Fas-mediated Apoptosis Research Articles

Interleukin-7 improves the fitness of regulatory T cells for adoptive transfer.

Adoptive regulatory T-cell (Treg) transfer has emerged as a promising therapeutic strategy for regulating immune responses in organ transplantation, graft versus host disease, and autoimmunity, including Type 1 diabetes. Traditionally, Treg for adoptive therapy have been sorted and expanded in vitro using high doses of IL-2, demonstrating stability and suppressive capabilities. However, limitations in their long-term survival post-infusion into patients have been observed. To address this challenge, we investigated a novel expansion protocol incorporating interleukin-7 (IL-7) alongside the traditional method utilizing IL-2 (referred to as IL-7 method, IL-7M). Our study revealed that naïve Treg express significant levels of CD127 and display robust responsiveness to IL-7, characterized by STAT-5 phosphorylation. Expanding naïve Treg with the IL-7M protocol led to a substantial enrichment of CD45RA+ CD62L+ CD95+ Treg but showing a reduction in the final cell yield and suppressive function. Moreover, Treg expanded with the IL-7M exhibited preserved telomere length and demonstrated enhanced resistance to cytokine withdrawal and fas-mediated apoptosis. When transferred into NSG mice IL-7M-Treg persisted longer and reduced the expansion of T cells, but did not significantly reduce the severity of xenoGvHD. In conclusion, our data demonstrate the feasibility of expanding naïve Treg in the presence of IL-7 to generate a Treg product enriched in poorly differentiated CD45RA+ cells with enhanced survival capabilities.

MiR-21 attenuates FAS-mediated cardiomyocyte apoptosis by regulating HIPK3 expression.

MicroRNA-21 (miR-21) plays an anti-apoptotic role following ischemia-reperfusion (I/R) injury (IRI) in vivo; however, its underlying mechanism remains unclear. This study explored the effects of miR-21 and homeodomain interacting protein kinase 3 (HIPK3) on cardiomyocyte apoptosis induced by hypoxia/reoxygenation (H/R) in vitro. To this end, the rat cardiomyocyte H9C2 cell line was exposed to H/R and the roles of miR-21 and HIPK3 in regulating cell viability and apoptosis were evaluated by cell counting kit-8 assay, terminal-deoxynucleotidyl-transferase -mediated dUTP nick end labeling, and flow cytometry. Immunofluorescence and western blotting were performed to detect the expression/phosphorylation of apoptosis-related proteins. miR-21 expression was measured with quantitative real-time polymerase chain reaction. The putative interaction between miR-21 and HIPK3 was evaluated using the luciferase reporter assay. Our results showed that: (i) miR-21 overexpression or HIPK3 downregulation significantly attenuated H9C2 cells apoptosis after H/R; (ii) suppression of miR-21 expression promoted apoptosis; (iii) miR-21 overexpression inhibited HIPK3 expression; (iv) HIPK3 was the direct and main target of miR-21; (v) miR-21/HIPK3 formed part of a reciprocal, negative feedback loop; and (vi) HIPK3 downregulation decreased FAS-mediated apoptosis by inhibiting the phosphorylation of FADD, which subsequently inhibited the expression of BAX and cleaved caspase-3 and increased the expression of BCL2. Our study indicates that miR-21 attenuates FAS-mediated cardiomyocyte apoptosis by regulating HIPK3 expression, which could eventually have important clinical implications for patients with acute myocardial infarction.

Levamisole suppresses activation and proliferation of human T cells by the induction of a p53-dependent DNA damage response.

Levamisole (LMS) is a small molecule used in the treatment of idiopathic nephrotic syndrome (INS). The pathogenesis of INS remains unknown, but evidence points toward an immunological basis of the disease. Recently, LMS has been shown to increase the relapse-free survival in INS patients. While LMS has been hypothesized to exert an immunomodulatory effect, its mechanism of action remains unknown. Here, we show that LMS decreased activation and proliferation of human T cells. T-cell activation-associated cytokines such as IL-2, TNF-α, and IFN-γ were reduced upon LMS treatment, whereas IL-4 and IL-13 were increased. Gene expression profiling confirmed that the suppressive effects of LMS as genes involved in cell cycle progression were downregulated. Furthermore, genes associated with p53 activation were upregulated by LMS. In agreement, LMS treatment resulted in p53 phosphorylation and increased expression of the p53 target gene FAS. Accordingly, LMS sensitized activated T cells for Fas-mediated apoptosis. LMS treatment resulted in a mid-S phase cell cycle arrest accompanied by γH2AX-foci formation and phosphorylation of CHK1. Our findings indicate that LMS acts as an immunosuppressive drug that directly affects the activation and proliferation of human T cells by induction of DNA damage and the activation of a p53-dependent DNA damage response.

Deacetylation via SIRT2 prevents keratin-mutation-associated injury and keratin aggregation.

Keratin (K) and other intermediate filament (IF) protein mutations at conserved arginines disrupt keratin filaments into aggregates and cause human epidermolysis bullosa simplex (EBS; K14-R125C) or predispose to mouse liver injury (K18-R90C). The challenge for more than 70 IF-associated diseases is the lack of clinically utilized IF-targeted therapies. We used high-throughput drug screening to identify compounds that normalized mutation-triggered keratin filament disruption. Parthenolide, a plant sesquiterpene lactone, dramatically reversed keratin filament disruption and protected cells and mice expressing K18-R90C from apoptosis. K18-R90C became hyperacetylated compared with K18-WT and treatment with parthenolide normalized K18 acetylation. Parthenolide upregulated the NAD-dependent SIRT2, and increased SIRT2-keratin association. SIRT2 knockdown or pharmacologic inhibition blocked the parthenolide effect, while site-specific Lys-to-Arg mutation of keratin acetylation sites normalized K18-R90C filaments. Treatment of K18-R90C-expressing cells and mice with nicotinamide mononucleotide had a parthenolide-like protective effect. In 2 human K18 variants that associate with human fatal drug-induced liver injury, parthenolide protected K18-D89H- but not K8-K393R-induced filament disruption and cell death. Importantly, parthenolide normalized K14-R125C-mediated filament disruption in keratinocytes and inhibited dispase-triggered keratinocyte sheet fragmentation and Fas-mediated apoptosis. Therefore, keratin acetylation may provide a novel therapeutic target for some keratin-associated diseases.

The TLR7/IRF-5 axis sensitizes memory CD4+ T cells to Fas-mediated apoptosis during HIV-1 infection.

HIV-1 infection is characterized by inflammation and a progressive decline in CD4+ T cell count. Despite treatment with antiretroviral therapy (ART), the majority of people living with HIV (PLWH) maintain residual levels of inflammation, a low degree of immune activation, and higher sensitivity to cell death in their memory CD4+ T cell compartment. To date, the mechanisms responsible for this high sensitivity remain elusive. We have identified the transcription factor IRF-5 to be involved in impairing the maintenance of murine CD4+ T cells during chronic infection. Here, we investigate whether IRF-5 also contributes to memory CD4+ T cell loss during HIV-1 infection. We show that TLR7 and IRF-5 were upregulated in memory CD4+ T cells from PLWH, when compared with naturally protected elite controllers and HIVfree participants. TLR7 was upstream of IRF-5, promoting Caspase 8 expression in CD4+ T cells from ART HIV-1+ but not from HIVfree donors. Interestingly, the TLR7/IRF-5 axis acted synergistically with the Fas/FasL pathway, suggesting that TLR7 and IRF-5 expression in ART HIV-1+ memory CD4+ T cells represents an imprint that predisposes cells to Fas-mediated apoptosis. This predisposition could be blocked using IRF-5 inhibitory peptides, suggesting IRF-5 blockade as a possible therapy to prevent memory CD4+ T cell loss in PLWH.

Neutrophils in Cancer and Potential Therapeutic Strategies Using Neutrophil-Derived Exosomes.

Neutrophils are the most abundant immune cells and make up about 70% of white blood cells in human blood and play a critical role as the first line of defense in the innate immune response. They also help regulate the inflammatory environment to promote tissue repair. However, in cancer, neutrophils can be manipulated by tumors to either promote or hinder tumor growth depending on the cytokine pool. Studies have shown that tumor-bearing mice have increased levels of neutrophils in peripheral circulation and that neutrophil-derived exosomes can deliver various cargos, including lncRNA and miRNA, which contribute to tumor growth and degradation of extracellular matrix. Exosomes derived from immune cells generally possess anti-tumor activities and induce tumor-cell apoptosis by delivering cytotoxic proteins, ROS generation, H2O2 or activation of Fas-mediated apoptosis in target cells. Engineered exosome-like nanovesicles have been developed to deliver chemotherapeutic drugs precisely to tumor cells. However, tumor-derived exosomes can aggravate cancer-associated thrombosis through the formation of neutrophil extracellular traps. Despite the advancements in neutrophil-related research, a detailed understanding of tumor-neutrophil crosstalk is still lacking and remains a major barrier in developing neutrophil-based or targeted therapy. This review will focus on the communication pathways between tumors and neutrophils, and the role of neutrophil-derived exosomes (NDEs) in tumor growth. Additionally, potential strategies to manipulate NDEs for therapeutic purposes will be discussed.

Exploiting Fas-mediated autocrine apoptosis of donor alloreactive T cells as an effective approach to prevent acute graft-versus-host disease in a humanized mouse model

Abstract Acute GVHD initiated by donor T cells is a barrier to allogeneic bone marrow transplantation. Pan T cell elimination from the graft is associated with failure of stem cell engraftment and delayed immune reconstitution. T cells upregulate the Fas receptor on their surface following activation and become susceptible to FasL-mediated apoptosis. The objective of this study was to target Fas-mediated apoptosis for the preferential elimination of alloreactive T cells. We previously reported a novel form of FasL protein chimeric with streptavidin (SA-FasL). Mouse and human lymphocytes were surface modified with biotin, followed by engineering with the SA-FasL protein taking advantage of biotin's high affinity to SA. SA-FasL-engineered cells were assessed for autocrine apoptosis of alloreactive T cells in vitro and in vivo. NSG mice transplanted with 10 7human PBMC were used as an acute GVHD xenogeneic model. SA-FasL induced autocrine apoptosis in alloreactive T cells in vitro and in vivo. Mice transplanted with SA-FasL-engineered PBMC had significantly lower numbers of human T cells in the spleen and liver analyzed five days post-transplantation compared to the recipients of SA-engineered cells. Recipients of SA-engineered PBMC developed acute GVHD with a median survival time of 15 days. In marked contrast, sixty percent of SA-FasL-engineered PBMCs recipients survived for a 60-day observation period and showed minimal clinical GVHD scores. Transient display of SA-FasL protein on the surface of donor cells provides a practical and clinically applicable effective approach to prevent acute GVHD with significant translational potential.

Persistent High Risk HPV Infection in the Presence of a Pathogenic FAS Mutation

IntroductionAutoimmune lymphoproliferative syndrome (ALPS) is characterized by immune dysregulation due to aberrant lymphocyte homeostasis. It mainly results from mutations in the Fas-mediated apoptotic pathway, with deleterious heterozygous mutations in the FAS gene being most common. While patients with pathogenic FAS mutations often demonstrate defective apoptosis in vitro, this is not sufficient to diagnose clinical disease and disease penetrance is less than 60%. Although lymphomas have been reported in FAS mutation positive, disease negative patients, to our knowledge persistent HPV infection has not previously been reported in this context. Case DescriptionA 58-year-old lady was evaluated for persistent human papillomavirus (HPV) infection. HPV 16 positive dysplasia of the cervical, vulvar, vaginal, and anal canal was identified at age 30. Despite treatment, subsequent smears and anoscopy examinations were without dysplasia but with persistent HPV 16 infection. Past medical history was notable for Bechet’s disease and Sjogren’s Syndrome. Family history was noncontributory. Complete blood count and lymphocyte enumeration were normal. Immunoglobulin levels (IgG and IgM) were normal with elevated IgA (397 mg/dL). Vitamin B12 level was increased (1,569 pg/mL). Double negative T cells (DNTs) were normal (28 cells/mcL). There was a mild increase in double negative T cells expressing B220 (15 cells/mcL; reference < 6 cells/ mcL). Fas-mediated apoptosis was decreased at 40% (reference >/ = 56%). Cytokine panel demonstrated normal IL-10 level. Prior imaging was without lymphadenopathy or hepatosplenomegaly. Whole exome sequencing was normal. Whole genome sequencing demonstrated a pathogenic variant in FAS [c.429dup (p.Asp144*)]. DiscussionChronic nonmalignant noninfectious lymphadenopathy and/or splenomegaly and elevated DNTs are required for the diagnosis of ALPS. Our patient does not meet the definition of clinical ALPS but has a confirmed pathogenic mutation, defective Fas-mediated apoptosis, and elevated serum B12 level. While ALPS is associated with an increased risk for both Hodgkin’s lymphoma and a non-Hodgkin’s lymphoma, the presence of a pathogenic FAS mutation has not been reported in association with persistent HPV infection previously to our knowledge. This case highlights the expanded clinical phenotype possible in the presence of pathogenic FAS mutations and highlights the need for close cancer surveillance in mutation positive, disease negative patients.

Clinical and immunological phenotype of patients caryying CASP10 variants

BackgroundALPS is a congenital disorder secondary to defects of FASmediated apoptosis, usually showing during the first years of life with variable phenotype, mainly characterized by lymphoproliferation and autoimmunity. Most patients carry mutation on FAS gene, more rarely defects on FASL/CASP10 are involved. Very few cases of ALPS-CASP10 have been reported, therefore clinical and immunological characteristics of such patients need to be better defined. Some mutations with a role in the pathogenesis of the disease have been reported over the years (Leu285Phe, Ile406Leu). Although others with conflicting interpretation have been afterwards classified as polymorphisms (Val410Ile, Tyr446Cys), nonetheless they have been associated with a defective apoptosis function. Aims/MethodsThe aim of this study is to describe the phenotype of a cohort of patients carrying CASP10 variants. The clinical and immunological parameters included in the ALPS diagnostic criteria were evaluated as CD3+TCRaβ+CD4-CD8- (DNTs), the biomarkers (IL10,IL-18,sFAS, Vitamin B12), the ALPS panel signature (high DNT, high DNTsB220+, and low CD27+, low CD3+CD25+/CD3+HLADR+ ratio), and the apoptosis test. ResultsThirty-two patients aged 0–53 years (median 10.9) were studied. Six carried the known Ile406Leu mutation, 11 presented some unreported variants as Cys401Leufs* (3), Pro501Leu (4), Ser239Cys (1), Arg104*(2), c684+4G>A (1). The remaining 16 carried polymorphic variants. Overall, only 18 patients (56%) showed lymphoproliferation and/or splenomegaly. Twenty-seven patients (84%) showed autoimmune features including autoimmune cytopenia (65%) in 19 cases. DNTs were raised in 64% of cases and apoptosis test was positive in 74% of patients including all cases with novel mutations. Five (15%) patients showed hypogammaglobulinemia. Twelve (37%) and 10/32 (31%) patients showed an ALPS and ALPS-like phenotype, respectively. The remaining 10 (31%) presented a different immune-dysregulation pattern. Two patient (6%) developed a lymphoma. Table 1 shows details of clinical and immunological characteristics. Median follow-up was 5.9 years (0.5–24). DiscussionThis is the largest reported cohort of patients carrying CASP10 variants showing that most of them have a clinical phenotype other than ALPS, mainly presenting with milder immune dysregulation showing at older age. Due to this different clinical pattern, ALPS diagnostic criteria should be reviewed and patients with CASP10 variants should be considered as having a different disorder.Table 1Clinical and immunological characteristics of patients carrying CASP10 variants.Total (З2)Casp10 polymorphic variants (16)Casp10 pathogenic variants (16)pALPS phenotype12 (37%)6 (37%)6 (37%)nsALPS-like phenotype10 (31%)6 (37%)4 (25%)nsOther immunedysregulation10 (31%)4 (25%)6 (37%)nsIL-1S >(500 pg/mL)10 (31%)7 (44%)3 (19%)<0.05B12(>663 pg/mL)11 (34%)5 (31%)6 (37%)nsIL-10 (>20 pg/mL)1 (3%)1 (6%)0 (0%)nssFAS ligand (>200 pg/mL)0 (0%)0 (0%)0 (0%)nsDNT cells (%CD3) (>1,5%)21 (65%)10 (62%)11 (69%)nsCD3CD25+/CD3HLADR+ ratio (<1%)21 (65%)10 (62%)11 (69%)nsTCR αβ+ B220+ (>60%)10 (31%)5 (31%)4 (25%)nsLinfoB CD27+ (CD19tot) (<15%)20 (62%)11 (69%)9 (56%)nsALPS Panel positive 4/45 (15%)3 (19%)2 (12%)ns

Abstract 4102: attIL12-T cell therapy destructs cancer-associated fibroblasts and extracellular matrix in heterogenous osteosarcoma xenograft models

Abstract The extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs) play major roles in tumor progression, metastasis, and the poor response of many solid tumors to immunotherapy. CAF-targeted CAR-T cell therapy does not impair ECM components such as collagen. In this study, we investigated whether the recently invented attIL12-T cells could destroy CAFs to disrupt the ECM and uncovered the underlying mechanism by which attIL12-T cells penetrate stroma-enriched osteosarcoma tumors. RNA sequencing demonstrated that attIL12-T cell treatment altered ECM-related gene expression. Immunohistochemistry staining revealed disruption or elimination of high-density CAFs and ECM in osteosarcoma xenograft tumors following attIL12-T cell treatment and that CAF/ECM density was inversely correlated with T-cell infiltration. Other IL12-armed T cells, such as wild-type IL12-T or ttIL12-T cells, did not disrupt the ECM because this effect depended on the engagement between cell-surface vimentin (CSV) on the tumor cell surface and its ligand on the attIL12 -T cells. Mechanistic studies found that attIL12-T cell treatment elevated IFNγ production upon interacting with CSV+ tumor cells to suppress TGFβ secretion and in turn upregulate FAS-mediated CAF apoptosis. CAF destruction reshaped the tumor stroma to favor T cell infiltration and tumor destruction. Thus, this study unveiled a novel therapy—attIL12-T cells—for targeting CAFs/ECM. These findings are highly relevant to humans because CAFs are abundant in human osteosarcoma. Citation Format: Jiemiao Hu, Alexander Lazar, Davis Ingram, Wei-Lien Wang, Wendong Zhang, Zhiliang Jia, Dristhi Ragoonanan, Jian Wang, Xueqing Xia, Kris Mahadeo, Richard Gorlick, Shulin Li. attIL12-T cell therapy destructs cancer-associated fibroblasts and extracellular matrix in heterogenous osteosarcoma xenograft models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4102.

Retraction.

Iwase M, Watanabe H, Kondo G, Ohashi M, Nagumo M. Enhanced susceptibility of oral squamous cell carcinoma cell lines to FAS-mediated apoptosis by cisplatin and 5-fluorouracil. Int J Cancer. 2003 Sep 10;106(4):619-25. doi:10.1002/ijc.11239 The above article, published online on May 30, 2003, in Wiley Online Library (https://onlinelibrary.wiley.com/doi/10.1002/ijc.11239), has been retracted by agreement between the journal Editor-in-Chief (Prof. Christoph Plass), the authors, and Wiley Periodicals LLC. An Expression of Concern (https://onlinelibrary.wiley.com/doi/10.1002/ijc.33825) was published during an earlier stage of this investigation. The retraction has been agreed following internal analyses and an investigation by the author's institution. The investigation concluded that data fabrication had taken place during compilation of the figures and that the manuscript was submitted without approval of the co-authors. As a result, the overall conclusions of this manuscript are considered invalid.

5-Fluorouracil Suppresses Colon Tumor through Activating the p53-Fas Pathway to Sensitize Myeloid-Derived Suppressor Cells to FasL+ Cytotoxic T Lymphocyte Cytotoxicity.

Myelosuppression is a major adverse effect of 5-fluorouracil (5-FU) chemotherapy. However, recent findings indicate that 5-FU selectively suppresses myeloid-derived suppressor cells (MDSCs), to enhance antitumor immunity in tumor-bearing mice. 5-FU-mediated myelosuppression may thus have a beneficial effect for cancer patients. The molecular mechanism underlying 5-FU's suppression of MDSCs is currently unknown. We aimed at testing the hypothesis that 5-FU suppresses MDSCs through enhancing MDSC sensitivity to Fas-mediated apoptosis. We observed that, although FasL is highly expressed in T cells, Fas is weakly expressed in myeloid cells in human colon carcinoma, indicating that downregulation of Fas is a mechanism underlying myeloid cell survival and accumulation in human colon cancer. 5-FU treatment upregulated expression of both p53 and Fas, and knocking down p53 diminished 5-FU-induced Fas expression in MDSC-like cells, in vitro. 5-FU treatment also increased MDSC-like cell sensitivity to FasL-induced apoptosis in vitro. Furthermore, we determined that 5-FU therapy increased expression of Fas on MDSCs, suppressed MDSC accumulation, and increased CTL tumor infiltration in colon tumor-bearing mice. In human colorectal cancer patients, 5-FU chemotherapy decreased MDSC accumulation and increased CTL level. Our findings determine that 5-FU chemotherapy activates the p53-Fas pathway, to suppress MDSC accumulation, to increase CTL tumor infiltration.

A novel role of Fas in delaying cellular senescence

Fas-mediated apoptosis is a major player of many physiological and pathological cellular processes. Fas-regulated immune regulation exhibits either the beneficial or the harmful effects which is associated with the onset or development of immune disorders. Alterations in apoptosis may contribute to age-associated changes. However, the role of apoptosis in the ageing process remains ambiguous. Here we demonstrated Fas signaling-mediated premature senescence in young mouse embryonic fibroblast (MEF) cells. Activated Fas signaling by agonist Jo-2 resulted in declined senescence in young and aged MEFs. Premature senescence induced the early activation of senescence markers, including the increase in the percentage of SA-β-galactosidase (SA-β-gal) cells, the induction of p53 phosphorylation, and the enhanced expression of p16 and p21 protein and elevated IL-6 pro-inflammatory cytokine in the absence of Fas. The elevated production of reactive oxygen species (ROS) in Fas-deficient MEFs was associated with dysfunctional mitochondria. Further, we determined that the known ROS scavenger NAC (N-acetyl-l-cysteine) could reverse the process of premature senescence in absence of Fas. Therefore, this study signifies a novel role of Fas in the control of cellular senescence.

Hepatocyte growth factor-mediated apoptosis mechanisms of cytotoxic CD8+ T cells in normal and cirrhotic livers

Intrahepatic stem/progenitor cells and cytotoxic CD8+ T cells (CD8+ T cells) in the cirrhotic liver undergo apoptosis, which potentially facilitates progression to cancer. Here, we report that hepatocyte growth factor (HGF) signaling plays an important role in promoting normal and damaged liver CD8+ T cell Fas-mediated apoptosis through its only receptor, c-Met. In addition to binding with HGF, c-Met also binds to Fas to form a complex. Using a diethylnitrosamine (DEN)-induced liver fibrosis/cirrhosis mouse model, immunostaining, and terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) staining, we found that HGF secretion was significantly higher at 10 weeks post-DEN, the liver cirrhotic phase (LCP), than at 3 weeks post-DEN, the liver fibrotic phase (LFP). Correspondingly, differences in CD8+ T cell proliferation and apoptosis were noted between the two phases. Interestingly, staining and TUNEL assays revealed lower smooth muscle actin (α-SMA)+ cell apoptosis, a marker for hepatic stellate cells (HSCs), in the LFP group than in the LCP group, which suggested a beneficial correlation among HGF, CD8+ T cells and HSCs in improving the fibrotic load during damaged liver repair. In cultures, when met different concentrations of recombinant HGF (rHGF), phytohemagglutinin (PHA)-stimulated naive mouse splenic CD8+ T cells (pn-msCD8+ T cells) responded differently; as increases in rHGF increased were associated with decreases in the clonal numbers of pn-msCD8+ T cells, and when the rHGF dose was greater than 200 ng/mL, the clonal numbers significantly decreased. In the presence of 400 ng/mL rHGF, the death-inducing signaling complex (DISC) can be directly activated in both nsCD8+ T cells and healthy human peripheral blood CD8+ T cells (hp-CD8+ T cells), as indicated by recruitment of FADD and caspase-8 because DISC forms via the recruitment of FADD and caspase-8, among others. These findings suggest that Fas-mediated apoptosis, may also indicate a regulatory role of HGF signaling in hepatic homeostasis.

CircFLNA promotes intestinal injury during abdominal sepsis through Fas-mediated apoptosis pathway by sponging miR-766-3p

BackgroundIntra-abdominal infections are the second most common cause of sepsis in the intensive care unit. Intestinal epithelial injury due to abdominal sepsis results in a variety of pathological changes, such as intestinal bacteria and toxins entering the blood, leading to persistent systemic inflammation and multiple organ dysfunction. The increased apoptosis of intestinal epithelial cells induced by sepsis further exacerbates the progression of sepsis. Although several studies have revealed that circRNAs are involved in intestinal epithelial injury in sepsis, few studies have identified the roles of circRNAs in intestinal epithelial apoptosis.MethodsWe used laser capture microdissection to obtain purified epithelial cells located in intestinal crypts from four patients with abdominal sepsis induced by intestinal perforation and four samples from age and sex-matched non-septic patients. Microarray analysis of circRNAs was conducted to assess differentially expressed circRNAs between patients with and without sepsis. Lastly, in vitro and in vivo assays were performed to study the mechanism of circFLNA in intestinal epithelial apoptosis during sepsis.ResultscircFLNA was upregulated in the intestinal epithelium after abdominal sepsis induced by intestinal perforation. Inhibition of miR-766-3p impaired si-circFLNA-mediated inhibition of apoptosis and inflammation factor levels in lipopolysaccharide (LPS)-treated HIEC-6 cells. circFLNA aggravated apoptosis and inflammation through the Fas-mediated apoptosis pathway in both LPS-treated HIEC-6 cells and a mouse cecal ligation and puncture model.ConclusionOur findings showed that circFLNA promotes intestinal injury in abdominal sepsis through the Fas-mediated apoptosis pathway by sponging miR-766-3p. The circFLNA/miR-766-3p/Fas axis has potential as a novel therapeutic target for treating intestinal injury in sepsis.

LCL161 enhances expansion and survival of engineered anti-tumor T cells but is restricted by death signaling.

The genesis of SMAC mimetic drugs is founded on the observation that many cancers amplify IAP proteins to facilitate their survival, and therefore removal of these pathways would re-sensitize the cells towards apoptosis. It has become increasingly clear that SMAC mimetics also interface with the immune system in a modulatory manner. Suppression of IAP function by SMAC mimetics activates the non-canonical NF-κB pathway which can augment T cell function, opening the possibility of using SMAC mimetics to enhance immunotherapeutics. We have investigated the SMAC mimetic LCL161, which promotes degradation of cIAP-1 and cIAP-2, as an agent for delivering transient costimulation to engineered BMCA-specific human TAC T cells. In doing so we also sought to understand the cellular and molecular effects of LCL161 on T cell biology. LCL161 activated the non-canonical NF-κB pathway and enhanced antigen-driven TAC T cell proliferation and survival. Transcriptional profiling from TAC T cells treated with LCL161 revealed differential expression of costimulatory and apoptosis-related proteins, namely CD30 and FAIM3. We hypothesized that regulation of these genes by LCL161 may influence the drug's effects on T cells. We reversed the differential expression through genetic engineering and observed impaired costimulation by LCL161, particularly when CD30 was deleted. While LCL161 can provide a costimulatory signal to TAC T cells following exposure to isolated antigen, we did not observe a similar pattern when TAC T cells were stimulated with myeloma cells expressing the target antigen. We questioned whether FasL expression by myeloma cells may antagonize the costimulatory effects of LCL161. Fas-KO TAC T cells displayed superior expansion following antigen stimulation in the presence of LCL161, suggesting a role for Fas-related T cell death in limiting the magnitude of the T cell response to antigen in the presence of LCL161. Our results demonstrate that LCL161 provides costimulation to TAC T cells exposed to antigen alone, however LCL161 did not enhance TAC T cell anti-tumor function when challenged with myeloma cells and may be limited due to sensitization of T cells towards Fas-mediated apoptosis.

Assessment Of Soluble Fas Ligand In Patients With Chronic Myeloid Leukemia

Background: Soluble FasL (sFasL) generating from Membrane-bound FasL (mFasL) cleaved by matrix metalloproteinases (MMPs). sFasL inhibits the apoptotic and inflammatory activity of mFasL because of sFasL competes with the mFasL and binds to Fas. To evaluate serum soluble Fas ligand (sFasL) level in patients with chronic myeloid leukemia.&#x0D; Materials and Method: Serum levels of sFasL were measured by ELISA method after venous blood was collected from 56 CML patients (newly diagnosed and optimally treated) and 28 healthy subjects as control group.&#x0D; Results: There were no significant increases in serum sFasL patient compared to healthy control with P=0.07. When the mean sFasL concentration was obviously highest in newly diagnosed (216.9pg/ml) followed by healthy control (152.5pg/ml) and lowest in optimally treated (147.7 pg/ml).&#x0D; Conclusion: Production of sFasL in tumor patients may be a key mechanism to inhibit Fas-mediated apoptosis.&#x0D;

The Functional Properties and Physiological Roles of Signal-Transducing Adaptor Protein-2 in the Pathogenesis of Inflammatory and Immune Disorders.

Adaptor molecules play a crucial role in signal transduction in immune cells. Several adaptor molecules, such as the linker for the activation of T cells (LAT) and SH2 domain-containing leukocyte protein of 76 kDa (SLP-76), are essential for T cell development and activation following T cell receptor (TCR) aggregation, suggesting that adaptor molecules are good therapeutic targets for T cell-mediated immune disorders, such as autoimmune diseases and allergies. Signal-transducing adaptor protein (STAP)-2 is a member of the STAP family of adaptor proteins. STAP-2 functions as a scaffold for various intracellular proteins, including BRK, signal transducer, and activator of transcription (STAT)3, STAT5, and myeloid differentiation primary response protein (MyD88). In T cells, STAP-2 is involved in stromal cell-derived factor (SDF)-1α-induced migration, integrin-dependent cell adhesion, and Fas-mediated apoptosis. We previously reported the critical function of STAP-2 in TCR-mediated T cell activation and T cell-mediated autoimmune diseases. Here, we review how STAP-2 affects the pathogenesis of T cell-mediated inflammation and immune diseases in order to develop novel STAP-2-targeting therapeutic strategies.

Immune dysregulation associated with co-occurring germline CBL and SH2B3 variants

BackgroundCBL syndrome is a RASopathy caused by heterozygous germline mutations of the Casitas B-lineage lymphoma (CBL) gene. It is characterized by heterogeneous clinical phenotype, including developmental delay, facial dysmorphisms, cardiovascular malformations and an increased risk of cancer development, particularly juvenile myelomonocytic leukemia (JMML). Although the clinical phenotype has been progressively defined in recent years, immunological manifestations have not been well elucidated to date.MethodsWe studied the genetic, immunological, coagulative, and clinical profile of a family with CBL syndrome that came to our observation after the diagnosis of JMML, with homozygous CBL mutation, in one of the members.ResultsVariant analysis revealed the co-occurrence of CBL heterozygous mutation (c.1141 T > C) and SH2B3 mutation (c.1697G > A) in two other members. Patients carrying both mutations showed an ALPS-like phenotype characterized by lymphoproliferation, cytopenia, increased double-negative T-cells, impaired Fas-mediated lymphocyte apoptosis, altered cell death in PBMC and low TRECs expression. A coagulative work-up was also performed and showed the presence of subclinical coagulative alterations in patients carrying both mutations.ConclusionIn the reported family, we described immune dysregulation, as part of the clinical spectrum of CBL mutation with the co-occurrence of SH2B3.

Early B cell factor 4 modulates FAS-mediated apoptosis and promotes cytotoxic function in human immune cells

Apoptosis is a genetically regulated program of cell death that plays a key role in immune disease processes. We identified EBF4, a little-studied member of the early B cell factor (EBF) family of transcription factors, in a whole-genome CRISPR screen for regulators of Fas/APO-1/CD95-mediated T cell death. Loss of EBF4 increases the half-life of the c-FLIP protein, and its presence in the Fas signaling complex impairs caspase-8 cleavage and apoptosis. Transcriptome analysis revealed that EBF4 regulates molecules such as TBX21, EOMES, granzyme, and perforin that are important for human natural killer (NK) and CD8+ T cell functions. Proximity-dependent biotin identification (Bio-ID) mass spectrometry analyses showed EBF4 binding to STAT3, STAT5, and MAP kinase 3 and a strong pathway relationship to interleukin-2 regulated genes, which are known to govern cytotoxicity pathways. Chromatin immunoprecipitation and DNA sequencing analysis defined a canonical EBF4 binding motif, 5'-CCCNNGG/AG-3', closely related to the EBF1 binding site; using a luciferase-based reporter, we found a dose-dependent transcriptional response of this motif to EBF4. We also conducted assay for transposase-accessible chromatin sequencing in EBF4-overexpressing cells and found increased chromatin accessibility upstream of granzyme and perforin and in topologically associated domains in human lymphocytes. Finally, we discovered that the EBF4 has basal expression in human but not mouse NK cells and CD8+ T cells and vanishes following activating stimulation. Together, our data reveal key features of a previously unknown transcriptional regulator of human cytotoxic immune function.