CD95L-mediated Apoptosis Research Articles

Targeting the epitope spreader Pep19 by naïve human CD45RA+ regulatory T cells dictates a distinct suppressive T cell fate in a novel form of immunotherapy.

PurposeBeyond the limited scope of non-specific polyclonal regulatory T cell (Treg)-based immunotherapy, which depends largely on serendipity, the present study explored a target Treg subset appropriate for the delivery of a novel epitope spreader Pep19 antigen as part of a sophisticated form of immunotherapy with defined antigen specificity that induces immune tolerance.MethodsHuman polyclonal CD4+CD25+CD127lo− Tregs (127-Tregs) and naïve CD4+CD25+CD45RA+ Tregs (45RA-Tregs) were isolated and were stimulated with target peptide 19 (Pep19)-pulsed dendritic cells in a tolerogenic milieu followed by ex vivo expansion. Low-dose interleukin-2 (IL-2) and rapamycin were added to selectively exclude the outgrowth of contaminating effector T cells (Teffs). The following parameters were investigated in the expanded antigen-specific Tregs: the distinct expression of the immunosuppressive Treg marker Foxp3, epigenetic stability (demethylation in the Treg-specific demethylated region), the suppression of Teffs, expression of the homing receptors CD62L/CCR7, and CD95L-mediated apoptosis. The expanded Tregs were adoptively transferred into an NOD/scid/IL-2Rγ−/− mouse model of collagen-induced arthritis.ResultsEpitope-spreader Pep19 targeting by 45RA-Tregs led to an outstanding in vitro suppressive T cell fate characterized by robust ex vivo expansion, the salient expression of Foxp3, high epigenetic stability, enhanced T cell suppression, modest expression of CD62L/CCR7, and higher resistance to CD95L-mediated apoptosis. After adoptive transfer, the distinct fate of these T cells demonstrated a potent in vivo immunotherapeutic capability, as indicated by the complete elimination of footpad swelling, prolonged survival, minimal histopathological changes, and preferential localization of CD4+CD25+ Tregs at the articular joints in a mechanistic and orchestrated way.ConclusionsWe propose human naïve CD4+CD25+CD45RA+ Tregs and the epitope spreader Pep19 as cellular and molecular targets for a novel antigen-specific Treg-based vaccination against collagen-induced arthritis.

Impaired survival of regulatory T cells in pulmonary sarcoidosis.

BackgroundImpaired regulatory T cell (Treg) function is thought to contribute to ongoing inflammatory responses in sarcoidosis, but underlying mechanisms remain unclear. Moreover, it is not known if increased apoptotic susceptibility of Tregs may contribute to an impaired immunosuppressive function in sarcoidosis. Therefore, the aim of this study is to analyze proportions, phenotype, survival, and apoptotic susceptibility of Tregs in sarcoidosis.MethodsPatients with pulmonary sarcoidosis (n = 58) were included at time of diagnosis. Tregs were analyzed in broncho-alveolar lavage fluid and peripheral blood of patients and healthy controls (HC).ResultsIn sarcoidosis patients no evidence was found for a relative deficit of Tregs, neither locally nor systemically. Rather, increased proportions of circulating Tregs were observed, most prominently in patients developing chronic disease. Sarcoidosis circulating Tregs displayed adequate expression of FoxP3, CD25 and CTLA4. Remarkably, in sarcoidosis enhanced CD95 expression on circulating activated CD45RO+ Tregs was observed compared with HC, and proportions of these cells were significantly increased. Specifically sarcoidosis Tregs - but not Th cells - showed impaired survival compared with HC. Finally, CD95L-mediated apoptosis was enhanced in sarcoidosis Tregs.ConclusionIn untreated patients with active pulmonary sarcoidosis, Tregs show impaired survival and enhanced apoptotic susceptibility towards CD95L. Increased apoptosis likely contributes to the insufficient immunosuppressive function of sarcoidosis Tregs. Further research into this field will help determine whether improvement of Treg survival holds a promising new therapeutic approach for chronic sarcoidosis patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-015-0265-8) contains supplementary material, which is available to authorized users.

Proapoptotic CD95L levels in normal human serum and sera of breast cancer patients.

The CD95 pathway is a critical apoptotic pathway used by immune cells to avoid cancer development. CD95 ligand (CD95L) is found in several forms, as a cell membrane-associated form, a soluble metalloprotease-cleaved form, and a soluble but membrane-bound CD95L released on cell-derived exosomes. In this study, we used a cell-based assay to evaluate the activity of proapoptotic CD95L in sera from healthy individuals and breast cancer patients. We confirmed that our cell-based assay using Jurkat cells was sensitive to the presence of proapoptotic CD95L in serum, and apoptosis induction by mechanisms other than CD95 was discriminated using apoptosis-resistant Jurkat subclones. Our results indicated a proapoptotic potential of normal serum that involved CD95L. Sera from breast cancer patients exhibited significantly decreased apoptosis induction, due to increased CD95 receptor levels compared with healthy women. Apoptotic potential tended to decrease as the Breast Imaging Reporting and Data System grade increased, and we observed restoration of proapoptotic potential after tumor removal. The CD95L in serum responsible for apoptotic induction was associated with high-molecular-weight particles, perhaps with exosomes. The sera of healthy individuals generally contain a proapoptotic environment, and this property is mainly maintained by the presence of CD95L. Furthermore, measurement of CD95L-mediated apoptosis induction by sera could be a useful parameter to be evaluated during cancer development and therapeutic response.

APG101 - a Novel Inhibitor of CD95/CD95L Signaling Prevents Graft-Versus-Host Disease without Disabling T Cell Effector Function and Anti-Tumor Activity,

Abstract 4019Allogeneic bone marrow transplantation (BMT) is a curative treatment modality for haematological malignancies since mature allogeneic T cells in the transplant efficiently eliminate residual tumor cells (graft-versus-tumor (GVT) effect). However, these donor T cells are also responsible for the induction of graft-versus-host disease (GVHD) by attacking recipient tissue leading to significant morbidity and mortality. Interfering with T cell effector mechanisms responsible for GVHD-induction without preventing T cell functions towards third-party or opportunistic pathogens and while maintaining anti-tumor cytotoxicity might lead to new treatment strategies after allogeneic BMT. The CD95/CD95L system significantly contributes to GVHD-associated tissue damage and intervention into this signaling pathway might therefore inhibit GVHD-development without disturbing the GVT-effect. APG101 is a human soluble fusion protein consisting of the extracellular domain of CD95 and the Fc portion of IgG blocking the interaction of CD95L with its cognate receptor. We tested the effect of APG101 on T cell function, GVHD development and anti-tumor cytotoxicity in an allogeneic parent into F1 (C57BL/6 (B6) (H-2b) into B6D2F1 (H-2bxd)) BMT model with a mismatch in HLA-Class I and II molecules. In vitro, APG101 efficiently inhibited CD95L-mediated apoptosis of primary naïve mouse CD4+ and CD8+ T cells but did not interfere with their proliferative or cytotoxic capacity. To test APG101 as a therapeutic agent in BMT we induced acute GVHD in lethally irradiated B6D2F1 mice by transplantation of B6-derived BM together with allogeneic spleen cells. APG101 treatment was conducted intraperitoneally twice a week until the end of the experiment, starting either one day before BMT (= prophylactic treatment) or starting six or thirteen days after BMT (= therapeutic treatment). Prophylactic and early (day 6), but not late therapeutic treatment (day 13), effectively protected recipients from clinical GVHD and significantly improved survival. Although APG101 efficiently prevented GVHD-induction, homing and proliferation of allogeneic T cells into lymphoid organs and GVHD-target organs was unaltered compared to non-treated animals. Since CD95L is also known as a reverse signaling co-stimulator in T cell activation, APG101 might interfere with T cell function in vivo. However, no differences in phenotype, proliferation or cytokine expression of transplanted allogeneic T cells was observed in APG101-treated mice compared to non-treated mice. Most importantly, the GVT effect was preserved in APG101-treated mice. Anti-tumor cytotoxicity of transplanted allogeneic T cells was not abrogated by APG101 injection independent whether the murine mastocytoma cell line P815 or Bcr-Abl transduced primary B-ALL cells were injected. In summary, our findings convincingly show that early APG101 treatment is a promising approach to prevent GVHD without abrogating T cell effector functions and while preserving the GvT effect after allogeneic BMT. Disclosures:Strauss:Apogenix: Research Funding. Hartmann:Apogenix: Research Funding. Kluge:Apogenix: Employment. Fricke:Apogenix GmbH: Employment, Equity Ownership. Debatin:Apogenix GmbH: Patents & Royalties, Research Funding.

Intracerebral human regulatory T cells: analysis of CD4+ CD25+ FOXP3+ T cells in brain lesions and cerebrospinal fluid of multiple sclerosis patients.

Impaired suppressive capacity of CD4+CD25+FOXP3+ regulatory T cells (Treg) from peripheral blood of patients with multiple sclerosis (MS) has been reported by multiple laboratories. It is, however, currently unresolved whether Treg dysfunction in MS patients is limited to reduced control of peripheral T cell activation since most studies analyzed peripheral blood samples only. Here, we assessed early active MS lesions in brain biopsies obtained from 16 patients with MS by FOXP3 immunohistochemistry. In addition, we used six-color flow cytometry to determine numbers of Treg by analysis of FOXP3/CD127 expression in peripheral blood and cerebrospinal fluid (CSF) of 17 treatment-naïve MS patients as well as quantities of apoptosis sensitive CD45ROhiCD95hi cells in circulating and CSF Treg subsets. Absolute numbers of FOXP3+ and CD4+ cells were rather low in MS brain lesions and Treg were not detectable in 30% of MS biopsies despite the presence of CD4+ cell infiltrates. In contrast, Treg were detectable in all CSF samples and Treg with a CD45ROhiCD95hi phenotype previously shown to be highly apoptosis sensitive were found to be enriched in the CSF compared to peripheral blood of MS patients. We suggest a hypothetical model of intracerebral elimination of Treg by CD95L-mediated apoptosis within the MS lesion.

Efficient and selective tumor cell lysis and induction of apoptosis in melanoma cells by a conditional replication‐competent CD95L adenovirus

The high mortality of melanoma demands the development of new strategies, and gene therapy may be considered provided improvements in efficacy and selectivity. Overexpression of the death ligand CD95L/FasL has been shown in previous studies as highly effective for apoptosis induction in melanoma cells. For efficient and selective targeting of melanoma, a conditional replication-competent adenoviral vector was constructed (Ad5-FFE-02), which drives CD95L expression by a tetracycline-inducible promoter. For restricting its replication to melanoma cells, the adenoviral E1A gene is controlled by a tyrosinase-derived promoter. Furthermore, adenoviral E1B was deleted and a mutated E1A was used to preferentially support replication in tumor cells. Proving its high selectivity and efficiency, strong expression of E1A and doxycycline-dependent induction of CD95L were characteristic for tyrosinase-positive melanoma cells after Ad5-FFE-02 transduction, whereas absent in non-melanoma cell lines. Importantly, Ad5-FFE-02-mediated cell lysis was restricted to melanoma cells, and induction of apoptosis was found only in tyrosinase and CD95 expressing cells. Finally, the combination of adenoviral replication and CD95L-mediated apoptosis resulted in an enhanced repression of melanoma cell growth. This new adenoviral vector may provide a basis for an efficient targeting of melanoma.

T1777 Activation of PPAR β/δ Reduces Copper-Dependant Oxidative Stress and Fasl (Cd95l) Mediated Apoptosis in HEPG2 Cells: Possible Implications in Clinical Practice

T1777 Activation of PPAR β/δ Reduces Copper-Dependant Oxidative Stress and Fasl (Cd95l) Mediated Apoptosis in HEPG2 Cells: Possible Implications in Clinical Practice

Suppression of cFLIP is sufficient to sensitize human melanoma cells to TRAIL- and CD95L-mediated apoptosis

Death ligands such as tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and certain forms of CD95L are attractive therapeutic options for metastatic melanoma. Since knowledge about the regulation of death receptor sensitivity in melanoma is sparse, we have analysed these signaling pathways in detail. The loss of CD95 or TRAIL-R1, but not of TRAIL-R2, surface expression correlated with apoptosis sensitivity in a panel of melanoma cell lines. In contrast, the expression of proteins of the apical apoptosis signaling cascade (FADD, initiator caspases-8 and cFLIP) did not predict apoptosis sensitivity. Since both TRAIL-R1 and -R2 transmit apoptotic signals, we asked whether cFLIP, highly expressed in several of the cell lines tested, is sufficient to maintain resistance to TRAIL-R2-mediated apoptosis. Downregulation of cFLIP in TRAIL-R2-positive, TRAIL-resistant IGR cells dramatically increased TRAIL sensitivity. Conversely ectopic expression of cFLIP in TRAIL-sensitive, TRAIL-R2-expressing RPM-EP melanoma cells inhibited TRAIL- and CD95L-mediated cell death. Thus, modulation of cFLIP is sufficient to sensitize TRAIL-R2-expressing cells for TRAIL. Taken together, albeit expressing all proteins necessary for death receptor-mediated apoptosis, TRAIL-R1 negative melanoma cells cannot undergo TRAIL- or CD95L-induced apoptosis due to expression of cFLIP. Hence, cFLIP represents an attractive therapeutic target for melanoma treatment, especially in combination with TRAIL receptor agonists.

Naive regulatory T cells: a novel subpopulation defined by resistance toward CD95L-mediated cell death

AbstractMost CD4+CD25hiFOXP3+ regulatory T cells (Tregs) from adult peripheral blood express high levels of CD45RO and CD95 and are prone to CD95L-mediated apoptosis in contrast to conventional T cells (Tconvs). However, a Treg subpopulation remained consistently apoptosis resistant. Gene microarray and 6-color flow cytometry analysis including FOXP3 revealed an increase in naive T-cell markers on the CD95L-resistant Tregs compared with most Tregs. In contrast to Tregs found in adult humans, most CD4+CD25+FOXP3+ T cells found in cord blood are naive and exhibit low CD95 expression. Furthermore, most of these newborn Tregs are not sensitive toward CD95L similar to naive Tregs from adult individuals. After short stimulation with anti-CD3/CD28 monoclonal antibodies (mAbs), cord blood Tregs strongly up-regulated CD95 and were sensitized toward CD95L. This functional change was paralleled by a rapid up-regulation of memory T-cell markers on cord blood Tregs that are frequently found on adult memory Tregs. In summary, we show a clear functional difference between naive and memory Tregs that could result in different survival rates of those 2 cell populations in vivo. This new observation could be crucial for the planning of therapeutic application of Tregs.

Similar sensitivity of regulatory T cells towards CD95L-mediated apoptosis in patients with multiple sclerosis and healthy individuals

Impaired suppressive function of CD4 +CD25 high regulatory T cells (T reg) has been reported as a novel pathogenetic mechanism in Multiple sclerosis (MS). We addressed if high apoptosis sensitivity of MS-T reg could explain this functional T reg defect. T reg from treatment-naïve MS patients showed high sensitivity towards CD95Ligand-mediated apoptosis and exhibited enhanced cell death to IL-2 and TCR-signal deprivation. Since susceptibility of T reg to cell death was similar in MS patients and healthy controls, this cannot explain the inhibitory dysfunction of T reg associated with MS. Furthermore, as cell death is not enhanced, therapeutic expansion of MS-T reg in vitro should be a reasonable and novel therapeutic option.

The role of receptor internalization in CD95 signaling

Activation of the cell surface CD95 receptor triggers a cascade of signaling events, including assembly of the death-inducing signaling complex (DISC), that culminate in cellular apoptosis. In this study, we demonstrate a general requirement of receptor internalization for CD95 ligand-mediated DISC amplification, caspase activation and apoptosis in type I cells. Recruitment of DISC components to the activated receptor predominantly occurs after the receptor has moved into an endosomal compartment and blockade of CD95 internalization impairs DISC formation and apoptosis. In contrast, CD95 ligand stimulation of cells unable to internalize CD95 results in activation of proliferative Erk and NF-kappaB signaling pathways. Hence, the subcellular localization and internalization pathways of CD95 play important roles in controlling activation of distinct signaling cascades to determine divergent cellular fates.

Effective Depletion of Alloreactive T Cells by CD95l-Transduced Cells with Preservation of FoxP3 Regulatory T-Cells.

Adoptive transfer of donor T-cells after stem cell transplantation across HLA barriers could bridge the period of prolonged immunodeficiency until new thymic derived T-cells develop. In such an approach, however, donor T-cells reactive to alloantigens of the recipient induce GvHD, necessitating their selective elimination, inactivation or modulation. Depletion of alloreactive cells has been attempted by monoclonal antibodies, but prevention of GvHD was insufficient in clinical application so far. We developed a system allowing efficient selective depletion of alloreactive cells by CD95L-mediated apoptosis, which in addition has the advantage of preserving FoxP3 regulatory T-cells. An apoptosis-resistant B-cell line (C1RA1) was transduced with lentiviral CD95L (C1RA1-CD95L). CD95L was stably expressed by 65–80% of the cells and was fully functional to induce apoptosis in Jurkat cells. MNC of donor A were stimulated with APC of donor B, and, after 5 days of culture, viable responder cells were enriched and cultured with C1RA1-CD95L for another 3 days. T cells obtained by this culture system had lost proliferative activity towards cells of donor B, while proliferation against 3rd-party cells was fully preserved. Likewise, cytotoxic activity against donor B was markedly reduced. Since CD4+CD25+ T-cells were still detectable at levels of 7 to 10% of MNC at the end of the culture despite effective depletion of alloactivated cells, we analyzed for the presence of FoxP3+ regulatory T-cells (Treg). While gene expression of FoxP3 was almost undetectable in MNC before culture, it was present after activation of MNC and was strongly expressed after induction of apoptosis with C1RA1-CD95L at the end of our culture system. Suppression of proliferation of about 50% by these cells was noted in a suppressor assay using fresh MNC from donor A stimulated with cells of donor B. When suppression was analyzed in a stimulation system with 3rd-party stimulatory cells, reduction of proliferation was less prominent, indicating the possibility that the suppressive activity of Treg preserved in our culture system could be alloantigen-specific. In summary, we have developed a culture system, allowing alloantigen-specific depletion of T cells while preserving FoxP3+ regulatory T-cells. This sequential system of alloantigen-specific stimulation followed by CD95L-mediated apoptosis of activated cells by an unrelated, modified cell line offers the advantage of broad applicability in clinical transplantation settings.

Impaired activation of caspases and prevention of mitochondrial dysfunction in the metastatic colon carcinoma CC531s-m2 cell line

In a previous paper we described the properties of a rapidly metastasizing cell line CC531s-m2 derived from the poorly metastasizing CC531s cell. The m2-cell line was relatively resistant to killing by NK cells. Both CD95L and TRAIL mediated apoptosis was decreased in the m2-cell line. Now, by flow cytometrical analysis of intra- and extra-cellular expressed receptors, we show that the localization of the receptors for CD95L and TRAIL was not altered in the CC531s-m2 cells as compared to the parental cell line. Subsequently caspase-activation and mitochondrial function were studied by enzymatic cleavage of fluorescent caspase-substrates and retention of the mitochondrial dye rhodamine-123, respectively. The activation of caspases as well as the loss of the mitochondrial membrane potential (MMP) was less in the CC531s-m2 cell line upon CD95L- and TRAIL-signalling. Furthermore, the sensitivity of the CC531-m2 towards cisplatin-induced apoptosis was strongly decreased. This was consistent with less mitochondrial damage, delayed caspase cleavage and decreased caspase activity. Altogether, we conclude that an Natural Killer-cell insensitive cell is less sensitive to CD95L- and TRAIL-induced apoptosis as well as anti-cancer drug induced apoptosis by prevention of mitochondrial damage and activation of caspases.

NF-kappaB-independent actions of sulfasalazine dissociate the CD95L- and Apo2L/TRAIL-dependent death signaling pathways in human malignant glioma cells.

Death receptor-mediated apoptosis of human malignant glioma cells triggered by CD95 ligand (CD95L) or Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) share several features, including processing of multiple caspases and mitochondrial cytochrome c release. We here report that CD95L-induced cell death is inhibited by sulfasalazine (SS) in all of four human glioma cell lines, both in the absence and presence of cycloheximide (CHX). Coexposure to CD95L and SS prevents the CD95L-evoked processing of caspases 2, 3, 8 and 9, the release of cytochrome c from mitochondria, and the loss of BCL-x(L) protein. This places the protective effect of SS proximal to most known events triggered by the CD95-dependent signaling pathway in glioma cells. CD95L promotes the accumulation of nuclear factor kappa B (NF-kappaB) in the nucleus and induces the DNA-binding activity of NF-kappaB assessed by electrophoretic mobility shift assay. The total levels of p50, p65 and IkappaBalpha remain unchanged, but the levels of phosphorylated IkappaBalpha and of nuclear p65 increase, in response to CD95L. IkappaBalpha phosphorylation as well as nuclear NF-kappaB translocation and DNA binding are blocked by SS. However, unlike SS, dominant-negative IkappaBalpha (IkappaBdn) does not block apoptosis, suggesting that SS inhibits CD95L-mediated apoptosis in an NF-kappaB-independent manner. In contrast to CD95L, the cytotoxic effects of Apo2L/TRAIL are enhanced by SS, and SS facilitates Apo2L/TRAIL-evoked caspase processing, cytochrome c release, and nuclear translocation of p65. These effects of SS are nullified in the presence of CHX, suggesting that the effects of SS and CHX are redundant or that enhanced apoptosis mediated by SS requires protein synthesis. IkappaBdn fails to modulate Apo2L/TRAIL-induced apoptosis. Similar effects of SS on CD95L- and Apo2L/TRAIL-induced apoptosis are observed in MCF-7 breast and HCT116 colon carcinoma cells. Interestingly, HCT cells lacking p21 (80S14(p21-/-)) are only slightly protected by SS from CD95L-induced apoptosis, but sensitized to Apo2L/TRAIL-induced apoptosis, indicating a link between the actions of SS and p21. Thus, SS modulates the death cascades triggered by CD95L and Apo2L/TRAIL in opposite directions in an NF-kappaB-independent manner, and SS may be a promising agent for the augmentation of Apo2L/TRAIL-based cancer therapies.

Interferon-γ Augments CD95(APO-1/Fas) and Pro-Caspase-8 Expression and Sensitizes Human Vascular Endothelial Cells to CD95-Mediated Apoptosis

We have examined the effects of interferon (IFN)-gamma on expression and function of CD95 (APO-1/Fas) and associated proteins in cultured human umbilical vein and dermal microvascular endothelial cells (HUVEC and HDMEC, respectively). Unstimulated cells express only low levels of CD95; IFN-gamma produces a time- and concentration-dependent increase of CD95 in both cell types at the mRNA and cell surface protein levels. IFN-gamma also produces an increase in expression of pro-caspase-8 (FLICE/MACH) but does not significantly change expression of either Fas-associated death domain (FADD) protein or cellular FLICE inhibitory protein (cFLIP), other proteins associated with the CD95 death-inducing signaling complex (DISC). Neither resting nor IFN-gamma-treated EC express detectable CD95L mRNA or protein. Untreated HUVEC and HDMEC show minimal apoptosis when transduced to express CD95L. Treatment of CD95L-transduced cells with IFN-gamma causes apoptosis within 24 to 36 hours that can be blocked by antagonistic anti-CD95 antibody or by the caspase-inhibitory peptide zVAD-FMK. The extent of apoptosis is increased by co-treatment with either the protein synthesis inhibitor cycloheximide or the phosphatidylinositol 3-kinase inhibitor LY294002. Untransduced HUVEC treated with IFN-gamma also undergo CD95-initiated apoptosis when mixed with CD95L-transduced HUVEC or when incubated with pharmacologically activated cytolytic T lymphocytes. Overexpression of CD95 in HUVEC confers sensitivity to CD95L in the absence of IFN-gamma-treatment. We conclude that IFN-gamma induces sensitivity of endothelium to CD95L-mediated apoptosis, and that this response may result from increased expression of CD95 and/or pro-caspase-8.

Hyperosmolarity triggers CD95 membrane trafficking and sensitizes rat hepatocytes toward CD95L-induced apoptosis

The effect of hyperosmolarity on CD95 membrane targeting and CD95 ligand (CD95L)–induced apoptosis was studied in rat hepatocytes. CD95 showed a predominant intracellular localization in normoosmotically exposed rat hepatocytes, whereas hyperosmotic exposure induced, within 1 hour, CD95 trafficking to the plasma membrane followed by activation of caspase-3 and -8. Hyperosmotic CD95 membrane targeting was sensitive to inhibition of c-Jun-N-terminal kinase (JNK), protein kinase C (PKC), and cyclic adenosine monophosphate, but not to inhibition of extracellular regulated kinases (Erks) or p38 mitogen activated protein kinase (p38 MAPK). Hyperosmotic CD95 targeting to the plasma membrane was dose-dependently diminished by glutamine or taurine, probably caused by an augmentation of volume regulatory increase. Despite CD95 trafficking to the plasma membrane and caspase activation, hyperosmolarity per se did not induce apoptosis. Hyperosmolarity, however, sensitized hepatocytes toward CD95L-induced apoptosis, as assessed by annexin V staining and terminal deoxynucleotidyl transferase-mediated X-dUTP nick-end labeling (TUNEL) assay. This sensitization was abolished when hyperosmotic CD95 membrane trafficking was prevented by cyclic adenosine monophosphate, PKC, or JNK inhibition, whereas these effectors had no effect on CD95L-induced apoptosis in normoosmotically exposed hepatocytes. CD95L addition under normoosmotic conditions caused CD95 membrane trafficking, which was sensitive to JNK inhibition, but not to cyclic adenosine monophosphate or inhibition of PKC, Erks, and p38 MAPK. In conclusion, multiple signaling pathways are involved in CD95 membrane trafficking. Hyperosmotic hepatocyte shrinkage induces CD95 trafficking to the plasma membrane, which involves JNK-, PKA-, and PKC-dependent mechanisms and sensitizes hepatocytes toward CD95L-mediated apoptosis. (H EPATOLOGY 2002;36:602-614.)

CD95 engagement induces disseminated endothelial cell apoptosis in vivo: immunopathologic implications

AbstractFas (CD95) is a death receptor involved in apoptosis induction on engagement by Fas ligand (CD95L). Although CD95L-mediated apoptosis has been proposed as a pathogenic mechanism in a wide range of diseases, including graft-versus-host disease, systemic CD95 engagement in mice by agonistic CD95-specific antibodies or by soluble multimeric CD95L (smCD95L), though lethal, has been reported to cause apoptosis only in a limited range of cell types, that is, hepatocytes, hepatic sinusoidal endothelial cells, and lymphocytes. Another member of the tumor necrosis factor (TNF)/CD95L family, TNF-α, induces disseminated vascular endothelial cell apoptosis, which precedes apoptosis of other cell types and lethal multiorgan failure. Here we show that systemic CD95 engagement in vivo by agonistic CD95-specific antibody or smCD95L causes rapid, extensive, and disseminated endothelial cell apoptosis throughout the body, by a mechanism that does not depend on TNF-α. Disseminated endothelial cell apoptosis was also the first detectable lesion in a murine model of acute tissue damage induced by systemic transfer of allogeneic lymphocytes and did not occur when allogeneic lymphocytes were from CD95L-defective mice. Both vascular and additional tissue lesions induced by agonistic CD95-specific antibody, smCD95L, or allogeneic lymphocytes were prevented by treatment with an inhibitor of caspase-8, the upstream caspase coupled to CD95 death signaling. Vascular lesions are likely to play an important role in the pathogenesis of allogeneic immune responses and of other diseases involving circulating CD95L-expressing cells or smCD95L, and the prevention of CD95-mediated death signaling in endothelial cells may have therapeutic implications in these diseases.

Rapid induction of CD95 ligand and CD4+ T cell-mediated apoptosis by CD137 (4-1BB) costimulation.

We investigated the cytolytic mechanism by CD4+ T cells in anti-CD3 mAb-induced redirected cytotoxicity against a murine Fc receptor-bearing mastocytoma (P815) transfected with either CD80 or CD137 ligand (CD137L). CD137 costimulation preferentially induced anti-CD3-induced redirected cytotoxicity within 4 h. This cytotoxicity was efficiently abrogated by the addition of anti-CD137L or anti-CD95L mAb, or by treatment with a broad caspase inhibitor, Z-VAD, suggesting that the induced cytotoxicity against CD137L-P815 is dependent on CD95L-mediated apoptosis. In contrast, the cytotoxicity against CD80-P815, but not CD137L-P815 was efficiently inhibited by an inhibitor of perforin-dependent cytotoxicity, concanamycin A. Involvement of CD95L in the CD137L-dependent cytotoxicity was confirmed by a failure of induction of cytotoxicity by CD4+ T cells from CD95L-gene mutated gid mice. A rapid and remarkable induction of CD95L transcription within 1 h was observed by CD137L costimulation. These results demonstrated that CD137L costimulation induces a rapid induction of CD95L on CD4+ T cells and leads to apoptosis of CD95-sensitive target cells. This biological function of CD137 in CD4+ T cells may play an important role for immune homeostasis.

Impaired CD95-(Fas,APO-1-)apoptosis regulation is a progression factor in early MALT-type lymphoma genesis

MALT-type lymphomas are B cell tumors arising for so far unknown reasons on the background of chronic inflammation, e.g. Helicobacter pylori-associated gastritis. T cells are supposed to have a positive impact on tumor growth, but fail in their control function. We therefore examined the interaction of T cells with malignant B cells in vitro and focused on T cell control which normally operates by CD95L/CD95-mediated apoptosis. Malignant B cells were isolated from tumor tissues of 7 patients with low-grade MALT-type lymphoma and 4 patients with DLBL and cocultured in vitro with activated T cells. Normal memory B cells were used as control. We developed a T/B coculture assay for investigation of CD95L/CD95-mediated apoptosis. The influence of T cells on CD95 expression and survival of B cells was measured by FACS analysis. Activated T cells induced CD95 expression and thus enhanced sensitivity to CD95L-mediated apoptosis in normal memory B cells. However, malignant B cells from 3 out of 7 low-grade MALT-type lymphomas and all 4 gastric DLBLs resisted apoptosis, although the cells showed enhanced CD95 expression. Resistance to CD95L/CD95-mediated apoptosis allows malignant B cells from MALT-type lymphoma to escape T cell control and leads to prolonged survival. This phenomenon acts as a progression factor in early lymphomagenesis.

Defining CD95 as a tumor suppressor gene.

The CD95 (Apo-1/Fas) receptor-ligand system is one of the key regulators of apoptosis and is particularly important for the maintenance of lymphocyte homeostasis. There is now broad evidence that susceptibility of tumor cells towards CD95-mediated apoptosis is largely reduced. In the human, germline and somatic mutations of the CD95 gene are associated with a high risk of both lymphoid and solid tumors. Based on these observations a new concept defining CD95 as a tumor suppressor gene is discussed. In addition to CD95, its natural ligand (CD95L) is also implicated in malignant progression. Compared to their nonmalignant counterparts, malignant cells frequently exhibit aberrant de novo expression of CD95L and are able to induce CD95L-mediated apoptosis in bystander cells. The role for neoplastic CD95L expression in local tissue destruction, invasion, and metastatic spread has been established for many tumor types. CD95L expression by malignant cells may counteract the host's antitumor immunity and favors immune escape of the tumor. On this basis, the significance of loss of CD95 and gain of CD95L expression for tumor progression is discussed.