Inhibition Of Activation-induced Cell Death Research Articles

A Novel Role for IL-27 in Mediating the Survival of Activated Mouse CD4 T Lymphocytes

IL-27, an IL-12 family cytokine, has pleiotropic functions in the differentiation and expansion of CD4(+) T cell subsets. In this study, we discovered a novel function of IL-27. CD4(+)CD45RB(high) T cells from mice deficient for the α-chain of IL-27 receptor failed to induce colitis in Rag(-/-) recipients, because of an inability of activated donor cells to survive. Interestingly, IL-27 was indispensable for the prevention of colitis by regulatory T cells, also because of a defect in long-term cell survival. IL-27 affected the survival of activated T lymphocytes, rather than promoting cell proliferation, by inhibiting Fas-mediated activation-induced T cell death, acting through the STAT3 signaling pathway. The addition of IL-27 during activation resulted in an increased cell number, which was correlated with decreased activation of both caspases 3 and 8. This prosurvival effect was attributed to downregulation of FasL and to the induction of the antiapoptotic protein cFLIP. Although activation induced cell death is an important mechanism for the maintenance of immunological homeostasis, protection of lymphocytes from excessive cell death is essential for effective immunity. Our data indicate that IL-27 has a crucial role in the inhibition of activation-induced cell death, thereby permitting Ag-driven T cell expansion.

Eicosanoids, Prostaglandins, and the Progression of Tuberculosis

Eicosanoids, Prostaglandins, and the Progression of Tuberculosis

Taming regulatory T cells by autologous T cell immunization: A potential new strategy for cancer immune therapy

Regulatory T cells (Tregs) play important roles in the maintenance of immune homeostasis, and is also involved in tumor immune tolerance. Dampening or elimination of Treg functions has been shown to lead to enhanced immune responses against tumors, and thus inhibition of tumor growth. Recently, we have developed a new immunization scheme, referred to as irradiated mitogen- activated autologous T cell vaccination (ATCV), and shown that such immunization could significantly enhance anti-tumor immunity in vivo. Mechanistically, the enhanced anti-tumor response appears to be due to reduced Treg functions and inhibition of activation-induced cell death (AICD) in effector T cells. Thus, ATCV may constitute a novel strategy in cancer immune therapy.

Signaling through ephrin-A ligand leads to activation of Src-family kinases, Akt phosphorylation, and inhibition of antigen receptor-induced apoptosis

Eph receptor tyrosine kinases and ephrins play important roles in diverse biological processes such as migration, adhesion, and angiogenesis. Forward and reverse signaling has been reported in receptor- and ligand-bearing cells. The ligands can be divided into the transmembrane ephrin-B family and the GPI-anchored ephrin-A family. Here, we show expression of ephrin-A ligands on CD4+ T cells cultured in medium with human serum and the T cell line Jurkat TAg and on cells isolated from patients with T cell lymphomas and T cell leukemias. Functional role and identification of proteins involved in ephrin-A signaling were investigated here in the T cell line Jurkat TAg. Signaling through ephrin-A induces phosphorylation of several proteins, including the Src kinases Lck and Fyn. In addition, PI-3K is activated, shown by induced phosphorylation of the Akt kinase. An ephrin-A signaling complex could be isolated, containing several phosphorylated proteins including Lck and Fyn. Interestingly, we show that signaling through ephrin-A in Jurkat TAg cells, initiated by interaction with the EphA2 receptor, leads to inhibition of activation-induced cell death. To conclude, ephrin-A signaling in Jurkat TAg cells leads to induced phosphorylation of several proteins including Lck, Fyn, and Akt. A consequence of ephrin-A signaling is inhibition of antigen receptor-induced apoptosis.

4-1BB Costimulation of Effector T Cells for Adoptive Immunotherapy of Cancer: Involvement of Bcl Gene Family Members

We previously reported that in vitro costimulation of murine MCA 205 tumor-draining lymph node (TDLN) cells through a third signal, 4-1BB (CD137), in addition to CD3 and CD28 engagement significantly increases T-cell yield and amplifies antitumor responses in adoptive therapy. The increased T-cell yield seemed to be related to inhibition of activation-induced cell death. In this study, using real time-polymerase chain reaction and intracellular staining, we tested our hypothesis that antiapoptotic Bcl gene members are modulated in 4-1BB ligated TDLN cells. TDLN cells activated through 4-1BB in conjunction with CD3/CD28 demonstrated elevated Bcl-2 and Bcl-xL gene and protein expression compared with CD3/CD28 activation. Furthermore, Bcl-2 and/or Bcl-xL inhibition abrogated 4-1BB-conferred rescue of activation-induced cell death in TDLN cells, and as a result, 4-1BB-enhanced TDLN cell yield was abolished. Congenic mice were used as donors for TDLN cells labeled with CFSE to evaluate proliferation and persistence of activated cells after intravenous adoptive transfer. The effector function of transferred cells was assessed by determining the incidence of interferon-gamma-producing cells in response to tumor stimulation in serial blood samples drawn from treated mice using intracellular cytokine staining. CD28 and CD28/4-1BB costimulation significantly enhanced in vivo proliferation and survival of the infused cells compared with CD3 activation. 4-1BB coligation augmented the proliferation and effector function of the infused cells compared with both CD3 and CD3/CD28-activated cells. Characterizing the function of signaling molecules involved in T-cell activation pathways may allow optimization of conditions in the generation of effector T cells for cancer immunotherapy.

Group I mGlu receptor stimulation inhibits activation‐induced cell death of human T lymphocytes

1. The effects of L-glutamate on activation-induced cell death (AICD) of human activated (1 microg ml(-1) phytohemagglutinin plus 2 U ml(-1) interleukin-2; 8 days) T lymphocytes were studied by measuring anti-CD3 monoclonal antibody (10 microg ml(-1); 18 h)-induced cell apoptosis (Annexin V and propidium iodide staining). 2. L-Glutamate (1 x 10(-8)-1 x 10(-4) M) significantly (P < or = 0.01) inhibited AICD in a concentration-dependent manner (EC50=6.3 x 10(-8) M; maximum inhibition 54.8+/-6.3% at 1 x 10(-6) M). 3. The L-glutamate inhibitory effect was pharmacologically characterized as mediated by group I mGlu receptors, since mGlu receptor agonists reproduced this effect. The EC50 values were: 3.2 x 10(-7) M for (1S,3R)-ACPD; 4.5 x 10(-8) M for quisqualate; 1.0 x 10(-6) M for (S)-3,5-DHPG; 2.0 x 10(-5) M for CHPG. 4. Group I mGlu receptor antagonists inhibited the effects of quisqualate 1.0 x 10(-6) M. The IC50 values calculated were: 8.7 x 10(-5), 4.3 x 10(-6) and 6.3 x 10(-7) M for AIDA, LY 367385 and MPEP, respectively. 5. L-Glutamate (1 x 10(-6) M; 18 h) significantly (P < or = 0.05) inhibited FasL expression (40.8+/-11.3%) (cytofluorimetric analysis), whereas it did not affect Fas signalling. 6. Expression of both mGlu1 and mGlu5 receptor mRNA by T lymphocytes and T-cell lines, as demonstrated by reverse transcriptase-PCR analysis, suggests that L-glutamate-mediated inhibition of AICD was exerted on T cells. 7. These data depict a novel role for L-glutamate in the regulation of the immune response through group I mGlu receptor-mediated mechanisms.

P38 MAPK Mediates Inhibition of Activation Induced Cell Death in T Lymphocytes Via Upregulation of Bcl-xL and FLIPshort upon Ligation of CD137/4-1BB or CD28.

Costimulation is essential for the induction of proliferation in naive T cells and for the inhibition of activation induced cell death (AICD) in activated T lymphocytes. While costimulatory signals mediated through the immunglobulin family member CD28 play a prominent role during primary T cell activation, ligation of the tumor necrosis factor receptor family member CD137/4-1BB is more important during late primary and secondary T cell activation. Signals mediated through either costimulatory protein block AICD. Inhibition of AICD through ligation of CD137/4-1BB or CD28 involves upregulation of Bcl-xL and FLIPshort (Eur J Immunol 2005, 35: 1257–66). We further demonstrated that costimulatory signals mediated through CD137/4-1BB or CD28 depend on the activity of phosphatidylinositol 3-kinase and AKT/protein kinase B, two kinases that had formerly been implied in CD28-induced signaling, indicating that CD28- and CD137/4-1BB-mediated signals share downstream signaling pathways. Here, we demonstrate that p38 mitogen-activated protein kinase (MAPK) mediates CD137/4-1BB-induced as well as CD28-mediated costimulation of cell proliferation and inhibition of AICD. This coincides with upregulation of Bcl-xL and FLIPshort. Inhibition of p38 MAPK abrogates T cell receptor induced proliferation and antagonizes costimulation mediated survival. Thus, p38 MAPK, which was previously thought to be primarily involved in CD137/4-1BB-mediated signaling, is similarly important for CD28-induced costimulation and survival. This indicates that, while involving different protein families, signal transduction by CD28 and CD137/4-1BB depends on a common upstream and downstream network of survival kinases.

CD4-Dependent Signaling Is Required for a Late Checkpoint during Th2 Development Associated with Resistance to Activation-Induced Cell Death

Previous studies have found that class II-restricted T cells from CD4-deficient mice reconstituted with a tail-less CD4 transgene have a specific defect in the development of Th2 effector cells; however, the reason for this defect was not clear. Following stimulation with a high potency peptide and exogenous IL-4, CD4-dependent signaling is required for optimal generation of a Th2 effector population. However, initial IL-4 and GATA-3 transcription is appropriately induced, suggesting that the initial stages of Th2 development are intact and independent of CD4 after priming with a strong agonist peptide. In addition to the defect in Th2 development, CD4 mutant T cells are also relatively resistant to activation-induced cell death (AICD). Furthermore, inhibition of AICD in wild-type T cells causes a defect in Th2 development similar to that seen in the CD4 mutant T cells. These data support the hypothesis that CD4-dependent signaling pathways regulate a distinct checkpoint in the expansion and commitment phase of Th2 development, which is related to dysregulation of AICD.

Glucocorticoids inhibit activation-induced cell death (AICD) via direct DNA-dependent repression of the CD95 ligand gene by a glucocorticoid receptor dimer

AbstractGlucocorticoids (GCs) play an important role in the regulation of peripheral T-cell survival. Their molecular mechanism of action and the question of whether they have the ability to inhibit apoptosis in vivo, however, are not fully elucidated. Signal transduction through the glucocorticoid receptor (GR) is complex and involves different pathways. Therefore, we used mice with T-cell-specific inactivation of the GR as well as mice with a function-selective mutation in the GR to determine the signaling mechanism. Evidence is presented for a functional role of direct binding of the GR to 2 negative glucocorticoid regulatory elements (nGREs) in the CD95 (APO-1/Fas) ligand (L) promoter. Binding of GRs to these nGREs reduces activation-induced CD95L expression in T cells. These in vitro results are fully supported by data obtained in vivo. Administration of GCs to mice leads to inhibition of activation-induced cell death (AICD). Thus, GC-mediated inhibition of CD95L expression of activated T cells might contribute to the anti-inflammatory function of steroid drugs. (Blood. 2005;106:617-625)

Costimulation by CD137/4–1BB inhibits T cell apoptosis and induces Bcl‐xL and c‐FLIPshort via phosphatidylinositol 3‐kinase and AKT/protein kinase B

Costimulation is essential for induction of T lymphocyte proliferation and inhibition of activation-induced cell death. While signaling pathways activated following the ligation of the costimulatory molecule CD28 are well defined, less is known about the molecular events induced by alternative costimulators. CD137/4-1BB, a costimulatory member of the tumor necrosis factor receptor family, plays an important role during late primary T cell stimulation. Here, we demonstrate for the first time that inhibition of activation-induced cell death by exposure to the CD137/4-1BB ligand involves up-regulation of the anti-apoptotic protein c-FLIP(short). Inhibition of T cell death by 4-1BB ligation and up-regulation of c-FLIP(short) and Bcl-x(L) were abolished by blocking the phosphatidylinositol 3-kinase or the AKT/protein kinase B, which also mediate CD28-induced inhibition of activation-induced cell death. Our findings, therefore, demonstrate that costimulatory molecules, although belonging to different protein families and participating in distinct upstream signaling pathways, employ common downstream signaling pathways.

VIP and PACAP inhibit activation induced apoptosis in T lymphocytes.

Apoptosis in T and B lymphocytes is a major element controlling the immune response. Activation induced cell death (AICD) in T cells is a main mechanism for maintaining peripheral tolerance and for limiting an ongoing immune response. AICD is initiated by antigen reengagement of the T cell receptor (TCR), and mediated through Fas/Fas ligand (FasL) interactions. VIP and PACAP are two multifunctional neuropeptides present in the lymphoid microenvironment that act primarily as anti-inflammatory agents. In this study we report on the role of VIP and PACAP on T cell AICD, and on the mechanisms involved. VIP and PACAP inhibit AICD in vivo and in vitro, in peripheral T cells and T cell hybridomas. The effect is dose dependent and is mediated through the specific receptors VPAC1 and VPAC2. The inhibition of AICD is achieved through reduction in FasL expression at protein and mRNA level. By affecting FasL expression, VIP and PACAP may play a physiological role in both the generation of memory T cells and the inhibition of FasL-mediated T cell cytotoxicity.

Activation-induced T cell death occurs at G1A phase of the cell cycle

Peripheral negative selection of cycling T cells after TCR engagement and deletion of activated T cells after an immune response occur by an apoptotic process termed activation-induced cell death (AICD). The cross-linking of TCR-CD3 complex with anti-CD3 monoclonal antibody led to significant apoptotic cell death in peripheral blood T cells. To further define cell cycle restriction points for triggering AICD in T cells, we evaluated the association between cell cycle progression and death signal transduction. Simultaneous DNA / RNA quantification analysis revealed that T cells entering G1A phase of the cell cycle may acquire sensitivity to AICD. The activation of caspase-3 was induced when T cells entered G1A phase. Up-regulation of cyclin-dependent kinases (Cdk4 and Cdk6) and cyclin D3 was initiated in TCR-stimulated T cells entering G1A phase and expression of these markers steadily increased as T cells progressed from G1A into G1B phase. Interestingly, caspase-3 inhibitors could inhibit the up-regulation of these G1 cell cycle regulators and induce G0 / G1A arrest as well as the inhibition of AICD. On the basis of these results, AICD signals are most likely transduced into TCR-stimulated T cells entering G1A phase. T cells that fail to progress from G1A into G1B phase undergo AICD.

Activation-induced T cell death occurs at G1A phase of the cell cycle.

Peripheral negative selection of cycling T cells after TCR engagement and deletion of activated T cells after an immune response occur by an apoptotic process termed activation-induced cell death (AICD). The cross-linking of TCR-CD3 complex with anti-CD3 monoclonal antibody led to significant apoptotic cell death in peripheral blood T cells. To further define cell cycle restriction points for triggering AICD in T cells, we evaluated the association between cell cycle progression and death signal transduction. Simultaneous DNA / RNA quantification analysis revealed that T cells entering G1A phase of the cell cycle may acquire sensitivity to AICD. The activation of caspase-3 was induced when T cells entered G1A phase. Up-regulation of cyclin-dependent kinases (Cdk4 and Cdk6) and cyclin D3 was initiated in TCR-stimulated T cells entering G1A phase and expression of these markers steadily increased as T cells progressed from G1A into G1B phase. Interestingly, caspase-3 inhibitors could inhibit the up-regulation of these G1 cell cycle regulators and induce G0 / G1A arrest as well as the inhibition of AICD. On the basis of these results, AICD signals are most likely transduced into TCR-stimulated T cells entering G1A phase. T cells that fail to progress from G1A into G1B phase undergo AICD.

Requirement for Shc in TCR-Mediated Activation of a T Cell Hybridoma

Engagement of the TCR determines the fate of T cells to activate their functional programs, proliferate, or undergo apoptosis. The intracellular signal transduction pathways that dictate the specific outcome of receptor engagement have only been partially elucidated. The adapter protein, Shc, is involved in cytokine production, mitogenesis, transformation, and apoptosis in different cell systems. We found that Shc becomes phosphorylated on tyrosine residues upon stimulation of the TCR in DO11.10 hybridoma T cells; therefore, we investigated the role of Shc in activation-induced cell death in these cells by creating a series of stably transfected cell lines. Expression of Shc-SH2 (the SH2 domain of Shc) or Shc-Y239/240F (full-length Shc in which tyrosines 239 and 240 have been mutated to phenylalanine) resulted in the inhibition of activation-induced cell death and Fas ligand up-regulation after TCR cross-linking. Expression of wild-type Shc or Shc-Y317F had no significant effect. In addition, we found that Shc-SH2 and Shc-Y239/240F, but not Shc-Y317F, inhibited phosphorylation of extracellular signal-regulated protein kinase and production of IL-2 after TCR cross-linking. These results indicate an important role for Shc in the early signaling events that lead to activation-induced cell death and IL-2 production after TCR activation.

NOD Mice are Resistant to Depletion of Thymic Cells Caused by Acute Stress or Infection

NOD mice spontaneously develop autoimmune diabetes; disease onset in females of our colony of NOD mice usually takes place around the 4th month of age. Diabetes of NOD mice can be modulated by different stress protocols, even though these animals were shown to be resistant to the effects of glucocorticoids on their lymphocytes. We have recently found that the early host inflammatory response to mycobacteria can be strongly modified by stress, the autoimmunity-prone NOD and NZB/W mice being particularly affected. These mice show reduced numbers of granulocytes in the inflammatory cavity after exposure to stress. Mycobacterium avium is an opportunistic agent that is responsible for disseminated infections seen in AIDS patients. Here, we investigated whether the early immune response to M. avium was altered by stress in NOD mice and we compared the stress response of these mice with a non-autoimmune strain, BALB/c mice. The effects of stress on infected BALB/c mice, which like AIDS patients are susceptible to M. avium infection, offers experimental evidence that M. avium infection, if coupled with stress of the host, may accelerate loss of T helper cells. In contrast, in NOD mice, stress or infection significantly increased the number of cells of the thymuses of the animals. Data obtained with NOD mice support the previously reported resistance of NOD mice lymphocytes to glucocorticoids and suggest that there are two distinct signalling pathways involved in the response of NOD lymphocytes to these stress hormones: one leading to apoptosis and the other mediating glucocorticoid inhibition of activation-induced cell death.