Apoptosis In Immature Thymocytes Research Articles

CD7 expression and galectin-1-induced apoptosis of immature thymocytes are directly regulated by NF-κB upon T-cell activation

CD7, one of the galectin-1 receptors, has crucial roles in galectin-1-mediated apoptosis of activated T-cells and T-lymphoma progression in peripheral tissues. In this study, we showed that CD7 promoter activity was increased by NF-κB and that this activity was synergistic when Sp1 was co-expressed in the immature T-cell line L7. Site-directed mutagenesis analysis of the CD7 promoter indicated that NF-κB specifically bound to the NF-κE2 site in cooperation with Sp1. Overexpression of E12 or Twist2 proteins negatively regulated NF-κB-mediated activity of the CD7 proximal promoter. In addition, CD7 expression was down-regulated by treatment with the p38 MAPK inhibitor SB20358, or the MSK1 inhibitor H-89. These signaling pathway inhibitors prevented galectin-1-mediated apoptosis of immature T-cells. From these results, we concluded that the regulation of CD7 gene expression through NF-κB activation induced by TCR/CD28 might have significant implications for T-cell homeostasis.

β-Catenin Expression Results in p53-Independent DNA Damage and Oncogene-Induced Senescence in Prelymphomagenic Thymocytes In Vivo

The expression of beta-catenin, a potent oncogene, is causally linked to tumorigenesis. Therefore, it was surprising that the transgenic expression of oncogenic beta-catenin in thymocytes resulted in thymic involution instead of lymphomagenesis. In this report, we demonstrate that this is because the expression of oncogenic beta-catenin induces DNA damage, growth arrest, oncogene-induced senescence (OIS), and apoptosis of immature thymocytes. In p53-deficient mice, the expression of oncogenic beta-catenin still results in DNA damage and OIS, but the thymocytes survive and eventually progress to thymic lymphoma. beta-Catenin-induced thymic lymphomas are distinct from lymphomas that arise in p53(-/-) mice. They are CD4(-) CD8(-), while p53-dependent lymphomas are largely CD4(+) CD8(+), and they develop at an earlier age and in the absence of c-Myc expression or Notch1 signaling. Thus, we report that oncogenic beta-catenin-induced, p53-independent growth arrest and OIS and p53-dependent apoptosis protect developing thymocytes from transformation by oncogenic beta-catenin.

Gene expression profiling of leukemic cells and primary thymocytes predicts a signature for apoptotic sensitivity to glucocorticoids

BackgroundGlucocorticoids (GC's) play an integral role in treatment strategies designed to combat various forms of hematological malignancies. GCs also are powerful inhibitors of the immune system, through regulation of appropriate cytokines and by causing apoptosis of immature thymocytes. By activating the glucocorticoid receptor (GR), GCs evoke apoptosis through transcriptional regulation of a complex, interactive gene network over a period of time preceding activation of the apoptotic enzymes. In this study we used microarray technology to determine whether several disparate types of hematologic cells, all sensitive to GC-evoked apoptosis, would identify a common set of regulated genes. We compared gene expression signatures after treatment with two potent synthetic GCs, dexamethasone (Dex) and cortivazol (CVZ) using a panel of hematologic cells. Pediatric CD4+/CD8+ T-cell leukemia was represented by 3 CEM clones: two sensitive, CEM-C7–14 and CEM-C1–6, and one resistant, CEM-C1–15, to Dex. CEM-C1–15 was also tested when rendered GC-sensitive by several treatments. GC-sensitive pediatric B-cell leukemia was represented by the SUP-B15 line and adult B-cell leukemia by RS4;11 cells. Kasumi-1 cells gave an example of the rare Dex-sensitive acute myeloblastic leukemia (AML). To test the generality of the correlations in malignant cell gene sets, we compared with GC effects on mouse non-transformed thymocytes.ResultsWe identified a set of genes regulated by GCs in all GC-sensitive malignant cells. A portion of these were also regulated in the thymocytes. Because we knew that the highly Dex-resistant CEM-C1–15 cells could be killed by CVZ, we tested these cells with the latter steroid and again found that many of the same genes were now regulated as in the inherently GC-sensitive cells. The same result was obtained when we converted the Dex-resistant clone to Dex-sensitive by treatment with forskolin (FSK), to activate the adenyl cyclase/protein kinase A pathway (PKA).ConclusionOur results have identified small sets of genes that correlate with GC-sensitivity in cells from several hematologic malignancies. Some of these are also regulated in normal mouse thymocytes.

Functional Role for IκBNS in T Cell Cytokine Regulation As Revealed by Targeted Gene Disruption

Triggering of the TCR by cognate peptide/MHC ligands induces expression of I kappa BNS, a member of the I kappa B family of NF-kappaB inhibitors whose expression is associated with apoptosis of immature thymocytes. To understand the role of I kappa BNS in TCR triggering, we created a targeted disruption of the I kappa BNS gene. Surprisingly, mice lacking I kappa BNS show normal thymic progression but both thymocytes and T cells manifest reduced TCR-stimulated proliferation. Moreover, I kappa BNS knockout thymocytes and T cells produce significantly less IL-2 and IFN-gamma than wild-type cells. Transfection analysis demonstrates that I kappa BNS and c-Rel individually increase IL-2 promoter activity. The effect of I kappa BNS on the IL-2 promoter, unlike c-Rel, is dependent on the NF-kappaB rather than the CD28RE site; mutation of the NF-kappaB site extinguishes the induction of transcription by I kappa BNS in transfectants and prevents association of I kappa BNS with IL-2 promoter DNA. Microarray analyses confirm the reduction in IL-2 production and some IFN-gamma-linked transcripts in I kappa BNS knockout T cells. Collectively, our findings demonstrate that I kappa BNS regulates production of IL-2 and other cytokines induced via "strong" TCR ligation.

Effects of Glucocorticoids on Apoptosis and Clearance of Apoptotic Cells

The glucocorticoid (GC) drugs are one of the most commonly prescribed and effective anti-inflammatory agents used for the treatment of many inflammatory disorders through their ability to attenuate phlogistic responses. The glucocorticoid receptor (GCR) primarily mediates GC actions via activation or repression of gene expression. GCs directly induce the expression of proteins displaying anti-inflammatory activities. However, the likely predominant effect of GCs is the repression of multiple inflammatory genes that invariably are overexpressed during nonresolving chronic inflammation. Although most GC actions are mediated through regulation of transcription, rapid nongenomic actions have also been reported. In addition, GCs modulate inflammatory cell survival, inducing apoptosis in immature thymocytes and eosinophils, while delaying constitutive neutrophil apoptosis. Importantly, GCs promote noninflammatory phagocytosis of apoptotic cell targets, a process important for the successful resolution of inflammation. Here, the effects and mechanisms of action of GC on inflammatory cell apoptosis and phagocytosis will be discussed.

Induction of IL-15 by TCR/CD3 aggregation depends on IFN-gamma and protects against apoptosis of immature thymocytes in vivo.

TCR/CD3 aggregation by injection of anti-CD3 Ab produces T cell activation, release of cytokines such as IFN-gamma, and apoptosis in the cortical region of the thymus. We show that anti-CD3 Ab induces IL-15 mRNA in spleens of wild-type but not IFN-gamma receptor-knock-out (IFN-gammaR KO) mice. The loss of IL-15 mRNA induction in IFN-gammaR KO mice was associated with increased thymocyte apoptosis. Pretreatment of wild-type mice with neutralizing anti-IL-15 Ab increased the anti-CD3-triggered thymocyte apoptosis, thus mimicking the sensitive phenotype of IFN-gammaR KO mice. Inversely, anti-CD3-induced apoptosis in IFN-gammaR KO mice was suppressed by administration of recombinant IL-15. In IFN-gammaR KO mice and in wild-type mice that were treated with anti-IL-15, augmented apoptosis affected mainly CD4+CD8+ immature thymocytes. IL-15 as well as IL-15Ralpha mRNA expression in thymocytes was not increased by anti-CD3. These data demonstrate that systemic IL-15 exerts anti-apoptotic activity on immature T cells and establish a regulatory mechanism whereby TCR/CD3 engagement induces IL-15 expression via an IFN-gamma-dependent pathway. The self-amplifying nature of this IFN-gamma/IL-15 connection may constitute a regulatory pathway in central tolerance to self.

Diet and Cancer Prevention Studies in p53-Deficient Mice

Progress in mechanism-based cancer prevention research may be facilitated by the use of animal models displaying specific genetic susceptibilities for cancer such as mice deficient in the p53 tumor suppressor gene, the most frequently altered gene in human cancer. We observed in p53-knockout (p53-/-) mice that calorie restriction (CR; 60% of the control group's intake of carbohydrate energy) increased the latency of spontaneous tumor development (mostly lymphomas) approximately 75%, decreased serum insulin-like growth factor (IGF)-1 and leptin levels, significantly slowed thymocyte cell cycle traverse and induced apoptosis in immature thymocytes. In heterozygous p53-deficient (p53+/-) mice, CR and 1 d/wk of food deprivation each significantly delayed spontaneous tumor development (a mix of lymphomas, sarcomas and epithelial tumors) and decreased serum IGF-1 and leptin levels even when begun late in life. We have also developed a rapid and relevant p53+/- mouse mammary tumor model by crossing p53-deficient mice with MMTV-Wnt-1 transgenic mice, and found that CR and 1 d/wk food deprivation significantly increased mammary tumor latency (greater than twofold) and reduced the mean serum IGF-1 and leptin levels to <50% of that of control mice (P < 0.0001). In addition, fluasterone, fenretinide and soy each delayed tumor development but had little effect on IGF-1 or leptin levels. We have capitalized on the susceptibility of p53+/- mice to chronic, low dose, aromatic amine-induced bladder carcinogenesis to develop a useful model for evaluating bladder cancer prevention approaches such as cyclooxygenase-2 inhibition. As demonstrated by these examples, mice with specific (and human-like) genetic susceptibilities for cancer provide powerful new tools for testing and characterizing interventions that may inhibit the process of carcinogenesis in humans.

Akting Upon Nurr77

The transcription factor Nurr77, an orphan nuclear receptor, can promote apoptosis in immature thymocytes. Nurr77-dependent transcription is partially controlled by phosphorylation of Ser 350 , which reduces the DNA binding activity of Nurr77. Pekarsky et al. observed that Akt, a serine-threonine kinase that lies in the phosphatidylinositol 3' kinase (PI3K)-dependent antiapoptotic pathway, coprecipitated with Nurr77 (in cells transfected with Akt and Nurr77), and phosphorylated Nurr77 on Ser 350 in vitro and in vivo. Further experiments confirmed that Akt-mediated phosphorylation decreased Nurr77-mediated gene expression as measured by gene reporter assays. These data suggest that one way Akt regulates antiapoptotic effects is by preventing the activation of Nurr77-dependent gene expression. Y. Pekarsky, C. Hallas, A. Palamarchuk, A. Koval, F. Bullrich, Y. Hirata, R. Bichi, J. Letofsky, C. M. Croce, Akt phosphorylates and regulates the orphan nuclear receptor Nurr77. Proc. Natl. Acad. Sci. U.S.A. 98 , 3690-3694 (2001). [Abstract] [Full Text]

Cancer prevention studies in p53-deficient mice.

Future progress in mechanism-based cancer prevention research may be facilitated by animal models displaying specific genetic susceptibilities for cancer, such as mice deficient in 1 (+/-) or both (-/-) alleles of the p53 tumor suppressor gene. We observed in p53-/- mice that calorie restriction (CR) increased the latency of spontaneous tumor development (mostly lymphomas) by approximately 75%, decreased serum insulin-like growth factor-1 (IGF-1) and leptin levels, slowed thymocyte cell cycle traverse, and induced apoptosis in immature thymocytes. In p53+/- mice, CR and a 1 d/wk fast each delayed spontaneous tumor development (a mix of lymphomas, sarcomas, and epithelial tumors) and decreased serum IGF-1 and leptin levels, even when begun late in life. In p53+/-Wnt-1 transgenic mice, a mammary tumor model, the same interventions increased mammary tumor latency and reduced mean serum IGF-1 and leptin levels to <50% of those of control mice. We capitalized on the susceptibility of p53+/- mice to chronic, low-dose aromatic amine-induced bladder carcinogenesis to develop a useful model for evaluating bladder cancer prevention approaches. These examples clearly indicate that mice with specific (and humanlike) genetic susceptibilities for cancer are powerful models for testing interventions that may inhibit carcinogenesis in humans.

Glucocorticoid-induced thymocyte apoptosis: protease-dependent activation of cell shrinkage and DNA degradation

Glucocorticoids are well known to stimulate apoptosis in immature thymocytes. Apoptosis in this and other cells is characterized by cell shrinkage, DNA fragmentation and activation of a class of proteases named caspases. We have utilized the flow cytometer to evaluate the coordinate regulation of cell shrinkage and DNA fragmentation in glucocorticoid-treated rat thymocytes and explore the role of caspases upstream of both changes. The results indicate that the activation of apoptosis by glucocorticoids in a cell population is an asynchronous event with only a percentage of the cells displaying apoptotic characteristics at any given time. Both cell shrinkage and chromatin degradation are tightly coupled with similar proportions of the cells displaying each characteristic. The coordinate appearance of these characteristics may suggest a similar mechanism of regulation. Incubation of thymocytes with the general caspase inhibitor Z-VAD-FMK completely blocked both cell shrinkage and DNA fragmentation in spontaneous and glucocorticoid-induced thymocyte apoptosis, implicating an early upstream role for proteases in the activation of thymocyte apoptosis.

7alpha-Hydroxylation and 3-dehydrogenation abolish the ability of 25-hydroxycholesterol and 27-hydroxycholesterol to induce apoptosis in thymocytes.

Oxygenated derivatives of sterols (oxysterols), including 25-hydroxycholesterol and 27-hydroxycholesterol, have immunosuppressive effects. Oxysterols can directly induce apoptosis in immature thymocytes, cells which are inherently sensitive to induction of programmed cell death. For that reason, the metabolism of 25-hydroxycholesterol and 27-hydroxycholesterol in mouse thymus has been studied. When incubated with thymic tissue, both oxysterols were found to be 7alpha-hydroxylated with subsequent oxidation to 7alpha-hydroxy-3-oxo-delta4 steroids. A minor fraction of 27-hydroxycholesterol was also metabolised to 3beta-hydroxy-5-cholestenoic, 3beta,7alpha-dihydroxy-5-cholestenoic and 7alpha-hydroxy-3-oxo-4-cholestenoic acids. The 7alpha-hydroxylase was found to be localised to the thymic epithelial cells and the reaction was stimulated by interleukin-1beta and inhibited by metyrapone and RU486. In contrast to 25-hydroxycholesterol and 27-hydroxycholesterol, the 7alpha-hydroxylated metabolites, 7alpha,25-dihydroxycholesterol, 7alpha,25-dihydroxy-4-cholesten-3-one and 7alpha,27-dihydroxy-4-cholesten-3-one did not induce thymocyte apoptosis. The results suggest that 7alpha-hydroxylation may be of regulatory importance, possibly by protecting the developing thymocytes against toxic effects by oxysterols.

CD2 Rescues T Cells From T-Cell Receptor/CD3 Apoptosis: A Role for the Fas/Fas-L System

AbstractAnti-CD3 monoclonal antibodies (MoAbs) and glucocorticoid hormones induce apoptosis in immature thymocytes and peripheral T lymphocytes. This process is inhibited by a number of growth factors, including interleukin-2 (IL-2), IL-3, and IL-4, as well as by triggering of the adhesion molecule CD44, which would indicate that signals generated by membrane receptors can modulate the survival of lymphoid cells. To investigate whether triggering of CD2 may also affect apoptosis in lymphoid cells, we analyzed the effect of stimu-lation with anti-CD2 MoAbs on T-cell apoptosis induced by two stimuli, anti-CD3 MoAbs and dexamethasone (DEX), using a hybridoma T-cell line and a T-helper cell clone. The results show that CD2 engagement decreased anti-CD3 MoAb-induced apoptosis, but did not influence DEX-induced cell death. Furthermore, the decrease appeared to be related to the expression of Fas/APO-1 (CD95) and Fas-ligand (Fas-L). In fact, we show that CD2 stimulation inhibits apoptosis by preventing the CD3-induced upregulation of Fas and Fas-L in a Fas-dependent experimental system. These data suggest that a costimulatory molecule may control a deletion pathway and may therefore contribute to the regulation of peripheral tolerance.

CD2 Rescues T Cells From T-Cell Receptor/CD3 Apoptosis: A Role for the Fas/Fas-L System

Anti-CD3 monoclonal antibodies (MoAbs) and glucocorticoid hormones induce apoptosis in immature thymocytes and peripheral T lymphocytes. This process is inhibited by a number of growth factors, including interleukin-2 (IL-2), IL-3, and IL-4, as well as by triggering of the adhesion molecule CD44, which would indicate that signals generated by membrane receptors can modulate the survival of lymphoid cells. To investigate whether triggering of CD2 may also affect apoptosis in lymphoid cells, we analyzed the effect of stimu-lation with anti-CD2 MoAbs on T-cell apoptosis induced by two stimuli, anti-CD3 MoAbs and dexamethasone (DEX), using a hybridoma T-cell line and a T-helper cell clone. The results show that CD2 engagement decreased anti-CD3 MoAb-induced apoptosis, but did not influence DEX-induced cell death. Furthermore, the decrease appeared to be related to the expression of Fas/APO-1 (CD95) and Fas-ligand (Fas-L). In fact, we show that CD2 stimulation inhibits apoptosis by preventing the CD3-induced upregulation of Fas and Fas-L in a Fas-dependent experimental system. These data suggest that a costimulatory molecule may control a deletion pathway and may therefore contribute to the regulation of peripheral tolerance.

Protection from anti-TCR/CD3-induced apoptosis in immature thymocytes by a signal through thymic shared antigen-1/stem cell antigen-2.

During T cell development in the thymus, the expression of thymic shared antigen-1 (TSA-1)/stem cell antigen-2 (Sca-2), a glycosylphosphatidylinositol (GPI)-anchored differentiation antigen, is developmentally regulated. The expression level of TSA-1 is the highest in most immature CD4- CD8- thymocytes, high in CD4+ CD8+ thymocytes, but barely detectable in mature CD4+ CD8- or CD4- CD8- thymocytes and peripheral T cells. We have previously shown that surface TSA-1 expression in peripheral T cells is induced upon activation and that anti-TSA-1 mAb inhibits the T cell receptor (TCR) signaling pathway in activated T cells. In the present study, we have analyzed a role of TSA-1 in thymic selection events, especially in TCR-mediated apoptosis. In in vitro experiments, anti-TSA-1 blocked anti-CD3-induced cell death of T cell hybridomas. When anti-TSA-1 was injected into newborn mice in vivo together with anti-CD3 epsilon or anti-TCR-beta, TCR/CD3-mediated apoptosis of thymocytes was almost completely blocked. The blockade of apoptosis was defined by the inhibition of, first, the decrease in total number of thymocytes; second, the decrease in percentages of CD4+ CD8+ thymocytes; and third, the induction of DNA fragmentation. However, anti-TSA-1 did not block either steroid- or radiation-induced apoptosis, indicating that a signal via TSA-1 does not inhibit a common pathway of thymocyte apoptosis. Since TCR-mediated apoptosis is pivotal in thymic ontogeny, these results suggest that TSA-1/Sca-2 is an important cell surface molecule regulating the fate of a developing T cell.

Regulation of the Nur77 orphan steroid receptor in activation-induced apoptosis.

T-cell receptor (TCR)-mediated apoptosis in immature thymocytes and T-cell hybridomas is calcium dependent and can be inhibited by cyclosporin A (CsA). Induction of the orphan steroid receptor Nur77 (NGFI-B) is required for activation-induced apoptosis. Here, we examined the regulation of Nur77 expression, in response to apoptotic TCR signals, which consists of kinase C and calcium pathways. We show that the major control of Nur77 induction is mediated by the calcium signaling pathway. In contrast, protein kinase C signals induce only a low level of Nur77 activity. Nur77 promoter activity parallels its protein levels. CsA decreases both Nur77 protein levels and promoter activity, and the kinetics of CsA inhibition of apoptosis correlates with a decrease in Nur77 protein levels. TCR signals and kinase C signals result in a similar level of Nur77 protein phosphorylation but mediate differential transactivation activity of Nur77. In addition, Nur77 promoter deletion analysis revealed two RSRF (related to serum-responsive factor) binding sites, which can confer calcium and CsA sensitivity on a heterologous promoter. Taken together, our data suggest that the levels of transcriptional induction of Nur77 play an important role during activation-induced apoptosis and that calcium signals regulate a novel CsA-sensitive nuclear factor required for Nur77 transcription in T cells.

CD44 (Pgp-1) Inhibits CD3 and Dexamethasone-Induced Apoptosis

Anti-CD3 monoclonal antibodies (MoAbs) and glucocorticoid hormones (GCH) induce apoptosis in immature thymocytes and peripheral T lymphocytes. This process is inhibited by a number of growth factors, including interleukin-2 (IL-2), IL-3, and IL-4, indicating that signals generated by membrane receptors can modulate the survival of lymphoid cells. To investigate whether signals activated by adhesion receptors have a similar activity, we analyzed the effect of CD44 (Pgp-1) adhesion molecule receptor stimulation on T-cell apoptosis induced by three stimuli (anti-CD3 MoAbs, dexamethasone [DEX] treatment, and exposure to ultraviolet irradiation [UV]) on a 3DO T-cell line. The results show that CD44 engagement, either by hyaluronic acid (HA) or anti-CD44 MoAbs, inhibits DNA fragmentation and apoptosis induced by DEX and anti-CD3 MoAbs, whereas that induced by UV, a p53-dependent phenomenon, was not inhibited. Furthermore, the antiapoptotic effect exerted through CD44 activation does not seem related to overexpression of bcl-2 or to have appreciable effects on cell proliferation. Our results indicate that adhesion molecules modulate T-cell survival by counteracting apoptosis induced by DEX or anti-CD3 MoAbs.

CD44 (Pgp-1) inhibits CD3 and dexamethasone-induced apoptosis

Anti-CD3 monoclonal antibodies (MoAbs) and glucocorticoid hormones (GCH) induce apoptosis in immature thymocytes and peripheral T lymphocytes. This process is inhibited by a number of growth factors, including interleukin-2 (IL-2), IL-3, and IL-4, indicating that signals generated by membrane receptors can modulate the survival of lymphoid cells. To investigate whether signals activated by adhesion receptors have a similar activity, we analyzed the effect of CD44 (Pgp-1) adhesion molecule receptor stimulation on T-cell apoptosis induced by three stimuli (anti-CD3 MoAbs, dexamethasone [DEX] treatment, and exposure to ultraviolet irradiation [UV]) on a 3DO T-cell line. The results show that CD44 engagement, either by hyaluronic acid (HA) or anti-CD44 MoAbs, inhibits DNA fragmentation and apoptosis induced by DEX and anti-CD3 MoAbs, whereas that induced by UV, a p53-dependent phenomenon, was not inhibited. Furthermore, the antiapoptotic effect exerted through CD44 activation does not seem related to overexpression of bcl-2 or to have appreciable effects on cell proliferation. Our results indicate that adhesion molecules modulate T-cell survival by counteracting apoptosis induced by DEX or anti-CD3 MoAbs.

Expression of the death-associated gene RP-8 in granule cell neurons undergoing postnatal cell death in the cerebellum of weaver mice

RP-8 is one of several mRNAs elevated during apoptosis in immature thymocytes. We used in situ hybridization to look for RP-8 mRNA in the cerebella of weaver mice. In the weaver mouse cerebellar granule cells fail to differentiate and instead die during the first two weeks of postnatal development [30]. We demonstrate that the dying cells exhibit DNA degradation, a characteristic feature of apoptosis. RP-8 is expressed in the weaver cerebellum during the period of granule cell death, and is limited to cells in the same region where apoptotic granule cells are located. Thus, RP-8 expression, first associated with PCD in thymocytes, correlates with granule cell death in the weaver cerebellum.

Cyclosporin A blocks apoptosis by inhibiting the DNA binding activity of the transcription factor Nur77.

Engagement of T-cell receptors (TCRs) on immature thymocytes by self-antigen-major histocompatibility complexes causes the death of self-reactive thymocytes via apoptosis, a phenomenon that establishes T-cell tolerance. Similarly, treatment of thymocytes with anti-TCR antibodies leads to TCR-mediated apoptosis, which can also be induced in T-cell hybridomas. TCR-mediated apoptosis in immature thymocytes and T-cell hybridomas requires the expression of a new set of genes. In particular, it has recently been shown that the expression of Nur77, a transcription factor which is a member of the steroid/thyroid receptor superfamily, is required for TCR-mediated apoptosis in T-cell hybridomas and perhaps in thymocytes. Cyclosporin A (CsA), an immunosuppressive drug, has been shown to interfere with clonal deletion of self-reactive T cells in vivo, partly by blocking TCR-mediated apoptosis. We report here that CsA inhibits the TCR-mediated activation of Nur77 protein in T-cell hybridomas by blocking the DNA binding activity of Nur77 protein rather than its de novo synthesis. We also show that CsA mediates its negative effects on the Nur77 DNA binding activity through the N-terminal region of the protein. This complete inhibition of Nur77 protein DNA binding activity may explain how CsA interferes with TCR-mediated apoptosis.

Co-receptor (CD4/CD8) engagement enhances CD3-induced apoptosis in thymocytes. Implications for negative selection.

Negative selection of self-reactive immature T cells is mediated by TCR engagement and is thought to occur via apoptosis (programmed cell death). The requirement for the co-receptors CD4 and CD8 in negative selection has been demonstrated, but the biochemical mechanisms underlying their involvement in this process remain undefined. Here we present evidence that co-receptor engagement dramatically enhances CD3-induced endonuclease activation and cell death characteristic of apoptosis in immature thymocytes. The responses are associated with increased tyrosine phosphorylation of a number of cellular substrates, including the gamma isoform of phospholipase C, and with increased association of tyrosine phosphoproteins, including the protein tyrosine kinase p56lck, with the TCR complex. Co-receptor engagement also potentiated CD3-mediated Ca2+ increases via a mechanism dependent upon tyrosine kinase activation. Sustained Ca2+ availability was found to be necessary for endonuclease activation and apoptosis to occur. We suggest that CD4 and CD8 may participate in negative selection by enhancing TCR/CD3-induced tyrosine kinase activation and sustained Ca2+ increases that lead to endonuclease activation and apoptosis in self-reactive CD4+ CD8+ thymocytes.