Critical function of death-associated protein 3 in T cell receptor-mediated apoptosis induction

Death-associated protein 3 (DAP3) is crucial for promoting apoptosis induced by various stimulations. This report demonstrates that DAP3 is also important for T cell receptor (TCR)-mediated apoptosis induction in immature thymocytes. Enforced expression of DAP3 accelerated the negative selection in developing thymocytes, using the reaggregate thymus organ culture system. In addition, expression of DAP3 accelerated TCR-mediated apoptosis induction in DO11.10 cells. We also demonstrated that DAP3 translocates into the nucleus during TCR-mediated apoptosis in a Nur77 dependent manner. It is concluded that DAP3 is critical for TCR-mediated induction of apoptosis at the downstream of Nur77.     

Retinoic acids regulate apoptosis of T lymphocytes through an interplay between RAR and RXR receptors

Vitamin A deficiency has been known for a long time to be accompanied with immune deficiency and susceptibility to a wide range of infectious diseases. Increasing evidence suggests that retinoic acids derived from vitamin A are involved in the functional regulation of the immune system. Of the two groups of retinoid receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs) all-trans and 9-cis retinoic acids are high affinity ligands for RARs and 9-cis retinoic acid additionally binds to RXRs. In cells, at high concentrations, all-trans retinoic acid can be converted to 9-cis retinoic acid by unknown mechanisms. Apoptosis plays a major role in shaping the T cell repertoire and one way in which retinoids may affect immune functions is to influence the various apoptosis pathways. Indeed, it has been shown that retinoic acids can induce apoptosis, increase the rate of dexamethasone-induced death and inhibit activation-induced death of thymocytes and T lymphocytes. Therefore, retinoids together with glucocorticoids may be involved in regulating positive and negative selection of T lymphocytes. Here we demonstrate that retinoids can induce apoptosis of T cells through the stimulation of RARgamma. Specific stimulation of RARalpha, on the other hand, prevents both RARgamma-dependent and TCR-mediated cell death. In all these functions 9-cis retinoic acid proved to be more effective than all-trans retinoic acid suggesting the involvement of RXRs. Based on these results a possible mechanism through which costimulation of RARs and RXRs might affect spontaneous and activation-induced death of T lymphocytes is proposed.     

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.     

The EphB6 receptor inhibits JNK activation in T lymphocytes and modulates T cell receptor-mediated responses

EphB6 is the most recently identified member of the Eph receptor tyrosine kinase family. EphB6 is primarily expressed in thymocytes and a subpopulation of T cells, suggesting that it may be involved in regulation of T lymphocyte differentiation and functions. We show here that overexpression of EphB6 in Jurkat T cells and stimulation with the EphB6 ligand, ephrin-B1, results in the selective inhibition of TCR-mediated activation of JNK but not the MAPK pathway. EphB6 appears to suppress the JNK pathway by preventing T cell receptor (TCR)-induced activation of the small GTPase Rac1, a critical event in initiating the JNK cascade. Furthermore, EphB6 blocked anti-CD3-induced secretion of IL-2 and CD25 expression in a ligand-dependent manner. Dominant negative EphB6 suppressed the inhibitory activity of the endogenous receptor and enhanced anti-CD3-induced JNK activation, CD25 expression, and IL-2 secretion, confirming the requirement for EphB6-specific signaling. Activation of the JNK pathway and the establishment of an IL-2/IL-2R autocrine loop have been shown to play a role in the negative selection of CD4(+)CD8(+) self-reacting thymocytes. In agreement, stimulation of murine thymocytes with ephrin-B1 not only blocked anti-CD3-induced CD25 up-regulation and IL-2 production, but also inhibited TCR-mediated apoptosis. Thus, EphB6 may play an important role in regulating thymocyte differentiation and modulating responses of mature T cells.     

The Functional Role of B7 Molecules on the Induction of Thymocyte Activation and Apoptosis

To evaluate the role of B7 on thymocyte activation and apoptosis, we took advantage of TCR transgenic mice in which the majority of thymocytes express a uniform TCR that is specific for ovalbumin. We also prepared Chinese hamster ovary (CHO) cells expressing B7 and appropriate class II molecules. We found that the apoptosis of double-positive thymocytes by TCR-mediated signaling, which presumably represents negative selection, requires a costimulatory signal provided by B7-1 or B7-2. The requirement of B7-1 costimulation for the apoptosis of thymocytes does not change in either low or high antigenic peptide loading. We also demonstrated that two signals through TCR and CD28 augmented the proliferation of thymocytes, and the requirement of CD28-mediated signal by B7-1 or B7-2 for thymocyte proliferation became less evident when high doses of antigenic peptide were loaded, indicating that the intensity of TCR-mediated signal determines the requirement of B7-mediated second signal for thymocyte proliferation.     

Cutting Edge: Pivotal function of Ubc13 in thymocyte TCR signaling

The Ubc13 E2 ubiquitin-conjugating enzyme is essential for BCR-, TLR-, and IL-1 receptor (IL-1R)-mediated immune responses. Although Ubc13-deficient mice show defects in BCR-, TLR/IL-1R-, or CD40-mediated activation of mitogen-activated protein kinases, the function of Ubc13 in TCR-mediated signaling and responses remains uncertain. To address this, we here generated T cell-specific conditional Ubc13-deficient mice. The frequency of T lymphocytes was severely reduced in spleens from Ubc13-deficient mice. Moreover, Ubc13-deficient thymocytes displayed defective proliferation in response to anti-CD3/CD28 or PMA/ionophore stimulation. Regarding the signal transduction, although NF-kappaB activation was modestly affected, PMA/ionophore-induced activation of Jnk and p38 was profoundly impaired in Ubc13-deficient thymocytes. In addition, PMA/ionophore-mediated ubiquitination of NF-kappaB essential modulator (NEMO)/IkappaB kinase gamma (IKKgamma) and phosphorylation of TGF-beta-activated kinase 1 (TAK1) were nearly abolished in Ubc13-deficient thymocytes. Thus, Ubc13 plays an important role in thymocyte TCR-mediated signaling and immune responses.     

Ionomycin-induced apoptosis of thymocytes is independent of Nur77 NBRE or NurRE binding, but is accompanied by Nur77 mitochondrial targeting

The induction of thymocyte apoptosis through the Nur77-mediated intrinsic pathway can be of physiological importance in the clonal deletion of autoreactive thymocytes during negative selection in the thymus and/or in thymocytes undergoing oncogenic transformation. Ionomycin treatment induces endogenous Nur77 expression as well as apoptosis and cytochrome c release in thymocytes. Here it is shown for the first time that in normal thymocytes undergoing apoptosis, ionomycin induces translocation of endogenous Nur77 not only to the nucleus, but also to mitochondria. Immunosuppressant FK506 inhibits Nur77 NBRE and NurRE binding activity but has no effect on thymocytes apoptosis, the subcellular localization of Nur77, or cytochrome c release. This indicates that thymocytes can undergo apoptosis through the intrinsic Nur77-mediated mitochondrial pathway and that the transactivation activity of Nur77 monomers or dimers is not necessary for thymocyte apoptosis.     

A requirement for IL-2/IL-2 receptor signaling in intrathymic negative selection

The nature of the signals that influence thymocyte selection and determine the fate of CD4(+)8(+) (double positive) thymocytes remains unclear. Cytokines produced locally in the thymus may modulate signals delivered by TCR-MHC/peptide interactions and thereby influence the fate of double-positive thymocytes. Because the IL-2/IL-2R signaling pathway has been implicated in thymocyte and peripheral T cell survival, we investigated the possibility that IL-2/IL-2R interactions contribute to the deletion of self-reactive, Ag-specific thymocytes. By using nontransgenic and transgenic IL-2-sufficient and -deficient animal model systems, we have shown that during TCR-mediated thymocyte apoptosis, IL-2 protein is expressed in situ in the thymus, and apoptotic thymocytes up-regulate expression of IL-2RS: IL-2R(+) double-positive and CD4 single-positive thymocytes undergoing activation-induced cell death bind and internalize IL-2. IL-2-deficient thymocytes are resistant to TCR/CD3-mediated apoptotic death, which is overcome by providing exogenous IL-2 to IL-2(-/-) mice. Furthermore, disruption or blockade of IL-2/IL-2R interactions in vivo during Ag-mediated selection rescues some MHC class II-restricted thymocytes from apoptosis. Collectively, these findings provide evidence for the direct involvement of the IL-2/IL-2R signaling pathway in the deletion of Ag-specific thymocyte populations and suggest that CD4 T cell hyperplasia and autoimmunity in IL-2(-/-) mice is a consequence of ineffective deletion of self-reactive T cells.     

Caspases in T-cell receptor-induced thymocyte apoptosis.

Apoptosis eliminates inappropriate or autoreactive T lymphocytes during thymic development. Intracellular mediators involved in T-cell receptor (TCR)-mediated apoptosis in developing thymocytes during negative selection are therefore of great interest. Caspases, cysteine proteases that mediate mature T-cell apoptosis, have been implicated in thymocyte cell death, but their regulation is not understood. We examined caspase activities in distinct thymocyte subpopulations that represent different stages of T-cell development. We found caspase activity in CD4+CD8+ double positive (DP) thymocytes, where selection involving apoptosis occurs. Earlier and later thymocyte stages exhibited no caspase activity. Only certain caspases, such as caspase-3 and caspase-8-like proteases, but not caspase-1, are active in DP thymocytes in vivo and can be activated when DP thymocytes are induced to undergo apoptosis in vitro by TCR-crosslinking. Thus, specific caspases appear to be developmentally regulated in thymocytes.     

Characterization of a mutant T-cell hybridoma line with defects in the TCR-mediated apoptotic pathway

A mutant T-cell hybridoma line named mutant 51 was developed that, unlike the parental line, did not die after T-cell receptor (TCR) engagement and demonstrated reduced death in response to dexamethasone. Intracellular calcium measurements showed that available calcium stores were markedly reduced in the mutant cell line. Unlike control cells, secretion of IL-2 from mutant cells was also greatly reduced, although addition of exogenous IL-2 did not facilitate increased apoptosis. Although levels of the cell death gene product Nur77 were equivalent, additional studies showed that mutant cells expressed Nur77 predominantly in the cytoplasm following TCR engagement, while parental cells displayed a nuclear translocalization of Nur77. In addition, Fas levels and Fas ligand dependant killing were both markedly reduced in the mutant clone. From these data we hypothesize a role for available calcium stores and Nur77 nuclear localization in TCR-mediated apoptosis in T-cell hybridomas.     

Thymocyte apoptosis induced by T cell activation is mediated by glucocorticoids in vivo

Glucocorticoids, administered in pharmacological doses, potently modulate immune system function and are a mainstay therapy for many common human diseases. Physiologic production of glucocorticoids may play a role in optimization of the immune repertoire both centrally and peripherally. Possible effects include alteration of lymphocyte development and down-regulation of cytokine responses, but essential roles remain unclear. To determine the part that endogenous glucocorticoids play in thymocyte development, we used fetal liver from mice lacking the glucocorticoid receptor GRko for immunological reconstitution of lethally irradiated wild-type (WT) mice. We find normal numbers and subset distribution of GRko thymocytes. GRko thymocytes also exhibit similar sensitivity to apoptosis induced by activating anti-CD3epsilon Ab as WT thymocytes in vitro. Surprisingly, GRko thymocytes are significantly more resistant than WT thymocytes to anti-CD3epsilon-mediated thymocyte apoptosis in vivo. Consistent with this finding, in vivo TCR complex activation induces sustained high levels of glucocorticoids that correlate strongly with thymocyte apoptosis in WT mice. We find that while direct engagement of the TCR complex may cause death of a subset of thymocytes, glucocorticoids are required for deletion of the majority of thymocytes. Thus, TCR stimulation by Ab administration may more accurately reflect polyclonal T cell activation than negative selection in vivo.     

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.     

Involvement of Rho GTPases and their effectors in the secretory process of PC12 cells

Intrathymic maturation of thymocytes is essential for the proper formation of T-cell repertoire. This process involves two major biochemical pathways, one initiated by the recognition of MHC/peptide by the T-cell receptor and the other mediated by glucocorticoids. These hormones seem to affect thymocyte maturation by increasing the threshold of TCR-mediated positive and negative selection, and by inducing apoptosis of nonselected thymocytes. We have previously reported that an SV40-immortalized murine thymic epithelial cell line, namely 2BH4, was able to protect thymocytes from dexamethasone-induced apoptosis. Here we show that this protection is independent of cell-to-cell contact and does not seem to involve a Bcl-2-mediated resistance, since incubation of thymocytes with 2BH4 cells or its supernatant does not interfere with the levels of this antiapoptotic molecule. The protection conferred by 2BH4 cells, or by a primary culture of thymic stromal cells, is specific for the CD4(+)CD8(-) and CD4(-)CD8(+) single-positive thymocytes, whereas the broad-spectrum caspase inhibitor z-VAD-fmk blocks apoptosis induced by dexamethasone in all thymocyte subpopulations. Our results suggest that positively selected single-positive thymocytes are still susceptible to glucocorticoid-induced apoptosis but are protected from it through the action of a heat-stable protein(s) released by thymic stromal cells.