Abstract

Introduction

Accumulating evidence suggests that high endothelial venule (HEV) density in the tumor stroma is a strong predictor of infiltration by CD4⁺ T, CD8⁺ T, and B cells. Although this statement seems obvious because the blood vessels carry oxygen, nutrients, and cells to the organs, selective infiltration by certain T cell subsets at the expense of others is a very complex process. Here, we describe recent data suggesting new roles for CD95L in this process and, more generally, new biological roles for CD95L in progression of cancer and autoimmune disorders such as systemic lupus erythematosus (SLE).

The immune system comprises specialized cells that protect the body from infection and cell transformation. Antigen (Ag)-presenting cells (APCs) survey tissues for non-self Ags and capture pathogenic agents. Ags are processed and presented at the cell surface in the context of major histocompatibility complex (MHC) molecules. Similar to macrophages and B lymphocytes, dendritic cells are professional APCs. APCs present Ags on MHC class II molecules and not only participate in activating non-self-Ag-recognizing T cells but also contribute to elimination of T-cells that recognize self-Ags with a too strong affinity; the latter process is called peripheral tolerance. αβ T cells display either CD4 or CD8 on their surface. CD4⁺ T helper (Th) lymphocytes bind Ag presented in the context of MHC class II molecules expressed by professional APCs and produce cytokines that drive the immune response. CD8⁺ T-lymphocytes recognize antigenic peptides presented by MHC class I molecules expressed on the surface of all cells (apart from some transformed or infected cells), resulting in elimination of these target cells.

The long list of Th subset lineages shows the complexity of the mechanisms developed by the immune system to respond to the diversity of infectious agents and cancers. Polarization of CD4⁺ T-cell subsets occurs via exposure to cytokines, microbiota, high salt concentrations, or high-fat diet (1) that induce expression of different transcription factors such as STAT1, STAT4, and T-bet (for Th1cells), STAT6 and GATA-3 (for Th2 cells), FoxP3 (for regulatory CD4⁺ T cells; Tregs), Bcl6 (for follicular Th cells; TFH) (2), RORγt, RORα, and STAT3 (for Th17cells), T-bet and AhR (for Th22 cells) (3), and PU-1 for Th9 cells (4). These T cells express a set of cytokines that organize the entire immune system (innate and adaptive immune cells), so that it responds adequately (or not) to infection and transformation. CD8⁺ cytotoxic T-lymphocytes destroy target cells and spare bystanders. Recent studies show that the immune landscape (intensity and distribution) within tumors may serve as a prognostic marker for the risk of disease progression and help to predict the efficiency of chemotherapeutic/radiotherapeutic/immunotherapeutic regimens (5).

To simplify, APCs present Ags to CD4⁺ Th cells, which in turn orchestrate activation of the immune response via a cellular (Th1/Th17) or humoral (Th2) response, or by downregulating the intensity/kinetics of the adaptive immune response by promoting differentiation of Tregs. CD4⁺ Th subsets regulate the adaptive immune response by finely tuning the response to particular infectious agents and cancer cells. In addition, the efficiency of the immune response relies on the ability of these cells to cross blood vessels and migrate within inflamed or transformed tissue to reach the target. Selectins, integrins, and chemokine receptors play a central role in T cell extravasation, which involves several different steps: arrest of cells on the endothelial surface, adhesion and spread, crawling, and transendothelial migration (Figure ​(Figure1).1). Once immune function has been achieved, effector T cells are eliminated by molecular mechanisms based on death receptors (DRs) or the BH3-only factor BIM (according to the affinity of the immune cell for the stimulating Ag, i.e., low versus high affinity) (6); however, some Ag-specific memory T cells persist.

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Figure 1

CD95L is a chemoattractant for inflammatory T cells and triple-negative breast cancer (TNBC) cells. Left panel: CD95L is expressed by endothelial cells in damaged organs in lupus patients and in TNBCs. Cleavage of CD95L by metalloproteases releases a soluble form of the ligand. In TNBCs, s-CD95L acts as a chemottractant for tumor cells promoting metastasis. Right panel: In chronic inflammatory pathologies like systemic lupus erythematosus, s-CD95L enhances the trafficking of Th17 T cells in damaged organs through the activation of sphingosine 1 phosphate (S1P) pathway. Infiltrating Th17cells release pro-inflammatory cytokines. Th17 recruitment can be blocked by S1P or CD95 inhibitors.

Because TCR activation induces de novo expression of CD95L by CD8⁺ T-cells, this ligand was regarded as a lethal weapon used by these cells to eliminate target cells (7–9). However, novel studies revisited the biological role of CD95 and found that this receptor also activates non-apoptotic signaling pathways, thereby enhancing pro-inflammatory functions that contribute to the severity of autoimmune disorders and cancers. For instance, CD95/CD95L participates in selective accumulation of pro-inflammatory Th17cells in the damaged organs of lupus patients (10).

DRs Belonging to the Tumor Necrosis Factor (TNF) Receptor (TNF-R) Family

Two CD95L and Many Cell Signaling Pathways

CD95L is a transmembrane cytokine whose ectodomain can be cleaved close to the plasma membrane by metalloproteases such as MMP3 (92), MMP7 (93), MMP9 (94), and ADAM10 (95, 96); CD95L is then released into the bloodstream as s-CD95L. Metalloprotease-mediated cleavage of CD95L releases a homotrimeric ligand that does not trigger the apoptotic program (35). Indeed, experiments show that hexameric CD95L represents the minimal level of self-association required to signal apoptosis (39). The homotrimeric ligand has long been considered an inert molecule that only competes with its membrane-bound counterpart to antagonize the death signal (34, 35). However, recent work shows that this metalloprotease-cleaved CD95L actively aggravates inflammation and autoimmunity in patients with SLE by inducing non-apoptotic signaling pathways, including the NF-κB and PI3K pathways (36, 97).

It is noteworthy that there are different forms of s-CD95L. Although the soluble form of CD95L generated by MMP7-driven cleavage of its ¹¹³ELR¹¹⁵ sequence (the CD95L stalk region) induces apoptosis (93), its counterpart (cleaved between serine 126 and leucine 127) does not (35, 36, 97). For instance, s-CD95L in the bronchoalveolar lavage (BAL) fluid of patients suffering from acute respiratory distress syndrome (ARDS) is oxidized at methionines 224 and 225, thereby promoting aggregation of the soluble ligand and boosting its cytotoxic activity (98). The stalk region of CD95L, corresponding to amino acids 103–136, contains all metalloprotease cleavage sites described in the literature. Surprisingly, this region is conserved in s-CD95L detected in ARDS patients, participates in CD95L multimerization, and accounts for ligand-mediated cytotoxicity and damage to the lung epithelium (98). Of note, in ARDS BAL fluid, additional oxidation of s-CD95L occurs at methionine 121, thereby preventing cleavage of CD95L by MMP7 and potentially explaining why this cytotoxic ligand retains its stalk region and contributes to disease pathology (98). Nonetheless, preservation of this region raises the question of whether an as-yet-unidentified MMP7-independent cleavage site exists in the juxtamembrane region of CD95L, or whether the ligand detected in ARDS patients corresponds in fact to the full-length form of CD95L embedded in exosomes (99, 100). Indeed, exosome-bound CD95L can be expressed by human prostate cancer cells (i.e., the LNCaP cell line) and evokes apoptosis in activated T lymphocytes (101). In addition to MMPs, plasmin can also cleave the m-CD95L expressed by neo-vessels in cancers (102). Plasminogen activator inhibitor-1 (PAI-1) inhibits the activity of urokinase-type plasminogen activator, which converts plasminogen into its active form, plasmin. PAI-1 is a good prognostic marker in patients affected by glioblastoma or breast and colon cancers (102). Plasmin cleaves CD95L between the amino acid residues Arg144 and Lys145 within its trimerization domain and releases a soluble ligand, which surprisingly triggers cell death in endothelial cells (102). Because no analysis of the s-CD95L stoichiometry has been realized in this study, it is difficult to decipher whether the soluble plasmin-generated CD95L corresponds to an aggregate of homotrimers or whether cleavage by plasmin releases a peculiar homotrimeric ligand, which interacts with other components accounting for its unexpected apoptotic effect.

Overall, these findings emphasize the importance of finely characterizing the stoichiometry of naturally processed CD95L in the serum of patients affected by cancers or chronic/acute inflammatory disorders to predict the biological role (i.e., apoptotic versus non-apoptotic) of this soluble ligand. Such investigations will improve our understanding of the molecular mechanisms set in motion by this ligand, and our appreciation of its downstream biological functions.

Interplay Between Caspase-8 and Kinases in the CD95 Signaling Pathways

As mentioned earlier, CD95 does not possess any enzymatic activity; however, by triggering a large number of PPIs, it activates different signaling pathways resulting in different clinical outcomes. Here, the aim is not to exhaustively describe the initial events of the CD95-mediated signaling pathway, but rather to focus on the role of kinases in these pathways. Unlike m-CD95L, whose interaction with CD95 leads to formation of the apoptotic complex Death-Inducing Signaling Complex (DISC), s-CD95L leads to formation of a molecular complex devoid of FADD and caspase-8, which instead recruits and activates the Src kinase c-yes via production of NADPH oxidase 3 and ROS (36, 78); this unconventional receptosome is designated the motility-inducing signaling complex (MISC) (36, 37). This process was also observed in apoptotic-resistant glioblastoma cells, in which CD95 stimulation triggers recruitment of a molecular complex that contains the Src tyrosine kinase c-yes (78). This complex promotes micrometastatic dissemination of glioblastoma cells. Similarly, upon injury to the central nervous system, neutrophils and macrophages overexpress CD95L, which promotes migration of these cells via activation of the src kinase Syk (76). Of note, while deleting CD95 from neural cells has no effect on pathology progression, deleting CD95L from myeloid cells accelerates spinal cord recovery by reducing the infiltration of neutrophils and macrophages and the magnitude of inflammation. These findings clearly show that the CD95-mediated apoptotic response is not responsible for CD95/CD95L function in this pathological context, but that the ligand/receptor pair is required for accumulation of inflammatory cells contributing to the degradation of the organ.

Although our data showed no trace of caspase-8 in the MISC of TNBC cells exposed to s-CD95L (37), we cannot rule out the presence of trivial amounts of this protease in MISC (undetectable by western blotting). Caspase-8 is known to participate in cell migration mainly through an enzymatic-independent mechanism. Src kinases abrogate the protease activity of caspase-8 by phosphorylating it at tyrosine 380 (Y380) (103); this favors recruitment of the PI3K adaptor p85 alpha subunit, thereby activating the PI3K signaling pathway (104) (Figure ​(Figure2).2). A study revealed that in hepatocytes, hyperosmolarity induces association between a receptor tyrosine kinase (RTK) [called epidermal growth factor receptor (EGFR)] and CD95 through a JNK and PKC-dependent mechanism that remains to be elucidated (105). Strikingly, this study showed that CD95/EGFR increases both DISC formation and the caspase-8-dependent apoptotic response in hepatocytes. More recent data show that EGFR phosphorylates caspase-8 at Y380 through activation of Src kinases, and that this RTK-mediated translational modification prevents triggering of the CD95-mediated apoptotic signaling pathway (103). Several reports confirm that phosphorylation of caspase-8 at Y380 inhibits its protease activity (106, 107). The death effector domains of caspase-8 are both sufficient and necessary for EGFR-dependent cell migration (108). Because caspase-8 phosphorylation at Y380 recruits Src kinases, including Fyn, Lyn, and Src kinases, it is tempting to speculate that this process constitutes a positive feedback loop, which in turn phosphorylates additional caspase-8 molecules (107) (Figure ​(Figure2).2). We recently observed that exposure of TNBC cells overexpressing EGFR to s-CD95L induces association of CD95 with EGFR, thereby promoting cell migration and metastatic dissemination of these cancer cells (37). RTK-driven Y380 phosphorylation of caspase-8 might prime certain cancer cells to become unresponsive to the apoptotic signal triggered by cytotoxic CD95L, thereby diverting this cell signaling pathway toward promoting cell migration, an essential step in cancer cell metastasis (Figure ​(Figure2).2). These data are in agreement with those published in a recent study showing that another RTK, PDGFR-β, is recruited by CD95 in colon cancer cells exposed to CD95L to trigger cancer cell metastasis (109). CD95 triggers phosphorylation of PDGFR at tyrosine 1021, thereby promoting recruitment of phospholipase Cγ1 (PLCγ1) (109). Of note, we recently showed that PLCγ1 directly docks onto the calcium-inducing domain of CD95 (10), suggesting that PDGFR-β-dependent recruitment of PLCγ1 might increase the amount of lipase recruited by CD95, and/or sustain its presence at the plasma membrane to increase production of inositol trisphosphate, which activates the Ca²⁺ response and diacylglycerol to stimulate conventional and novel PKCs (Figure ​(Figure2).2). CD95 induces T cell migration by activating PLCγ1 (36), which necessitates its Src kinase-driven phosphorylation at tyrosine 783 (Y783) (110). Also, the RTK called hepatocyte growth factor receptor or c-Met binds and sequesters CD95 to impair its interaction with CD95L, highlighting another molecular mechanism used by RTKs to prevent apoptosis (111).

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Figure 2

Non-apoptotic signaling pathway induced by CD95. Left panel: Cancer cells exposed to s-CD95L induce the recruitment of RTKs (i.e., EGFR or PDGFR) by CD95 leading to the binding and activation of src. In turn, src kinases can phosphorylate caspase-8 neutralizing its enzymatic activity and promoting its role in the activation of the PI3K signaling pathway (i.e., recruitment of p85) and cell migration. Right panel: CD95 favors RTK activation leading to Src recruitment. s-CD95L interaction with CD95 induces binding of PLCγ1, which is activated through a src-dependent phosphorylation. Activated PLCγ1 hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2), releasing inositol triphosphate (IP3), which activates IP3Rs on endoplasmic reticulum promoting calcium release into the cytoplasm that contributes to the CD95-mediated cell migration signaling pathway.

These findings show that CD95-mediated recruitment of RTKs, or vice versa, may correspond to a physiological process that simultaneously inhibits the apoptotic signal by phosphorylating caspase-8, thereby promoting cell motility by activating the PI3K signaling pathway. However, it is unclear how CD95 interacts with RTKs.

Conclusion and Perspectives

Although the CD95/CD95L interaction can eliminate malignant cells by promoting formation of the DISC, it can also promote carcinogenesis by maintaining inflammation and/or by inducing metastatic dissemination (36, 76–78, 97, 112, 113). The molecular mechanisms underlying the switch between these different signaling pathways remain enigmatic. An important question to be addressed is how the magnitude of CD95 aggregation regulates formation of “Death”- versus “Motility”-ISCs. Answering these questions will lead to development of new therapeutic agents with the ability to prevent the spread and chronicity of inflammation, or to impinge on carcinogenesis, at least with respect to pathologies associated with increased s-CD95L, such as cancers [e.g., pancreatic cancer (114), large granular lymphocytic leukemia, breast cancer (37), and NK cell lymphoma (115)] and autoimmune disorders [e.g., rheumatoid arthritis and osteoarthritis (116), and graft-versus-host-disease (117, 118), or SLE (36, 119)].

This review highlights that CD95L is frequently detected overexpressed at the surface of endothelial cells covering blood vessels in tissues of patients affected by inflammatory disorders or cancers. Although its role remains to be elucidated, growing evidence suggest that this ectopic expression can be a biological marker whose combination with a “lethal” metalloprotease might turn out to enhance the severity of autoimmune disorders and cancers. An extensive review of the CD95/CD95L role in the function of endothelial cells indicates that besides its role in the immune system, CD95 can directly regulate angiogenesis either by implementing cell death when encountering its transmembrane ligand or by promoting survival and migration of these endothelial cells when exposed to s-CD95L. Finally, CD95 can also stimulate the production of nitric oxide and thereby control blood pressure (120) revealing its complex biological role in the context of the endothelium survival, proliferation, and function.

Author Contributions

All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Footnotes

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