Signal transduction via CD4, CD8, and CD28 in mature and immature thymocytes. Implications for thymic selection.

The positive and negative selection of immature thymocytes that shapes the mature T cell repertoire appears to occur at an intermediate stage of development when the cells express low levels of TCR/CD3. These cells are also CD4+CD8+ and CD28+ (dull), and signals delivered by these three accessory molecules have been implicated in the selection process. We have examined the regulatory function of these accessory molecules on responses of immature thymocytes stimulated through the TCR/CD3 complex. Cross-linking CD4 or CD8 with CD3 strongly enhanced signal transduction via CD3 as assessed by protein tyrosine phosphorylation and calcium mobilization. Subsequent cell proliferation could be induced by soluble anti-CD28 mAb, which was comitogenic for cells stimulated with CD3 x CD4 or CD3 x CD8 cross-linking, but was without effect on cells stimulated with CD3 x CD3 cross-linking. A potential role for CD28 signal transduction in thymic maturation is suggested by the demonstration that the BB-1 molecule, a natural ligand for CD28, is expressed on thymic stromal cells. Taken together, our data suggest a model of thymic development in which CD4 or CD8 may enhance TCR/CD3 signaling upon coligation by an MHC molecule. If the CD28 surface receptor is simultaneously stimulated by a BB-1 expressing stromal cell, this set of interactions could lead to proliferation and positive selection. In the absence of CD28 stimulation the enhanced TCR/CD3 signals might lead to apoptosis and negative selection.     

CD4 and CD8 are positive regulators of T cell receptor signal transduction in early T cell differentiation.

Although cortical (CD4+CD8+) thymocytes mobilize intracellular calcium poorly when CD3/TCR is ligated, we have found that murine cortical thymocytes can transduce strong biochemical signals in response to ligation of the CD3/Ti TCR complex (CD3/TCR) and that the signals are regulated by CD4 and CD8 interactions with CD3/TCR. Striking increases in intracellular calcium were observed in cortical thymocytes from transgenic mice containing productively rearranged alpha and beta TCR genes, when CD3 or TCR was cross-linked with CD4 or CD8 using heteroconjugated mAb. However, in mature T cells derived from lymph nodes of these mice, identical stimuli elicited calcium responses that were significantly smaller in magnitude. A thymocyte cell line that expresses a low level of the transgenic TCR and has a phenotype characteristic of cortical thymocytes (CD4+CD8+J11d+Thy-1+) was established from a female alpha beta TCR transgenic mouse. Cross-linking of CD4 or CD8 molecules to CD3/TCR induced strong calcium responses in these cells. Responses were weak or absent when CD3 or TCR were aggregated alone. Heteroconjugates of Thy-1xCD3 did not increase the intracellular calcium concentration in transgenic thymocytes or in the thymocyte cell line, although Thy-1 is highly expressed on immature cells. Enhanced tyrosine phosphorylation was observed when CD3 or TCR was cross-linked with CD4 or CD8 on transgenic thymocytes or on the thymocyte cell line, in comparison with aggregation of CD3/TCR alone. Taken together, these data show that CD4 and CD8 molecules allow the weakly expressed CD3/TCR of cortical thymocytes to transduce strong intracellular signals upon receptor ligation. These signals may be involved in selection processes at the CD4+CD8+ stage of differentiation.     

A Novel TCR Transgenic Model Reveals That Negative Selection Involves an Immediate,Bim-Dependent Pathway and a Delayed,Bim-Independent Pathway

A complete understanding of negative selection has been elusive due to the rapid apoptosis and clearance of thymocytes in vivo. We report a TCR transgenic model in which expression of the TCR during differentiation occurs only after V(D)J-like recombination. TCR expression from this transgene closely mimics expression of the endogenous TCRα locus allowing for development that is similar to wild type thymocytes. This model allowed us to characterize the phenotypic changes that occurred after TCR-mediated signaling in self-reactive thymocytes prior to their deletion in a highly physiological setting. Self-reactive thymocytes were identified as being immature, activated and CD4^loCD8^lo. These cells had upregulated markers of negative selection and were apoptotic. Elimination of Bim reduced the apoptosis of self-reactive thymocytes, but it did not rescue their differentiation and the cells remained at the immature CD4^loCD8^lo stage of development. These cells upregulate Nur77 and do not contribute to the peripheral T cell repertoire in vivo. Remarkably, development past the CD4^loCD8^lo stage was possible once the cells were removed from the negatively selecting thymic environment. In vitro development of these cells occurred despite their maintenance of high intracellular levels of Nur77. Therefore, in vivo, negatively selected Bim-deficient thymocytes are eliminated after prolonged developmental arrest via a Bim-independent pathway that is dependent on the thymic microenvironment. These data newly reveal a layering of immediate, Bim-dependent, and delayed Bim-independent pathways that both contribute to elimination of self-reactive thymocytes in vivo.     

The affinity/avidity and length of exposure to the deleting ligand determine dependence on CD28 for the efficient deletion of self-specific CD4+CD8+ thymocytes

Whether the CD28/B7 signaling pathway is essential for the negative selection of immature CD4+CD8+ (DP) thymocytes expressing self-specific alphabeta TCRs is a controversial issue. In this study we examined the role of CD28 in the deletion of thymocytes that express either the H-Y or the 2C transgenic TCR. In H-2(b) male mice that expressed the H-Y TCR, negative selection of DP H-Y TCR+ thymocytes occurred very efficiently and this deletion was unaffected by the CD28(-/-) mutation. In H-2(b) 2C mice, where the deletion of DP 2C TCR+ thymocytes occurred less efficiently, the CD28(-/-) mutation led to a higher recovery of DP thymocytes. Using an in vitro deletion assay, a requirement for the CD28 signaling pathway in the deletion of DP H-Y TCR+ thymocytes was evident at low, but not high, densities of the antigenic ligand. Similar results were also observed in an in vivo assay for the deletion of these thymocytes. Intraperitoneal administration of an anti-CD3epsilon mAb led to the intrathymic deletion of DP H-Y TCR+ thymocytes in a CD28-dependent manner at the 24-h time point. However, the CD28 dependence was less evident at the 40-h time point. These results indicate that the dependence on CD28 for the efficient deletion of self-specific thymocytes is determined by the concentration, affinity/avidity, and length of exposure to the deleting ligand.     

T-cell receptor ligation by peptide/MHC induces activation of a caspase in immature thymocytes: the molecular basis of negative selection.

T-cell receptors (TCRs) are created by a stochastic gene rearrangement process during thymocyte development, generating thymocytes bearing useful, as well as unwanted, specificities. Within the latter group, autoreactive thymocytes arise which are subsequently eliminated via a thymocyte-specific apoptotic mechanism, termed negative selection. The molecular basis of this deletion is unknown. Here, we show that TCR triggering by peptide/MHC ligands activates a caspase in double-positive (DP) CD4+ CD8+ thymocytes, resulting in their death. Inhibition of this enzymatic activity prevents antigen-induced death of DP thymocytes in fetal thymic organ culture (FTOC) from TCR transgenic mice as well as apoptosis induced by anti-CD3epsilon monoclonal antibody and corticosteroids in FTOC of normal C57BL/6 mice. Hence, a common caspase mediates immature thymocyte susceptibility to cell death.     

Immature CD4+CD8+ thymocytes and mature T cells regulate Nur77 distinctly in response to TCR stimulation

The orphan steroid receptor, Nur77, is thought to be a central participant in events leading to TCR-mediated clonal deletion of immature thymocytes. Interestingly, although both immature and mature murine T cell populations rapidly up-regulate Nur77 after TCR stimulation, immature CD4+CD8+ thymocytes respond by undergoing apoptosis, whereas their mature descendants respond by dividing. To understand these developmental differences in susceptibility to the proapoptotic potential of Nur77, we compared its regulation and compartmentalization and show that mature, but not immature, T cells hyperphosphorylate Nur77 in response to TCR signals. Nur77 resides in the nucleus of immature CD4+CD8+ thymocytes throughout the course of its expression and is not found in either the organellar or cytoplasmic fractions. However, hyperphosphorylation of Nur77 in mature T cells, which is mediated by both the MAPK and PI3K/Akt pathways, shifts its localization from the nucleus to the cytoplasm. The failure of immature CD4+CD8+ thymocytes to hyperphosphorylate Nur77 in response to TCR stimulation may be due in part to decreased Akt activity at this developmental stage.     

In vivo administration of monoclonal antibodies to the CD3 T cell receptor complex induces cell death (apoptosis) in immature thymocytes.

Some thymocytes, upon activation via the TCR complex in vitro, undergo apoptotic cell death. In this report, we examine the cell death induced in the thymus after administration of anti-CD3 or anti-TCR antibodies. We found that shortly after antibody injection, cortical thymocytes undergo apoptosis as characterized by morphologic changes and DNA fragmentation. Anti-CD3 administration led to depletion of nearly all CD4+CD8+ thymocytes, and approximately 50% of CD4+CD8- thymocytes. This depletion predominantly affected cells bearing low levels of CD3, although some depletion also occurred among cells expressing intermediate and high levels. Administration of an anti-TCR antibody also induced apoptosis, but affected significantly fewer thymocytes than anti-CD3. This effect was probably not due to different binding affinities for the two antibodies, because both antibodies show similar dose response effects in an in vitro model of activation-induced apoptosis. This work demonstrates that findings on activation-induced apoptosis in vitro can be extended to the in vivo situation, and further, that the activation of cortical thymocytes, in situ, results in apoptosis and removal of the activated cells. The possible relationships between this activation-induced cell death in immature thymocytes and the process of negative selection of autoreactive T cells is discussed.     

Retinoic acids inhibit activation-induced apoptosis in T cell hybridomas and thymocytes.

Apoptosis is induced in immature thymocytes and T cell hybridomas upon stimulation via the TCR/CD3 complex. This phenomenon appears to be related to negative selection of T cell clones in the thymus. In T cell hybridomas, it has been shown that glucocorticoids inhibit TCR/CD3-mediated apoptosis, whereas glucocorticoids alone induce apoptosis. All-trans-retinoic acid (RA) at 0.1 to 10 microM also inhibited TCR/CD3-mediated apoptosis assessed by DNA fragmentation and cytolysis, but RA alone hardly induced apoptosis. RA enhanced the effects of glucocorticoids to induce apoptosis and to inhibit TCR/CD3-mediated apoptosis. TCR/CD3-mediated stimulation can be mimicked by the combination of ionomycin, a calcium ionophore, and PMA, an activator of protein kinase C, and the combination-induced DNA fragmentation was also inhibited by RA. RA, however, failed to inhibit the combination-induced increase in intracellular Ca2+ concentration or the combination-induced translocation of protein kinase C from the cytosolic fraction to the particulate fraction. Time course studies of RA addition into the culture indicated that a 3- to 6-h delay in the addition of RA did not reduce its inhibitory effect on anti-CD3-induced DNA fragmentation. These results suggest that RA interferes with the apoptotic process at some point after its initiation stage. It has been suggested that negative selection involves not only TCR/CD3-mediated signals but also LFA-1-mediated signals. RA at 0.01 to 1 microM significantly inhibited the induction of thymocyte apoptosis by co-immobilized mAb to CD3 and LFA-1 molecules. RA by itself hardly induced apoptosis, but enhanced glucocorticoid-induced apoptosis. The results suggest that thymic selection might be influenced by RA at near-physiologic concentrations. The receptors of glucocorticoids and RA belong to the erbA oncogene-related steroid hormone receptor superfamily. Thyroid hormones and 1 alpha,25-dihydroxy vitamin D3, whose receptors also belong to the superfamily, failed to modulate apoptosis in both T cell hybridomas and thymocytes.     

Maturation versus death of developing double-positive thymocytes reflects competing effects on Bcl-2 expression and can be regulated by the intensity of CD28 costimulation

Immature double-positive (DP) thymocytes mature into CD4(+)CD8(-) cells in response to coengagement of TCR with any of a variety of cell surface "coinducer" receptors, including CD2. In contrast, DP thymocytes are signaled to undergo apoptosis by coengagement of TCR with CD28 costimulatory receptors, but the molecular basis for DP thymocyte apoptosis by TCR plus CD28 coengagement is not known. In the present study, we report that TCR plus CD28 coengagement does not invariably induce DP thymocyte apoptosis but, depending on the intensity of CD28 costimulation, can induce DP thymocyte maturation. We demonstrate that distinct but interacting signal transduction pathways mediate DP thymocyte maturation signals and DP thymocyte apoptotic signals. Specifically, DP maturation signals are transduced by the extracellular signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway and up-regulate expression of the antiapoptotic protein Bcl-2. In contrast, the apoptotic response stimulated by CD28 costimulatory signals is mediated by ERK/MAPK-independent pathways. Importantly, when TCR-activated thymocytes are simultaneously coengaged by both CD28 and CD2 receptors, CD28 signals can inhibit ERK/MAPK-dependent Bcl-2 protein up-regulation. Thus, there is cross-talk between the signal transduction pathways that transduce apoptotic and maturation responses, enabling CD28-initiated signal transduction pathways to both stimulate DP thymocyte apoptosis and also negatively regulate maturation responses initiated by TCR plus CD2 coengagement.     

CD28 ligation costimulates cell death but not maturation of double-positive thymocytes due to defective ERK MAPK signaling

The differentiation of double-positive (DP) CD4(+)CD8(+) thymocytes to single-positive CD4(+) or CD8(+) T cells is regulated by signals that are initiated by coengagement of the Ag (TCR) and costimulatory receptors. CD28 costimulatory receptors, which augment differentiation and antiapoptotic responses in mature T lymphocytes, have been reported to stimulate both differentiation and apoptotic responses in TCR-activated DP thymocytes. We have used artificial APCs that express ligands for TCR and CD28 to show that CD28 signals increase expression of CD69, Bim, and cell death in TCR-activated DP thymocytes but do not costimulate DP thymocytes to initiate the differentiation program. The lack of a differentiation response is not due to defects in CD28-initiated TCR proximal signaling events but by a selective defect in the activation of ERK MAPK. To characterize signals needed to initiate the death response, a mutational analysis was performed on the CD28 cytoplasmic domain. Although mutation of all of CD28 cytoplasmic domain signaling motifs blocks cell death, the presence of any single motif is able to signal a death response. Thus, there is functional redundancy in the CD28 cytoplasmic domain signaling motifs that initiate the thymocyte death response. In contrast, immobilized Abs can initiate differentiation responses and cell death in DP thymocytes. However, because Ab-mediated differentiation occurs through CD28 receptors with no cytoplasmic domain, the response may be mediated by increased adhesion to immobilized anti-TCR Abs.     

Tonic ubiquitylation controls T‐cell receptor:CD3 complex expression during T‐cell development

Expression of the T‐cell receptor (TCR):CD3 complex is tightly regulated during T‐cell development. The mechanism and physiological role of this regulation are unclear. Here, we show that the TCR:CD3 complex is constitutively ubiquitylated in immature double positive (DP) thymocytes, but not mature single positive (SP) thymocytes or splenic T cells. This steady state, tonic CD3 monoubiquitylation is mediated by the CD3ε proline‐rich sequence, Lck, c‐Cbl, and SLAP, which collectively trigger the dynamin‐dependent downmodulation, lysosomal sequestration and degradation of surface TCR:CD3 complexes. Blocking this tonic ubiquitylation by mutating all the lysines in the CD3 cytoplasmic tails significantly upregulates TCR levels on DP thymocytes. Mimicking monoubiquitylation by expression of a CD3ζ‐monoubiquitin (monoUb) fusion molecule significantly reduces TCR levels on immature thymocytes. Moreover, modulating CD3 ubiquitylation alters immunological synapse (IS) formation and Erk phosphorylation, thereby shifting the signalling threshold for positive and negative selection, and regulatory T‐cell development. Thus, tonic TCR:CD3 ubiquitylation results in precise regulation of TCR expression on immature T cells, which is required to maintain the fidelity of T‐cell development.     

Developmental regulation of transmembrane signaling via the T cell antigen receptor/CD3 complex in human T lymphocytes.

We have examined transmembrane signaling events via the TCR/CD3 complex (TCR/CD3) at various stages of T cell development for evidence of developmental regulation. Engagement of TCR/CD3 induced defective activation of phospholipase C (PLC) in thymocytes relative to peripheral blood T lymphocytes. The defect in PLC activation via TCR/CD3 was restricted to immature thymocytes (CD3low, CD4+CD8+). Mature thymocytes (CD3high, CD4+CD8-/CD8+CD4-) were similar to PBL in signaling via TCR/CD3. Both immature and mature thymocytes expressed a similar profile of PLC isoenzyme mRNA species, indicating that the defect in signaling in immature thymocytes was not due to altered expression of PLC isoenzymes. Activation of tyrosine phosphorylation pathways implicated in the coupling of TCR/CD3 to PLC was impaired in immature thymocytes, as evidenced by depressed phosphorylation of CD3 zeta subunit after stimulation with anti TCR/CD3 mAb. This was associated with lower levels of p59fyn tyrosine kinase and minimal or undetectable stimulus-induced kinase activation in immature thymocytes relative to mature thymocytes. We conclude that the capacity to signal via TCR/CD3 is regulated during T cell development by mechanisms acting at the level of TCR/CD3-associated tyrosine phosphorylation pathways.     

MEK activity regulates negative selection of immature CD4+CD8+ thymocytes

CD4+CD8+ thymocytes are either positively selected and subsequently mature to CD4 single positive (SP) or CD8 SP T cells, or they die by apoptosis due to neglect or negative selection. This clonal selection is essential for establishing a functional self-restricted T cell repertoire. Intracellular signals through the three known mitogen-activated protein (MAP) kinase pathways have been shown to selectively guide positive or negative selection. Whereas the c-Jun N-terminal kinase and p38 MAP kinase regulate negative selection of thymocytes, the extracellular signal-regulated kinase (ERK) pathway is required for positive selection and T cell lineage commitment. In this paper, we show that the MAP/ERK kinase (MEK)-ERK pathway is also involved in negative selection. Thymocytes from newborn TCR transgenic mice were cultured with TCR/CD3epsilon-specific Abs or TCR-specific agonist peptides to induce negative selection. In the presence of the MEK-specific pharmacological inhibitors PD98059 or UO126, cell recovery was enhanced and deletion of DP thymocytes was drastically reduced. Furthermore, development of CD4 SP T cells was blocked, but differentiation of mature CD8 SP T cells proceeded in the presence of agonist peptides when MEK activity was blocked. Thus, our data indicate that the outcome between positively and negatively selecting signals is critically dependent on MEK activity.     

B7 costimulates proliferation of CD4-8+ T lymphocytes but is not required for the deletion of immature CD4+8+ thymocytes.

In addition to TCR-derived signals, costimulatory signals derived from stimulation of the CD28 molecule by its natural ligand, B7, have been shown to be required for CD4+8- T cell activation. We investigate the ability of B7 to provide costimulatory signals necessary to drive proliferation and differentiation of virgin CD4-8+ T-cells that express a transgenic TCR specific for the male (H-Y) Ag presented by H-2Db class I MHC molecules. Virgin male-specific CD4-8+ T cells can be activated either with B7 transfected chinese hamster ovary (CHO) cells and T3.70, a mAb specific for the transgenic TCR-alpha chain that is associated with male-reactivity, or by male dendritic cells (DC). Activated CD4-8+ T cells proliferated in the absence of exogenously added IL-2. IL-2 activity was detected in supernatants of CD4-8+T3.70+ cells that were stimulated with T3.70 and B7+CHO cells. The response of CD4-8+T3.70+ cells to T3.70/B7+CHO or to male DC stimulation were inhibited by CTLA4Ig, a fusion protein comprising the extracellular portion of CTLA4 and human IgG C gamma 1. It has been previously shown that CTLA4Ig binds B7 with high affinity. Staining with CTLA4Ig revealed that DC express about 50 times more B7 than CD4-8+ T cells. CTLA4Ig also specifically blocked the proliferation of male-reactive cells in vivo. We have also used an in vitro deletion assay whereby immature CD4+8+ thymocytes expressing the transgenic male-specific TCR are deleted by overnight incubation with either immobilized T3.70 or male DC to investigate the participation of the CD28/B7 pathway in the negative selection of immature thymocytes. Staining with B7Ig established that both immature murine CD4+8+ and mature CD4-8+ thymocytes express a high level of CD28. However, despite the high expression of CD28 on CD4+8+ thymocytes, it was found that deletion of CD4+8+ thymocytes expressing the male-specific TCR by the T3.70 mAb was not inhibited by B7+CHO cells. Furthermore, the deletion of these thymocytes by DC also was not inhibited by CTLA4Ig. These findings provide evidence that although signaling through CD28 can costimulate a primary anti-male response in mature CD4-8+ T cells, the CD28/B7 pathway does not appear to participate in the negative selection of immature CD4+8+ thymocytes.     

CAST, a novel CD3epsilon-binding protein transducing activation signal for interleukin-2 production in T cells.

Antigen recognition through T cell receptor (TCR)-CD3 complex transduces signals into T cells, which regulate activation, function, and differentiation of T cells. The TCR-CD3 complex is composed of two signaling modules represented by CD3zeta and CD3epsilon. Signaling through CD3zeta has been extensively analyzed, but that via CD3epsilon, which is also crucial in immature thymocyte development, is still not clearly understood. We isolated cDNA encoding a novel CD3epsilon-binding protein CAST. CAST specifically interacts in vivo and in vitro with CD3epsilon but not with CD3zeta or FcRgamma via a unique membrane-proximal region of CD3epsilon. CAST is composed of 512 amino acids including a single tyrosine and undergoes tyrosine phosphorylation upon TCR stimulation. Overexpression of two dominant-negative types of CAST, a minimum CD3epsilon-binding domain and a tyrosine-mutant, strongly suppressed NFAT activation and interleukin-2 production. These results demonstrate that CAST serves as a component of preformed TCR complex and transduces activation signals upon TCR stimulation and represents a new signaling pathway via the CD3epsilon-containing TCR signaling module.     

TCR-independent and caspase-independent apoptosis of murine thymocytes by CD24 cross-linking

CD24, also referred to as the heat-stable Ag, is a T cell differentiation Ag that is highly expressed on both CD4-CD8- double negative and CD4+CD8+ double positive thymocytes. Here, we report that CD24 ligation by a new anti-CD24 Ab, mT-20, induced the apoptosis of both double negative and double positive thymocytes, as well as the Scid.adh thymic lymphoma cell line, in the absence of TCR/CD3 engagement. CD24-mediated apoptosis of mouse thymocytes and its signaling pathway appeared not to be associated with p53, CD95, TNFR, or caspases. Furthermore, we found that cell death was blocked by the addition of scavengers of reactive oxygen species or by Bcl-2 overexpression, implying the role of CD24 signaling in the mitochondrial regulation. In this study, we suggest that CD24 ligation induced the apoptosis of immature thymocytes independently of both caspase and TCR.     

The CD3epsilon proline-rich sequence, and its interaction with Nck, is not required for T cell development and function

The CD3epsilon proline-rich sequence (PRS) binds to the cytosolic adaptor molecule Nck after TCR ligation. It has been proposed that this interaction is essential for immunological synapse formation and T cell activation. To assess the physiological importance of the CD3epsilon PRS, we have generated mice that lack this motif (CD3epsilon.PRS(M)). Pull-down experiments demonstrated the inability of Nck to bind to the CD3epsilon PRS in thymocytes from mutant mice after TCR ligation. Surprisingly, no differences were observed in the number and percentage of T cell subsets in the thymus and spleen, and there was no apparent defect in positive or negative selection. Furthermore, the proliferative response of CD3epsilon.PRS(M) T cells to staphylococcal enterotoxin B and anti-CD3 Ab was normal. TCR surface expression, constitutive internalization, and Ag-induced down-modulation were also normal. These data suggest that the interaction between the CD3epsilon PRS and Nck, or any other Src homology 3 domain-containing molecule, is not essential for T cell development and function.