Patients affected by chronic inflammatory disorders display high amounts of soluble CD95L. This homotrimeric ligand arises from the cleavage by metalloproteases of its membrane-bound counterpart, a strong apoptotic inducer. In contrast, the naturally processed CD95L is viewed as an apoptotic antagonist competing with its membrane counterpart for binding to CD95. Recent reports pinpointed that activation of CD95 may attract myeloid and tumoral cells, which display resistance to the CD95-mediated apoptotic signal. However, all these studies were performed using chimeric CD95Ls (oligomerized forms), which behave as the membrane-bound ligand and not as the naturally processed CD95L. Herein, we examine the biological effects of the metalloprotease-cleaved CD95L on CD95-sensitive activated T-lymphocytes. We demonstrate that cleaved CD95L (cl-CD95L), found increased in sera of systemic lupus erythematosus (SLE) patients as compared to that of healthy individuals, promotes the formation of migrating pseudopods at the leading edge of which the death receptor CD95 is capped (confocal microscopy). Using different migration assays (wound healing/Boyden Chamber/endothelial transmigration), we uncover that cl-CD95L promotes cell migration through a c-yes/Ca2+/PI3K-driven signaling pathway, which relies on the formation of a CD95-containing complex designated the MISC for Motility-Inducing Signaling Complex. These findings revisit the role of the metalloprotease-cleaved CD95L and emphasize that the increase in cl-CD95L observed in patients affected by chronic inflammatory disorders may fuel the local or systemic tissue damage by promoting tissue-filtration of immune cells.

The death receptor CD95 plays a pivotal role in immune surveillance and immune tolerance. Binding of CD95L to CD95 leads to recruitment of the adaptor protein Fas-associated death domain protein (FADD), which in turn aggregates caspase-8 and caspase-10. Efficient formation of the CD95/FADD/caspase complex, known as the death-inducing signaling complex (DISC), culminates in the induction of apoptosis. We show that cells exposed to CD95L undergo a reorganization of the plasma membrane in which the Ca 2+ release-activated Ca 2+ channel Orai1 and the endoplasmic reticulumresident activator stromal interaction molecule 1 colocalize with CD95 into a micrometer-sized cluster in which the channel elicits a polarized entry of calcium. Orai1 knockdown and expression of a dominant negative construct (Orai1E106A) reveal that on CD95 engagement, the Orai1-driven localized Ca 2+ influx is fundamental to recruiting the Ca 2+ -dependent protein kinase C (PKC) β2 to the DISC. PKCβ2 in turn transiently holds the complex in an inactive status, preventing caspase activation and transmission of the apoptotic signal. This study identifies a biological role of Ca 2+ and the Orai1 channel that drives a transient negative feedback loop, introducing a lag phase in the early steps of the CD95 signal. We suggest that these localized events provide a time of decision to prevent accidental cell death.Fas | lymphocytes T he ubiquitously expressed death receptor CD95 (Fas/APO1) belongs to the TNF receptor family. CD95 and its cognate ligand CD95L are instrumental in immune surveillance and tolerance (1) and in the elimination of tumor cells exposed to radiotherapeutic and chemotherapeutic treatments (2). From a molecular standpoint, binding of CD95L to CD95 produces receptor clustering and formation of a polarized plasma membrane structure known as CD95-Cap (3). This membrane platform is crucial to promoting the recruitment of the adaptor protein Fas-associated death domain protein (FADD), which in turn aggregates caspase-8 and caspase-10. The CD95/FADD/caspase-8 complex is known as the death-inducing signaling complex (DISC) (4). The close proximity of these initiator caspases elicits their autoactivation and induction of the apoptotic signal.Calcium ions (Ca 2+ ) participate in cell signaling as a second messenger that relies on intensity (cytosolic concentration), temporal parameters (i.e., duration and frequency), and spatial localization to trigger various cellular responses. In nonexcitable cells, Ca 2+ responses occur mainly through a biphasic signal caused by activation of inositol 1,4,5-triphosphate (IP 3 ) receptors and the release of Ca 2+ from the endoplasmic reticulum (ER), followed by a sustained Ca 2+ entry across the plasma membrane (5). This store-operated Ca 2+ entry (SOCE), mediated by Ca 2+ release-activated Ca 2+ (CRAC) channels, plays pivotal roles in both replenishment of the ER store and lymphocyte activation, leading to proliferation and cytokine production (6). Immune cells express a functional network of ion cha...

cis-Diamminedichloroplatinum(II) (CDDP), which is mostly referred to as cisplatin, is a widely used antineoplastic. The efficacy of cisplatin can be improved by combining it with the vitamin B6 precursor pyridoxine. Here, we evaluated the putative synergistic interaction of CDDP with pyridoxine in the treatment of an orthotopic mouse model of non-small-cell lung cancer (NSCLC). CDDP and pyridoxine exhibited hyperadditive therapeutic effects. However, this synergy was only observed in the context of an intact immune system and disappeared when the otherwise successful drug combination was applied to the same NSCLC cancer implanted in the lungs of athymic mice (which lack T lymphocytes). Immunocompetent mice that had been cured from NSCLC by the combined regimen of CDDP plus pyridoxine became resistant against subcutaneous rechallenge with the same (but not with an unrelated) cancer cell line. In vitro, CDDP and pyridoxine did not only cause synergistic killing of NSCLC cells but also elicited signs of immunogenic cell death including an endoplasmic reticulum stress response and exposure of calreticulin at the surface of the NSCLC cells. NSCLC cells treated with CDDP plus pyridoxine in vitro elicited a protective anticancer immune response upon their injection into immunocompetent mice. Altogether, these results suggest that the combined regimen of cisplatin plus pyridoxine mediates immune-dependent antineoplastic effects against NSCLC.

Death receptors play a crucial role in immune surveillance and cellular homeostasis, two processes circumvented by tumor cells. CD95 (also termed Fas or APO1) is a transmembrane receptor, which belongs to the tumor necrosis factor receptor superfamily, and induces a potent apoptotic signal. Initial steps of the CD95 signal take place through protein/protein interactions that bring zymogens such as caspase-8 and caspase-10 closer. Aggregation of these procaspases leads to their autoprocessing, to the release of activated caspases in the cytosol, which causes a caspase cascade, and to the transmission of the apoptotic signal. In parallel, CD95 engagement drives an increase in the intracellular calcium concentration (Ca(2+))i whose origin and functions remain controversial. Although Ca(2+) ions play a central role in apoptosis/necrosis induction, recent studies have highlighted a protective role of Ca(2+) in death receptor signaling. In the light of these findings, we discuss the role of Ca(2+) ions as modulators of CD95 signaling.