Cell-intrinsic PD-L1 ablation sustains effector CD8+ T cell responses and promotes antitumor T cell therapy.

Adoptive cell therapies are emerging forms of immunotherapy that reprogram T cells for enhanced antitumor responses. Although surface programmed cell death-ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) engagement inhibits antitumor immunity, the role of cell-intrinsic PD-L1 in adoptive T cell therapy remains unknown. Here, we found that intracellular PD-L1 was enriched in tumor-infiltrating CD8+ T cells of cancer patients. PD-L1 ablation promoted antitumor immune responses and the maintenance of an effector-like state of therapeutic CD8+ T cells, while blockade of surface PD-L1 was unable to impact on their expansion and function. Moreover, cell-intrinsic PD-L1 impeded CD8+ T cell activity, which partially relied on mTORC1 signaling. Furthermore, endogenous tumor-reactive CD8+ T cells were motivated by BATF3-driven dendritic cells after adoptive transfer of PD-L1-deficient therapeutic CD8+ T cells. This role of cell-intrinsic PD-L1 in therapeutic CD8+ T cell dysfunction highlights that disrupting cell-intrinsic PD-L1 in CD8+ T cells represents a viable approach to improving T cell-based cancer immunotherapy.

Noncanonical MAVS signaling restrains dendritic cell-driven antitumor immunity by inhibiting IL-12.

Mitochondrial antiviral signaling protein (MAVS)-mediated cytosolic RNA sensing plays a central role in tumor immunogenicity. However, the effects of host MAVS signaling on antitumor immunity remain unclear. Here, we demonstrate that the host MAVS pathway supports tumor growth and impairs antitumor immunity, whereas MAVS deficiency in dendritic cells (DCs) promotes tumor-reactive CD8+ T cell responses. Specifically, CD8+ T cell priming capacity was enhanced by MAVS ablation in a type I interferon-independent, but IL-12-dependent, manner. Mechanistically, loss of the RIG-I/MAVS cascade activated the noncanonical NF-κB pathway and in turn induced IL-12 production by DCs. MAVS-restrained IL-12 promoted cross-talk between CD8+ T cells and DCs, which was licensed by IFN-γ. Moreover, ablation of host MAVS sensitized tumors to immunotherapy and attenuated radiation resistance, thereby facilitating the maintenance of effector CD8+ T cells. These findings demonstrate that the host MAVS pathway acts as an immune regulator of DC-driven antitumor immunity and support the development of immunotherapies that antagonize MAVS signaling in DCs.

STING signaling promotes NK cell antitumor immunity and maintains a reservoir of TCF-1+ NK cells.

Natural killer (NK) cells are cytotoxic innate lymphocytes that eradicate tumor cells. Inducing durable antitumor immune responses by NK cells represents a major priority of cancer immunotherapy. While cytosolic DNA sensing plays an essential role in initiating antitumor immunity, the role of NK cell-intrinsic STING signaling remains unclear. Here, we find that NK cell-intrinsic STING promotes antitumor responses and maintains a reservoir of TCF-1+ NK cells. In contrast, tumor cell-intrinsic cGAS and mtDNA are required for NK cell antitumor activity, indicating that tumor mtDNA recognition by cGAS partially triggers NK cell-intrinsic STING activation. Moreover, addition of cGAMP enables STING activation and type I interferon production in NK cells, thereby supporting the activation of NK cells in vitro. In humans, STING agonism promotes the expansion of TCF-1+ NK cells. This study provides insight into understanding how STING signaling drives NK cell antitumor immunity and the development of NK cell-based cancer immunotherapy.

Androgen receptor-mediated CD8+ T cell stemness programs drive sex differences in antitumor immunity.

The incidence and mortality rates of many non-reproductive human cancers are generally higher in males than in females. However, the immunological mechanism underlying sexual differences in cancers remains elusive. Here, we demonstrated that sex-related differences in tumor burden depended on adaptive immunity. Male CD8+ T cells exhibited impaired effector and stem cell-like properties compared with female CD8+ T cells. Mechanistically, androgen receptor inhibited the activity and stemness of male tumor-infiltrating CD8+ T cells by regulating epigenetic and transcriptional differentiation programs. Castration combined with anti-PD-L1 treatment synergistically restricted tumor growth in male mice. In humans, fewer male CD8+ T cells maintained a stem cell-like memory state compared with female counterparts. Moreover, AR expression correlated with tumor-infiltrating CD8+ T cell exhaustion in cancer patients. Our findings reveal sex-biased CD8+ T cell stemness programs in cancer progression and in the responses to cancer immunotherapy, providing insights into the development of sex-based immunotherapeutic strategies for cancer treatment.

5-HT3A receptors maintain hippocampal LTP in a CB1 and GABAA receptor- dependent manner for spatial memory.

BACKGROUND AND PURPOSE: As the only ionotropic receptor in the 5-HT receptor family, the 5-HT3 receptor (5-HT3 R) is involved in psychiatric disorders and its modulators have potential therapeutic effects for cognitive impairment in these disorders. However, it remains unclear how 5-HT3 Rs shape synaptic plasticity for memory function. EXPERIMENTAL APPROACH: Extracellular as well as whole-cell electrophysiological recordings were used to monitor hippocampal LTP and synaptic transmission in hippocampal slices in 5-HT3 AR knockout or 5-HT3 AR-GFP mice. Immunocytochemistry, qRT-PCR and western blotting were used to measure receptor expression. We also assessed hippocampal dependent cognition and memory, using the Morris water maze (MWM) and novel object recognition. KEY RESULTS: We found that 5-HT3 R dysfunction impaired hippocampal LTP in Schaffer collateral (SC)-CA1 pathway in hippocampal slices, by facilitating GABAergic inputs in pyramidal cells. This effect was dependent on 5-HT3 Rs on axon terminals. It resulted from reduced expression and function of the cannabinoid receptor 1 (CB1 R) co-localized with 5-HT3 Rs on axon terminals, and then led to diminishment of tonic inhibition of GABA release by CB1 Rs. Inhibition of CB1 Rs mimicked the facilitation of GABAergic transmission by 5-HT3 R disruption. Consequently, mice with hippocampal 5-HT3 R disruption exhibited impaired spatial memory in MWM tasks. CONCLUSION AND IMPLICATIONS: These results suggest that 5-HT3 Rs are crucial in enabling hippocampal synaptic plasticity via a novel CB1 R-GABAA -dependent pathway to regulate spatial memory.

ZBP1-MLKL necroptotic signaling potentiates radiation-induced antitumor immunity via intratumoral STING pathway activation.

Necroptosis, a form of regulated necrosis, participates in tumor development and dying cell immunogenicity. However, it remains unclear how tumor cell­intrinsic necroptotic signaling contributes to radiation-induced antitumor immunity. Here, we found that the ZBP1-MLKL necroptotic cascade in irradiated tumor cells was essential for antitumor immunity. ZBP1-dependent activation of MLKL potentiated type I interferon responses following tumor cell irradiation. Mechanistically, the ZBP1-MLKL necroptotic cascade induced cytoplasmic DNA accumulation in irradiated tumor cells and, in turn, autonomously activated cGAS-STING signaling, thus creating a positive feedback loop between those two pathways to drive persistent inflammation. Accordingly, ablation of caspase-8 enhanced STING pathway activation and the antitumor effects of radiation by activating MLKL. These findings reveal that ZBP1-MLKL necroptosis signaling maximized radiation-induced antitumor immunity through mutual interaction with the tumor cell­intrinsic STING pathway. This study provides insight into how radiotherapy bridges tumor cell damage to antitumor immune responses and an alternative strategy to improve radiotherapy.

Therapy-Induced Transdifferentiation Promotes Glioma Growth Independent of EGFR Signaling.

EGFR is frequently amplified, mutated, and overexpressed in malignant gliomas. Yet the EGFR-targeted therapies have thus far produced only marginal clinical responses, and the underlying mechanism remains poorly understood. Using an inducible oncogenic EGFR-driven glioma mouse model system, our current study reveals that a small population of glioma cells can evade therapy-initiated apoptosis and potentiate relapse development by adopting a mesenchymal-like phenotypic state that no longer depends on oncogenic EGFR signaling. Transcriptome analyses of proximal and distal treatment responses identified TGFß/YAP/Slug signaling cascade activation as a major regulatory mechanism that promotes therapy-induced glioma mesenchymal lineage transdifferentiation. Following anti-EGFR treatment, TGFß secreted from stressed glioma cells acted to promote YAP nuclear translocation that stimulated upregulation of the pro-mesenchymal transcriptional factor SLUG and subsequent glioma lineage transdifferentiation toward a stable therapy-refractory state. Blockade of this adaptive response through suppression of TGFß-mediated YAP activation significantly delayed anti-EGFR relapse and prolonged animal survival. Together, our findings shed new insight into EGFR-targeted therapy resistance and suggest that combinatorial therapies of targeting both EGFR and mechanisms underlying glioma lineage transdifferentiation could ultimately lead to deeper and more durable responses. SIGNIFICANCE: This study demonstrates that molecular reprogramming and lineage transdifferentiation underlie anti-EGFR therapy resistance and are clinically relevant to the development of new combinatorial targeting strategies against malignant gliomas with aberrant EGFR signaling.

cGAS-STING-mediated DNA sensing maintains CD8+ T cell stemness and promotes antitumor T cell therapy.

Although cGAS-STING-mediated DNA sensing in tumor cells or phagocytes is central for launching antitumor immunity, the role of intrinsic cGAS-STING activation in T cells remains unknown. Here, we observed that peripheral blood CD8+ T cells from patients with cancer showed remarkably compromised expression of the cGAS-STING cascade. We demonstrated that the cGAS-STING cascade in adoptively transferred CD8+ T cells was essential for antitumor immune responses in the context of T cell therapy in mice. Mechanistically, cell-autonomous cGAS and STING promoted the maintenance of stem cell-like CD8+ T cells, in part, by regulating the transcription factor TCF1 expression. Moreover, autocrine cGAS-STING-mediated type I interferon signaling augmented stem cell-like CD8+ T cell differentiation program mainly by restraining Akt activity. In addition, genomic DNA was selectively enriched in the cytosol of mouse CD8+ T cells upon in vitro and in vivo stimulation. STING agonism enhanced the formation of stem-like central memory CD8+ T cells from patients with cancer and potentiated antitumor responses of CAR-T cell therapy in a xenograft model. These findings advance our understanding of inherent cGAS-STING activation in T cells and provide insight into the development of improved T cell therapy by harnessing the cGAS-STING pathway for cancer immunotherapy.

Targeting hypoxia downstream signaling protein, CAIX, for CAR T-cell therapy against glioblastoma.

BACKGROUND: Glioblastoma survival remains unchanged despite continuing therapeutic innovation. Herein, we aim to (i) develop chimeric antigen receptor (CAR) T cells with a specificity to a unique antigen, carbonic anhydrase IX (CAIX), which is expressed in the hypoxic microenvironment characteristic of glioblastoma, and (ii) demonstrate its efficacy with limited off-target effects. METHODS: First we demonstrated expression of CAIX in patient-derived glioblastoma samples and available databases. CAR T cells were generated against CAIX and efficacy was assessed in 4 glioblastoma cell lines and 2 glioblastoma stem cell lines. Cytotoxicity of anti-CAIX CAR T cells was assessed via interferon gamma, tumor necrosis factor alpha, and interleukin-2 levels when co-cultured with tumor cells. Finally, we assessed efficacy of direct intratumoral injection of the anti-CAIX CAR T cells on an in vivo xenograft mouse model using the U251 luciferase cell line. Tumor infiltrating lymphocyte analyses were performed. RESULTS: We confirm that CAIX is highly expressed in glioblastoma from patients. We demonstrate that CAIX is a suitable target for CAR T-cell therapy using anti-CAIX CAR T cells against glioblastoma in vitro and in vivo. In our mouse model, a 20% cure rate was observed without detectable systemic effects. CONCLUSIONS: By establishing the specificity of CAIX under hypoxic conditions in glioblastoma and highlighting its efficacy as a target for CAR T-cell therapy, our data suggest that anti-CAIX CAR T may be a promising strategy to treat glioblastoma. Direct intratumoral injection increases anti-CAIX CAR T-cell potency while limiting its off-target effects.

A signature based on survival-related genes identifies high-risk glioblastomas harboring immunosuppressive and aggressive ECM characteristics.

OBJECTIVE: To seek survival-related genes in glioblastoma and establish a survival-gene signature for predicting prognoses of glioblastoma using public databases.
 Methods: Three independent glioma databases (GEO GSE53733, CGGA, TCGA) with whole genome expression data were included for analysis. Survival-related genes were obtained by comparing the long-term (>36 months) and short-term (<12 months) survivors in the database GSE53733. CGGA was used as the training set to develop the signature and TCGA was used as the validation set. Cox regression analysis and linear risk score assessment were conducted to look for prognostic signatures with survival-related genes. Principal components analysis, gene set enrichment analysis (GSEA), gene ontology (GO) and protein-protein interaction (PPI) analysis were performed to explore distinct expression profiles between risk grouped glioblastoma.
 Results: We totally found 211 survival-related genes and developed a signature with 17 survival-related genes for prognosis of glioblastoma. Based on this signature, the low-risk group had longer survival time while the high-risk group had shorter survival time. Additionally, the expression profiles between the high-risk and low-risk glioblastoma were different. Functional annotations revealed that the genes enriched in the high-risk glioblastoma were involved in immune systems and processes of extracellular matrix (ECM).
 Conclusion: The novel survival-gene signature can predict high-risk glioblastoma with shorter survival time, enhance immunosuppressive features, and increased invasion preferences.

Amphotericin B suppresses M2 phenotypes and B7-H1 expression in macrophages to prevent Raji cell proliferation.

BACKGROUND: Macrophages in the tumor microenvironment play a critical role in tumorigenesis and anti-cancer drug resistance. Burkitt's lymphoma (BL) is a B-cell non-Hodgkin's lymphoma with dense macrophage infiltration. However, the role for macrophages in BL remains largely unknown. METHODS: B7-H1, a transmembrane glycoprotein in the B7 family, suppresses T cell activation and proliferation and induces the apoptosis of activated T cells. The expression of B7-H1 in BL clinical tissues was determined by streptavidin-peroxidase immunohistochemistry. The mutual regulation between macrophages and BL Raji cells was investigated in a co-culture system. The cell proliferation and cell cycle distribution of Raji cells were determined using BrdU staining coupled with flow cytometry. CD163, CD204 and B7-H1 expression was assessed by flow cytometry and Western blot. Cell invasion was analyzed by Transwell assay. The expression of cytokines was detected by quantitative RT-PCR. Immunofluorescence and allogeneic T-cell proliferation assays were used to compare the expression of B7-H1, p-STAT6, or p-STAT3 and CD3+ T cell proliferation treated with or without amphotericin B. RESULTS: B7-H1 was highly expressed in tumor infiltration macrophages in most clinical BL tissues. In vitro, Raji cells synthesized IL-4, IL-6, IL-10 and IL-13 to induce CD163, CD204 and B7-H1 expression in co-cultured macrophages, which in turn promoted Raji cell proliferation and invasion. Interestingly, antifungal agent amphotericin B not only inhibited STAT6 phosphorylation to suppress the M2 polarization of macrophages, but also promoted CD3+ T cell proliferation by regulating B7-H1 protein expression in macrophages. CONCLUSION: Amphotericin B might represent a novel immunotherapeutic approach to treat patients with BL.

Far Upstream Element-Binding Protein 1 Regulates LSD1 Alternative Splicing to Promote Terminal Differentiation of Neural Progenitors.

Loss of a cell's ability to terminally differentiate because of mutations is a selected genetic event in tumorigenesis. Genomic analyses of low-grade glioma have reported recurrent mutations of far upstream element-binding protein 1 (FUBP1). Here, we show that FUBP1 expression is dynamically regulated during neurogenesis and that its downregulation in neural progenitors impairs terminal differentiation and promotes tumorigenesis collaboratively with expression of IDH1R132H. Mechanistically, collaborative action between SRRM4 and FUBP1 is necessary for mini-exon splicing of the neurospecific LSD1+8a isoform. LSD1+8a was downregulated upon loss of FUBP1 in neural progenitors, thereby impairing terminal neuronal differentiation and maturation. Reinforcing LSD1+8a expression in FUBP1-downregulated neural progenitors restored terminal differentiation and suppressed tumorigenesis; hence, LSD1+8a is an obligatory effector of FUBP1-dependent neuronal differentiation. These findings establish a direct role for FUBP1 in neuronal differentiation and also explain its tumor-suppressor function in the nervous system.

Hydrogen Sulfide Regulates the [Ca2+]i Level in the Primary Medullary Neurons.

In the present study, we attempted to elucidate mechanisms for the regulation of intracellular calcium levels by H2S in primary rat medullary neurons. Our results showed that NaHS significantly increased the level of [Ca2+]i in rat medullary neurons in a concentration-dependent manner. L-Cysteine and SAM significantly raised the level of [Ca2+]i in the medullary neurons while HA and/or AOAA produced a reversal effect. In addition, L-cysteine and SAM significantly increased but HA and/or AOAA decreased the production of H2S in the cultured neurons. The [Ca2+]i elevation induced by H2S was significantly diminished by EGTA-Ca2+-free solutions, and this elevation was also reduced by nifedipine or nimodipine and mibefradil, suggesting the role of L-type and/or T-type Ca2+ channels. Moreover, the effect of H2S on [Ca2+]i level in neurons was significantly attenuated by BAPTA-AM and thapsigargin, suggesting the source of Ca2+. Therefore, we concluded that both exogenous and endogenous H2S elevates [Ca2+]i level in primarily cultured rat medullary neurons via both increasing calcium influx and mobilizing intracellular Ca2+ stores from ER.

[The study on significance of detecting the hepatitis B virus large proteins in public health physical examination].

OBJECTIVE: It was to explore the application of the hepatitis B virus large proteins (HBV-LP) in public health physical examination through clinical testing of HBV-LP in Catering practitioners, students and soldiers of New requisition etc. METHODS: 709 samples of HBV infection people were collected in public health physical examination and all collected samples were detected. ELISA was used to detect HBV serum markers (HBV-M) and HBV-LP. Real-time PCR was applied to detect quantity of HBV DNA. RESULTS: (1) The detectable results of HBV-LP and HBV DNA were no statistical significance in 709 samples. (2) The detectable results of HBV-LP and HBV DNA were also no statistical significance in different models of HBV infection patients. The values of chi2 and P were respectively 2.67, 0.60, 0.00 and 0.10, 0.44, 1.00. S/A of HBV-LP and the copies of HBV-DNA had a positive relationship (r = 0.959, P < 0.05). CONCLUSION: HBV-LP could reflect the level of hepatitis B virus replication and be used to judge the level of hepatitis B virus replication in people of public health physical examination. HBV-LP may be worth using widely in physical examination.