Glycogen synthase kinase 3 controls T-cell exhaustion by regulating NFAT activation.

Cellular immunity mediated by CD8+ T cells plays an indispensable role in bacterial and viral clearance and cancers. However, persistent antigen stimulation of CD8+ T cells leads to an exhausted or dysfunctional cellular state characterized by the loss of effector function and high expression of inhibitory receptors during chronic viral infection and in tumors. Numerous studies have shown that glycogen synthase kinase 3 (GSK3) controls the function and development of immune cells, but whether GSK3 affects CD8+ T cells is not clearly elucidated. Here, we demonstrate that mice with deletion of Gsk3α and Gsk3ß in activated CD8+ T cells (DKO) exhibited decreased CTL differentiation and effector function during acute and chronic viral infection. In addition, DKO mice failed to control tumor growth due to the upregulated expression of inhibitory receptors and augmented T-cell exhaustion in tumor-infiltrating CD8+ T cells. Strikingly, anti-PD-1 immunotherapy substantially restored tumor rejection in DKO mice. Mechanistically, GSK3 regulates T-cell exhaustion by suppressing TCR-induced nuclear import of NFAT, thereby in turn dampening NFAT-mediated exhaustion-related gene expression, including TOX/TOX2 and PD-1. Thus, we uncovered the molecular mechanisms underlying GSK3 regulation of CTL differentiation and T-cell exhaustion in anti-tumor immune responses.

Universal immunotherapeutic strategy for hepatocellular carcinoma with exosome vaccines that engage adaptive and innate immune responses.

BACKGROUND: Personalized immunotherapy utilizing cancer vaccines tailored to the tumors of individual patients holds promise for tumors with high genetic heterogeneity, potentially enabling eradication of the tumor in its entirety. METHODS: Here, we demonstrate a general strategy for biological nanovaccines that trigger tailored tumor-specific immune responses for hepatocellular carcinoma (HCC). Dendritic cell (DC)-derived exosomes (DEX) are painted with a HCC-targeting peptide (P47-P), an α-fetoprotein epitope (AFP212-A2) and a functional domain of high mobility group nucleosome-binding protein 1 (N1ND-N), an immunoadjuvant for DC recruitment and activation, via an exosomal anchor peptide to form a "trigger" DEX vaccine (DEXP&A2&N). RESULTS: DEXP&A2&N specifically promoted recruitment, accumulation and activation of DCs in mice with orthotopic HCC tumor, resulting in enhanced cross-presentation of tumor neoantigens and de novo T cell response. DEXP&A2&N elicited significant tumor retardation and tumor-specific immune responses in HCC mice with large tumor burdens. Importantly, tumor eradication was achieved in orthotopic HCC mice when antigenic AFP peptide was replaced with the full-length AFP (A) to form DEXP&A&N. Supplementation of Fms-related tyrosine kinase 3 ligand greatly augmented the antitumor immunity of DEXP&A&N by increasing immunological memory against tumor re-challenge in orthotopic HCC mice. Depletion of T cells, cross-presenting DCs and other innate immune cells abrogated the functionality of DEXP&A&N. CONCLUSIONS: These findings demonstrate the capacity of universal DEX vaccines to induce tumor-specific immune responses by triggering an immune response tailored to the tumors of each individual, thus presenting a generalizable approach for personalized immunotherapy of HCC, by extension of other tumors, without the need to identify tumor antigens.

Mitochondrial C1qbp promotes differentiation of effector CD8+ T cells via metabolic-epigenetic reprogramming.

Early-activated CD8+ T cells increase both aerobic glycolysis and mitochondrial oxidative phosphorylation (OXPHOS). However, whether and how the augmentation of OXPHOS regulates differentiation of effector CD8+ T cell remains unclear. Here, we found that C1qbp was intrinsically required for such differentiation in antiviral and antitumor immune responses. Activated C1qbp-deficient CD8+ T cells failed to increase mitochondrial respiratory capacities, resulting in diminished acetyl­coenzyme A as well as elevated fumarate and 2-hydroxyglutarate. Consequently, hypoacetylation of H3K27 and hypermethylation of H3K27 and CpG sites were associated with transcriptional down-regulation of effector signature genes. The effector differentiation of C1qbp-sufficient or C1qbp-deficient CD8+ T cells was reversed by fumarate or a combination of histone deacetylase inhibitor and acetate. Therefore, these findings identify C1qbp as a pivotal positive regulator in the differentiation of effector CD8+ T cells and highlight a metabolic-epigenetic axis in this process.

Erratum: Serum exosomes can restore cellular function in vitro and be used for diagnosis in dysferlinopathy: Erratum.

[This corrects the article DOI: 10.7150/thno.22856.].

Exosome-mediated improvement in membrane integrity and muscle function in dystrophic mice.

Duchenne muscular dystrophy (DMD) is a devastating genetic disorder that leads to compromised cellular membranes, caused by the absence of membrane-bound dystrophin protein. Muscle membrane leakage results in disrupted intracellular homeostasis, protein degradation, and muscle wasting. Improving muscle membrane integrity may delay disease progression and extend the lifespan of DMD patients. Here, we demonstrate that exosomes, membranous extracellular vesicles, can elicit functional improvements in dystrophic mice by improving muscle membrane integrity. Systemic administration of exosomes from different sources induced phenotypic rescue and mitigated pathological progression in dystrophic mice without detectable toxicity. Improved membrane integrity conferred by exosomes inhibited intracellular calcium influx and calcium-dependent activation of calpain proteases, preventing the degradation of the destabilized dystrophin-associated protein complex. We show that exosomes, particularly myotube-derived exosomes, induced functional improvements and alleviated muscle deterioration by stabilizing damaged muscle membrane in dystrophic mice. Our findings suggest that exosomes may have therapeutic implications for DMD and other diseases with compromised membranes.

Erratum: Fructose Promotes Uptake and Activity of Oligonucleotides with Different Chemistries in a Context-Dependent Manner in mdx Mice.

[This corrects the article DOI: 10.1038/mtna.2016.46.].

Erratum: Long-Term Morpholino Oligomers in Hexose Elicit Long-Lasting Therapeutic Improvements in mdx Mice.

[This corrects the article DOI: 10.1016/j.omtn.2018.06.005.].

Alarmin-painted exosomes elicit persistent antitumor immunity in large established tumors in mice.

Treating large established tumors is challenging for dendritic cell (DC)-based immunotherapy. DC activation with tumor cell-derived exosomes (TEXs) carrying multiple tumor-associated antigen can enhance tumor recognition. Adding a potent adjuvant, high mobility group nucleosome-binding protein 1 (HMGN1), boosts DCs' ability to activate T cells and improves vaccine efficiency. Here, we demonstrate that TEXs painted with the functional domain of HMGN1 (TEX-N1ND) via an exosomal anchor peptide potentiates DC immunogenicity. TEX-N1ND pulsed DCs (DCTEX-N1ND) elicit long-lasting antitumor immunity and tumor suppression in different syngeneic mouse models with large tumor burdens, most notably large, poorly immunogenic orthotopic hepatocellular carcinoma (HCC). DCTEX-N1ND show increased homing to lymphoid tissues and contribute to augmented memory T cells. Importantly, N1ND-painted serum exosomes from cancer patients also promote DC activation. Our study demonstrates the potency of TEX-N1ND to strengthen DC immunogenicity and to suppress large established tumors, and thus provides an avenue to improve DC-based immunotherapy.

Effects of exosome-mediated delivery of myostatin propeptide on functional recovery of mdx mice.

Duchenne muscular dystrophy (DMD) is a devastating disorder caused by loss of functional dystrophin protein, resulting in muscle wasting. Enhancing muscle growth by inhibiting myostatin, a growth factor negatively regulating skeletal muscle mass, is a promising approach to slow disease progression. Direct administration of myostatin propeptide, a natural inhibitor of mature myostatin, has shown limited efficacy probably due to low serum stability. Here, we demonstrate that serum stability, delivery efficiency and efficacy of propeptide can be significantly enhanced by anchoring propeptide to the surface of exosomes by fusing the inhibitory domain of myostatin propeptide into the second extracellular loop of CD63 (EXOpro). Repeated administrations of EXOpro accelerated muscle regeneration and growth, resulting in significantly increased muscle mass and functional rescue without any detectable toxicity in mdx mice. Importantly, EXOpro partially rehabilitated bone structure and promoted bone regeneration in mdx mice. Our findings demonstrate that anchoring to exosomes increased delivery and serum stability of propeptide and augmented the inhibitory efficacy of myostatin propeptide and thus provide a delivery platform for propeptide-based intervention in DMD.

Hexose Potentiates Peptide-Conjugated Morpholino Oligomer Efficacy in Cardiac Muscles of Dystrophic Mice in an Age-Dependent Manner.

Insufficient delivery of oligonucleotides to muscle and heart remains a barrier for clinical implementation of antisense oligonucleotide (AO)-mediated exon-skipping therapeutics in Duchenne muscular dystrophy (DMD), a lethal monogenic disorder caused by frame-disrupting mutations in the DMD gene. We previously demonstrated that hexose, particularly an equal mix of glucose:fructose (GF), significantly enhanced oligonucleotide delivery and exon-skipping activity in peripheral muscles of mdx mice; however, its efficacy in the heart remains limited. Here we show that co-administration of GF with peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO, namely, BMSP-PMO) induced an approximately 2-fold higher level of dystrophin expression in cardiac muscles of adult mdx mice compared to BMSP-PMO in saline at a single injection of 20 mg/kg, resulting in evident phenotypic improvement in dystrophic mdx hearts without any detectable toxicity. Dystrophin expression in peripheral muscles also increased. However, GF failed to potentiate BMSP-PMO efficiency in aged mdx mice. These findings demonstrate that GF is applicable to both PMO and PPMO. Furthermore, GF potentiates oligonucleotide activity in mdx mice in an age-dependent manner, and, thus, it has important implications for its clinical deployment for the treatment of DMD and other muscular disorders.

Alarmin augments the antitumor immunity of lentiviral vaccine in ectopic, orthotopic and autochthonous hepatocellular carcinoma mice.

It is a daunting therapeutic challenge to completely eradicate hepatocellular carcinoma (HCC) from patients. Alpha-fetoprotein (AFP) -based vaccines appear promising, however the efficacy needs to be improved. Methods: Here, we explore if fusing high-mobility group nucleosome binding protein 1 (HMGN1), a potent immunoadjuvant, to AFP (lenti-HA) can augment the antitumor immunity of AFP-expressing lentiviral vector (lenti-AFP), a vehicle extensively employed for genetic immunization with high transduction efficacy and good safety profiles. The antitumor immunity of Lenti-HA was systemically assessed in ectopic, orthotopic and autochthonous HCC models. Results: Lenti-HA elicited strong anti-HCC effects in mice and amplified the antitumor immunity of lenti-AFP by reducing effective dose 6-fold. Importantly, lenti-HA induced a robust antitumor immune response with prolonged survival rate and improved the immune and tumor microenvironment in mice with carcinogen-induced autochthonous HCC. Lenti-HA localized primarily to lymphoid organs with no preference for specific immune cell types. Activated dendritic cells (DCs), particularly CD103+CD11b- DCs, were also actively recruited to lymph nodes in lenti-HA-treated HCC mice. Moreover, lenti-HA-transduced human DCs elicited stronger immune response than lenti-AFP against HCC cells in vitro. Conclusion: Our study demonstrates that HMGN1 augments the antitumor immunity of AFP-expressing lentiviral vaccines in HCC mice and human cells in vitro and thus provides a new therapeutic strategy for HCC.

Correction: Intrahepatic Tissue Implantation Represents a Favorable Approach for Establishing Orthotopic Transplantation Hepatocellular Carcinoma Mouse Models.

[This corrects the article DOI: 10.1371/journal.pone.0148263.].

Use of Glucose-Fructose to Enhance the Exon Skipping Efficacy.

Exon-skipping antisense oligonucleotides (AOs) are promising treatments for muscle-related genetic ailments including Duchenne muscular dystrophy (DMD), but clinical translation is unfortunately hampered by insufficient systemic delivery. Here we describe that how one can employ a glucose-fructose injection mixture to improve muscle uptake and functional outcomes of DMD AOs in energy-deficient peripheral muscles of mdx mice. The potentiating effect of glucose-fructose on AOs in energy-deficient muscles offers a simple and economical method for enhancing AO potency, reducing screening costs for researchers and accelerating the translation of nucleic acid-based therapeutics in DMD and other muscular dystrophies.

Long-Term Morpholino Oligomers in Hexose Elicits Long-Lasting Therapeutic Improvements in mdx Mice.

Approval of antisense oligonucleotide eteplirsen highlights the promise of exon-skipping therapeutics for Duchenne muscular dystrophy patients. However, the limited efficacy of eteplirsen underscores the importance to improve systemic delivery and efficacy. Recently, we demonstrated that a glucose and fructose (GF) delivery formulation effectively potentiates phosphorodiamidate morpholino oligomer (PMO). Considering the clinical potential of GF, it is important to determine the long-term compatibility and efficacy with PMO in mdx mice prior to clinical translation. Here, we report that yearlong administration of a clinically applicable PMO dose (50 mg/kg/week for 3 weeks followed by 50 mg/kg/month for 11 months) with GF elicited sustainably high levels of dystrophin expression in mdx mice, with up to 45% of the normal level of dystrophin restored in most peripheral muscles without any detectable toxicity. Importantly, PMO-GF resulted in phenotypical rescue and mitochondrial biogenesis with functional improvement. Carbohydrate metabolites measurements revealed improved metabolic and energetic conditions after PMO-GF treatment in mdx mice without metabolic anomaly. Collectively, our study shows PMO-GF's ability to elicit long-lasting therapeutic effects with tolerable toxicity and represents a new treatment modality for Duchenne muscular dystrophy, and provides guidelines for antisense oligonucleotides with GF in clinical use.

Anchor peptide captures, targets, and loads exosomes of diverse origins for diagnostics and therapy.

Exosomes are circulating nanovesicular carriers of macromolecules, increasingly used for diagnostics and therapeutics. The ability to load and target patient-derived exosomes without altering exosomal surfaces is key to unlocking their therapeutic potential. We demonstrate that a peptide (CP05) identified by phage display enables targeting, cargo loading, and capture of exosomes from diverse origins, including patient-derived exosomes, through binding to CD63-an exosomal surface protein. Systemic administration of exosomes loaded with CP05-modified, dystrophin splice-correcting phosphorodiamidate morpholino oligomer (EXOPMO) increased dystrophin protein 18-fold in quadriceps of dystrophin-deficient mdx mice compared to CP05-PMO. Loading CP05-muscle-targeting peptide on EXOPMO further increased dystrophin expression in muscle with functional improvement without any detectable toxicity. Our study demonstrates that an exosomal anchor peptide enables direct, effective functionalization and capture of exosomes, thus providing a tool for exosome engineering, probing gene function in vivo, and targeted therapeutic drug delivery.

Serum exosomes can restore cellular function in vitro and be used for diagnosis in dysferlinopathy.

Purpose: It is challenging to deliver the full-length dysferlin gene or protein to restore cellular functions of dysferlin-deficient (DYSF-/-) myofibres in dysferlinopathy, a disease caused by the absence of dysferlin, which is currently without effective treatment. Exosomes, efficient membranous nanoscale carriers of biological cargoes, could be useful. Experimental design: Myotube- and human serum-derived exosomes were investigated for their capabilities of restoring dysferlin protein and cellular functions in murine and human DYSF-/- cells. Moreover, dysferlinopathic patient serum- and urine-derived exosomes were assessed for their abilities as diagnostic tools for dysferlinopathy. Results: Here we show that exosomes from dysferlin-expressing myotubes carry abundant dysferlin and enable transfer of full-length dysferlin protein to DYSF-/- myotubes. Exogenous dysferlin correctly localizes on DYSF-/- myotube membranes, enabling membrane resealing in response to injury. Human serum exosomes also carry dysferlin protein and improve membrane repair capabilities of human DYSF-/- myotubes irrespective of mutations. Lack of dysferlin in dysferlinopathic patient serum and urine exosomes enables differentiation between healthy controls and dysferlinopathic patients. Conclusions: Our findings provide evidence that exosomes are efficient carriers of dysferlin and can be employed for the treatment and non-invasive diagnosis of dysferlinopathy.

Feature Selection for Object-Based Classification of High-Resolution Remote Sensing Images Based on the Combination of a Genetic Algorithm and Tabu Search.

In object-based image analysis of high-resolution images, the number of features can reach hundreds, so it is necessary to perform feature reduction prior to classification. In this paper, a feature selection method based on the combination of a genetic algorithm (GA) and tabu search (TS) is presented. The proposed GATS method aims to reduce the premature convergence of the GA by the use of TS. A prematurity index is first defined to judge the convergence situation during the search. When premature convergence does take place, an improved mutation operator is executed, in which TS is performed on individuals with higher fitness values. As for the other individuals with lower fitness values, mutation with a higher probability is carried out. Experiments using the proposed GATS feature selection method and three other methods, a standard GA, the multistart TS method, and ReliefF, were conducted on WorldView-2 and QuickBird images. The experimental results showed that the proposed method outperforms the other methods in terms of the final classification accuracy.

Fluorescent peptide highlights micronodules in murine hepatocellular carcinoma models and humans in vitro.

Early detection and clear delineation of microscopic lesions during surgery are critical to the prognosis and survival of patients with hepatocellular carcinoma (HCC), a devastating malignancy without effective treatments except for resection. Tools to specifically identify and differentiate micronodules from normal tissue in HCC patients can have a positive impact on survival. Here, we discovered a peptide that preferentially binds to HCC cells through phage display. Significant accumulation of the fluorescence-labeled peptide in tumor from ectopic and orthotopic HCC mice was observed within 2 hours of systemic injection. Contrast between tumor and surrounding liver is up to 6.5-fold, and useful contrast lasts for 30 hours. Micronodules (0.03 cm in diameter) in liver and lung can clearly be distinguished from normal tissue with this fluorescence-labeled peptide in orthotopic HCC mice and HCC patients. Compared to indocyanine green, a Food and Drug Administration-approved imaging contrast agent, an up to 8.7-fold higher differentiation ratio of tumor to fibrosis is achieved with this fluorescence-labeled peptide. Importantly, this peptide enables up to 10-fold differentiation between HCC and peritumoral tissue in human tissues and the complete removal of tumor in HCC mice with surgical navigation. No abnormalities in behavior or activity are observed after systemic treatment, indicating the absence of overt toxicity. The peptide is metabolized with a half-life of approximately 4 hours in serum. CONCLUSION: Our findings demonstrate that micronodules can be specifically differentiated with high sensitivity from surrounding tissue with this molecule, opening clinical possibilities for early detection and precise surgery of HCC. (Hepatology 2018).