Study of the potential role of CASPASE-10 mutations in the development of autoimmune lymphoproliferative syndrome.

Autoimmune lymphoproliferative syndrome (ALPS) is a primary disorder of lymphocyte homeostasis, leading to chronic lymphoproliferation, autoimmune cytopenia, and increased risk of lymphoma. The genetic landscape of ALPS includes mutations in FAS, FASLG, and FADD, all associated with apoptosis deficiency, while the role of CASP10 defect in the disease remains debated. In this study, we aimed to assess the impact of CASP10 variants on ALPS pathogenesis. We benefit from thousands of genetic analysis datasets performed in our Institute's genetic platform to identify individuals carrying CASP10 variants previously suspected to be involved in ALPS outcome: p.C401LfsX15, p.V410I and p.Y446C, both at heterozygous and homozygous state. Clinical and laboratory features of the six included subjects were variable but not consistent with ALPS. Two individuals were healthy. Comprehensive analyses of CASP10 protein expression and FAS-mediated apoptosis were conducted and compared to healthy controls and ALPS patients with FAS mutations. Missense CASP10 variants (p.V410I and p.Y446C), which are common in the general population, did not disrupt CASP10 expression, nor FAS-mediated apoptosis. In contrast, homozygous p.C401LfsX15 CASP10 variant lead to a complete abolished CASP10 expression but had no impact on FAS-mediated apoptosis function. At heterozygous state, this p.C401LfsX15 variant lead to a reduced CASP10 protein levels but remained associated with a normal FAS-mediated apoptosis function. These findings demonstrate that CASPASE 10 is dispensable for FAS-mediated apoptosis. In consequences, CASP10 defect unlikely contribute to ALPS pathogenesis, since they did not result in an impairment of FAS-mediated apoptosis nor in clinical features of ALPS in human. Moreover, the absence of FAS expression up-regulation in subjects with CASP10 variants rule out any compensatory mechanisms possibly involved in the normal apoptosis function observed. In conclusion, this study challenges the notion that CASP10 variants contribute to the development of ALPS.

Enhanced CD95 and interleukin 18 signalling accompany T cell receptor Vß21.3+ activation in multi-inflammatory syndrome in children.

Multisystem inflammatory syndrome in children is a post-infectious presentation SARS-CoV-2 associated with expansion of the T cell receptor Vß21.3+ T-cell subgroup. Here we apply muti-single cell omics to compare the inflammatory process in children with acute respiratory COVID-19 and those presenting with non SARS-CoV-2 infections in children. Here we show that in Multi-Inflammatory Syndrome in Children (MIS-C), the natural killer cell and monocyte population demonstrate heightened CD95 (Fas) and Interleuking 18 receptor expression. Additionally, TCR Vß21.3+ CD4+ T-cells exhibit skewed differentiation towards T helper 1, 17 and regulatory T cells, with increased expression of the co-stimulation receptors ICOS, CD28 and interleukin 18 receptor. We observe no functional evidence for NLRP3 inflammasome pathway overactivation, though MIS-C monocytes show elevated active caspase 8. This, coupled with raised IL18 mRNA expression in CD16- NK cells on single cell RNA sequencing analysis, suggests interleukin 18 and CD95 signalling may trigger activation of TCR Vß21.3+ T-cells in MIS-C, driven by increased IL-18 production from activated monocytes and CD16- Natural Killer cells.

Effects of miR-129-5p on inflammation and nucleus pulposus cell apoptosis in rats with intervertebral disc degeneration through JNK signaling pathway.

This study aimed to explore the effects of miR-129-5p on inflammation and nucleus pulposus (NP) cell apoptosis in rats with intervertebral disc degeneration (IVDD) through the c-Jun N-terminal kinase (JNK) signaling pathway. A total of 20 rats were randomly divided into control group (n=10) or IVDD group (n=10). The mRNA expressions of miR-129-5p and apoptosis index Fas in IVDD tissues were determined using RT-PCR. NP cell apoptosis rate was detected via TUNEL assay. NP cells were extracted from IVDD tissues for primary culture. Subsequently, the cells were transfected with miR-129-5p inhibitor or mimic to inhibit or overexpress miR-129-5p, respectively. Furthermore, the changes in the JNK pathway indexes and apoptosis indexes were detected using Western blotting. In IVDD group, the expression of miR-129-5p was significantly down-regulated, while the transcriptional level of Fas was up-regulated compared with those in control group. Pearson correlation analysis revealed a negative correlation between the expressions of miR-129-5p and Fas mRNA (r=-0.75, P<0.05). IVDD group exhibited significantly higher levels of serum TNF-α, IL-6 and IL-1 than control group. Subsequent TUNEL assay indicated that the apoptosis rate was evidently higher in IVDD group (60.6%) than control group (2.5%). The results of Western blotting showed that the protein expressions of JNK1, JNK2 and Fas remarkably rose in IVDD group compared with those in control group. However, they declined remarkably in miR-129-5p mimic group compared with those in control group. Furthermore, such trends were significantly reversed in miR-129-5p inhibitor group. MiR-129-5p was significantly down-regulated in IVDD, whose overexpression has anti-inflammatory and anti-apoptotic effects.

Autoimmune lymphoproliferative immunodeficiencies (ALPIDs): A proposed approach to redefining ALPS and other lymphoproliferative immune disorders.

Chronic nonmalignant lymphoproliferation and autoimmune cytopenia are relevant manifestations of immunohematologic diseases of childhood. Their diagnostic classification is challenging but important for therapy. Autoimmune lymphoproliferative syndrome (ALPS) is a genetically defined inborn error of immunity combining these manifestations, but it can explain only a small proportion of cases. Diagnostic categories such as ALPS-like disease, common variable immunodeficiency, or Evans syndrome have therefore been used. Advances in genetics and increasing availablity of targeted therapies call for more therapy-oriented disease classification. Moreover, recent discoveries in the (re)analysis of genetic conditions affecting FAS signaling ask for a more precise definition of ALPS. In this review, we propose the term autoimmune lymphoproliferative immunodeficiencies for a disease phenotype that is enriched for patients with genetic diseases for which targeted therapies are available. For patients without a current molecular diagnosis, this term defines a subgroup of immune dysregulatory disorders for further studies. Within the concept of autoimmune lymphoproliferative immunodeficiencies, we propose a revision of the ALPS classification, restricting use of this term to conditions with clear evidence of perturbation of FAS signaling and resulting specific biologic and clinical consequences. This proposed approach to redefining ALPS and other lymphoproliferative conditions provides a framework for disease classification and diagnosis that is relevant for the many specialists confronted with these diseases.

Combined germline and somatic human FADD mutations cause autoimmune lymphoproliferative syndrome.

BACKGROUND: The autoimmune lymphoproliferative syndrome (ALPS) is a noninfectious and nonmalignant lymphoproliferative disease frequently associated with autoimmune cytopenia resulting from defective FAS signaling. We previously described germline monoallelic FAS (TNFRSF6) haploinsufficient mutations associated with somatic events, such as loss of heterozygosity on the second allele of FAS, as a cause of ALPS-FAS. These somatic events were identified by sequencing FAS in DNA from double-negative (DN) T cells, the pathognomonic T-cell subset in ALPS, in which the somatic events accumulated. OBJECTIVE: We sought to identify whether a somatic event affecting the FAS-associated death domain (FADD) gene could be related to the disease onset in 4 unrelated patients with ALPS carrying a germline monoallelic mutation of the FADD protein inherited from a healthy parent. METHODS: We sequenced FADD and performed array-based comparative genomic hybridization using DNA from sorted CD4+ or DN T cells. RESULTS: We found homozygous FADD mutations in the DN T cells from all 4 patients, which resulted from uniparental disomy. FADD deficiency caused by germline heterozygous FADD mutations associated with a somatic loss of heterozygosity was a phenocopy of ALPS-FAS without the more complex symptoms reported in patients with germline biallelic FADD mutations. CONCLUSIONS: The association of germline and somatic events affecting the FADD gene is a new genetic cause of ALPS.

Abnormal biomarkers predict complex FAS or FADD defects missed by exome sequencing.

BACKGROUND: Elevated TCRαß+CD4-CD8- double-negative T cells (DNT) and serum biomarkers help identify FAS mutant patients with autoimmune lymphoproliferative syndrome (ALPS). However, in some patients with clinical features and biomarkers consistent with ALPS, germline or somatic FAS mutations cannot be identified on standard exon sequencing (ALPS-undetermined: ALPS-U). OBJECTIVE: We sought to explore whether complex genetic alterations in the FAS gene escaping standard sequencing or mutations in other FAS pathway-related genes could explain these cases. METHODS: Genetic analysis included whole FAS gene sequencing, copy number variation analysis, and sequencing of FAS cDNA and other FAS pathway-related genes. It was guided by FAS expression analysis on CD57+DNT, which can predict somatic loss of heterozygosity (sLOH). RESULTS: Nine of 16 patients with ALPS-U lacked FAS expression on CD57+DNT predicting heterozygous "loss-of-expression" FAS mutations plus acquired somatic second hits in the FAS gene, enriched in DNT. Indeed, 7 of 9 analyzed patients carried deep intronic mutations or large deletions in the FAS gene combined with sLOH detectable in DNT; 1 patient showed a FAS exon duplication. Three patients had reduced FAS expression, and 2 of them harbored mutations in the FAS promoter, which reduced FAS expression in reporter assays. Three of the 4 ALPS-U patients with normal FAS expression carried heterozygous FADD mutations with sLOH. CONCLUSION: A combination of serum biomarkers and DNT phenotyping is an accurate means to identify patients with ALPS who are missed by routine exome sequencing.

NGAL Mediates Anaplastic Thyroid Carcinoma Cells Survival Through FAS/CD95 Inhibition.

Neutrophil gelatinase-associated lipocalin (NGAL), a siderophore-mediated iron binding protein, is highly expressed in human anaplastic thyroid carcinomas (ATCs) where it plays pleiotropic protumorigenic roles including that of a prosurvival protein. Here we show that NGAL inhibits FAS/CD95 death receptor to control ATC cell survival. FAS/CD95 expression in human specimens from patients with ATC and in ATC-derived cell lines negatively correlate with NGAL expression. Silencing of NGAL in ATC cells leads to FAS/CD95 upregulation, whereas NGAL overexpression determines the opposite effect. As a result, an agonist anti-FAS/CD95 antibody induces cell death in NGAL-silenced cells while it is ineffective on NGAL-overexpressing cells. Interestingly, the inhibitory activity of NGAL on FAS/CD95 is due to its iron carrier property given that perturbing iron homeostasis of NGAL-proficient and -deficient ATC cells directly influences FAS/CD95 expression. Accordingly, conditioned media containing a mutant form of NGAL unable to bind siderophores cannot rescue cells from FAS/CD95-dependent death, whereas NGAL wild type-containing conditioned media abolish the effects of the agonist antibody. We also find that downregulation of FAS/CD95 expression is mediated by iron-dependent NGAL suppression of p53 transcriptional activity. Our results indicate that NGAL contributes to ATC cell survival by iron-mediated inhibition of p53-dependent FAS/CD95 expression and suggest that restoring FAS/CD95 by NGAL suppression could be a helpful strategy to kill ATC cells.

Characterizing the regulatory Fas (CD95) epitope critical for agonist antibody targeting and CAR-T bystander function in ovarian cancer.

Receptor clustering is the most critical step to activate extrinsic apoptosis by death receptors belonging to the TNF superfamily. Although clinically unsuccessful, using agonist antibodies, the death receptors-5 remains extensively studied from a cancer therapeutics perspective. However, despite its regulatory role and elevated function in ovarian and other solid tumors, another tumor-enriched death receptor called Fas (CD95) remained undervalued in cancer immunotherapy until recently, when its role in off-target tumor killing by CAR-T therapies was imperative. By comprehensively analyzing structure studies in the context of the binding epitope of FasL and various preclinical Fas agonist antibodies, we characterize a highly significant patch of positively charged residue epitope (PPCR) in its cysteine-rich domain 2 of Fas. PPCR engagement is indispensable for superior Fas agonist signaling and CAR-T bystander function in ovarian tumor models. A single-point mutation in FasL or Fas that interferes with the PPCR engagement inhibited apoptotic signaling in tumor cells and T cells. Furthermore, considering that clinical and immunological features of the autoimmune lymphoproliferative syndrome (ALPS) are directly attributed to homozygous mutations in FasL, we reveal differential mechanistic details of FasL/Fas clustering at the PPCR interface compared to described ALPS mutations. As Fas-mediated bystander killing remains vital to the success of CAR-T therapies in tumors, our findings highlight the therapeutic analytical design for potentially effective Fas-targeting strategies using death agonism to improve cancer immunotherapy in ovarian and other solid tumors.

Digital spatial profiling of melanoma shows CD95 expression in immune cells is associated with resistance to immunotherapy.

Although immune checkpoint inhibitor (ICI) therapy has dramatically improved outcome for metastatic melanoma patients, many patients do not benefit. Since adverse events may be severe, biomarkers for resistance would be valuable, especially in the adjuvant setting. We performed high-plex digital spatial profiling (DSP) using the NanoString GeoMx® on 53 pre-treatment specimens from ICI-treated metastatic melanoma cases. We interrogated 77 targets simultaneously in four molecular compartments defined by S100B for tumor, CD68 for macrophages, CD45 for leukocytes, and nonimmune stromal cells defined as regions negative for all three compartment markers but positive for SYTO 13. For DSP validation, we confirmed the results obtained for some immune markers, such as CD8, CD4, CD20, CD68, CD45, and PD-L1, by quantitative immunofluorescence (QIF). In the univariable analysis, 38 variables were associated with outcome, 14 of which remained significant after multivariable adjustment. Among them, CD95 was further validated using multiplex immunofluorescence in the Discovery immunotherapy (ITX) Cohort and an independent validation cohort with similar characteristics, showing an association between high levels of CD95 and shorter progression-free survival. We found that CD95 in stroma was associated with resistance to ICI. With further validation, this biomarker could have value to select patients that will not benefit from immunotherapy.

Understanding the Spectrum of Immune Dysregulation Manifestations in Autoimmune Lymphoproliferative Syndrome and Autoimmune Lymphoproliferative Syndrome-like Disorders.

As a disorder of immune dysregulation, autoimmune lymphoproliferative syndrome (ALPS) stems from pathogenic variants in the first apoptosis signal-mediated apoptosis (Fas) and Fas-ligand pathway that result in elevations of CD3+ TCRαß+ CD4- CD8- T cells along with chronic lymphoproliferation, a heightened risk for malignancy, and importantly for the rheumatologist, increased risk of autoimmunity. While immune cytopenias are the most encountered autoimmune phenomena, there is increasing appreciation for ocular, musculoskeletal, pulmonary and renal inflammatory manifestations similar to more common rheumatology diseases. Additionally, ALPS-like conditions that share similar clinical features and opportunities for targeted therapy are increasingly recognized via genetic testing, highlighting the need for rheumatologists to be facile in the recognition and diagnosis of this spectrum of disorders. This review will focus on clinical and laboratory features of both ALPS and ALPS-like disorders with the intent to provide a framework for rheumatologists to understand the pathophysiologic drivers and discriminate between diagnoses.

Autoimmune lymphoproliferative syndrome: A disorder of immune dysregulation.

Autoimmune Lymphoproliferative Syndrome (ALPS) is an autoimmune disease that has been reported in over 2200 patients. It is a rare, genetic disease where pathogenic variants occur in the extrinsic pathway of apoptosis. Various mutations in different genes, such as FAS, FASL, and CASP10, can result in ALPS. Most commonly, pathogenic variants occur in the FAS receptor. This malfunctioning pathway allows for the abnormal accumulation of lymphocytes, namely CD3 + TCRαß+CD4 - CD8- (double negative (DN) T) cells, which are a hallmark of the disease. This disease usually presents in childhood with lymphadenopathy and splenomegaly as a result of lymphoproliferation. Over time, these patients may develop cytopenias or lymphomas because of irregularities in the immune system. Current treatments include glucocorticoids, mycophenolate mofetil, sirolimus, immunoglobulin G, and rituximab. These medications serve to manage the symptoms and there are no standardized recommendations for the management of ALPS. The only curative therapy is a bone marrow transplant, but this is rarely done because of the complications. This review serves to broaden the understanding of ALPS by discussing the mechanism of immune dysregulation, how the symptoms manifest, and the mechanisms of treatment. Additionally, we discuss the epidemiology, comorbidities, and medications relating to ALPS patients across the United States using data from Cosmos.

The Role of FAS Receptor Methylation in Osteosarcoma Metastasis.

Osteosarcoma is the most frequent primary malignant bone tumor with an annual incidence of about 400 cases in the United States. Osteosarcoma primarily metastasizes to the lungs, where FAS ligand (FASL) is constitutively expressed. The interaction of FASL and its cell surface receptor, FAS, triggers apoptosis in normal cells; however, this function is altered in cancer cells. DNA methylation has previously been explored as a mechanism for altering FAS expression, but no variability was identified in the CpG island (CGI) overlapping the promoter. Analysis of an expanded region, including CGI shores and shelves, revealed high variability in the methylation of certain CpG sites that correlated significantly with FAS mRNA expression in a negative manner. Bisulfite sequencing revealed additional CpG sites, which were highly methylated in the metastatic LM7 cell line but unmethylated in its parental non-metastatic SaOS-2 cell line. Treatment with the demethylating agent, 5-azacytidine, resulted in a loss of methylation in CpG sites located within the FAS promoter and restored FAS protein expression in LM7 cells, resulting in reduced migration. Orthotopic implantation of 5-azacytidine treated LM7 cells into severe combined immunodeficient mice led to decreased lung metastases. These results suggest that DNA methylation of CGI shore sites may regulate FAS expression and constitute a potential target for osteosarcoma therapy, utilizing demethylating agents currently approved for the treatment of other cancers.

Synthetic receptor platform to identify loss-of-function single nucleotide variants and designed mutants in the death receptor Fas/CD95.

Synthetic biology has emerged as a useful technology for studying cytokine signal transduction. Recently, we described fully synthetic cytokine receptors to phenocopy trimeric receptors such as the death receptor Fas/CD95. Using a nanobody as an extracellular-binding domain for mCherry fused to the natural receptor's transmembrane and intracellular domain, trimeric mCherry ligands were able to induce cell death. Among the 17,889 single nucleotide variants in the SNP database for Fas, 337 represent missense mutations that functionally remained largely uncharacterized. Here, we developed a workflow for the Fas synthetic cytokine receptor system to functionally characterize missense SNPs within the transmembrane and intracellular domain of Fas. To validate our system, we selected five functionally assigned loss-of-function (LOF) polymorphisms and included 15 additional unassigned SNPs. Moreover, based on structural data, 15 gain-of-function or LOF candidate mutations were additionally selected. All 35 nucleotide variants were functionally investigated through cellular proliferation, apoptosis and caspases 3 and 7 cleavage assays. Collectively, our results showed that 30 variants resulted in partial or complete LOF, while five lead to a gain-of-function. In conclusion, we demonstrated that synthetic cytokine receptors are a suitable tool for functional SNPs/mutations characterization in a structured workflow.

The role of death receptor signaling pathways in mouse Sertoli cell avoidance of apoptosis during LPS- and IL-18-induced inflammatory conditions.

Lipopolysaccharide (LPS) triggers infectious acute inflammation, and interleukin (IL)-18 is an inflammasome-mediated cytokine. We previously demonstrated that endogenous IL-18 induces testicular germ cell apoptosis during acute inflammation when plasma IL-18 levels are high. Additionally, high-dose recombinant IL-18 (rIL-18) induced Leydig cell apoptosis. The blood-testis barrier formed by Sertoli cells protects testicular germ cells from both exogenous and endogenous harmful substances. However, the impact of LPS and IL-18 on Sertoli cells remained unclear. We stimulated TM4 cells, a mouse Sertoli cell line, with LPS (200 or 1000 ng/mL) or rIL-18 (0.1-100 ng/mL) at levels that induced Leydig cell apoptosis in our previous study and assessed caspase 3 cleavage and the mRNA expression of inflammatory cytokines and markers of apoptotic pathways (Tnfr1, Fasl, Fas, Fadd) after stimulation. Il6 mRNA was increased by LPS stimulation. Tnfα mRNA was increased by 200 ng/mL LPS but not 1000 ng/mL LPS. Fas was increased, but Fasl was decreased, by LPS. LPS had little influence on Tnfr1 or Fadd mRNA expression and did not induce apoptosis. Il18 mRNA was not increased, and Il18r1 was significantly decreased following LPS treatment. Treatment with rIL-18 increased Il18r1 mRNA and induced inflammation, but decreased Tnfr1 and had little influence on apoptosis, as indicated by Tnfα, Fasl, Fas, Fadd and cleaved caspase 3. These results suggested that Sertoli cells do not easily undergo apoptosis despite strong inflammatory stimuli. Additionally, Sertoli cells may resist inflammation and play a larger role in protecting testicular homeostasis than other component cells of the testis.

Revisiting autoimmune lymphoproliferative syndrome caused by Fas ligand mutations.

BACKGROUND: Fas ligand (FasL) is expressed by activated T cells and induces death in target cells upon binding to Fas. Loss-of-function FAS or FASLG mutations cause autoimmune-lymphoproliferative syndrome (ALPS) characterized by expanded double-negative T cells (DNT) and elevated serum biomarkers. While most ALPS patients carry heterozygous FAS mutations, FASLG mutations are rare and usually biallelic. Only 2 heterozygous variants were reported, associated with an atypical clinical phenotype. OBJECTIVE: We revisited the significance of heterozygous FASLG mutations as a cause of ALPS. METHODS: Clinical features and biomarkers were analyzed in 24 individuals with homozygous or heterozygous FASLG variants predicted to be deleterious. Cytotoxicity assays were performed with patient T cells and biochemical assays with recombinant FasL. RESULTS: Homozygous FASLG variants abrogated cytotoxicity and resulted in early-onset severe ALPS with elevated DNT, raised vitamin B12, and usually no soluble FasL. In contrast, heterozygous variants affected FasL function by reducing expression, impairing trimerization, or preventing Fas binding. However, they were not associated with elevated DNT and vitamin B12, and they did not affect FasL-mediated cytotoxicity. The dominant-negative effects of previously published variants could not be confirmed. Even Y166C, causing loss of Fas binding with a dominant-negative effect in biochemical assays, did not impair cellular cytotoxicity or cause vitamin B12 and DNT elevation. CONCLUSION: Heterozygous loss-of-function mutations are better tolerated for FASLG than for FAS, which may explain the low frequency of ALPS-FASLG.

Bifidobacterium animalis sup F1-7 Acts as an Effective Activator to Regulate Immune Response Via Casepase-3 and Bak of FAS/CD95 Pathway.

Intestinal microecology was closely related to immune regulation, but the related mechanism was still unclear. This study aimed to reveal how microorganisms improved immune response via casepase-3 and Bak of FAS/CD95 pathway. Bifidobacterium animalis F1-7 inhibited the melanoma B16-F10 cells in vitro effectively; had a potent anticancer effect of lung cancer mice; effectively improved the spleen immune index and CD3+ (75.8%) and CD8+ (19.8%) expression level; strengthened the phagocytosis of macrophages; inhibited the overexpression of inflammatory factors IL-6 (319.10 ± 2.46 pg/mL), IL-8 (383.05 ± 9.87 pg/mL), and TNF-α (2003.40 ± 11.42 pg/mL); and promoted the expression of anti-inflammatory factor IL-10 (406.00 ± 3.59 pg/mL). This process was achieved by promoting caspase-8/3 and BH3-interacting domain death agonist (Bid), Bak genes, and protein expression. This study confirmed the B. animalis F1-7 could act as an effective activator to regulate immune response by promoting the expression of caspase-8/3, Bid and Bak genes, and proteins and by activating the FAS/CD95 pathway. Our study provided a data support for the application of potentially beneficial microorganisms of B. animalis F1-7 as an effective activator to improve immunity.

Unusual Polyclonal IgM in an IgG Deficient Patient with Autoimmune Lymphoproliferative Syndrome: A Case Study and Literature Review.

Autoimmune lymphoproliferative syndrome (ALPS) is a rare genetic disease caused germline mutation of FAS gene, gene encoding Fas ligand or Caspase 10 gene. However, in 20% of all ALPS patients, genetic defect is unknown. We presented a case of a 20-year-old male with a history of autoimmune lymphoproliferative syndrome (ALPS; confirmed by genetic study) who came to our medical center with a concern for malignancy. Although no malignancy was detected, his lack of IgA, very low level of IgG (requiring therapy with intravenous IgG) and highly elevated polyclonal IgM (hyperimmunoglobulin M syndrome) were unusual findings because ALPS patients with hypergammaglobulinemia usually demonstrate elevated IgA or IgG.

CRISPR/Cas9-mediated abrogation of CD95L/CD95 signaling-induced glioma cell growth and immunosuppression increases survival in murine glioma models.

PURPOSE: Glioblastoma is the most common brain tumor in adults and is virtually incurable. Therefore, new therapeutic strategies are urgently needed. Over the last decade, multiple growth-promoting functions have been attributed to CD95, a prototypic death receptor well characterized as an apoptosis mediator upon CD95L engagement. Strategic targeting of non-apoptotic or apoptotic CD95 signaling may hold anti-glioblastoma potential. Due to its antithetic nature, understanding the constitutive role of CD95 signaling in glioblastoma is indispensable. METHODS: We abrogated constitutive Cd95 and Cd95l gene expression by CRISPR/Cas9 in murine glioma models and characterized the consequences of gene deletion in vitro and in vivo. RESULTS: Expression of canonical CD95 but not CD95L was identified in mouse glioma cells in vitro. Instead, a soluble isoform-encoding non-canonical Cd95l transcript variant was detected. In vivo, an upregulation of the membrane-bound canonical CD95L form was revealed. Cd95 or Cd95l gene deletion decreased cell growth in vitro. The growth-supporting role of constitutive CD95 signaling was validated by Cd95 re-transfection, which rescued growth. In vivo, Cd95 or Cd95l gene deletion prolonged survival involving tumor-intrinsic and immunological mechanisms in the SMA-497 model. In the GL-261 model, that expresses no CD95, only CD95L gene deletion prolonged survival, involving a tumor-intrinsic mechanism. CONCLUSION: Non-canonical CD95L/CD95 interactions are growth-promoting in murine glioma models, and glioma growth and immunosuppression may be simultaneously counteracted by Cd95l gene silencing.