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Abstract
Despite studies implicating adipose tissue T cells (ATT) in the initiation and persistence of adipose tissue inflammation, fundamental gaps in knowledge regarding ATT function impedes progress towards understanding how obesity influences adaptive immunity. We hypothesized ATT activation and function would have tissue-resident specific properties and that obesity would potentiate their inflammatory properties. We assessed ATT activation and inflammatory potential within mouse and human stromal vascular fraction (SVF). Surprisingly, murine and human ATTs from obese visceral white adipose tissue exhibited impaired inflammatory characteristics. Both environmental and cell-intrinsic factors are implicated in ATT dysfunction. Soluble factors from obese SVF inhibit ATTs activation. Additionally, chronic signaling through the T cell receptor is necessary for ATT impairment in obese adipose tissue but is independent of increased PD1 expression. To assess intracellular signaling mechanisms responsible for ATT inflammation impairments, single-cell RNA sequencing of ATTs was performed. ATTs in obese adipose tissue exhibit gene expression resembling T cell exhaustion and increased expression of co-inhibitory receptor Btla. In sum, this work suggests that obesity-induced ATT cells have functional characteristics and gene expression resembling T cell exhaustion, which is dependent upon localized soluble factors and cell-to-cell interactions in adipose tissue.
Authors
Cara E. Porsche, Jennifer B. DelProposto, Lynn Geletka, Robert O’Rourke, Carey N. Lumeng
Abstract
After 9/11, threat of nuclear attack on American urban centers prompted government agencies to develop medical radiation countermeasures to mitigate hematopoietic-acute radiation syndrome (H-ARS) and higher-dose gastrointestinal-ARS (GI-ARS) lethality. While re-purposing leukemia drugs that enhance bone marrow repopulation successfully treats H-ARS in pre-clinical models, no mitigator potentially deliverable under mass casualty conditions preserves GI tract. Here we generate anti-ceramide 6B5 single-chain variable fragment (scFv) and show subcutaneous 6B5 scFv delivery at 24h after a 90% lethal GI-ARS dose of 15Gy mitigates mouse lethality, despite administration after DNA repair is complete. We define an alternate target to DNA repair, an evolving pattern of ceramide-mediated endothelial apoptosis post-radiation, which when disrupted by 6B5 scFv, initiates a durable program of tissue repair, permitting crypt, organ and mouse survival. We posit successful pre-clinical development will render anti-ceramide 6B5 scFv a candidate for inclusion in the Strategic National Stockpile for distribution after a radiation catastrophe.
Authors
Jimmy A. Rotolo, Chii Shyang Fong, Sahra Bodo, Prashanth K. B. Nagesh, John D. Fuller, Thivashnee Sharma, Alessandra Piersigilli, Zhigang Zhang, Zvi Fuks, Vijay K. Singh, Richard Kolesnick
Abstract
Resistance to AR signaling inhibitors (ARSi) in a subset of metastatic castration-resistant prostate cancers (mCRPC) occurs with emergence of AR-negative Neuroendocrine Prostate Cancer (NEPC), coupled with mutations/deletions in PTEN, TP53, RB1, and overexpression of DNMTs, EZH2, and/or SOX2. To resolve whether lack of AR is the driving factor for the emergence of the NE phenotype, molecular, cell, and tumor biology analyses were performed on 23 prostate cancer patient-derived xenografts recapitulating the full spectrum of genetic alterations proposed to drive NE differentiation. Additionally, phenotypic response to CRISPR-Cas9-mediated AR knockout in AR-positive CRPC cells was evaluated. These analyses document that: 1) ARSi-resistant NEPC can develop without androgen deprivation treatment; 2) AR signaling in ARSi-resistant AR+/NE+ double positive “amphicrine” mCRPCs does not suppress NE differentiation; 3) lack of AR expression does not necessitate acquiring a NE phenotype despite concomitant mutations/deletions in PTEN and TP53, and loss of RB1, but can occur via emergence of an AR-/NE- double negative prostate cancer (DNPC); 4) despite DNPC cells having homogeneous genetic driver mutations, they are phenotypically heterogeneous, expressing basal lineage markers alone or in combination with luminal lineage markers; and 5) AR loss is associated with AR promoter hypermethylation in NEPCs but not in DNPCs.
Authors
W. Nathaniel Brennen, Yezi Zhu, Ilsa M. Coleman, Susan L. Dalrymple, Lizamma Antony, Radhika A. Patel, Brian Hanratty, Roshan Chikarmane, Alan K. Meeker, S. Lilly Zheng, Jody E. Hooper, Jun Luo, Angelo M. De Marzo, Eva Corey, Jianfeng Xu, Srinivasan Yegnasubramanian, Michael C. Haffner, Peter S. Nelson, William G. Nelson, William B. Isaacs, John T. Isaacs
Abstract
Pre-existing cross-reactivity to SARS-CoV-2 may occur in absence of prior viral exposure. However, this has been difficult to quantify at the population level due to a lack of reliably defined seroreactivity thresholds. Using an orthogonal antibody testing approach, we estimated that ~0.6% of non-triaged adults from the greater Vancouver area, Canada between May 17th and June 19th 2020 showed clear evidence of a prior SARS-CoV-2 infection, after adjusting for false-positive and false-negative test results. Using a highly sensitive multiplex assay and positive/negative thresholds established in infants in whom maternal antibodies have waned, we determine that more than 90% of uninfected adults showed antibody reactivity against the spike, receptor-binding domain (RBD), N-terminal domains (NTD) or the nucleocapsid (N) protein from SARS-CoV-2. This sero-reactivity was evenly distributed across age and sex, correlated with circulating coronaviruses reactivity, and was partially outcompeted by soluble circulating coronaviruses’ spike. Using a custom SARS-CoV-2 peptide mapping array, we found that this antibody reactivity broadly mapped to spike, and to conserved non-structural viral proteins. We conclude that most adults display pre-existing antibody cross-reactivity against SARS-CoV-2, which further supports investigation of how this may impact the clinical severity of COVID-19 or SARS-CoV-2 vaccine responses.
Authors
Abdelilah Majdoubi, Christina Michalski, Sarah E. O’Connell, Sarah Dada, Sandeep R. Narpala, Jean P. Gelinas, Disha Mehta, Claire Cheung, Dirk F.H. Winkler, Manjula Basappa, Aaron C. Liu, Matthias Görges, Vilte E. Barakauskas, Michael A. Irvine, Jennifer Mehalko, Dominic Esposito, Inna Sekirov, Agatha N. Jassem, David M. Goldfarb, Steven Pelech, Daniel C. Douek, Adrian B. McDermott, Pascal M Lavoie
Abstract
Morphologic examination of tissue biopsies is essential for histopathological diagnosis. However, accurate and scalable cellular quantification in human samples remains challenging. Here, we present a deep learning-based approach for antigen-specific cellular morphometrics in human kidney biopsies, which combines indirect immunofluorescence imaging with U-Net-based architectures for image-to-image translation and dual segmentation tasks, achieving human-level accuracy. In the kidney, podocyte loss represents a hallmark of glomerular injury and can be estimated in diagnostic biopsies. Thus, we profiled over 27,000 podocytes from 110 human samples, including patients with anti-neutrophil cytoplasmic antibody-associated glomerulonephritis (ANCA-GN), an immune-mediated disease with aggressive glomerular damage and irreversible loss of kidney function. We identified previously unknown morphometric signatures of podocyte depletion in patients with ANCA-GN, which allowed patient classification and, in combination with routine clinical tools, showed potential for risk stratification. Our approach enables robust and scalable molecular morphometric analysis of human tissues, yielding deeper biological insights into the human kidney pathophysiology.
Authors
Marina Zimmermann, Martin Klaus, Milagros N. Wong, Ann-Katrin Thebille, Lukas Gernhold, Christoph Kuppe, Maurice Halder, Jennifer Kranz, Nicola Wanner, Fabian Braun, Sonia Wulf, Thorsten Wiech, Ulf Panzer, Christian F. Krebs, Elion Hoxha, Rafael Kramann, Tobias B. Huber, Stefan Bonn, Victor G. Puelles
Abstract
Compromised regenerative capacity of lung epithelial cells can lead to cellular senescence, which may precipitate fibrosis. While increased markers of senescence have been reported in idiopathic pulmonary fibrosis (IPF), the origin and identity of these senescent cells remain unclear, and tools to characterize context-specific cellular senescence in human lung are lacking. We observed that the senescent marker p16 is predominantly localized to bronchiolized epithelial structures in scarred regions of IPF and systemic sclerosis associated interstitial lung disease ILD (SSc-ILD) lung tissue, overlapping with the basal epithelial markers Keratin 5 and Keratin 17. Using in vitro models, we derived transcriptional signatures of senescence programming specific to different types of lung epithelial cells, and interrogated these signatures in a single-cell RNA-seq data set derived from control, IPF, and SSc-ILD lung tissue. We identified a population of basal epithelial cells defined by, and enriched for, markers of cellular senescence, and identified candidate markers specific to senescent basal epithelial cells in ILD that can enable future functional studies. Notably, gene expression of these cells significantly overlaps with terminally differentiating cells in stratified epithelia, where it is driven by p53 activation as part of the senescence program.
Authors
Daryle J. DePianto, Jason A. Vander Heiden, Katrina B. Morshead, Kai-Hui Sun, Zora Modrusan, Grace Teng, Paul J. Wolters, Joseph R. Arron
Abstract
Currently, no effective therapies exist for fibrodysplasia ossificans progressiva (FOP), a rare congenital syndrome in which heterotopic bone is formed in soft tissues due to dysregulated activity of the bone morphogenetic protein (BMP) receptor kinase ALK2/ACVR1. From a screen of known biologically active compounds, we identified saracatinib as a potent ALK2 kinase inhibitor. In enzymatic and cell-based assays, saracatinib preferentially inhibited ALK2 compared with other receptors of the BMP/TGFb signaling pathway, and induced dorsalization in zebrafish embryos consistent with BMP antagonism. We further tested the efficacy of saracatinib using an inducible ACVR1Q207D transgenic mouse line, which provides a model of heterotopic ossification, as well as an inducible ACVR1R206H knock-in, which serves as a genetically and physiologically faithful model of FOP. In both models, saracatinib was well tolerated and potently inhibited the development of heterotopic ossification even when administered transiently following soft tissue injury. Together, these data suggest that saracatinib is an efficacious clinical candidate for repositioning in the treatment of FOP, offering an accelerated path to clinical proof of efficacy studies and potentially significant benefits to individuals with this devastating condition.
Authors
Eleanor P. Williams, Jana Bagarova, Georgina Kerr, Dong-Dong Xia, Elsie S. Place, Devaveena Dey, Yue Shen, Geoffrey A. Bocobo, Agustin H. Mohedas, Xiuli Huang, Philip E. Sanderson, Arthur Lee, Wei Zheng, Aris N. Economides, James C. Smith, Paul B. Yu, Alex N. Bullock
Abstract
With the advent of cancer immunology, mass cytometry has been increasingly employed to characterize the responses to cancer therapies and the tumor microenvironment (TME). One of its most notable applications is efficient multiplexing of samples into batches by dedicating a number of metal isotope channels to barcodes, enabling robust data acquisition and analysis. Barcoding is most effective when markers are present in all cells of interest. While CD45 has been shown to be a reliable marker for barcoding all immune cells in a given sample, a strategy to reliably barcode mouse cancer cells has not been demonstrated. To this end, we identified CD29 and CD98 as markers widely expressed by commonly used mouse cancer cell lines. We conjugated anti-CD29 and anti-CD98 antibodies to cadmium or indium metals and validated their utility in 10-plex barcoding of live cells. Finally, we established a novel barcoding system incorporating the combination of CD29, CD98, and CD45 to multiplex ten tumors from subcutaneous MC38 and KPC tumor models, while successfully recapitulating the known contrast in the PD1-PDL1 axis between the two models. The ability to barcode tumor cells along with immune cells empowers the interrogation of the tumor-immune interactions in mouse TME studies.
Authors
Soren Charmsaz, Nicole Gross, Elizabeth Jaffee, Won Jin Ho
Abstract
The increased incidence of whooping cough worldwide suggests that current vaccination against Bordetella pertussis infection has limitations in quality and duration of protection. The resurgence of infection has been linked to the introduction of acellular vaccines (aP) which has an improved safety profile compared to the previously used whole-cell (wP) vaccines. To determine immunological differences between aP vs. wP priming in infancy, we performed a systems approach of the immune response to booster vaccination. Transcriptomic, proteomic, cytometric, and serologic profiling revealed multiple shared immune responses with different kinetics across cohorts, including an increase of blood monocyte frequencies, and strong antigen-specific IgG responses. Additionally, we found a prominent subset of aP-primed individuals (30%) with a strong differential signature, including higher levels of expression for CCL3, NFKBIA, and ICAM1. Contrary to the wP individuals, this subset displayed increased PT-specific IgE responses postboost and higher antigen-specific IgG4 and IgG3 antibodies against FHA and FIM2/3 at baseline and post-boost. Overall, the results show that, while broad immune response patterns to Tdap boost overlap between aP- and wP-primed individuals, a subset of aP-primed individuals present a divergent response. These findings provide candidate targets to study the causes and correlates of waning immunity after aP vaccination.
Authors
Ricardo da Silva Antunes, Ferran Soldevila, Mikhail Pomaznoy, Mariana Babor, Jason Bennett, Yuan Tian, Natalie N. Khalil, Yu Qian, Aishwarya Mandava, Richard H. Scheuermann, Mario Cortese, Bali Pulendran, Christopher D. Petro, Adrienne P. Gilkes, Lisa A. Purcell, Alessandro Sette, Bjoern Peters
Abstract
Vaccine delivery technologies are mainly designed to minimally invasively deliver vaccines to target tissues with little or no adjuvant effects. This study presents a prototype laser-based powder delivery (LPD) with inherent adjuvant effects for more immunogenic vaccination without incorporation of external adjuvants. LPD takes advantage of aesthetic ablative fractional laser to generate skin microchannels to support high-efficient vaccine delivery and at the same time creates photothermal stress in microchannel-surrounding tissues to boost vaccination. LPD could significantly enhance pandemic influenza 2009 H1N1 vaccine immunogenicity and protective efficacy as compared to needle-based intradermal delivery in murine models. The ablative fractional laser was found to induce host DNA release, activate NLR family pyrin domain containing 3 (NLRP3) inflammasome, and stimulate interleukin 1β release despite of their dispensability for laser adjuvant effects. Instead, the ablative fractional laser activated MyD88 to mediate its adjuvant effects by potentiation of antigen uptake, maturation, and migration of dendritic cells. LPD also induced minimal local or systemic adverse reactions due to the micro-fractional and sustained vaccine delivery. Our data support the development of self-adjuvanted vaccine delivery technologies by intentional induction of well-controlled tissue stress to alert innate immune systems for more immunogenic vaccination.
Authors
Zhuofan Li, Yan Cao, Yibo Li, Yiwen Zhao, Xinyuan Chen
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