Recent transcriptomic-based analysis of diffuse large B cell lymphoma (DLBCL) has highlighted the clinical relevance of lymph node (LN) fibroblast and tumor-infiltrating lymphocyte (TIL) signatures within the tumor microenvironment (TME). However, the immunomodulatory role of fibroblasts in lymphoma remains unclear. Here, by studying human and mouse DLBCL-LNs, we identify the presence of an aberrantly remodeled fibroblastic reticular cell (FRC) network, expressing elevated fibroblast activated protein (FAP). RNA-sequencing analyses reveal that exposure to DLBCL reprograms key immunoregulatory pathways in FRCs, including a switch from homeostatic to inflammatory chemokine expression and elevated antigen presentation molecules. Functional assays show that DLBCL-activated FRCs (DLBCL-FRCs) hinder optimal TIL and chimeric antigen receptor T cell (CAR-T) migration. Moreover, DLBCL-FRCs inhibited CD8+ TIL cytotoxicity in an antigen-specific manner. Notably, the interrogation of patient LNs with imaging mass cytometry identified distinct environments differing in their CD8+ TIL-FRC composition and spatial organization that associated with survival outcomes. We further demonstrate the potential to target inhibitory FRCs to rejuvenate interacting TILs. Co-treating organotypic cultures with FAP-targeted immunostimulatory drugs and a bispecific antibody (glofitamab) augmented anti-lymphoma TIL cytotoxicity. Together, our study reveals an immunosuppressive role of FRCs in DLBCL, with implications for immune evasion, disease pathogenesis and optimizing immunotherapy for patients.
Benedetta Apollonio, Filomena Spada, Nedyalko Petrov, Domenico Cozzetto, Despoina Papazoglou, Peter Jarvis, Shichina Kannambath, Manuela Terranova-Barberio, Rose-Marie Amini, Gunilla Enblad, Charlotte E. Graham, Reuben Benjamin, Elizabeth H. Phillips, Richard J. Ellis, Rosamond Nuamah, Mansoor Saqi, Dinis P. Calado, Richard Rosenquist, Lesley A. Sutton, Jonathan R. Salisbury, Georgios Zacharioudakis, Anna Vardi, Patrick R. Hagner, Anita K. Gandhi, Marina Bacac, Christina Claus, Pablo Umana, Ruth F. Jarrett, Christian Klein, Alexander J.A. Deutsch, Alan G. Ramsay
Multiple sclerosis (MS) is the most common chronic inflammatory disease of the central nervous system (CNS). The individual course is highly variable with complete remission in some patients and relentless courses in others. We generated induced pluripotent stem cells (iPSCs) to investigate possible mechanisms in benign MS (BMS), compared to progressive MS (PMS). We differentiated neurons and astrocytes that were then stressed with inflammatory cytokines typically associated with MS. TNFα/IL-17A treatment increased neurite damage in MS neurons irrespective of clinical phenotypes. In contrast, TNFα/IL-17A-reactive BMS astrocytes cultured with healthy control (HC) neurons exhibited significantly decreased axonal damage, compared to PMS astrocytes. Accordingly, single cell transcriptomic analysis of BMS-astrocyte co-cultured neurons demonstrated upregulated pathways of neuronal resilience, namely these astrocytes revealed differential growth factor expression. Moreover, supernatants from BMS astrocyte-neuron co-cultures rescued TNFα/IL-17-induced neurite damage. This process was associated with the unique expression of the growth factors, LIF and TGF-β1, as induced by TNFα/IL-17 and JAK-STAT activation. Our findings highlight a potential therapeutic role of modulating astrocyte phenotypes that generate a neuroprotective milieu preventing permanent neuronal damage.
Janis Kerkering, Bakhrom Muinjonov, Kamil Sebastian Rosiewicz, Sebastian Diecke, Charlotte Biese, Juliane Schiweck, Claudia Chien, Dario Zocholl, Thomas Conrad, Friedemann Paul, Marlen Alisch, Volker Siffrin
BACKGROUND. SARS-CoV-2 infection in Africa has been characterized by less severe disease than elsewhere but the profile of SARS-CoV-2 specific adaptive immunity in this largely asymptomatic spread has not been studied. METHODS. We collected blood and nasopharyngeal samples from rural Kenyans (n=80) without respiratory symptoms since 2019, had no contact with COVID-19 cases or received COVID-19 vaccines and were negative for current SARS-CoV-2 infection. We analyzed spike-specific antibodies and T cells specific for SARS-CoV-2 structural (membrane, nucleocapsid and spike) and accessory (ORF3a, ORF7, ORF8) proteins. Pre-pandemic samples collected in urban Nairobi, Kenya (n=13) between 2015-2016 and samples of mild-moderately symptomatic COVID-19 convalescents (n=36) living in the urban environment of Singapore were also studied. RESULTS. Among asymptomatic Kenyans, we detected anti-spike antibodies in 41.0% and T cell responses against ≥2 SARS-CoV-2 proteins in 82.5%. The pre-pandemic samples from Nairobi had low-level, monospecific responses. Furthermore, distinct from cellular immunity in European and Asian COVID-19 convalescents, strong T cell immunogenicity was observed against viral accessory proteins (ORF3a, ORF8) and not structural proteins, as well as a higher IL-10/IFN-γ ratio cytokine profile. CONCLUSIONS. The high incidence of T cell responses against different SARS-CoV-2 proteins in largely seronegative participants suggests that serosurveys underestimate SARS-CoV-2 prevalence in settings where asymptomatic infections prevail. Similar observations have been made with other coronavirus infections such as MERS and SARS-CoV-1. The functional and antigen-specific profile of SARS-CoV-2 specific T cells in these African individuals suggests that genetic or environmental factors play a role in the development of protective antiviral immunity. FUNDINGS. U.S. Centers for Disease Control and Prevention, Division of Global Health Protection; the Singapore Ministry of Health’s National Medical Research Council.
Taraz Samandari, Joshua Ongalo, Kimberly McCarthy, Richard K. Biegon, Philister Madiega, Anne Mithika, Joseph Orinda, Grace M. Mboya, Patrick Mwaura, Omu Anzala, Clayton Onyango, Fredrick O. Oluoch, Eric M. Osoro, Charles-Antoine Dutertre, Nicole Tan, Shou Kit Hang, Smrithi Hariharaputran, David C. Lye, Amy Herman-Roloff, Nina Le Bert, Antonio Bertoletti
The ADP ribosyl transferases (PARPs 1–17) regulate diverse cellular processes, including DNA damage repair. PARPs are classified based on their ability to catalyze poly-ADP-ribosylation (PARylation) or mono-ADP-ribosylation (MARylation). While PARP9 mRNA expression is significantly increased in progressive human tuberculosis (TB), its participation in host immunity to TB is unknown. Here, we show that PARP9 mRNA encoding the MARylating PARP9 enzyme is upregulated during TB in humans and mice and provide evidence of a critical modulatory role for PARP9 in DNA damage, cGAS and type I IFN production during TB. Thus, Parp9-deficient mice are susceptible to Mtb infection and exhibit increased TB disease, cGAS expression, cGAMP and type I IFN production along with upregulation of complement and coagulation pathways. Enhanced Mtb susceptibility is type I IFN-dependent, as blockade of IFNAR signaling reversed the enhanced susceptibility of Parp9-/- mice. Thus, in sharp contrast with PARP9 enhancement of type I IFN production in viral infections, this member of the MAR family plays a protective role by limiting type I IFN responses during TB.
Shyamala Thirunavukkarasu, Mushtaq Ahmed, Bruce A. Rosa, Mark Boothby, Sung Hoon Cho, Javier Rangel-Moreno, Stanley K. Mbandi, Valérie Schreiber, Ananya Gupta, Joaquin Zúñiga, Makedonka Mitreva, Deepak Kaushal, Thomas J. Scriba, Shabaana A. Khader
Heterogeneity in human immune responses is difficult to model in standard laboratory mice. To understand how host variation affects BCG-induced immunity against Mycobacterium tuberculosis, we studied 24 unique Collaborative Cross (CC) mouse strains, which differ primarily in the genes and alleles they inherit from founder strains. The CC strains were vaccinated with or without BCG, and then challenged with aerosolized M. tuberculosis. As BCG protects only half of the CC strains tested, we conclude that host genetics has a major influence on BCG-induced immunity against M. tuberculosis infection, making it an important barrier to vaccine-mediated protection. Importantly, BCG efficacy is dissociable from inherent susceptibility to TB. T cell immunity was extensively characterized to identify components associated with protection that were stimulated by BCG and recalled after Mtb infection. Although considerable diversity is observed, BCG has little impact on the composition of T cells in the lung after infection. Instead, variability is largely shaped by host genetics. BCG-elicited protection against TB correlated with changes in immune function. Thus, CC mice can be used to define correlates of protection and to identify vaccine strategies that protect a larger fraction of genetically diverse individuals instead of optimizing protection for a single genotype.
Rocky Lai, Diana N. Gong, Travis Williams, Abiola F. Ogunsola, Kelly Cavallo, Cecilia S. Lindestam Arlehamn, Sarah Acolatse, Gillian Beamer, Martin T. Ferris, Christopher M. Sassetti, Douglas A. Lauffenburger, Samuel M. Behar
Hypertrophic cardiomyopathy (HCM) is the most prominent cause of sudden cardiac death in young individuals. Due to heterogeneity in the clinical manifestations, conventional HCM drugs have limitations for mitochondrial hypertrophic cardiomyopathy. Discovering more effective compounds would be of substantial benefit for further elucidating the pathogenic mechanisms of HCM and treating patients with this condition. We previously reported the MT-RNR2 variant associated with HCM that results in mitochondrial dysfunction. Here, we screened a mitochondria-associated compound library by quantifying the mitochondrial membrane potential of HCM cybrids and the survival rate of HCM induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) in galactose media. 1-Deoxynojirimycin (DNJ) was identified to rescue mitochondrial function by targeting optic atrophy protein 1 (OPA1) to promote its oligomerization, leading to reconstruction of the mitochondrial cristae. DNJ treatment further recovered the physiological properties of HCM iPSC-CMs by improving Ca2+ homeostasis and electrophysiological properties. An angiotensin II-induced cardiac hypertrophy mouse model further verified the efficacy of DNJ in promoting cardiac mitochondrial function and alleviating cardiac hypertrophy in vivo. These results demonstrated that DNJ could be a potential mitochondrial rescue agent for mitochondrial hypertrophic cardiomyopathy. Our findings will help elucidate the mechanism of HCM and provide a potential therapeutic strategy.
Qianqian Zhuang, Fengfeng Guo, Lei Fu, Yufei Dong, Shaofang Xie, Xue Ding, Shuangyi Hu, Xuanhao D. Zhou, Yangwei Jiang, Hui Zhou, Yue Qiu, Zhaoying Lei, Mengyao Li, Huajian Cai, Mingjie Fan, Lingjie Sang, Yong Fu, Dong Zhang, Aifu Lin, Xu Li, Tilo Kunath, Ruhong Zhou, Ping Liang, Zhong Liu, Qingfeng Yan
During emergency hematopoiesis, hematopoietic stem cells (HSCs) rapidly proliferate to produce myeloid and lymphoid effector cells, a response that is critical against infection or tissue injury. If unresolved, this process leads to sustained inflammation which can cause life-threatening diseases and cancer. We have identified a novel role of Dpf2 in inflammation. Dpf2 is a defining subunit of the hematopoietic-specific BAF (SWI/SNF) chromatin-remodeling complex, and it is mutated in multiple cancers and neurological disorders. We uncover that hematopoietic-specific Dpf2 knock-out mice develop leukopenia, severe anemia and lethal systemic inflammation characterized by histiocytic and fibrotic tissue infiltration, resembling a clinical hyper-inflammatory state. Dpf2 loss impairs the polarization of macrophages responsible for tissue repair, induces unrestrained activation of T helper cells, and generates an emergency-like state of HSC hyperproliferation and myeloid-biased differentiation. Mechanistically, Dpf2 deficiency results in the loss of the BAF catalytic subunit Brg1 from Nrf2-controlled enhancers, impairing the anti-oxidant and anti-inflammatory transcriptional response needed to modulate inflammation. Finally, pharmacological reactivation of Nrf2 can suppress the inflammation-mediated phenotypes and lethality of Dpf2Δ/Δ mice. Our work establishes the essential role of the Dpf2/BAF complex in licensing Nrf2-dependent gene expression in HSCs and immune effector cells to prevent chronic inflammation.
Gloria Mas, Na Man, Yuichiro Nakata, Concepcion Martinez-Caja, Daniel L. Karl, Felipe Beckedorff, Francesco Tamiro, Chuan Chen, Stephanie Duffort, Hidehiro Itonaga, Adnan K. Mookhtiar, Kranthi Kunkalla, Alfredo M. Valencia, Clayton K. Collings, Cigall Kadoch, Francisco Vega, Scott C. Kogan, Lluis Morey, Daniel Bilbao, Stephen D. Nimer
ATRX is one of the most frequently altered genes in solid tumors, and mutation is especially frequent in soft tissue sarcomas. However, the role of ATRX in tumor development and response to cancer therapies remains poorly understood. Here, we developed a primary mouse model of soft tissue sarcoma and showed that Atrx deleted tumors were more sensitive to radiation therapy and to oncolytic herpesvirus. In the absence of Atrx, irradiated sarcomas had increased persistent DNA damage, telomere dysfunction, and mitotic catastrophe. Our work also showed that Atrx deletion resulted in downregulation of the CGAS/STING signaling pathway at multiple points in the pathway, and was not driven by mutations or by transcriptional downregulation of the CGAS/STING pathway components. We found that both human and mouse models of Atrx deleted sarcoma had a reduced adaptive immune response, markedly impaired CGAS/STING signaling, and increased sensitivity to TVEC, an oncolytic herpesvirus that is currently FDA approved for the treatment of aggressive melanomas. Translation of these results to patients with ATRX mutant cancers could enable genomically-guided cancer therapeutic approaches that improve patient outcomes.
Warren Floyd, Matthew Pierpoint, Chang Su, Rutulkumar Patel, Lixia Luo, Katherine Deland, Amy J. Wisdom, Daniel Zhu, Yan Ma, Suzanne Bartholf DeWitt, Nerissa T. Williams, Alexander L. Lazarides, Jason A. Somarelli, David L. Corcoran, William C. Eward, Diana M. Cardona, David G. Kirsch
Increased levels and diversity of human endogenous retrovirus (HERV) transcription characterizes most cancer types, linked with disease outcomes. However, the underlying processes are incompletely understood. We show that elevated transcription of HERVH proviruses predicts survival of lung squamous cell carcinoma (LUSC) and identify an isoform of CALB1, encoding Calbindin, ectopically driven by an upstream HERVH provirus under the control of KLF5, as the mediator of this effect. HERVH-CALB1 expression initiates in pre-invasive lesions and associates with their progression. Calbindin loss in LUSC cell lines impairs in vitro and in vivo growth and triggers senescence, consistent with a pro-tumor effect. However, Calbindin also directly controls the senescence-associated secretory phenotype (SASP), marked by secretion of CXCL8 and other neutrophil chemoattractants. In established carcinomas, CALB1-negative cancer cells become the dominant source of CXCL8, correlating with neutrophil infiltration and worse prognosis. Thus, HERVH-CALB1 expression in LUSC may display antagonistic pleiotropy, whereby the benefits of escaping senescence early during cancer initiation and clonal competition are offset by the prevention of SASP and pro-tumor inflammation at later stages.
Jan Attig, Judith Pape, Laura Doglio, Anastasiya Kazachenka, Eleonora Ottina, George R. Young, Katey S.S. Enfield, Iker Valle Aramburu, Kevin W. Ng, Nikhil Faulkner, William Bolland, Venizelos Papayannopoulos, Charles Swanton, George Kassiotis
Dravet syndrome (DS), an intractable childhood epileptic encephalopathy with a high fatality rate, is typically caused by loss-of-function mutations in one allele of SCN1A, which encodes NaV1.1, a 250-kDa voltage-gated sodium channel. In contrast to other epilepsies, pharmaceutical treatment for DS is limited. Here, we demonstrate that viral vector-mediated delivery of a codon-modified SCN1A open reading frame into the brain improves DS comorbidities in juvenile and adolescent DS mice (Scn1aA1783V/WT). Notably, bilateral vector injections into the hippocampus and/or the thalamus of DS mice increased survival, reduced the occurrence of epileptic spikes, provided protection from thermally-induced seizures, corrected background electrocorticography activity and behavioral deficits, and restored hippocampal inhibition. Together, our results provide a proof-of-concept for the potential of SCN1A delivery as a therapeutic approach for infants and adolescents with DS-associated comorbidities.
Saja Fadila, Bertrand Beucher, Iria González Dopeso-Reyes, Anat Mavashov, Marina Brusel, Karen Anderson, Caroline Ismeurt, Ethan M. Goldberg, Ana Ricobaraza, Ruben Hernandez-Alcoceba, Eric J. Kremer, Moran Rubinstein
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