Research Article
Abstract
A key mechanism of tumor resistance to immune cells is mediated by expression of peptide-loaded HLA class I molecule (HLA-E) in tumor cells, which suppresses NK cell activity via ligation of the NK inhibitory receptor CD94/NK group 2 member A (NKG2A). Gene expression data from approximately 10,000 tumor samples showed widespread HLAE expression, with levels correlating with those of KLRC1 (NKG2A) and KLRD1 (CD94). To bypass HLA-E inhibition, we developed a way to generate highly functional NK cells lacking NKG2A. Constructs containing a single-chain variable fragment derived from an anti-NKG2A antibody were linked to endoplasmic reticulum–retention domains. After retroviral transduction in human peripheral blood NK cells, these NKG2A protein expression blockers (PEBLs) abrogated NKG2A expression. The resulting NKG2Anull NK cells had higher cytotoxicity against HLA-E–expressing tumor cells. Transduction of anti-NKG2A PEBL produced more potent cytotoxicity than interference with an anti-NKG2A antibody and prevented de novo NKG2A expression without affecting NK cell proliferation. In immunodeficient mice, NKG2Anull NK cells were substantially more powerful than NKG2A+ NK cells against HLA-E–expressing tumors. Thus, NKG2A downregulation evades the HLA-E cancer immune checkpoint and increases the antitumor activity of NK cell infusions. Because this strategy is easily adaptable to current protocols for clinical-grade immune cell processing, its clinical testing is feasible and warranted.
Authors
Takahiro Kamiya, See Voon Seow, Desmond Wong, Murray Robinson, Dario Campana
Abstract
The Epstein-Barr virus (EBV) is one of the predominant tumor viruses in humans, but so far no therapeutic or prophylactic vaccination against this transforming pathogen is available. We demonstrated that heterologous prime-boost vaccination with the nuclear antigen 1 of EBV (EBNA1), either targeted to the DEC205 receptor on DCs or expressed from a recombinant modified vaccinia virus Ankara (MVA) vector, improved priming of antigen-specific CD4+ T cell help. This help supported the expansion and maintenance of EBNA1-specific CD8+ T cells that are most efficiently primed by recombinant adenoviruses that encode EBNA1. These combined CD4+ and CD8+ T cell responses protected against EBNA1-expressing T and B cell lymphomas, including lymphoproliferations that emerged spontaneously after EBNA1 expression. In particular, the heterologous EBNA1-expressing adenovirus, boosted by EBNA1-encoding MVA vaccination, demonstrated protection as a prophylactic and therapeutic treatment for the respective lymphoma challenges. Our study shows that such heterologous prime-boost vaccinations against EBV-associated malignancies as well as symptomatic primary EBV infection should be further explored for clinical development.
Authors
Julia Rühl, Carmen Citterio, Christine Engelmann, Tracey Haigh, Andrzej Dzionek, Johannes Dreyer, Rajiv Khanna, Graham S. Taylor, Joanna B. Wilson, Carol S. Leung, Christian Münz
Abstract
Diabetic individuals are at considerable risk for invasive infection by Staphylococcus aureus, however, the mechanisms underlying this enhanced susceptibility to infection are unclear. We observed increased mortality following i.v. S. aureus infection in diabetic mice compared with nondiabetic controls, correlating with increased numbers of low-density neutrophils (LDNs) and neutrophil extracellular traps (NETs). LDNs have been implicated in the inflammatory pathology of diseases such as lupus, given their release of large amounts of NETs. Our goal was to describe what drives LDN increases during S. aureus infection in the diabetic host and mechanisms that promote increased NET production by LDNs. LDN development is dependent on TGF-β, which we found to be more activated in the diabetic host. Neutralization of TGF-β, or the TGF-β–activating integrin αvβ8, reduced LDN numbers and improved survival during S. aureus infection. Targeting S. aureus directly with MEDI4893*, an α toxin–neutralizing monoclonal antibody, blocked TGF-β activation, reduced LDNs and NETs, and significantly improved survival. A comparison of gene and protein expression in high-density neutrophils and LDNs identified increased GPCRs and elevated phosphatase and tensin homolog (PTEN) in the LDN subset. Inhibition of PTEN improved the survival of infected diabetic mice. Our data identify a population of neutrophils in infected diabetic mice that correlated with decreased survival and increased NET production and describe 3 therapeutic targets, a bacterial target and 2 host proteins, that prevented NET production and improved survival.
Authors
Taylor S. Cohen, Virginia Takahashi, Jessica Bonnell, Andrey Tovchigrechko, Raghothama Chaerkady, Wen Yu, Omari Jones-Nelson, Young Lee, Rajiv Raja, Sonja Hess, C. Kendall Stover, John J. Worthington, Mark A. Travis, Bret R. Sellman
Abstract
Alveolar epithelium plays a pivotal role in protecting the lungs from inhaled infectious agents. Therefore, the regenerative capacity of the alveolar epithelium is critical for recovery from these insults in order to rebuild the epithelial barrier and restore pulmonary functions. Here, we show that sublethal infection of mice with Streptococcus pneumoniae, the most common pathogen of community-acquired pneumonia, led to exclusive damage in lung alveoli, followed by alveolar epithelial regeneration and resolution of lung inflammation. We show that surfactant protein C–expressing (SPC-expressing) alveolar epithelial type II cells (AECIIs) underwent proliferation and differentiation after infection, which contributed to the newly formed alveolar epithelium. This increase in AECII activities was correlated with increased nuclear expression of Yap and Taz, the mediators of the Hippo pathway. Mice that lacked Yap/Taz in AECIIs exhibited prolonged inflammatory responses in the lung and were delayed in alveolar epithelial regeneration during bacterial pneumonia. This impaired alveolar epithelial regeneration was paralleled by a failure to upregulate IκBa, the molecule that terminates NF-κB–mediated inflammatory responses. These results demonstrate that signals governing resolution of lung inflammation were altered in Yap/Taz mutant mice, which prevented the development of a proper regenerative niche, delaying repair and regeneration of alveolar epithelium during bacterial pneumonia.
Authors
Ryan LaCanna, Daniela Liccardo, Peggy Zhang, Lauren Tragesser, Yan Wang, Tongtong Cao, Harold A. Chapman, Edward E. Morrisey, Hao Shen, Walter J. Koch, Beata Kosmider, Marla R. Wolfson, Ying Tian
Abstract
Vacuolar H+-ATPase–dependent (V-ATPase–dependent) functions are critical for neural proteostasis and are involved in neurodegeneration and brain tumorigenesis. We identified a patient with fulminant neurodegeneration of the developing brain carrying a de novo splice site variant in ATP6AP2 encoding an accessory protein of the V-ATPase. Functional studies of induced pluripotent stem cell–derived (iPSC-derived) neurons from this patient revealed reduced spontaneous activity and severe deficiency in lysosomal acidification and protein degradation leading to neuronal cell death. These deficiencies could be rescued by expression of full-length ATP6AP2. Conditional deletion of Atp6ap2 in developing mouse brain impaired V-ATPase–dependent functions, causing impaired neural stem cell self-renewal, premature neuronal differentiation, and apoptosis resulting in degeneration of nearly the entire cortex. In vitro studies revealed that ATP6AP2 deficiency decreases V-ATPase membrane assembly and increases endosomal-lysosomal fusion. We conclude that ATP6AP2 is a key mediator of V-ATPase–dependent signaling and protein degradation in the developing human central nervous system.
Authors
Takuo Hirose, Alfredo Cabrera-Socorro, David Chitayat, Thomas Lemonnier, Olivier Féraud, Carmen Cifuentes-Diaz, Nicolas Gervasi, Cedric Mombereau, Tanay Ghosh, Loredana Stoica, Jeanne d’Arc Al Bacha, Hiroshi Yamada, Marcel A. Lauterbach, Marc Guillon, Kiriko Kaneko, Joy W. Norris, Komudi Siriwardena, Susan Blasér, Jérémie Teillon, Roberto Mendoza-Londono, Marion Russeau, Julien Hadoux, Sadayoshi Ito, Pierre Corvol, Maria G. Matheus, Kenton R. Holden, Kohji Takei, Valentina Emiliani, Annelise Bennaceur-Griscelli, Charles E. Schwartz, Genevieve Nguyen, Matthias Groszer
Abstract
Idiopathic pulmonary fibrosis (IPF) is a deadly disease with a poor prognosis and few treatment options. Pathological remodeling of the extracellular matrix (ECM) is a key factor that drives the disease pathogenesis, although the underlying mechanisms remain unknown. Alternative polyadenylation (APA) has recently been shown to play a major role in cellular responses to stress by driving the expression of fibrotic factors through the alteration of miRNA sensitivity, but a connection to IPF has not been established. Here, we demonstrated that CFIm25, a global regulator of APA, was downregulated in the lungs of patients with IPF and mice with pulmonary fibrosis, with its expression selectively reduced in α–smooth muscle actin–positive (α-SMA–positive) fibroblasts. Following CFIm25 knockdown in healthy human lung fibroblasts, we identified 808 genes with shortened 3′-UTRs, including those involved in the TGF-β signaling pathway, the Wnt signaling pathway, and cancer pathways. The expression of key profibrotic factors was suppressed by CFIm25 overexpression in IPF fibroblasts. Finally, we demonstrated that deletion of CFIm25 in fibroblasts or myofibroblast precursors using either the Col1a1 or the Foxd1 promoter enhanced pulmonary fibrosis after bleomycin exposure. Collectively, our results identified CFIm25 downregulation as an important mechanism for elevating profibrotic gene expression in pulmonary fibrosis.
Authors
Tingting Weng, Junsuk Ko, Chioniso P. Masamha, Zheng Xia, Yu Xiang, Ning-yuan Chen, Jose G. Molina, Scott Collum, Tinne C. Mertens, Fayong Luo, Kemly Philip, Jonathan Davies, Jingjing Huang, Cory Wilson, Rajarajan A. Thandavarayan, Brian A. Bruckner, Soma S.K. Jyothula, Kelly A. Volcik, Lei Li, Leng Han, Wei Li, Shervin Assassi, Harry Karmouty-Quintana, Eric J. Wagner, Michael R. Blackburn
Abstract
The mesenchymal (MES) subtype of glioblastoma (GBM) stem cells (GSCs) represents a subpopulation of cancer cells that are notorious for their highly aggressive nature and resistance to conventional therapy. Aldehyde dehydrogenase 1A3 (ALDH1A3) has been recently suggested as a key determinant for the maintenance of MES features of GSCs. However, the mechanisms underpinning aberrant ALDH1A3 expression remain elusive. Here, we identified ubiquitin-specific protease 9X (USP9X) as a bona fide deubiquitinase of ALDH1A3 in MES GSCs. USP9X interacted with, depolyubiquitylated, and stabilized ALDH1A3. Moreover, we showed that FACS-sorted USP9Xhi cells were enriched for MES GSCs with high ALDH1A3 activity and potent tumorigenic capacity. Depletion of USP9X markedly downregulated ALDH1A3, resulting in a loss of self-renewal and tumorigenic capacity of MES GSCs, which could be largely rescued by ectopic expression of ALDH1A3. Furthermore, we demonstrated that the USP9X inhibitor WP1130 induced ALDH1A3 degradation and showed marked therapeutic efficacy in MES GSC–derived orthotopic xenograft models. Additionally, USP9X strongly correlated with ALDH1A3 expression in primary human GBM samples and had a prognostic value for patients with the MES subgroup. Collectively, our findings unveil USP9X as a key deubiquitinase for ALDH1A3 protein stabilization and a potential target for GSC-directed therapy.
Authors
Zhengxin Chen, Hong-Wei Wang, Shuai Wang, Ligang Fan, Shuang Feng, Xiaomin Cai, Chenghao Peng, Xiaoting Wu, Jiacheng Lu, Dan Chen, Yuanyuan Chen, Wenting Wu, Daru Lu, Ning Liu, Yongping You, Huibo Wang
Abstract
Neuromyelitis optica (NMO) is an autoimmune CNS disorder mediated by pathogenic aquaporin-4 (AQP4) water channel autoantibodies (AQP4-IgG). Although AQP4-IgG–driven complement-dependent cytotoxicity (CDC) is critical for the formation of NMO lesions, the molecular mechanisms governing optimal classical pathway activation are unknown. We investigated the molecular determinants driving CDC in NMO using recombinant AQP4–specific autoantibodies (AQP4 rAbs) derived from affected patients. We identified a group of AQP4 rAbs targeting a distinct extracellular loop C epitope that demonstrated enhanced CDC on target cells. Targeted mutations of AQP4 rAb Fc domains that enhance or diminish C1q binding or antibody Fc-Fc interactions showed that optimal CDC was driven by the assembly of multimeric rAb platforms that increase multivalent C1q binding and facilitate C1q activation. A peptide that blocks antibody Fc-Fc interaction inhibited CDC induced by AQP4 rAbs and polyclonal NMO patient sera. Super-resolution microscopy revealed that AQP4 rAbs with enhanced CDC preferentially formed organized clusters on supramolecular AQP4 orthogonal arrays, linking epitope-dependent multimeric assembly with enhanced C1q binding and activation. The resulting model of AQP4-IgG CDC provides a framework for understanding classical complement activation in human autoantibody–mediated disorders and identifies a potential new therapeutic avenue for treating NMO.
Authors
John Soltys, Yiting Liu, Alanna Ritchie, Scott Wemlinger, Kristin Schaller, Hannah Schumann, Gregory P. Owens, Jeffrey L. Bennett
Abstract
DCs undergo metabolic reprogramming from a predominantly oxidative phosphorylation (OXPHOS) to glycolysis to mount an immunogenic response. The mechanism underpinning the metabolic reprogramming remains elusive. We demonstrate that miRNA-142 (miR-142) is pivotal for this shift in metabolism, which regulates the tolerogenic and immunogenic responses of DCs. In the absence of miR-142, DCs fail to switch from OXPHOS and show reduced production of proinflammatory cytokines and the ability to activate T cells in vitro and in in vivo models of sepsis and alloimmunity. Mechanistic studies demonstrate that miR-142 regulates fatty acid (FA) oxidation, which causes the failure to switch to glycolysis. Loss- and gain-of-function experiments identified carnitine palmitoyltransferase -1a (CPT1a), a key regulator of the FA pathway, as a direct target of miR-142 that is pivotal for the metabolic switch. Thus, our findings show that miR-142 is central to the metabolic reprogramming that specifically favors glycolysis and immunogenic response by DCs.
Authors
Yaping Sun, Katherine Oravecz-Wilson, Sydney Bridges, Richard McEachin, Julia Wu, Stephanie H. Kim, Austin Taylor, Cynthia Zajac, Hideaki Fujiwara, Daniel Christopher Peltier, Thomas Saunders, Pavan Reddy
Abstract
T cell heterogeneity is highly relevant to allergic disorders. We resolved the heterogeneity of human tissue CD3+ T cells during allergic inflammation, focusing on a tissue-specific allergic disease, eosinophilic esophagitis (EoE). We investigated 1088 single T cells derived from patients with a spectrum of disease activity. Eight disparate tissue T cell subtypes (designated T1–T8) were identified, with T7 and T8 enriched in the diseased tissue. The phenotypes of T7 and T8 resemble putative Treg (FOXP3+) and effector Th2-like (GATA3+) cells, respectively. Prodigious levels of IL-5 and IL-13 were confined to HPGDS+ CRTH2+IL-17RB+FFAR3+CD4+ T8 effector Th2 cells. EoE severity closely paralleled a lipid/fatty acid–induced activation node highlighted by the expression of the short-chain fatty acid receptor FFAR3. Ligands for FFAR3 induced Th2 cytokine production from human and murine T cells, including in an in vivo allergy model. Therefore, we elucidated the defining characteristics of tissue-residing CD3+ T cells in EoE, a specific enrichment of CD4+ Treg and effector Th2 cells, confinement of type 2 cytokine production to the CD4+ effector population, a highly likely role for FFAR3 in amplifying local Th2 responses in EoE, and a resource to further dissect tissue lymphocytes and allergic responses.
Authors
Ting Wen, Bruce J. Aronow, Yrina Rochman, Mark Rochman, Kiran KC, Phil J. Dexheimer, Philip Putnam, Vincent Mukkada, Heather Foote, Kira Rehn, Sam Darko, Daniel Douek, Marc E. Rothenberg
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Copyright © 2019 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)