There is an unmet need for monoclonal antibodies (mAbs) for prevention or as adjunctive treatment of herpes simplex virus (HSV) disease. Most vaccine and mAb efforts focus on neutralizing antibodies, but for HSV this strategy has proven ineffective. Preclinical studies with a candidate HSV vaccine strain, ΔgD-2, demonstrated that non-neutralizing antibodies that activate Fc-gamma receptors (FcɣRs) to mediate antibody-dependent cellular cytotoxicity (ADCC) provide active and passive protection against HSV-1 and HSV-2. We hypothesized that this vaccine provides a tool to identify and characterize protective mAbs. We isolated HSV-specific mAbs from germinal center and memory B cells and bone marrow plasmacytes of ΔgD-2 vaccinated mice and evaluated these mAbs for binding, neutralizing and FcɣR-activating activity and for protective efficacy in mice. The most potent protective mAb, BMPC-23, was not neutralizing but activated murine FcɣRIV, a biomarker of ADCC. The cryo-EM structure of the Fab-glycoprotein B (gB) assembly identified domain IV of gB as the epitope. A single dose of BMPC-23 administered 24 hours before or after viral challenge provided significant protection when configured as mouse IgG2c and protected mice expressing human FcɣRIII when engineered as a human IgG1. These results highlight the importance of FcɣR-activating antibodies in protecting against HSV.
Masayuki Kuraoka, Clare Burn Aschner, Ian W. Windsor, Aakash Mahant Mahant, Scott J. Garforth, Susan Luozheng Kong, Jacqueline M. Achkar, Steven C. Almo, Garnett Kelsoe, Betsy C. Herold
Comprehensive cis-regulatory landscapes are essential for accurate enhancer prediction and disease variant mapping. Although cis-regulatory element (CRE) resources exist for most tissues and organs, many rare – yet functionally important – cell types remain overlooked. Despite representing only a small fraction of the heart’s cellular biomass, the cardiac conduction system (CCS) unfailingly coordinates every life-sustaining heartbeat. To globally profile the mouse CCS cis-regulatory landscape, we genetically tagged CCS component-specific nuclei for comprehensive ATAC-seq analysis. Thus, we established a global CCS-enriched CRE database (CCS-ATAC) as a key resource for studying CCS-wide and component-specific regulatory functions. Using transcription factor (TF) motifs to construct CCS component-specific gene regulatory networks (GRNs), we identify and independently confirm several novel TF sub-networks. Highlighting the functional importance of CCS-ATAC, we also validate numerous CCS-enriched enhancer elements and suggest gene targets based on CCS single-cell RNA-seq data. Furthermore, we leverage CCS-ATAC to improve annotation of existing human variants related to cardiac rhythm and nominate a potential enhancer-target pair dysregulated by a specific SNP. Collectively, our results establish a CCS regulatory compendium, identify novel CCS enhancer elements, and illuminate potential functional associations between human genomic variants and CCS component-specific CREs.
Samadrita Bhattacharyya, Rahul K. Kollipara, Gabriela Orquera-Tornakian, Sean Goetsch, Minzhe Zhang, Cameron Perry, Boxun Li, John M. Shelton, Minoti Bhakta, Jialei Duan, Yang Xie, Guanghua Xiao, Bret M. Evers, Gary C. Hon, Ralf Kittler, Nikhil V. Munshi
The emergence of the novel henipavirus, Langya virus, received global attention earlier this month after the virus sickened over three dozen people in China. There is heightened concern henipaviruses as respiratory pathogens could spark another pandemic, most notably the deadly Nipah virus (NiV). NiV causes near annual outbreaks in Bangladesh and India and induces a highly fatal respiratory disease and encephalitis in humans. No licensed countermeasures against this pathogen exist. An ideal NiV vaccine would confer both fast-acting and long-lived protection. Recently, we reported the generation of a recombinant vesicular stomatitis virus (rVSV)-based vaccine expressing the NiV glycoprotein (rVSV-ΔG-NiVBG) that protected 100% of nonhuman primates from NiV-associated lethality within a week. Here, to evaluate the durability of rVSV-ΔG-NiVBG, we vaccinated African green monkeys (AGMs) one year prior to challenge with a uniformly lethal dose of NiV. The rVSV-ΔG-NiVBG vaccine induced stable and robust humoral responses, whereas cellular responses were modest. All immunized AGMs (whether receiving a single dose or prime-boosted) survived with no detectable clinical signs or NiV replication. Transcriptomic analyses indicated adaptive immune signatures correlated with vaccine-mediated protection. While vaccines for certain respiratory infections (e.g., COVID-19) have yet to provide durable protection, our results suggest rVSV-ΔG-NiVBG elicits long-lasting immunity.
Courtney Woolsey, Viktoriya Borisevich, Alyssa C. Fears, Krystle N. Agans, Daniel J. Deer, Abhishek N. Prasad, Rachel O'Toole, Stephanie L. Foster, Natalie S. Dobias, Joan B. Geisbert, Karla A. Fenton, Robert W. Cross, Thomas Geisbert
Absence of Interferon-γ Receptor (IFNGR) or Signal Transducer and Activator of Transcription 1 (STAT1) signaling in donor cells has been shown to result in reduced acute GVHD induction. In this study, we unexpectedly observed increased activation and expansion of donor lymphocytes in both lymphohematopoietic organs and GVHD target tissues of IFNGR/STAT1-deficient recipient mice, leading to rapid mortality following the induction of GVHD. Lipopolysaccharide (LPS)-matured bone marrow-derived Ifngr1-/-/Stat1-/- dendritic cells (BMDCs) were more potent allogeneic stimulators and expressed increased levels of MHC II and costimulatory molecules. Similar effects were observed in human APCs with knockdown of Stat1 by CRISPR/Cas9 and treatment with a JAK1/2 inhibitor. Furthermore, we demonstrated that the absence of IFNGR/STAT1 signaling in hematopoietic APCs impaired the presentation of exogenous antigens while promoting the presentation of endogenous antigens. In contrast, the indirect presentation of host antigens to donor lymphocytes was defective in IFNGR/STAT1-deficient donor-derived APCs in fully donor chimeric mice. The differential effects of IFNGR/STAT1 signaling on endogenous and exogenous antigen presentation could provide further insight into the roles of the IFN-γ/STAT1 signal pathway in the pathogenesis of GVHD, organ rejection, and autoimmune diseases.
Caisheng Lu, Huihui Ma, Liangsong Song, Hui Wang, Lily Wang, Shirong Li, Stephen M. Lagana, Antonia R. Sepulveda, Kasper Hoebe, Samuel S. Pan, Yong-Guang Yang, Suzanne Lentzsch, Markus Y. Mapara
KRAS is one of the most frequently activated oncogenes in human cancers. While the role of KRAS mutation in tumorigenesis and tumor maintenance has been extensively studied, the relationship between KRAS and the tumor immune microenvironment is not fully understood. Herein, we identified a novel role of KRAS in driving tumor evasion from innate immune surveillance. In lung adenocarcinoma patient samples and Kras-driven genetic mouse models of lung cancer, mutant KRAS activated the expression of cluster of differentiation 47 (CD47), an antiphagocytic signal in cancer cells, leading to decreased phagocytosis of cancer cells by macrophages. Mechanistically, mutant KRAS activated PI3K-STAT3 signaling, which restrained miR-34a expression and relieved the post-transcriptional repression of miR-34a on CD47. In three independent lung cancer patient cohorts, KRAS mutation status positively correlated with CD47 expression. Therapeutically, disruption of the KRAS-CD47 signaling axis with KRAS siRNA, the KRASG12C inhibitor AMG 510 or miR-34a mimic suppressed CD47 expression, enhanced the phagocytic capacity of macrophages and restored innate immune surveillance. Our results revealed a direct mechanistic link between active KRAS and innate immune evasion and identified CD47 as a major effector underlying KRAS-mediated immunosuppressive tumor microenvironment.
Huanhuan Hu, Rongjie Cheng, Yanbo Wang, Xiaojun Wang, Jianzhuang Wu, Yan Kong, Shoubin Zhan, Zhen Zhou, Hongyu Zhu, Ranran Yu, Gaoli Liang, Qingyan Wang, Xiaoju Zhu, Chen-Yu Zhang, Rong Yin, Chao Yan, Xi Chen
Repeated or prolonged, but not short-term, general anesthesia during the early postnatal period causes long-lasting impairments in memory formation in various species. The mechanisms underlying long-lasting impairment in cognitive function are poorly understood. Here we showed that repeated general anesthesia in postnatal mice induces preferential apoptosis and subsequent loss of parvalbumin-positive inhibitory interneurons in the hippocampus. Each parvalbumin interneuron controls the activity of multiple pyramidal excitatory neurons, thereby regulating neuronal circuits and memory consolidation. Preventing the loss of parvalbumin neurons by deleting a pro-apoptotic protein MAPL (Mitochondrial Anchored Protein Ligase) selectively in parvalbumin neurons rescued anesthesia-induced deficits in pyramidal cell inhibition, and hippocampus-dependent long-term memory. Conversely, partial depletion of parvalbumin neurons in neonates was sufficient to engender long-lasting memory impairment. Thus, loss of parvalbumin interneurons in postnatal mice following repeated general anesthesia critically contributes to memory deficits in adulthood.
Patricia Soriano Roque, Carolina Thörn Perez, Mehdi Hooshmandi, Calvin Wong, Mohammad Javad Eslamizade, Shilan Heshmati, Nicole Brown, Vijendra Sharma, Kevin C. Lister, Vanessa Magalie Goyon, Laura E. Neagu-Lund, Cathy Shen, Nicolas Daccache, Hiroaki Sato, Tamaki Sato, Jeffrey S. Mogil, Karim Nader, Christos G. Gkogkas, Mihaela D. Iordanova, Masha Prager-Khoutorsky, Heidi M. McBride, Jean-Claude Lacaille, Linda Wykes, Thomas Schricker, Arkady Khoutorsky
Glioblastoma ranks among the most aggressive and lethal of all human cancers. Self-renewing, highly tumorigenic glioblastoma stem cells (GSCs) contribute to therapeutic resistance and maintain cellular heterogeneity. Here, we interrogated superenhancer landscapes of primary glioblastoma specimens and patient-derived GSCs, revealing a kelch domain-containing gene (KLHDC8A) with a previously unknown function as an epigenetically-driven oncogene. Targeting KLHDC8A decreased GSC proliferation and self-renewal, induced apoptosis, and impaired in vivo tumor growth. Transcription factor control circuitry analyses revealed that the master transcriptional regulator SOX2 stimulated KLHDC8A expression. Mechanistically, KLHDC8A bound Chaperonin-Containing TCP1 (CCT) to promote assembly of primary cilia to activate Hedgehog signaling. KLHDC8A expression correlated with Aurora B/C Kinase inhibitor activity, which induced primary cilia and Hedgehog signaling. Combinatorial targeting of Aurora B/C Kinase and Hedgehog displayed augmented benefit against GSC proliferation. Collectively, superenhancer-based discovery revealed KLHDC8A as a novel molecular target of cancer stem cells that promotes ciliogenesis to activate the Hedgehog pathway, offering insights into therapeutic vulnerabilities for glioblastoma treatment.
Derrick Lee, Ryan C. Gimple, Xujia Wu, Briana C. Prager, Zhixin Qiu, Qiulian Wu, Vikas Daggubati, Aruljothi Mariappan, Jay Gopalakrishnan, Matthew R. Sarkisian, David R. Raleigh, Jeremy N. Rich
WEE1 has emerged as an attractive target in epithelial ovarian cancer (EOC), but how EOC cells may alter sensitivity to WEE1 inhibition remains unclear. Here, through a cell cycle machinery-related gene RNAi screen, we found that targeting ODF2L is synthetic lethal with WEE1 kinase inhibition in EOC cells. Knockdown of ODF2L robustly sensitized cells to treatment of the WEE1 inhibitor AZD1775 in EOC cell lines in vitro, as well as xenografts in vivo. Mechanistically, the increased sensitivity to WEE1 inhibition upon ODF2L loss was accompanied by accumulated DNA damage. ODF2L licensed the recruitment of PKMYT1, a functionally redundant kinase of WEE1, to the CDK1/cyclin B complex and thus restricted the activity of CDK1 when WEE1 was inhibited. Clinically, upregulation of ODF2L correlated with CDK1 activity, DNA damage level, and sensitivity to WEE1 inhibition in patient-derived EOC cells. Moreover, the ODF2L level predicted the response to WEE1 inhibition in an EOC patient-derived xenograft model. Combination treatment with tumor-targeted lipid nanoparticles that package ODF2L siRNA and AZD1775 led to the synergistic attenuation of tumor growth in the ID8 ovarian cancer syngeneic mouse model. These data suggest that WEE1 inhibition is a promising precision therapeutic strategy for ODF2L-low-expressing EOC cells.
Jie Li, Jingyi Lu, Manman Xu, Shiyu Yang, Tiantian Yu, Cuimiao Zheng, Xi Huang, Yuwen Pan, Yangyang Chen, Junming Long, Chunyu Zhang, Hua Huang, Qingyuan Dai, Bo Li, Wei Wang, Shuzhong Yao, Chaoyun Pan
T cell exhaustion is a state of T cell dysfunction associated with expression of programmed death 1 (PD-1). Exhausted CD8 T cells are maintained by self-renewing stem-like T (TSL) cells that provide differentiated TIM3+ cells, a part of which possesses effector-like properties. PD-1-targeted therapies enhance T cell response by promoting differentiation of TSL cells toward TIM3+ cells, but the role of mTOR during T cell exhaustion remains elusive. Here, we show that mTOR inhibition has distinct outcomes during the beginning of and after the establishment of chronic viral infection. Blocking mTOR during the T cell expansion phase enhanced the T cell response by accumulating TSL cells, leading to improved efficacy of PD-1 immunotherapy. Whereas, after exhaustion progressed, mTOR inhibition caused immunosuppression characterized by decreased TIM3+ cells and increased viral load with minimal changes in TSL cells. Mechanistically, a cell-intrinsic mTOR signal was vital for differentiation of TSL cells into the TIM3+ state in the early and late phases of chronic infection as well as during PD-1 immunotherapy. Thus, PD-1 blockade worked after cessation of mTOR inhibition but simultaneous treatment failed to induce functional TIM3+ cells, reducing efficacy of PD-1 immunotherapy. Our data demonstrate that mTOR regulates T cell exhaustion and have important implications for combination cancer therapies with PD-1 blockade
Satomi Ando, Charles Perkins, Yamato Sajiki, Chase Chastain, Rajesh M. Valanparambil, Andreas Wieland, William H. Hudson, Masao Hashimoto, Suresh S. Ramalingam, Gordon J. Freeman, Rafi Ahmed, Koichi Araki
Three principal ER quality-control mechanisms, namely, unfolded protein response (UPR), ER-associated degradation (ERAD) and ER-phagy are each important for the maintenance of ER homeostasis, yet how they are integrated to regulate ER homeostasis and organellar architecture in vivo is largely unclear. Here we report intricate crosstalk among the three pathways, centered around the SEL1L-HRD1 protein complex of ERAD, in the regulation of organellar organization in β-cells. SEL1L-HRD1 ERAD deficiency in β-cells triggers activation of autophagy via IRE1α [an endogenous ERAD substrate]. In the absence of functional SEL1L-HRD1 ERAD, proinsulin is retained in the ER as high molecular weight conformers, which are subsequently cleared via ER-phagy. A combined loss of both SEL1L and autophagy in β-cells leads to diabetes in mice shortly after weaning, with premature death by ~11 weeks of age, associated with marked ER retention of proinsulin and β-cell loss. Using focus-ion beam scanning electron microscopy (FIB-SEM) powered by deep-learning automated image segmentation and 3D reconstruction, our data demonstrate a profound organellar restructuring with a massive expansion of ER volume and network in β-cells lacking both SEL1L and autophagy. These data reveal at an unprecedented detail the intimate crosstalk among the three ER quality-control mechanisms in the dynamic regulation of organellar architecture and β-cell function.
Neha Shrestha, Mauricio Torres, Jason Zhang, You Lu, Leena Haataja, Rachel B. Reinert, Jeffrey Knupp, Yu-Jie Chen, Gunes Parlakgul, Ana Paula Arruda, Billy Tsai, Peter Arvan, Ling Qi
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