgenic mice results from the premature expression of both receptor chains, and does not reflect a difference in the timing or mechanism of negative selection for Mls antigens as against the allo- and MHC class 1-restricted antigens used in the other studies.     

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.     

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.     

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.     

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.     

Self-reactive gamma delta T cells are eliminated in the thymus

The genes encoding a gamma delta T-cell receptor specific for a major histocompatibility complex class I molecule encoded by the TIa locus have been inserted into the mouse germ line. In mice that do not express the TIa-encoded determinant, transgenic gamma delta T cells are a functional component of the CD4-CD8- 'double-negative' T cells in the thymus and peripheral lymphoid organs. In mice that express the TIa-encoded determinant, there are no transgenic gamma delta T cells in peripheral lymphoid organs, and there are no thymocytes expressing normal levels of the transgenic gamma delta T-cell receptor.     

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.     

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.     

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.     

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.