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Across clinical trials, T cell expansion and persistence following adoptive cell transfer (ACT) have correlated with superior patient outcomes. Herein, we undertook a pan-cancer analysis to identify actionable ligand-receptor pairs capable of compromising T cell durability following ACT. We discovered that FASLG, the gene encoding the apoptosis-inducing ligand FasL, is overexpressed within the majority of human tumor microenvironments (TMEs). Further, we uncovered that Fas, the receptor for FasL, is highly expressed on patient-derived T cells used for clinical ACT. We hypothesized that a cognate Fas-FasL interaction within the TME might limit both T cell persistence and antitumor efficacy. We discovered that genetic engineering of Fas variants impaired in the ability to bind FADD functioned as dominant negative receptors (DNRs), preventing FasL-induced apoptosis in Fas-competent T cells. T cells coengineered with a Fas DNR and either a T cell receptor or chimeric antigen receptor exhibited enhanced persistence following ACT, resulting in superior antitumor efficacy against established solid and hematologic cancers. Despite increased longevity, Fas DNR-engineered T cells did not undergo aberrant expansion or mediate autoimmunity. Thus, T cell-intrinsic disruption of Fas signaling through genetic engineering represents a potentially universal strategy to enhance ACT efficacy across a broad range of human malignancies.

Muscle cell death in polymyositis is induced by CD8 + cytotoxic T lymphocytes. We hypothesized that the injured muscle fibers release pro-inflammatory molecules, which would further accelerate CD8 + cytotoxic T lymphocytes-induced muscle injury, and inhibition of the cell death of muscle fibers could be a novel therapeutic strategy to suppress both muscle injury and inflammation in polymyositis. Here, we show that the pattern of cell death of muscle fibers in polymyositis is FAS ligand-dependent necroptosis, while that of satellite cells and myoblasts is perforin 1/granzyme B-dependent apoptosis, using human muscle biopsy specimens of polymyositis patients and models of polymyositis in vitro and in vivo. Inhibition of necroptosis suppresses not only CD8 + cytotoxic T lymphocytes-induced cell death of myotubes but also the release of inflammatory molecules including HMGB1. Treatment with a necroptosis inhibitor or anti-HMGB1 antibodies ameliorates myositis-induced muscle weakness as well as muscle cell death and inflammation in the muscles. Thus, targeting necroptosis in muscle cells is a promising strategy for treating polymyositis providing an alternative to current therapies directed at leukocytes. Polymyositis (PM) is a chronic inflammatory myopathy characterized by progressive muscle weakness. Here the authors showed that muscle fibers in PM undergo necroptosis and aggravate inflammation via releasing pro-inflammatory molecules such as HMGB1.

Tumor-associated B7-H1 molecules inhibit antitumor immunity in some malignancies. We found that B7-H1 expression on patient myeloma cells and human myeloma cell lines (HMCLs) was upregulated by cultivating the cells with autologous stromal cells and the human stromal cell line HS-5. Among major cytokines produced by HS-5 cells, interleukin (IL)-6-induced B7-H1 expression on HMCLs. Moreover, HS-5 cell-mediated B7-H1 expression was downregulated by inhibiting IL-6. B7-H1 + HMCLs were more proliferative and less susceptible to antimyeloma chemotherapy compared with B7-H1 − HMCLs. Moreover, the former cells showed higher levels of Bcl-2 and FasL expression than the latter. Finally, B7-H1 molecules on HMCLs induced T-cell apoptosis and anergy of tumor-specific T cells. Consistent with these in vitro observations, patients whose myeloma cells expressed high levels of B7-H1 had higher myeloma cell percentages in the bone marrow (BM) and higher serum lactate dehydrogenase levels compared with other myeloma patients. In addition, B7-H1 expression levels were often upregulated after myeloma patients relapsed or became refractory to therapy. Our data indicate that the BM microenvironment upregulates B7-H1 expression on myeloma cells, which links to the two biological actions of inducing T-cell downregulation and enhancing aggressive myeloma-cell characteristics. Modulating the B7-H1 pathway may be worthwhile in myeloma.

Background Cluster of differentiation 95 (CD95/Fas/Apo1) as part of the Tumor-necrosis factor (TNF) receptor family is a prototypic trigger of the ‘extrinsic’ apoptotic pathway and its activation by the trimeric ligand CD95L is of high interest. However, CD95L, when presented in solution, exhibits a low efficiency to induce apoptosis signaling in human cells. Results Here, we design a recombinant CD95L exhibiting an isoleucine zipper (IZ) motif at the N-terminus for stabilization of the trimerized CD95L and demonstrate its high apoptosis initiation efficiency. This efficiency is further enhanced by antibody-mediated crosslinking of IZ-CD95L.A cysteine amino acid fused behind the IZ is used as a versatile coupling site for bionanotechnological applications or for the development of biomedical assays. A fast, cheap, and efficient production of CD95L via the HEK293T secretory expression system is presented, along with CD95L affinity purification and functionalization. We verified the biological activity of the purified protein and identified a stabilized trimeric CD95L structure as the most potent inducer of apoptosis signaling. Conclusions The workflow and the findings reported here will streamline a wide array of future low- or high-throughput TNF-ligand screens, and their modification towards improving apoptosis induction efficiency and, potentially, anticancer therapy.

FasL's non-apoptotic functions The transmembrane protein known as FasL (Fas ligand) is a member of the tumour necrosis factor family with an important role in immune regulation. The binding of FasL with its receptor induces apoptosis, but it has not been clear how important cell death is in FasL's cellular functions. Experiments using gene-targeted mice that either lack secreted FasL but express normal levels of membrane-bound FasL or, that lack membrane-bound FasL but can still produce secreted FasL, show that soluble FasL promotes autoimmunity and tumorigenesis through mechanisms that do not involve apoptosis. Fas ligand (FasL) and its receptor Fas are critical for the shutdown of chronic immune responses and prevention of autoimmunity. FasL function is regulated by deposition in the plasma membrane and metalloprotease-mediated shedding, but it is unclear what the respective roles of these secreted and membrane-bound forms are. Gene-targeted mice that selectively lack either secreted FasL or membrane-bound FasL are now generated, shedding light on this problem. Fas ligand (FasL), an apoptosis-inducing member of the TNF cytokine family, and its receptor Fas are critical for the shutdown of chronic immune responses 1 , 2 , 3 and prevention of autoimmunity 4 , 5 . Accordingly, mutations in their genes cause severe lymphadenopathy and autoimmune disease in mice 6 , 7 and humans 8 , 9 . FasL function is regulated by deposition in the plasma membrane and metalloprotease-mediated shedding 10 , 11 . Here we generated gene-targeted mice that selectively lack either secreted FasL (sFasL) or membrane-bound FasL (mFasL) to resolve which of these forms is required for cell killing and to explore their hypothesized non-apoptotic activities. Mice lacking sFasL ( FasL Δs/Δs ) appeared normal and their T cells readily killed target cells, whereas T cells lacking mFasL ( FasL Δm/Δm ) could not kill cells through Fas activation. FasL Δm/Δm mice developed lymphadenopathy and hyper-gammaglobulinaemia, similar to FasL gld/gld mice, which express a mutant form of FasL that cannot bind Fas, but surprisingly, FasL Δm/Δm mice (on a C57BL/6 background) succumbed to systemic lupus erythematosus (SLE)-like autoimmune kidney destruction and histiocytic sarcoma, diseases that occur only rarely and much later in FasL gld/gld mice. These results demonstrate that mFasL is essential for cytotoxic activity and constitutes the guardian against lymphadenopathy, autoimmunity and cancer, whereas excess sFasL appears to promote autoimmunity and tumorigenesis through non-apoptotic activities.

Despite sharing >98% genomic similarity, humans are more likely to develop cancers than our closest living ancestors, the nonhuman primates. Here, we unexpectedly discover that, unlike chimpanzee and other primates, a critical embryonic development, immune homeostasis, and general cell-death regulator protein called Fas Ligand (FasL) contains a Pro153-Ser153 evolutionary substitution in humans. The latter renders human FasL preferentially susceptible to cleavage by plasmin, an overly elevated protease in solid tumors. Since FasL-mediated killing of tumor cells by activated T-lymphocytes and chimeric antigen receptor T-cells (CAR-T) is critical for therapeutic efficacy, we find that elevated plasmin levels in certain ovarian tumors interfere with the T-lymphocyte-expressed FasL death signaling. Either targeted inhibition or blocking plasmin accessibility to membrane FasL rescues the FasL cell-death function of activated T-lymphocytes in response to immune-checkpoint receptor targeting antibodies. These findings of evolutionary significance highlight that elevated plasmin in metastatic tumors potentially contributes to differential outcomes of T-cell-based immunotherapies in solid tumors. Fas ligand (FasL) regulates immunotherapeutic cancer-cell death. Here, the authors show a human-specific amino-acid substitution which renders human FasL more susceptible for plasmin cleavage and is relevant for the efficacy of T-cell-based immunotherapies.

Parkinson disease (PD) is a neurodegenerative disorder characterized by a loss of dopamine-containing neurons. Mounting evidence suggests that dopaminergic cell death is influenced by the innate immune system. However, the pathogenic role of the adaptive immune system in PD remains enigmatic. Here we showed that CD8+ and CD4+ T cells but not B cells had invaded the brain in both postmortem human PD specimens and in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD during the course of neuronal degeneration. We further demonstrated that MPTP-induced dopaminergic cell death was markedly attenuated in the absence of mature T lymphocytes in 2 different immunodeficient mouse strains (Rag1-/- and Tcrb-/- mice). Importantly, similar attenuation of MPTP-induced dopaminergic cell death was seen in mice lacking CD4 as well as in Rag1-/- mice reconstituted with FasL-deficient splenocytes. However, mice lacking CD8 and Rag1-/- mice reconstituted with IFN-gamma-deficient splenocytes were not protected. These data indicate that T cell-mediated dopaminergic toxicity is almost exclusively arbitrated by CD4+ T cells and requires the expression of FasL but not IFNgamma. Further, our data may provide a rationale for targeting the adaptive arm of the immune system as a therapeutic strategy in PD.

B cells are prominent components of human solid tumours, but activation status and functions of these cells in human cancers remain elusive. Here we establish that over 50% B cells in hepatocellular carcinoma (HCC) exhibit an FcγRII low/− activated phenotype, and high infiltration of these cells positively correlates with cancer progression. Environmental semimature dendritic cells, but not macrophages, can operate in a CD95L-dependent pathway to generate FcγRII low/− activated B cells. Early activation of monocytes in cancer environments is critical for the generation of semimature dendritic cells and subsequent FcγRII low/− activated B cells. More importantly, the activated FcγRII low/− B cells from HCC tumours, but not the resting FcγRII high B cells, without external stimulation suppress autologous tumour-specific cytotoxic T-cell immunity via IL-10 signals. Collectively, generation of FcγRII low/− activated B cells may represent a mechanism by which the immune activation is linked to immune tolerance in the tumour milieu. Activation and biological function of B cells in cancer are still unclear. Here, the authors show that hepatocarcinoma cells drive the formation of semimature dendritic cells that in turn activate FcγRII low/− tumour B cells through the CD95L/CD95 axis, leading to the production of IL-10 and suppression of CD8 T cells.

Approximately 30% of patients with Epstein-Barr virus (EBV)-positive advanced nasopharyngeal carcinoma (NPC) display chemoresistance to cisplatin-based regimens, but the underlying mechanisms are unclear. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), a functional homologue of the tumor necrosis factor receptor family, contributes substantially to the oncogenic potential of EBV through the activation of multiple signaling pathways, and it is closely associated with a poorer prognosis for NPC. Recent studies show that EBV infection can induce the expression of many cellular miRNAs, including microRNA-21, a biomarker for chemoresistance. However, neither a link between LMP1 expression and miR-21 upregulation nor their cross talk in affecting chemoresistance to cisplatin have been reported. Here, we observed that stable LMP1-transformed NPC cells were less sensitive to cisplatin treatment based on their proliferation, colony formation, the IC50 value of cisplatin and the apoptosis index. Higher levels of miR-21 were found in EBV-carrying and LMP1-positive cell lines, suggesting that LMP1 may be linked to miR-21 upregulation. These data were confirmed by our results that exogenous LMP1 increased miR-21 in both transiently and stably LMP1-transfected cells, and the knock down of miR-21 substantially reversed the resistance of the NPC cells to cisplatin treatment. Moreover, the proapoptotic factors programmed cell death 4 (PDCD4) and Fas ligand (Fas-L), which were negatively regulated by miR-21, were found to play an important role in the program of LMP1-dependent cisplatin resistance. Finally, we demonstrated that LMP1 induced miR-21 expression primarily by modulating the PI3K/AKT/FOXO3a signaling pathway. Taken together, we revealed for the first time that viral LMP1 triggers the PI3K/Akt/FOXO3a pathway to induce human miR-21 expression, which subsequently decreases the expression of PDCD4 and Fas-L, and results in chemoresistance in NPC cells.

Presbycusis, or age-related hearing loss (ARHL), is a progressive condition that involves a steady decline in auditory function, primarily caused by the physiological alterations that occur with aging. This disorder arises from the combined effect of multiple interconnected factors that progressively affect the auditory system over time. Genome-wide association studies (GWAS) and transcriptomic analyses in human populations are valuable approaches for identifying potential genes associated with ARHL. This research seeks to assess the potential of protective drugs or strategies for treating ARHL by analyzing target gene-miRNA interactions identified in human blood and associated with presbycusis. We performed RNA sequencing to analyze the transcriptomes of peripheral blood leukocytes from ARHL patients. To identify genes associated with ARHL, the RNA-sequencing data from the peripheral blood leukocytes were compared and further validated by real-time polymerase chain reaction (RT-qPCR) using whole blood samples from the same ARHL patients. To explore the involvement of target genes and microRNAs (miRNAs) in ARHL, we examined miRNA expression patterns using RT-qPCR and reporter gene assays. We found that four genes were up-expressed in ARHL serum: Fas Ligand ( FASLG ), Neural Cell Adhesion Molecule 1 ( NCAM1 ), Nectin Cell Adhesion Molecule 1 ( NECTIN1 ), Macrophage Receptor with Collagenous Structure ( MARCO ). The up-expressed FASLG is associated with cell apoptosis and aging. Additionally, we showed that miR‑5195 and miR-3941 regulated FASLG expression in House Ear Institute-Organ of Corti 1 (HEI-OC-1) and HeLa cells via targeting of FASLG using luciferase reporter assays. Finally, the over-expression of the FASLG gene may be associated with the development of ARHL, and the inhibitory role of miR-5195 and miR-3941 could be a key factor in the prevention or protection against ARHL.