Signaling through MHC class II molecules blocks CD95-induced apoptosis

B cells are induced to express CD95 upon interaction with T cells. This interaction renders the B cells sensitive to CD95-mediated apoptosis, but ligation of proviability surface receptors is able to inhibit apoptosis induction. MHC class II is a key molecule required for Ag presentation to Th cells, productive T cell-B cell interaction, and B cell activation. We demonstrate here for the first time that MHC class II ligation also confers a rapid resistance to CD95-induced apoptosis, an affect that does not require de novo protein synthesis. Signaling through class II molecules blocks the activation of caspase 8, but does not affect the association of CD95 and Fas-associated death domain-containing protein. MHC class II ligation thus blocks proximal signaling events in the CD95-mediated apoptotic pathway.     

HLA-DR-mediated apoptosis susceptibility discriminates differentiation stages of dendritic/monocytic APC

Professional APC are characterized by their ability to present peptide via HLA class II in the presence of costimulatory molecules (CD40, CD80, and CD86). The efficiency of Ag presentation can be classed as follows: mature dendritic cells (DC) are most efficient, immature DC and macrophages are intermediate, and monocytes are considered poor APC. There is a large body of evidence demonstrating that HLA-DR transmits signals in the APC. In this study, we have addressed the question of the outcome of HLA-DR signals on APC of the monocyte/DC lineages throughout their differentiation from immature to mature APC. DC were generated from both monocytes and CD34+ cells of the same individual, macrophages were differentiated from monocytes. Immunophenotypical analysis clearly distinguished these populations. HLA-DR-mediated signals led to marked apoptosis in mature DC of either CD34 or monocytic origin. Significantly less apoptosis was observed in immature DC of either origin. Nonetheless, even immature DC were more susceptible to HLA-DR-mediated apoptosis than macrophages, whereas monocytes were resistant to HLA-DR-mediated apoptosis. The mechanism of HLA-DR-mediated apoptosis was independent of caspase activation. Taken together, these data lead to the notion that signals generated via HLA-DR lead to the demise of mature professional APC, thereby providing a means of limiting the immune response.     

Aberrant expression of Fas ligand in mice deficient for the MHC class II transactivator

The MHC class II transactivator (CIITA) is a critical regulator of MHC class II genes and other genes involved in the Ag presentation pathway. CIITA-deficient mice lack MHC class II expression on almost all APCs. In this study, we show that these mice also have aberrant Fas ligand expression on both CD4 T cells and B cells. We found that Fas ligand expression was greatly increased on CIITA-deficient CD4 T cells during the Th1 differentiation process. However, both CIITA-deficient and control Th1 effector cells up-regulated Fas ligand to similar levels if cells were reactivated. The introduction of CIITA into primary CD4 T cells via retroviral infection resulted in a reduction in the level of Fas ligand and delay in apoptosis after activation. Interestingly, activated B cells from the CIITA-deficient mice also showed increased levels of Fas ligand that could be to some degree inhibited by the introduction of IL-4.     

Triggering HLA-DR molecules on human peripheral monocytes induces their death

Although engagement of MHC class II molecules on human monocytes triggers various cellular events, their possible role in monocyte death is yet unknown. We demonstrate that ligation of MHC class II on primary monocytes induces a rapid cell death that has all the characteristics of monocyte apoptosis, does not require de novo protein synthesis, is independent from both Fas and TNF-alpha systems, and is not rescued by ligation of CD40. However, cell-cell interactions that involve the beta2-integrin CD18 seem to be critical for the execution of this monocyte death. Priming monocytes with IFN-gamma enhances significantly their HLA-DR-mediated death whereas LPS treatment effectively reverses this death process. Thus, our results describe the MHC class II molecules, in particular HLA-DR, as mediators of monocyte death and suggest that this novel pathway of monocyte death might have an important role in controlling the outcome of inflammatory process and the regulation of monocyte hemostasis.     

Requirement for efficient interactions between CD4 and MHC class II molecules for survival of resting CD4+ T lymphocytes in vivo and for activation-induced cell death

Regulation of homeostasis in the immune system includes mechanisms that promote survival of resting T lymphocytes, and others that control activation-induced cell death (AICD). In this study, we report on the use of a transgenic mouse model to test the role of CD4-MHC class II interactions for the susceptibility of CD4+ T lymphocytes to AICD, and for the survival of resting CD4+ T cells in peripheral lymphoid organs. The only I-Abeta gene expressed in these mice is an Abetak transgene with a mutation that prevents MHC class II molecules from interacting with CD4. We show increased apoptosis in CD4+ T lymphocytes derived from wild-type, but not from mutant Abetak transgenic mice following stimulation with staphylococcal enterotoxin A. Therefore, AICD may be impaired in CD4+ T cells derived from mutant Abetak transgenic mice. Importantly, we observed much higher apoptosis in resting CD4+ T cells from mutant Abetak transgenic mice than from wild-type mice. Furthermore, resting CD4+ T cells from mutant Abetak transgenic mice expressed higher levels of cell surface CD95 (Fas, APO-1). Ab-mediated cross-linking of CD95 further increased apoptosis in CD4+ T cells from mutant Abetak transgenic mice, but not from wild-type mice, suggesting apoptosis involved CD95 signaling. When cocultured with APC-expressing wild-type MHC class II molecules, apoptosis in resting CD4+ T lymphocytes from mutant Abetak transgenic mice was reduced. Our results show for the first time that interactions between CD4 and MHC class II molecules are required for the survival of resting CD4+ T cells in peripheral lymphoid organs.     

Differential susceptibility to CD95 (Apo-1/Fas) and MHC class II-induced apoptosis during murine dendritic cell development

Disappearance of antigen presenting cells (APC) from the lymph node occurs following antigen specific interactions with T cells. We have investigated the regulation of CD95 (Apo-1/Fas) induced apoptosis during murine dendritic cell (DC) development. Consistent with the moderate levels of CD95 surface expression and low, or absent, MHC class II expression, immature DC in bone marrow cultures were highly sensitive to CD95 induced apoptosis, but insensitive to class II mediated apoptosis. In contrast, mature splenic, epidermal and bone marrow derived DC were fully resistant to CD95 induced cell death, but sensitive to class II induced apoptosis. Although caspase 3 and 8 activation was detected in immature DC undergoing CD95L-induced apoptosis, the pan-caspase inhibitor zVAD-fmk did not inhibit the early events of CD95-induced mitochondrial depolarisation or phosphatidyl serine exposure and only partially inhibited the killing of immature DC. In contrast, zVAD-fmk was completely effective in preventing CD95L mediated death of murine thymocytes. Collectively, these data do not support a major role of CD95: CD95L ligation in apoptosis of mature DC, but rather emphasise the existence of distinct pathways for the elimination of DC at different stages of maturation.     

Cognate B cell signaling via MHC class II: differential regulation of B cell antigen receptor and MHC class II/Ig-alpha beta signaling by CD22

Recent studies demonstrate that MHC class II molecules can signal via associated Ig-alphabeta dimers, signal transducers previously thought to function only in B cell Ag receptor (BCR) signaling. Surprisingly, the biologic outputs of MHC class II and BCR ligation (by thymus-dependent Ags) differ, e.g., MHC class II signaling leads to robust proliferation and extension of pseudopods. It seemed possible that these differences might be due, at least in part, to differential use of inhibitory coreceptors thought to modulate membrane Ig signals. In this study, we demonstrate that CD22, an inhibitory BCR coreceptor, neither associates with nor functions in MHC class II/Ig-alphabeta signaling. Interestingly, CD22 is actively excluded from cell surface MHC class II aggregates.     

Function of the CD4 and CD8 molecules on human cytotoxic T lymphocytes: regulation of T cell triggering

The function of the T cell differentiation antigens CD4 (Leu-3/T4) and CD8 (Leu-2/T8) on human cytotoxic T lymphocytes (CTL) is presently seen only in conjugate formation between CTL and target cell via class II or class I MHC antigens rather than in the later killing steps. In this study, human CD4+ and CD8+ CTL clones were used to investigate the effects of monoclonal antibodies against these differentiation antigens on nonspecific triggering of cytotoxicity. Cytotoxicity was induced either by antibodies against the CD3 (T3) antigen or by the lectins Con A and PHA. Anti-CD4 or anti-CD8 antibodies specifically inhibited all types of cytotoxicity of CD4+ or CD8+ CTL, respectively, regardless of the specificity of the CTL for class I or class II HLA antigens and regardless of whether target cells expressed class I or class II antigens. These results are incompatible with an exclusive role of the CD4 and CD8 molecules in MHC class recognition and are discussed with respect to a function as negative signal receptors for these molecules on CTL.     

CD4同源二聚化或寡聚化的间接检测及其介导的生物学功能的初步分析

近年来的研究表明 ,CD4分子于细胞膜表面不仅以单体形式存在还可以通过其D4和D1形成同源二聚化及寡聚化 ,并且二聚化及寡聚化的CD4分子才能稳定地与MHC Ⅱ类分子结合。通过分析CD4分子以及CD4缺失突变体分子融合Fas基因片段所诱导的转染靶细胞的凋亡 ,以及携带绿色荧光蛋白的CD4分子转染HEK2 93细胞所筛选出的稳定克隆的不同荧光强度和与MHC Ⅱ类分子阳性细胞Raji之间的不同黏附效应间接鉴定CD4同源二聚体或寡聚体的存在 ,并对二聚化或寡聚化CD4分子所介导的生物学功能进行初步分析。     

Recognition of a shared human prostate cancer-associated antigen by nonclassical MHC-restricted CD8+ T cells

To identify prostate cancer-associated Ags, tumor-reactive T lymphocytes were generated using iterative stimulations of PBMC from a prostate cancer patient with an autologous IFN-gamma-treated carcinoma cell line in the presence of IL-2. A CD8+ T cell line and TCR alphabeta+ T cell clone were isolated that secreted IFN-gamma and TNF-alpha in response to autologous prostate cancer cells but not to autologous fibroblasts or lymphoblastoid cells. However, these T cells recognized several normal and malignant prostate epithelial cell lines without evidence of shared classical HLA molecules. The T cell line and clone also recognized colon cancers, but not melanomas, sarcomas, or lymphomas, suggesting recognition of a shared epithelium-associated Ag presented by nonclassical MHC or MHC-like molecules. Although Ag recognition by T cells was inhibited by mAb against CD8 and the TCR complex (anti-TCR alphabeta, CD3, Vbeta12), it was not inhibited by mAb directed against MHC class Ia or MHC class II molecules. Neither target expression of CD1 molecules nor HLA-G correlated with T cell recognition, but beta2-microglobulin expression was essential. Ag expression was diminished by brefeldin A, lactacystin, and cycloheximide, but not by chloroquine, consistent with an endogenous/cytosolic Ag processed through the classical class I pathway. These results suggest that prostate cancer and colon cancer cells can process and present a shared peptidic Ag to TCR alphabeta+ T cells via a nonclassical MHC I-like molecule yet to be defined.     

Phosphatidylinositol 3-kinase-dependent pathways oppose Fas-induced apoptosis and limit chloride secretion in human intestinal epithelial cells. Implications for inflammatory diarrheal states

The epithelial lining of the intestine serves as a barrier to lumenal bacteria and can be compromised by pathologic Fas-mediated epithelial apoptosis. Phosphatidylinositol (PI)3-kinase signaling has been described to limit apoptosis in other systems. We hypothesized that PI3-kinase-dependent pathways regulate Fas-mediated apoptosis and barrier function in intestiynal epithelial cells (IEC). IEC lines (HT-29 and T84) were exposed to agonist anti-Fas antibody in the presence or absence of chemical inhibitors of PI3-kinase (LY294002 and wortmannin). Apoptosis, barrier function, changes in short circuit current (DeltaI(sc)), and expression of adhesion molecules were assessed. Inhibition of PI3-kinase strongly sensitized IEC to Fas-mediated apoptosis. Expression of constitutively active Akt, a principal downstream effector of the PI3-kinase pathway, protected against Fas-mediated apoptosis to an extent that was comparable with expression of a genetic caspase inhibitor, p35. PI3-kinase inhibition sensitized to apoptosis by increasing and accelerating Fas-mediated caspase activation. Inhibition of PI3-kinase combined with cross-linking Fas was associated with increased permeability to molecules that were <400 Da but not those that were >3,000 Da. Inhibition of PI3-kinase resulted in chloride secretion that was augmented by cross-linking Fas. Confocal analyses revealed polymerization of actin and maintenance of epithelial cell adhesion molecule-mediated interactions in monolayers exposed to anti-Fas antibody in the context of PI3-kinase inhibition. PI3-kinase-dependent pathways, especially Akt, protect IEC against Fas-mediated apoptosis. Inhibition of PI3-kinase in the context of Fas signaling results in increased chloride secretion and barrier dysfunction. These findings suggest that agonists of PI3-kinase such as growth factors may have a dual effect on intestinal inflammation by protecting epithelial cells against immune-mediated apoptosis and limiting chloride secretory diarrhea.     

Amplification of Fas-mediated apoptosis in type II cells via microdomain recruitment

Fas triggers apoptosis via the caspase cascade when bound to its ligand FasL. In type I cells, Fas is concentrated into the plasma membrane lipid rafts, and these domains are required for the apoptotic signal to occur. In contrast, Fas is excluded from the microdomains in type II cells. We report that the coligation with Fas of the membrane receptor CD28 strongly increases Fas-induced apoptosis in type II T lymphocytes, whereas it has no effect in a type I cell line. The effect of CD28 is independent of its intracellular region and requires the recruitment of the microdomains. Indeed, upon CD28 costimulation, Fas is redistributed in the lipid rafts, and their disruption with a cholesterol chelator abrogates the effect of CD28. The microdomain-mediated cell death amplification does not alter death-induced signaling complex formation and is mediated by the enhancement of the mitochondrial apoptotic pathway. These findings indicate that the sensitivity to Fas-induced apoptosis of type II cells can be amplified in vivo by the recruitment of lipid rafts following interactions between nonapoptotic ligand/receptor pairs during cell-to-cell contacts.     

Toxoplasma gondii inhibits Fas/CD95-triggered cell death by inducing aberrant processing and degradation of caspase 8

Ligation of the death receptor Fas/CD95 activates an apoptotic cascade and plays critical roles during infectious diseases. Previous work has established that infection with the intracellular parasite Toxoplasma gondii renders cells resistant to multiple inducers of apoptosis. However, the effect of T. gondii on the death receptor pathway is poorly characterized. Here we have determined the impact of the parasite on apoptosis in type I cells that transduce Fas/CD95 engagement via the death receptor pathway without the need of a mitochondrial amplification loop. The results have shown that T. gondii significantly reduced Fas/CD95-triggered apoptosis by impairing activation of the initiator caspase 8. Parasitic infection diminished the cellular amount of procaspase 8, resulting in its decreased recruitment to the death-inducing signalling complex and the impaired activation of effector caspases. Remarkably, downregulation of caspase 8 protein in T. gondii-infected cells also occurred in the absence of Fas/CD95 engagement and was associated with the appearance of non-canonical caspase 8 cleavage fragments. Distinct parasite proteins were associated with caspase 8 and its proteolytic fragments. These findings indicate that T. gondii aberrantly processes and finally degrades the initiator caspase 8, thereby, blocking Fas/CD95-mediated apoptosis which signals independently of the apoptogenic function of host cell mitochondria.     

Fas ligand-independent, FADD-mediated activation of the Fas death pathway by anticancer drugs

Trimerization of the Fas receptor (CD95, APO-1), a membrane bound protein, triggers cell death by apoptosis. The main death pathway activated by Fas receptor involves the adaptor protein FADD (for Fas-associated death domain) that connects Fas receptor to the caspase cascade. Anticancer drugs have been shown to enhance both Fas receptor and Fas ligand expression on tumor cells. The contribution of Fas ligand-Fas receptor interactions to the cytotoxic activity of these drugs remains controversial. Here, we show that neither the antagonistic anti-Fas antibody ZB4 nor the Fas-IgG molecule inhibit drug-induced apoptosis in three different cell lines. The expression of Fas ligand on the plasma membrane, which is identified in untreated U937 human leukemic cells but remains undetectable in untreated HT29 and HCT116 human colon cancer cell lines, is not modified by exposure to various cytotoxic agents. These drugs induce the clustering of Fas receptor, as observed by confocal laser scanning microscopy, and its interaction with FADD, as demonstrated by co-immunoprecipitation. Overexpression of FADD by stable transfection sensitizes tumor cells to drug-induced cell death and cytotoxicity, whereas down-regulation of FADD by transient transfection of an antisense construct decreases tumor cell sensitivity to drug-induced apoptosis. These results were confirmed by transient transfection of constructs encoding either a FADD dominant negative mutant or MC159 or E8 viral proteins that inhibit the FADD/caspase-8 pathway. These results suggest that drug-induced cell death involves the Fas/FADD pathway in a Fas ligand-independent fashion.     

Primary T cell expansion and differentiation in vivo requires antigen presentation by B cells

B cells are well documented as APC; however, their role in supporting and programming the T cell response in vivo is still unclear. Studies using B cell-deficient mice have given rise to contradictory results. We have used mixed BM chimeric mice to define the contribution that B cells make as APC. When the B cell compartment is deficient in MHC class II, while other APC are largely normal, T cell clonal expansion is significantly reduced and the differentiation of T cells into cytokine-secreting effector cells is impaired (in particular, Th2 cells). The development of the memory T cell populations is also decreased. Although MHC class II-mediated presentation by B cells was crucial for an optimal T cell response, neither a B cell-specific lack of CD40 (influencing costimulation) nor lymphotoxin alpha (influencing lymphoid tissue architecture) had any effect on the T cell response. We conclude that in vivo B cells provide extra and essential Ag presentation capacity over and above that provided by dendritic cells, optimizing expansion and allowing the generation of memory and effector T cells.     

Role of complement-binding CD21/CD19/CD81 in enhancing human B cell protection from Fas-mediated apoptosis

Defective expression of Fas leads to B cell autoimmunity, indicating the importance of this apoptotic pathway in eliminating autoreactive B cells. However, B cells with anti-self specificities occasionally escape such regulation in individuals with intact Fas, suggesting ways of precluding this apoptosis. Here, we examine whether coligation of the B cell Ag receptor (BCR) with the complement (C3)-binding CD21/CD19/CD81 costimulatory complex can enhance the escape of human B cells from Fas-induced death. This was warranted given that BCR-initiated signals induce resistance to Fas apoptosis, some (albeit not all) BCR-triggered events are amplified by coligation of BCR and the co-stimulatory complex, and several self Ags targeted in autoimmune diseases effectively activate complement. Using a set of affinity-diverse surrogate Ags (receptor-specific mAb:dextran conjugates) with varying capacity to engage CD21, it was established that BCR:CD21 coligation lowers the BCR engagement necessary for inducing protection from Fas apoptosis. Enhanced protection was associated with altered expression of several molecules known to regulate Fas apoptosis, suggesting a unique molecular model for how BCR:CD21 coligation augments protection. BCR:CD21 coligation impairs the generation of active fragments of caspase-8 via dampened expression of membrane Fas and augmented expression of FLIP(L). This, in turn, diminishes the generation of cells that would be directly triggered to apoptosis via caspase-8 cleavage of caspase 3 (type I cells). Any attempt to use the mitochondrial apoptotic protease-activating factor 1 (Apaf-1)-dependent pathway for apoptosis (as type II cells) is further blocked because BCR:CD21 coligation promotes up-regulation of the mitochondrial antiapoptotic molecule, Bcl-2.     

High levels of Fas ligand and MHC class II in the absence of CD80 or CD86 expression and a decreased CD4<Superscript>+</Superscript> T cell Infiltration,enables murine skin tumours to progress

It is still not clear why some tumours will be recognized and destroyed by the immune system, and others will persist, grow, and eventually kill the host. It has been hypothesized that tumour cells might evade immunological destruction by expressing Fas ligand (FasL), a molecule which induces apoptosis in Fas(+) target cells. However, the role of FasL in creating an immune privileged status within a tumour remains controversial. To determine whether FasL is associated with skin tumour progression, we developed a tumour model enabling us to compare two squamous cell carcinomas (SCC). One is a regressor SCC which spontaneously regresses after injection into syngeneic mice. The other is a progressor SCC which evades immunological destruction. Detailed flow cytometric analysis was used to study tumour cell expression of FasL, Fas, CD80, CD86 and MHC class II. We also analysed the percentage of apoptotic tumour cells in vivo using annexin V and correlated skin tumour progression with CD4 and CD8 T cell infiltration. Progressor tumours expressed high levels of FasL in vivo, which was virtually absent from regressor tumours. The percentage of progressor tumours expressing MHC II was significantly greater than regressor tumours, while neither tumour expressed CD80 or CD86 costimulatory molecules. Consistent with a regressor phenotype, the percentage of viable tumour cells was significantly lower for regressor compared to progressor tumours which coincided with a significantly larger CD4(+) T cell infiltrate into the tumour mass. The results suggest that progression of skin tumours occurs if tumour cells express high levels of MHC II but not costimulatory molecules such as CD80 or CD86. This implies that tumours may induce anergy in CD4(+) T cells via MHC II antigen presentation in the absence of costimulation. To ensure escape from the immune system, tumours may then kill these T cells via a FasL-dependent mechanism.     

Caspase-independent Cell Killing by Fas-associated Protein with Death Domain

The binding of Fas ligand to Fas recruits caspase 8 to Fas via an adaptor, FADD/MORT1, and activates a caspase cascade leading to apoptosis. Here, we describe a human Jurkat-derived cell line (JB-6) that is deficient in caspase 8. This cell line was resistant to the apoptosis triggered by Fas engagement. However, the multimerization of Fas-associated protein with death domain, through the use of a dimerizing system, killed the JB-6 cells. This killing process was not accompanied by the activation of caspases or DNA fragmentation. The dying cells showed neither condensation nor fragmentation of cells and nuclei, but the cells and nuclei swelled in a manner similar to that seen in necrosis. These results suggested that Fas-associated protein with death domain can kill the cells via two pathways, one mediated by caspases and another that does not involve them.     

Prolonged activation of ERK1,2 induces FADD-independent caspase 8 activation and cell death

Prolonged ERK/MAPK activation has been implicated in neuronal cell death in vitro and in vivo. We found that HEK293 cells, recently reported to express neuronal markers, are exquisitely sensitive to long term ERK stimulation. Activation of an inducible form of Raf-1 (Raf-1:ER) in HEK293 cells induced massive apoptosis characterized by DNA degradation, loss of plasma membrane integrity and PARP cleavage. Cell death required MEK activity and protein synthesis and occurred via the death receptor pathway independently of the mitochondrial pathway. Accordingly, prolonged ERK stimulation activated caspase 8 and strongly potentiated Fas signaling. The death receptor adaptator FADD was found to be rapidly induced upon ERK activation. However using RNA interference and ectopic expression, we demonstrated that neither FADD nor Fas were necessary for caspase 8 activation and cell death. These findings reveal that prolonged ERK/MAPK stimulation results in caspase 8 activation and cell death. This work was supported by grant from Association pour la Recherche sur le Cancer (CNRS6543/ARC). S. Cagnol is supported by a fellowship from the Ligue Nationale contre le Cancer.     

MEK1 activation rescues Jurkat T cells from Fas-induced apoptosis.

Although the protease cascade initiated by Fas (CD95, Apo-1) is well characterized, there remains little known about how kinase pathways may impact on Fas-mediated apoptosis. We recently observed that in T lymphocytes Fas strongly induced activation of JNK (c-Jun N-terminal kinase) but not of second messengers leading to activation of ERK (extracellular regulated kinase). Additionally, Fas-mediated apoptosis was significantly inhibited with PMA, a potent activator of the ERK signaling pathway. This suggested a model whereby activation of the ERK pathway might attenuate Fas-mediated apoptosis. This was confirmed in the current study by showing that activation of MEK1, the upstream regulator of ERK, reduces Fas-mediated apoptosis, whereas inhibition of MEK1 augments apoptosis by Fas. Furthermore, Fas-mediated apoptosis of Jurkat T cells is not affected by constitutively active or dominant negative variants that modulate the JNK pathway. These results demonstrate that Fas-induced JNK activation is not required for apoptosis by Jurkat T cells, but rather is more likely secondary to cell stress during the early phases of apoptosis. This is supported by the ability of the caspase blocker zVAD to inhibit both apoptosis and JNK activation by Fas.