Neutrophil elastase selectively kills cancer cells and attenuates tumorigenesis.

Cancer cell genetic variability and similarity to host cells have stymied development of broad anti-cancer therapeutics. Our innate immune system evolved to clear genetically diverse pathogens and limit host toxicity; however, whether/how innate immunity can produce similar effects in cancer is unknown. Here, we show that human, but not murine, neutrophils release catalytically active neutrophil elastase (ELANE) to kill many cancer cell types while sparing non-cancer cells. ELANE proteolytically liberates the CD95 death domain, which interacts with histone H1 isoforms to selectively eradicate cancer cells. ELANE attenuates primary tumor growth and produces a CD8+T cell-mediated abscopal effect to attack distant metastases. Porcine pancreatic elastase (ELANE homolog) resists tumor-derived protease inhibitors and exhibits markedly improved therapeutic efficacy. Altogether, our studies suggest that ELANE kills genetically diverse cancer cells with minimal toxicity to non-cancer cells, raising the possibility of developing it as a broad anti-cancer therapy.

Fas Promotes T Helper 17 Cell Differentiation and Inhibits T Helper 1 Cell Development by Binding and Sequestering Transcription Factor STAT1.

The death receptor Fas removes activated lymphocytes through apoptosis. Previous transcriptional profiling predicted that Fas positively regulates interleukin-17 (IL-17)-producing T helper 17 (Th17) cells. Here, we demonstrate that Fas promoted the generation and stability of Th17 cells and prevented their differentiation into Th1 cells. Mice with T-cell- and Th17-cell-specific deletion of Fas were protected from induced autoimmunity, and Th17 cell differentiation and stability were impaired. Fas-deficient Th17 cells instead developed a Th1-cell-like transcriptional profile, which a new algorithm predicted to depend on STAT1. Experimentally, Fas indeed bound and sequestered STAT1, and Fas deficiency enhanced IL-6-induced STAT1 activation and nuclear translocation, whereas deficiency of STAT1 reversed the transcriptional changes induced by Fas deficiency. Thus, our computational and experimental approach identified Fas as a regulator of the Th17-to-Th1 cell balance by controlling the availability of opposing STAT1 and STAT3 to have a direct impact on autoimmunity.

IRE1 RNase controls CD95-mediated cell death.

Signalling by the Unfolded Protein Response (UPR) or by the Death Receptors (DR) are frequently activated towards pro-tumoral outputs in cancer. Herein, we demonstrate that the UPR sensor IRE1 controls the expression of the DR CD95/Fas, and its cell death-inducing ability. Both genetic and pharmacologic blunting of IRE1 activity increased CD95 expression and exacerbated CD95L-induced cell death in glioblastoma (GB) and Triple-Negative Breast Cancer (TNBC) cell lines. In accordance, CD95 mRNA was identified as a target of Regulated IRE1-Dependent Decay of RNA (RIDD). Whilst CD95 expression is elevated in TNBC and GB human tumours exhibiting low RIDD activity, it is surprisingly lower in XBP1s-low human tumour samples. We show that IRE1 RNase inhibition limited CD95 expression and reduced CD95-mediated hepatic toxicity in mice. In addition, overexpression of XBP1s increased CD95 expression and sensitized GB and TNBC cells to CD95L-induced cell death. Overall, these results demonstrate the tight IRE1-mediated control of CD95-dependent cell death in a dual manner through both RIDD and XBP1s, and they identify a novel link between IRE1 and CD95 signalling.

Impact of human CD95 mutations on cell death and autoimmunity: a model.

CD95/Fas/APO-1 can trigger apoptotic as well as nonapoptotic pathways in immune cells. CD95 signaling in humans can be inhibited by several mechanisms, including mutations in the gene encoding CD95. CD95 mutations lead to autoimmune disorders, such as autoimmune lymphoproliferative syndrome (ALPS). Gaining further insight into the reported mutations of CD95 and resulting alterations of its signaling networks may provide further understanding of their presumed role in certain autoimmune diseases. For illustrative purposes and to better understand the potential outcomes of CD95 mutations, here we assign their positions to the recently determined 3D structures of human CD95. Based on this, we make certain predictions and speculate on the putative role of CD95 mutation defects in CD95-mediated signaling for certain autoimmune diseases.

CD95-Mediated Calcium Signaling Promotes T Helper 17 Trafficking to Inflamed Organs in Lupus-Prone Mice.

CD95 ligand (CD95L) is expressed by immune cells and triggers apoptotic death. Metalloprotease-cleaved CD95L (cl-CD95L) is released into the bloodstream but does not trigger apoptotic signaling. Hence, the pathophysiological role of cl-CD95L remains unclear. We observed that skin-derived endothelial cells from systemic lupus erythematosus (SLE) patients expressed CD95L and that after cleavage, cl-CD95L promoted T helper 17 (Th17) lymphocyte transmigration across the endothelial barrier at the expense of T regulatory cells. T cell migration relied on a direct interaction between the CD95 domain called calcium-inducing domain (CID) and the Src homology 3 domain of phospholipase Cγ1. Th17 cells stimulated with cl-CD95L produced sphingosine-1-phosphate (S1P), which promoted endothelial transmigration by activating the S1P receptor 3. We generated a cell-penetrating CID peptide that prevented Th17 cell transmigration and alleviated clinical symptoms in lupus mice. Therefore, neutralizing the CD95 non-apoptotic signaling pathway could be an attractive therapeutic approach for SLE treatment.

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.

A Novel Tetravalent CD95/Fas Fusion Protein With Superior CD95L/FasL Antagonism.

Inhibition of CD95/Fas activation is currently under clinical investigation as a therapy for glioblastoma multiforme and preclinical studies suggest that disruption of the CD95-CD95L interaction could also be a strategy to treat inflammatory and neurodegenerative disorders. Besides neutralizing anti-CD95L/FasL antibodies, mainly CD95ed-Fc, a dimeric Fc fusion protein of the extracellular domain of CD95 (CD95ed), is used to prevent CD95 activation. In view of the fact that full CD95 activation requires CD95L-induced CD95 trimerization and clustering of the resulting liganded CD95 trimers, we investigated whether fusion proteins of the extracellular domain of CD95 with a higher valency than CD95ed-Fc have an improved CD95L-neutralization capacity. We evaluated an IgG1(N297A)-based tetravalent CD95ed fusion protein which was obtained by replacing the variable domains of IgG1(N297A) with CD95ed (CD95ed-IgG1(N297A)) and a hexavalent variant obtained by fusion of CD95ed with a TNC-Fc(DANA) scaffold (CD95ed-TNC-Fc(DANA)) promoting hexamerization. The established N297A and DANA mutations were used to minimize FcγR binding of the constructs under maintenance of neonatal Fc receptor (FcRn) binding. Size exclusion high-performance liquid chromatography indicated effective assembly of CD95ed-IgG1(N297A). More important, CD95ed-IgG1(N297A) was much more efficient than CD95ed-Fc in protecting cells from cell death induction by human and murine CD95L. Surprisingly, despite its hexavalent structure, CD95ed-TNC-Fc(DANA) displayed an at best minor improvement of the capacity to neutralize CD95L suggesting that besides valency, other factors, such as spatial organization and agility of the CD95ed domains, play also a role in neutralization of CD95L trimers by CD95ed fusion proteins. More studies are now required to evaluate the superior CD95L-neutralizing capacity of CD95ed-IgG1(N297A) in vivo.

Membrane-bound CD95 ligand modulates CD19-mediated B cell receptor signaling and EBV activation.

Post-transplant lymphoproliferative disorders (PTLDs) are associated with Epstein-Barr virus (EBV) infection in transplant recipients. Most of lymphoblastoid cell lines (LCLs) derived from EBV-immortalized B cells or PTLDs are sensitive to CD95-mediated apoptosis and cytotoxic T cell (CTL) killing. CD95 ligand (CD95L) exists as a transmembrane ligand (mCD95L) or a soluble form (sCD95L). Using recombinant mCD95L and sCD95L, we observed that sCD95L does not affect LCLs. While high expression of mCD95L in CTLs promotes apoptosis of LCLs, low expression induces clathrin-dependent CD19 internalization, caspase-dependent CD19 cleavage, and proteasomal/lysosomal-dependent CD19 degradation. The CD95L/CD95-mediated CD19 degradation impairs B cell receptor (BCR) signaling and inhibits BCR-mediated EBV activation. Interestingly, although inhibition of the caspase activity restores CD19 expression and CD19-mediated BCR activation, it fails to rescue BCR-mediated EBV lytic gene expression. EBV-specific CTLs engineered to overexpress mCD95L exhibit a stronger killing activity against LCLs. This study highlights that engineering EBV-specific CTLs to express a higher level of mCD95L could represent an attractive therapeutic approach to improve T cell immunotherapy for PTLDs.

Unsolved mystery of Fas: mononuclear cells may have trouble dying in patients with Sjögren's syndrome.

BACKGROUND: Patients with Sjögren's syndrome, like other patients with autoimmune disorders, display dysregulation in the function of their immune system. Fas and Fas Ligand (FasL) are among the dysregulated proteins. METHODS: We studied Fas and FasL on IL-2Rα+ cells and in serum of patients with Sjögren's syndrome (n = 16) and healthy individuals (n = 16); both from same ethnic and geographical background. We used flow cytometry and enzyme-linked immunosorbent for this purpose. We also measured the expression of Bcl-2 and Bax by reverse transcription quantitative real-time PCR (RT-qPCR) and percentage of apoptotic and dead cells using Annexin V and 7-AAD staining in lymphocytes. RESULTS: FasL was increased in patients' T and B cells while Fas was increased in patients' monocytes, T and B cells. No signs of increased apoptosis were found. sFas and sFasL in patients' serum were increased, although the increase in sFasL was not significant. We suspect an effect of non-steroidal anti-inflammatory therapy on B cells, explaining the decrease of the percentage Fas+ B cells found within our samples. In healthy individuals, there was a noticeable pattern in the expression of FasL which mutually correlated to populations of mononuclear cells; this correlation was absent in the patients with Sjögren's syndrome. CONCLUSIONS: Mononuclear cells expressing IL-2Rα+ had upregulated Fas in Sjögren's syndrome. However, the rate of apoptosis based on Annexin V staining and the Bcl-2/Bax expression was not observed in mononuclear cells. We suspect a functional role of abnormal levels of Fas and FasL which has not been cleared yet.

High activation levels maintained in receptor-binding domain-specific memory B cells in people with severe coronavirus disease 2019.

The long-term health consequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are still being understood. The molecular and phenotypic properties of SARS-CoV-2 antigen-specific T cells suggest a dysfunctional profile that persists in convalescence in those who were severely ill. By contrast, the antigen-specific memory B-cell (MBC) population has not yet been analyzed to the same degree, but phenotypic analysis suggests differences following recovery from mild or severe coronavirus disease 2019 (COVID-19). Here, we performed single-cell molecular analysis of the SARS-CoV-2 receptor-binding domain (RBD)-specific MBC population in three patients after severe COVID-19 and four patients after mild/moderate COVID-19. We analyzed the transcriptomic and B-cell receptor repertoire profiles at ~2 months and ~4 months after symptom onset. Transcriptomic analysis revealed a higher level of tumor necrosis factor-alpha (TNF-α) signaling via nuclear factor-kappa B in the severe group, involving CD80, FOS, CD83 and TNFAIP3 genes that was maintained over time. We demonstrated the presence of two distinct activated MBCs subsets based on expression of CD80hi TNFAIP3hi and CD11chi CD95hi at the transcriptome level. Both groups revealed an increase in somatic hypermutation over time, indicating progressive evolution of humoral memory. This study revealed distinct molecular signatures of long-term RBD-specific MBCs in convalescence, indicating that the longevity of these cells may differ depending on acute COVID-19 severity.

Resistance of Human Liver Mesenchymal Stem Cells to FAS-Induced Cell Death.

Mesenchymal stem cells (MSCs) have a pronounced therapeutic potential in various pathological conditions. Though therapeutic effects of MSC transplantation have been studied for a long time, the underlying mechanisms are still not clear. It has been shown that transplanted MSCs are rapidly eliminated, presumably by apoptosis. As the mechanisms of MSC apoptosis are not fully understood, in the present work we analyzed MSC sensitivity to Fas-induced apoptosis using MSCs isolated from the biopsies of liver fibrosis patients (L-MSCs). The level of cell death was analyzed by flow cytometry in the propidium iodide test. The luminescent ATP assay was used to measure cellular ATP levels; and the mitochondrial membrane potential was assessed using the potential-dependent dye JC-1. We found that human L-MSCs were resistant to Fas-induced cell death over a wide range of FasL and anti-Fas mAb concentrations. At the same time, intrinsic death signal inducers CoCl2 and staurosporine caused apoptosis of L-MSCs in a dose-dependent manner. Despite the absence of Fas-induced cell death treatment of L-MSCs with low concentrations of FasL or anti-Fas mAb resulted in a cellular ATP level decrease, while high concentrations of the inducers caused a decline of the mitochondrial membrane potential. Pre-incubation of L-MSCs with the pro-inflammatory cytokine TNF-α did not promote L-MSC cell death. Our data indicate that human L-MSCs have increased resistance to receptor-mediated cell death even under inflammatory conditions.

Optimizing NK-92 serial killers: gamma irradiation, CD95/Fas-ligation, and NK or LAK attack limit cytotoxic efficacy.

BACKGROUND: The NK cell line NK-92 and its genetically modified variants are receiving attention as immunotherapies to treat a range of malignancies. However, since NK-92 cells are themselves tumors, they require irradiation prior to transfer and are potentially susceptible to attack by patients' immune systems. Here, we investigated NK-92 cell-mediated serial killing for the effects of gamma-irradiation and ligation of the death receptor Fas (CD95), and NK-92 cell susceptibility to attack by activated primary blood NK cells. METHODS: To evaluate serial killing, we used 51Cr-release assays with low NK-92 effector cell to target Raji, Daudi or K562 tumor cell (E:T) ratios to determine killing frequencies at 2-, 4-, 6-, and 8-h. RESULTS: NK-92 cells were able to kill up to 14 Raji cells per NK-92 cell in 8 h. NK-92 cells retained high cytotoxic activity immediately after irradiation with 10 Gy but the cells surviving irradiation lost > 50% activity 1 day after irradiation. Despite high expression of CD95, NK-92 cells maintained their viability following overnight Fas/CD95-ligation but lost some cytotoxic activity. However, 1 day after irradiation, NK-92 cells were more susceptible to Fas ligation, resulting in decreased cytotoxic activity of the cells surviving irradiation. Irradiated NK-92 cells were also susceptible to killing by both unstimulated and IL-2 activated primary NK cells (LAK). In contrast, non-irradiated NK-92 cells were more resistant to attack by NK and LAK cells. CONCLUSIONS: Irradiation is deleterious to both the survival and cytotoxicity mediated by NK-92 cells and renders the NK-92 cells susceptible to Fas-initiated death and death initiated by primary blood NK cells. Therefore, replacement of irradiation as an antiproliferative pretreatment and genetic deletion of Fas and/or NK activation ligands from adoptively transferred cell lines are indicated as new approaches to increase therapeutic efficacy.

Clusters of apoptotic signaling molecule-enriched rafts, CASMERs: membrane platforms for protein assembly in Fas/CD95 signaling and targets in cancer therapy.

Mammalian cells show the ability to commit suicide through the activation of death receptors at the cell surface. Death receptors, among which Fas/CD95 is one of their most representative members, lack enzymatic activity, and depend on protein-protein interactions to signal apoptosis. Fas/CD95 death receptor-mediated apoptosis requires the formation of the so-called death-inducing signaling complex (DISC), bringing together Fas/CD95, Fas-associated death domain-containing protein and procaspase-8. In the last two decades, cholesterol-rich lipid raft platforms have emerged as scaffolds where Fas/CD95 can be recruited and clustered. The co-clustering of Fas/CD95 and rafts facilitates DISC formation, bringing procaspase-8 molecules to be bunched together in a limited membrane region, and leading to their autoproteolytic activation by oligomerization. Lipid raft platforms serve as a specific region for the clustering of Fas/CD95 and DISC, as well as for the recruitment of additional downstream signaling molecules, thus forming the so-called cluster of apoptotic signaling molecule-enriched rafts, or CASMER. These raft/CASMER structures float in the membrane like icebergs, in which the larger portion lies inside the cell and communicates with other subcellular structures to facilitate apoptotic signal transmission. This allows an efficient spatiotemporal compartmentalization of apoptosis signaling machinery during the triggering of cell death. This concept of proapoptotic raft platforms as a basic chemical-biological structure in the regulation of cell death has wide-ranging implications in human biology and disease, as well as in cancer therapy. Here, we discuss how these raft-centered proapoptotic hubs operate as a major linchpin for apoptosis signaling and as a promising target in cancer therapy.

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.

Human UDP-galactose 4'-epimerase (GALE) is required for cell-surface glycome structure and function.

Glycan biosynthesis relies on nucleotide sugars (NSs), abundant metabolites that serve as monosaccharide donors for glycosyltransferases. In vivo, signal-dependent fluctuations in NS levels are required to maintain normal cell physiology and are dysregulated in disease. However, how mammalian cells regulate NS levels and pathway flux remains largely uncharacterized. To address this knowledge gap, here we examined UDP-galactose 4'-epimerase (GALE), which interconverts two pairs of essential NSs. Using immunoblotting, flow cytometry, and LC-MS-based glycolipid and glycan profiling, we found that CRISPR/Cas9-mediated GALE deletion in human cells triggers major imbalances in NSs and dramatic changes in glycolipids and glycoproteins, including a subset of integrins and the cell-surface death receptor FS-7-associated surface antigen. In particular, we observed substantial decreases in total sialic acid, galactose, and GalNAc levels in glycans. These changes also directly impacted cell signaling, as GALE-/- cells exhibited FS-7-associated surface antigen ligand-induced apoptosis. Our results reveal a role of GALE-mediated NS regulation in death receptor signaling and may have implications for the molecular etiology of illnesses characterized by NS imbalances, including galactosemia and metabolic syndrome.

Systemic treatment with alpha-tocopherol and/or sodium selenite decreases the progression of experimental periodontitis.

OBJECTIVE: To investigate the effects of sodium selenite (Se) and/or α-tocopherol (αT) applications on the alveolar bone loss (ABL), the number of gingival collagen fibers, inducible nitric oxide synthase (iNOS)+ and CD95+ cell numbers, and serum cytokine concentrations in experimental periodontitis in rats. MATERIALS AND METHODS: Forty Sprague Dawley rats were divided into four groups of ten as follows: group A: Se group, group B: αT group, group C: Se and αT combined group, and group D: control group (intraperitoneal (IP) saline injection applied). Using the image analysis method in the connective tissue under the connective epithelium, the numbers of iNOS, CD95 positive cells, and collagen fibers were counted. ELISA kits were used to test the concentrations of serum interleukin (IL)-1ß, IL-6, and IL-4. RESULTS: The combination of Se and αT (group C) suppressed ABL compared with the control group (group D) (P < 0.05). In group A (Se), the number of iNOS+ cells was smaller than in group D (P < 0.05). CONCLUSION: Se has been concluded to inhibit inflammation of the gum due to iNOS. Se and αT can have a remarkable important role in preventing alveolar bone loss, and particularly in combination. CLINICAL RELEVANCE: Se and/or αT application may be useful in preventing the destruction of periodontal tissue and treatment of periodontal disease.

Lipid rafts as signaling hubs in cancer cell survival/death and invasion: implications in tumor progression and therapy: Thematic Review Series: Biology of Lipid Rafts.

Cholesterol/sphingolipid-rich membrane domains, known as lipid rafts or membrane rafts, play a critical role in the compartmentalization of signaling pathways. Physical segregation of proteins in lipid rafts may modulate the accessibility of proteins to regulatory or effector molecules. Thus, lipid rafts serve as sorting platforms and hubs for signal transduction proteins. Cancer cells contain higher levels of intracellular cholesterol and lipid rafts than their normal non-tumorigenic counterparts. Many signal transduction processes involved in cancer development (insulin-like growth factor system and phosphatidylinositol 3-kinase-AKT) and metastasis [cluster of differentiation (CD)44] are dependent on or modulated by lipid rafts. Additional proteins playing an important role in several malignant cancers (e.g., transmembrane glycoprotein mucin 1) are also being detected in association with lipid rafts, suggesting a major role of lipid rafts in tumor progression. Conversely, lipid rafts also serve as scaffolds for the recruitment and clustering of Fas/CD95 death receptors and downstream signaling molecules leading to cell death-promoting raft platforms. The partition of death receptors and downstream signaling molecules in aggregated lipid rafts has led to the formation of the so-called cluster of apoptotic signaling molecule-enriched rafts, or CASMER, which leads to apoptosis amplification and can be pharmacologically modulated. These death-promoting rafts can be viewed as a linchpin from which apoptotic signals are launched. In this review, we discuss the involvement of lipid rafts in major signaling processes in cancer cells, including cell survival, cell death, and metastasis, and we consider the potential of lipid raft modulation as a promising target in cancer therapy.

Slow progressors to type 1 diabetes lose islet autoantibodies over time, have few islet antigen-specific CD8+ T cells and exhibit a distinct CD95hi B cell phenotype.

AIMS/HYPOTHESIS: The aim of this study was to characterise islet autoantibody profiles and immune cell phenotypes in slow progressors to type 1 diabetes. METHODS: Immunological variables were compared across peripheral blood samples obtained from slow progressors to type 1 diabetes, individuals with newly diagnosed or long-standing type 1 diabetes, and healthy individuals. Polychromatic flow cytometry was used to characterise the phenotypic attributes of B and T cells. Islet autoantigen-specific B cells were quantified using an enzyme-linked immunospot (ELISpot) assay and islet autoantigen-specific CD8+ T cells were quantified using peptide-HLA class I tetramers. Radioimmunoassays were used to detect islet autoantibodies. Sera were assayed for various chemokines, cytokines and soluble receptors via ELISAs. RESULTS: Islet autoantibodies were lost over time in slow progressors. Various B cell subsets expressed higher levels of CD95 in slow progressors, especially after polyclonal stimulation, compared with the corresponding B cell subsets in healthy donors (p < 0.05). The phenotypic characteristics of CD4+ and CD8+ T cells were similar in slow progressors and healthy donors. Lower frequencies of CD4+ T cells with a central memory phenotype (CD27int, CD127+, CD95int) were observed in slow progressors compared with healthy donors (mean percentage of total CD4+ T cells was 3.00% in slow progressors vs 4.67% in healthy donors, p < 0.05). Autoreactive B cell responses to proinsulin were detected at higher frequencies in slow progressors compared with healthy donors (median no. of spots was 0 in healthy donors vs 24.34 in slow progressors, p < 0.05) in an ELISpot assay. Islet autoantigen-specific CD8+ T cell responses were largely absent in slow progressors and healthy donors. Serum levels of DcR3, the decoy receptor for CD95L, were elevated in slow progressors compared with healthy donors (median was 1087 pg/ml in slow progressors vs 651 pg/ml in healthy donors, p = 0.06). CONCLUSIONS/INTERPRETATION: In this study, we found that slow progression to type 1 diabetes was associated with a loss of islet autoantibodies and a distinct B cell phenotype, consistent with enhanced apoptotic regulation of peripheral autoreactivity via CD95. These phenotypic changes warrant further studies in larger cohorts to determine their functional implications.

Increased CD95 (Fas) and PD-1 expression in peripheral blood T lymphocytes in COVID-19 patients.

A low count of CD4+ and CD8+ lymphocytes is a hallmark laboratory finding in the coronavirus disease 2019 (COVID-19). Using flow cytometry, we observed significantly higher CD95 (Fas) and PD-1 expression on both CD4+ T and CD8+ T cells in 42 COVID-19 patients when compared to controls. Higher CD95 expression in CD4+ cells correlated with lower CD4+ counts. A higher expression of CD95 in CD4+ and CD8+ lymphocytes correlated with a lower percentage of naive events. Our results might suggest a shift to antigen-activated T cells, expressing molecules increasing their propensity to apoptosis and exhaustion during COVID-19 infection.

Modulation of CD95-mediated signaling by post-translational modifications: towards understanding CD95 signaling networks.

CD95 is a member of the death receptor family and is well-known to promote apoptosis. However, accumulating evidence indicates that in some context CD95 has not only the potential to induce apoptosis but also can trigger non-apoptotic signal leading to cell survival, proliferation, cancer growth and metastasis. Despite extensive investigations focused on alterations in the expression level of CD95 and associated signal molecules, very few studies, however, have investigated the effects of post-translational modifications such as glycosylation, phosphorylation, palmitoylation, nitrosylation and glutathionylation on CD95 function. Post-translational modifications of CD95 in mammalian systems are likely to play a more prominent role than anticipated in CD95 induced cell death. In this review we will focus on the alterations in CD95-mediated signaling caused by post-translational modifications of CD95.