Regulation of T-cell death-associated gene 51 (TDAG51) expression in human T-cells

T-cell death-associated gene 51 (TDAG51) has been described to regulate T-cell receptor/CD3-dependent induction of CD95/Fas and subsequent activation-induced cell death (AICD) in a murine T-cell hybridoma. Using well-defined pharmacological inhibitors, we investigated the regulation of TDAG51 expression in human T-cells and the correlation with cell death. TDAG51 was induced in resting T-cells, lymphoid cell lines and AICD-susceptible as well as AICD-resistant T-cell clones, and induction was inhibited by MAP-kinase inhibitors and PKC inhibitor G?6983. No correlation between the effects of inhibitors on TDAG51 expression and cell death was observed. The constitutive TDAG51 expression in five pancreatic carcinoma cell lines was reduced by MAP-kinase inhibitors but not by G?6983. Furthermore, the inducible overexpression of TDAG51 in TetOn Jurkat cells did not modulate cellular proliferation, phorbolester/ionomycin-induced growth arrest, or the expression of various cell surface molecules. Our results indicate that the expression of TDAG51 in human T-cells does not correlate with AICD.     

Resistance of chimpanzee T cells to human immunodeficiency virus type 1 Tat-enhanced oxidative stress and apoptosis.

CD4+ T-cell depletion in AIDS patients involves induction of apoptosis in human immunodeficiency virus (HIV)-infected and noninfected T cells. The HIV type 1 (HIV-1)-transactivating protein Tat enhances apoptosis and activation-induced cell death (AICD) of human T cells. This effect is mediated by the CD95 (APO-1/Fas) receptor-CD95 ligand (CD95L) system and may be linked to the induction of oxidative stress by Tat. Here we show that HIV-1 Tat-induced oxidative stress is necessary for sensitized AICD in T cells caused by CD95L expression. Tat-enhanced apoptosis and CD95L expression in T cells are inhibited by neutralizing anti-Tat antibodies, antioxidants, and the Tat inhibitor Ro24-7429. Chimpanzees infected with HIV-1 show viral replication resembling early infection in humans but do not show T-cell depletion or progression towards AIDS. The cause for this discrepancy is unknown. Here we show that unlike Tat-treated T cells in humans, Tat-treated chimpanzee T cells do not show downregulation of manganese superoxide dismutase or signs of oxidative stress. Chimpanzee T cells are also resistant to Tat-enhanced apoptosis, AICD, and CD95L upregulation.     

Activation or suppression of NFkappaB by HPK1 determines sensitivity to activation-induced cell death

Restimulation of the T-cell receptor (TCR) in activated T cells induces CD95 (Fas/Apo-1)-mediated activation-induced cell death (AICD). The TCR-proximal mechanisms leading to AICD are elusive. Here we characterize hematopoietic progenitor kinase 1 (HPK1) as a differentially regulated TCR-proximal signaling protein involved in AICD of primary T cells. We show that HPK1 is a functional component of the endogenous IkappaB kinase (IKK) complex and is crucial for TCR-mediated NFkappaB activation. While full-length HPK1 enhances IKKbeta phosphorylation, siRNA-mediated knockdown of HPK1 blunts TCR-mediated NFkappaB activation and increases cell death. We also demonstrate proteolytic processing of HPK1 into HPK1-C, specifically in AICD-sensitive primary T cells. The cleavage product HPK1-C sequesters the inactive IKK complex and suppresses NFkappaB upon TCR restimulation by binding to IKKalpha and IKKbeta. T cells of HPK1-C transgenic mice are sensitized towards TCR-mediated AICD. Consequently, preventing HPK1-C generation in primary T cells by siRNA-mediated knockdown results in decreased AICD. Thus, these results show a novel mechanism of sensitization of T lymphocytes towards AICD by suppression of NFkappaB, and propose that HPK1 is a life/death switch in T lymphocytes.     

Regulation of activation-induced cell death of mature T-lymphocyte populations

Resting mature T lymphocytes are activated when triggered via their antigen-specific T-cell receptor (TCR) to elicit an appropriate immune response. In contrast, preactivated T cells may undergo activation-induced cell death (AICD) in response to the same signals. along with cell death induced by growth factor deprivation, AICD followed by the elimination of useless or potentially harmful cells preserves homeostasis, leads to the termination of cellular immune responses and ensures peripheral tolerance. T-cell apoptosis and AICD are controlled by survival cytokines such as interleukin-2 (IL-2) and by death factors such as tumor necrosis factor (TNF) and CD95 ligand (CD95L). In AICD-sensitive T cells, stimulation upregulates expression of one or several death factors, which in turn engage specific death receptors on the same or a neighboring cell. Death receptors are activated by oligomerization to rapidly assemble a number of adapter proteins and enzymes to result in an irreversible activation of proteases and nucleases that culminates in cell death by apoptosis. Increased knowledge of the molecular mechanisms that regulate AICD of lymphocytes opens new immunotherapeutic perspectives for the treatment of certain autoimmune diseases, and has implications in other areas such as transplantation medicine and AIDS research.     

TLR4/MYD88-dependent, LPS-induced synthesis of PGE2 by macrophages or dendritic cells prevents anti-CD3-mediated CD95L upregulation in T cells

Antigen-presenting cells (APCs) control T-cell responses by multiple mechanisms, including the expression of co-stimulatory molecules and the production of cytokines and other mediators that control T-cell proliferation, survival and differentiation. Here, we demonstrate that soluble factor(s) produced by Toll-like receptor (TLR)-activated APCs suppress activation-induced cell death (AICD). This effect was observed in non-stimulated APCs, but it was significantly increased after lipopolysaccharide (LPS) treatment. Using different KO mice, we found that the LPS-induced protective factor is dependent on TLR4/MyD88. We identified the protective factor as prostaglandin E(2) (PGE(2)) and showed that both APC-derived supernatants and PGE(2) prevented CD95L upregulation in T cells in response to TCR/CD3 stimulation, thereby avoiding both AICD and activated T cell killing of target macrophages. The PGE(2) receptors, EP2 and EP4, appear to be involved since pharmacological stimulation of these receptors mimics the protective effect on T cells and their respective antagonists interfere with the protection induced by either APCs derived or synthetic PGE(2). Finally, the engagement of EP2 and EP4 synergistically activates protein kinase A (PKA) and exchange protein directly activated by cAMP pathways to prevent AICD. Taken together, these results indicate that APCs can regulate T-cell levels of CD95L by releasing PGE(2) in response to LPS through a TLR4/MyD88-dependent pathway, with consequences for both T cell and their own survival.     

Identification of interaction partners of the cytosolic polyproline region of CD95 ligand (CD178)

The CD95/Fas/Apo-1 ligand (CD95L, CD178) induces apoptosis through the death receptor CD95. CD95L was also described as a co-stimulatory receptor for T-cell activation in mice in vivo. The molecular basis for the bidirectional signaling capacity and directed expression of CD95L is unknown. In the present study we identify proteins that precipitate from T-cell lysates with constructs containing fragments of the CD95L cytosolic tail. The determined peptide mass fingerprints correspond to Grb2, actin, beta-tubulin, formin binding protein 17 (FBP17) and PACSIN2. Grb2 had been identified as a putative mediator of T-cell receptor-to-CD95L signaling before. FBP17 and PACSIN2 may be associated with expression and trafficking of CD95L. When overexpressed, CD95L co-precipitates with FBP17 and PACSIN. Protein-protein interactions are mediated via Src homology 3 (SH3) domain binding to the polyproline region of CD95L and can be abolished by mutation or deletion of the respective SH3 domain.     

A novel role for sphingolipid intermediates in activation-induced cell death in T cells

Activation-induced cell death (AICD), a process mediated by CD95 and CD95 ligand (CD95L), plays a critical role in regulating homeostasis of the immune system. Although the role of sphingolipids such as ceramides has been suggested to participate in CD95-mediated apoptosis, the exact role of these molecules in this process remains controversial. We employed myriocin, a specific inhibitor of serine palmitoyl-CoA transferase that mediates the first commitment step in sphingolipid synthesis. We found that myriocin could effectively block AICD in T-cell hybridomas and T-cell blasts. However, fumonisin B1, an inhibitor of the final step of ceramide synthesis, or inhibitors of sphingomyelinases did not prevent AICD. Furthermore, ceramide analogues, such as C2 and C6, could not reverse the inhibitory effect of myriocin. Interestingly, sphinganine, an intermediate of ceramide synthesis, completely reversed the inhibitory effect of myriocin, indicating a critical role of sphinganine. Myriocin did not modulate the expression of CD95 or CD95L, instead, it interfered with the early steps of CD95-mediated caspase activation. Therefore, we have uncovered a novel mechanism by which sphingolipid intermediates regulate CD95-mediated apoptosis.     

Novel role for mitochondria: protein kinase Ctheta-dependent oxidative signaling organelles in activation-induced T-cell death

Reactive oxygen species (ROS) play a key role in regulation of activation-induced T-cell death (AICD) by induction of CD95L expression. However, the molecular source and the signaling steps necessary for ROS production are largely unknown. Here, we show that the proximal T-cell receptor-signaling machinery, including ZAP70 (zeta chain-associated protein kinase 70), LAT (linker of activated T cells), SLP76 (SH2 domain-containing leukocyte protein of 76 kDa), PLCgamma1 (phospholipase Cgamma1), and PKCtheta (protein kinase Ctheta), are crucial for ROS production. PKCtheta is translocated to the mitochondria. By using cells depleted of mitochondrial DNA, we identified the mitochondria as the source of activation-induced ROS. Inhibition of mitochondrial electron transport complex I assembly by small interfering RNA (siRNA)-mediated knockdown of the chaperone NDUFAF1 resulted in a block of ROS production. Complex I-derived ROS are converted into a hydrogen peroxide signal by the mitochondrial superoxide dismutase. This signal is essential for CD95L expression, as inhibition of complex I assembly by NDUFAF1-specific siRNA prevents AICD. Similar results were obtained when metformin, an antidiabetic drug and mild complex I inhibitor, was used. Thus, we demonstrate for the first time that PKCtheta-dependent ROS generation by mitochondrial complex I is essential for AICD.     

CD95 ligand--death factor and costimulatory molecule?

The CD95 ligand is involved as a death factor in the regulation of activation-induced cell death, establishment of immune privilege and tumor cell survival. In addition, CD95L may serve as a costimulatory molecule for T-cell activation. Alterations in expression or shedding of membrane and soluble CD95L are associated with numerous diseases, and underscore the pathophysiological relevance of the CD95/CD95L system. In most cases, the causal link between altered CD95L expression and pathophysiology is unknown. Given the potency of the molecule to regulate death and survival of many different cell types, the control of CD95L production, transport, storage, shedding and inactivation is of tremendous biological and clinical interest. This review summarizes the current knowledge, hypotheses and controversies about CD95L as a multifunctional ligand and receptor. It considers the different roles of membrane and soluble forms of CD95L and the complex networks of intracellular dynamics of protein trafficking, as well as the potential bidirectional signal transduction capacity of CD95L, with a focus on molecular interactions that have been worked out over the past years.     

Differential role of tyrosine phosphorylation in the induction of apoptosis in T cell clones via CD95 or the TCR/CD3-complex

Activated T cells undergo apoptosis when the Fas-antigen (APO-1, CD95) is ligated by Fas Ligand (FasL) or agonistic anti-Fas antibodies. Repeated stimulation of T lymphocytes via the TCR/CD3-complex induces activation-induced cell death (AICD) associated with FasL surface expression. FasL binding to Fas molecules triggers the Fas-dependent death signaling cascade. Since it is still controversial whether Fas-induced cell death is associated with tyrosine kinase activity, we investigated the tyrosine kinase activation requirements in anti-Fas antibody-induced cell death and AICD in human T cell clones. We report that cell death triggered by anti-Fas antibody is not accompanied by an increase in tyrosine phosphorylation and cannot be blocked by inhibitors of protein tyrosine kinases (PTK). Under the same conditions, AICD of T cell clones is clearly associated with tyrosine kinase activation. In fact, semiquantitative RT-PCR analysis of FasL mRNA expression triggered in T cell clones via the TCR/CD3-complex revealed that tyrosine phosphorylation is required for functional FasL mRNA and surface expression.     

Impaired Fas signaling pathway is involved in defective T cell apoptosis in autoimmune murine arthritis

Proteoglycan (PG)-induced arthritis (PGIA) is a novel autoimmune murine model for rheumatoid arthritis induced by immunization with cartilage PG in susceptible BALB/c mice. In this model, hyperproliferation of peripheral CD4(+) T cells has been observed in vitro with Ag stimulation, suggesting the breakdown of peripheral tolerance. Activation-induced cell death (AICD) is a major mechanism for peripheral T cell tolerance. A defect in AICD may result in autoimmunity. We report in this study that although CD4(+) T cells from both BALB/c and B6 mice, identically immunized with human cartilage PG or OVA, express equally high levels of Fas at the cell surface, CD4(+) T cells from human cartilage PG-immunized BALB/c mice, which develop arthritis, fail to undergo AICD. This defect in AICD in PGIA may lead to the accumulation of autoreactive Th1 cells in the periphery. The impaired AICD in PGIA might be ascribed to an aberrant expression of Fas-like IL-1beta-converting enzyme-inhibitory protein, which precludes caspase-8 activation at the death-inducing signaling complex, and subsequently suppresses the caspase cascade initiated by Fas-Fas ligand interaction. Moreover, this aberrant expression of Fas-like IL-1beta-converting enzyme-inhibitory protein may also mediate TCR-induced hyperproliferation of CD4(+) T cells from arthritic BALB/c mice. Our data provide the first insight into the molecular mechanism(s) of defective AICD in autoimmune arthritis.     

Activation of CD4+ T-lymphocytes in asymptomatic HIV infected patients induce the program action of lymphocyte death by apoptosis]

Activation-induced programmed cell death, or apoptosis, is a physiological cell suicide process involved in the negative thymic selection of the T-cell repertoire. We have proposed that the inappropriate re-emergence in the mature CD4+ T-cell population of such a death program could explain both the early dysfunction and the late depletion of CD4+ T-cells from human immunodeficiency virus (HIV)-infected individuals. We present evidence showing that the selective failure of T-cells from 10 HIV-infected asymptomatic individuals (with normal CD4+ T-cell counts) to proliferate in vitro to pokeweed mitogen and to self-major histocompatibility complex class-II T-cell receptor mobilization by superantigens is due to the induction by these stimuli of CD4+ T-cell death. This death process has characteristic features of apoptosis, including DNA fragmentation into multiples of a 200 base pair unit, and the preventive effect of the protein synthesis inhibitor cycloheximide. These findings suggest that in vivo CD4+ T-cell suicide upon activation might account, independently of any HIV-mediated cytopathic effect, for the progressive depletion of CD4+ T-cells that leads to AIDS.     

An IL-2-dependent switch between CD95 signaling pathways sensitizes primary human T cells toward CD95-mediated activation-induced cell death

The CD95 (APO-1/Fas) system plays a critical role in activation-induced cell death (AICD) of T cells. We previously described two distinct CD95 (APO-1/Fas) signaling pathways: 1) type I cells show strong death-inducing signaling complex (DISC) formation and mitochondria-independent apoptosis and 2) DISC formation is reduced in type II cells, leading to mitochondria-dependent apoptosis. To investigate the relevance of these pathways, we set up an in vitro model that mimics the initiation and the down phase of an immune response, respectively. Freshly activated human T cells (initiation) are resistant toward CD95-mediated AICD despite high expression of CD95. We previously reported that these T cells show reduced DISC formation. In this study, we show that freshly activated T cells are CD95-type II cells that show high expression levels of Bcl-x(L) and display a block in the mitochondrial apoptosis pathway. Furthermore, we show that, upon prolonged culture (down phase), human T cells undergo a switch from type II to type I cells that renders T cells sensitive to CD95-mediated AICD. Finally, we demonstrate that this switch is dependent on the presence of IL-2. Our observations reveal for the first time that the existence of coexisting CD95 signaling pathways is of physiological relevance.     

Viral IFN-regulatory factors inhibit activation-induced cell death via two positive regulatory IFN-regulatory factor 1-dependent domains in the CD95 ligand promoter.

The CD95 (also called APO-1/Fas) system plays a major role in the induction of apoptosis in lymphoid and nonlymphoid tissues. The CD95 ligand (CD95L) is induced in response to a variety of signals, including IFN-gamma and TCR/CD3 stimulation. Here we report the identification of two positive regulatory IFN-regulatory factor-dependent domains (PRIDDs) in the CD95L promoter and its 5' untranslated region, respectively. EMSAs demonstrate specific binding of IFN-gamma-induced IFN-regulatory factor 1 (IRF-1) to the PRIDD sequences. Ectopic IRF-1 expression induces CD95L promoter activity. Furthermore, we demonstrate that PRIDDs play an important role in TCR/CD3-mediated CD95L induction. Most interestingly, viral IRFs of human herpes virus 8 (HHV8) totally abolish IRF-1-mediated and strongly reduce TCR/CD3-mediated CD95L induction. We demonstrate here for the first time that viral IRFs inhibit activation-induced cell death. Thus, these results demonstrate an important mechanism of HHV8 to modulate the immune response by down-regulation of CD95L expression. Inhibition of CD95-dependent T cell function might contribute to the immune escape of HHV8.     

Caspase-dependent and -independent T-cell death pathways in pathogenic simian immunodeficiency virus infection: relationship to disease progression

Studies of human immunodeficiency virus (HIV) and nonhuman primate models of pathogenic and nonpathogenic simian immunodeficiency virus (SIV) infections have suggested that enhanced ex vivo CD4 T-cell death is a feature of pathogenic infection in vivo. However, the relative contributions of the extrinsic and intrinsic pathways to programmed T-cell death in SIV infection have not been studied. We report here that the spontaneous death rate of CD4+ T cells from pathogenic SIVmac251-infected rhesus macaques ex vivo is correlated with CD4 T-cell depletion and plasma viral load in vivo. CD4+ T cells from SIVmac251-infected macaques showed upregulation of the death ligand (CD95L) and of the proapoptotic proteins Bim and Bak, but not of Bax. Both CD4+ and CD8+ T cells from SIVmac251-infected macaques underwent caspase-dependent death following CD95 ligation. The spontaneous death of CD4+ and CD8+ T cells was not prevented by a decoy CD95 receptor or by a broad-spectrum caspase inhibitor (zVAD-fmk), suggesting that this form of cell death is independent of CD95/CD95L interaction and caspase activation. IL-2 and IL-15 prevented the spontaneous death of CD4+ and CD8+ T cells, whereas IL-10 prevented only CD8 T-cell death and IL-7 had no effect on T-cell death. Our results indicate that caspase-dependent and caspase-independent pathways are involved in the death of T cells in pathogenic SIVmac251-infected primates.