Abstract
The adoptive cell transfer (ACT) of T cells targeting mutated neoantigens can cause objective responses in varieties of metastatic cancers, but the development of new T cell–based treatments relies on accurate animal models. To investigate the therapeutic effect of targeting a neoantigen with ACT, we used T cells from pmel-1 T cell receptor–transgenic mice, known to recognize a WT peptide, gp100, and a mutated version of the peptide that has higher avidity. We gene-engineered B16 cells to express the WT or mutated gp100 epitopes and found that pmel-1–specific T cells targeting a neoantigen tumor target augmented recognition as measured by IFN-γ production. Neoantigen expression by B16 also enhanced the capacity of pmel-1 T cells to trigger the complete and durable regression of large, established, vascularized tumor and required less lymphodepleting conditioning. Targeting neoantigen uncovered the possibility of using enforced expression of the IL-2Rα chain (CD25) in mutation-reactive CD8+ T cells to improve their antitumor functionality. These data reveal that targeting of “mutated-self” neoantigens may lead to improved efficacy and reduced toxicities of T cell–based cellular immunotherapies for patients with cancer.
Authors
Ken-ichi Hanada, Zhiya Yu, Gabrielle R. Chappell, Adam S. Park, Nicholas P. Restifo
Abstract
Zebrafish are increasingly utilized to model cardiomyopathies and regeneration. Current methods evaluating cardiac function have known limitations, fail to reliably detect focal mechanics, and are not readily feasible in zebrafish. We developed a semiautomated, open-source method — displacement analysis of myocardial mechanical deformation (DIAMOND) — for quantitative assessment of 4D segmental cardiac function. We imaged transgenic embryonic zebrafish in vivo using a light-sheet fluorescence microscopy system with 4D cardiac motion synchronization. Our method permits the derivation of a transformation matrix to quantify the time-dependent 3D displacement of segmental myocardial mass centroids. Through treatment with doxorubicin, and by chemically and genetically manipulating the myocardial injury–activated Notch signaling pathway, we used DIAMOND to demonstrate that basal ventricular segments adjacent to the atrioventricular canal display the highest 3D displacement and are also the most susceptible to doxorubicin-induced injury. Thus, DIAMOND provides biomechanical insights into in vivo segmental cardiac function scalable to high-throughput research applications.
Authors
Junjie Chen, Yichen Ding, Michael Chen, Jonathan Gau, Nelson Jen, Chadi Nahal, Sally Tu, Cynthia Chen, Steve Zhou, Chih-Chiang Chang, Jintian Lyu, Xiaolei Xu, Tzung K. Hsiai, René R. Sevag Packard
Abstract
Extracellular mRNAs (ex-mRNAs) potentially supersede extracellular miRNAs (ex-miRNAs) and other RNA classes as biomarkers. We performed conventional small-RNA-sequencing (sRNA-seq) and sRNA-seq with T4 polynucleotide kinase (PNK) end-treatment of total exRNA isolated from serum and platelet-poor EDTA, ACD, and heparin plasma to study the effect on ex-mRNA capture. Compared to conventional sRNA-seq PNK-treatment increased the detection of informative ex-mRNAs reads up to 50-fold. The exRNA pool was dominated by hematopoietic cells and platelets, with additional contribution from the liver. About 60% of the 15- to 42-nt reads originated from the coding sequences, in a pattern reminiscent of ribosome-profiling. Blood sample type had a considerable influence on the exRNA profile. On average approximately 350 to 1,100 distinct ex-mRNA transcripts were detected depending on plasma type. In serum, additional transcripts from neutrophils and hematopoietic cells increased this number to near 2,300. EDTA and ACD plasma showed a destabilizing effect on ex mRNA and non-coding RNA ribonucleoprotein complexes compared to other plasma types. In a proof-of-concept study, we investigated differences between the exRNA profiles of patients with acute coronary syndrome (ACS) and healthy controls. The improved tissue resolution of ex mRNAs after PNK-treatment enabled us to detect a neutrophil-signature in ACS that escaped detection by ex miRNA analysis.
Authors
Kemal M. Akat, Youngmin A. Lee, Arlene Hurley, Pavel Morozov, Klaas E.A. Max, Miguel Brown, Kimberly Bogardus, Anuoluwapo Sopeyin, Kai Hildner, Thomas G. Diacovo, Markus F. Neurath, Martin Borggrefe, Thomas Tuschl
Abstract
The heterogeneity of individual cells in a tissue has been well characterized, largely using ex vivo approaches that do not permit longitudinal assessments of the same tissue over long periods of time. We demonstrate a potentially novel application of adaptive optics fluorescence microscopy to visualize and track the in situ mosaicism of retinal pigment epithelial (RPE) cells directly in the human eye. After a short, dynamic period during which RPE cells take up i.v.-administered indocyanine green (ICG) dye, we observed a remarkably stable heterogeneity in the fluorescent pattern that gradually disappeared over a period of days. This pattern could be robustly reproduced with a new injection and follow-up imaging in the same eye out to at least 12 months, which enabled longitudinal tracking of RPE cells. Investigation of ICG uptake in primary human RPE cells and in a mouse model of ICG uptake alongside human imaging corroborated our findings that the observed mosaicism is an intrinsic property of the RPE tissue. We demonstrate a potentially novel application of fluorescence microscopy to detect subclinical changes to the RPE, a technical advance that has direct implications for improving our understanding of diseases such as oculocutaneous albinism, late-onset retinal degeneration, and Bietti crystalline dystrophy.
Authors
HaeWon Jung, Jianfei Liu, Tao Liu, Aman George, Margery G. Smelkinson, Sarah Cohen, Ruchi Sharma, Owen Schwartz, Arvydas Maminishkis, Kapil Bharti, Catherine Cukras, Laryssa A. Huryn, Brian P. Brooks, Robert Fariss, Johnny Tam
Abstract
In clinical breast cancer intervention, selection of the optimal treatment protocol based on predictive biomarkers remains an elusive goal. Here, we present a modeling tool to predict the likelihood of breast cancer response to neoadjuvant chemotherapy using patient specific tumor vasculature biomarkers. A semi-automated analysis was implemented and performed on 3990 histological images from 48 patients, with 10–208 images analyzed for each patient. We applied a histology-based model to resected primary breast cancer tumors (n = 30), and then evaluated a cohort of patients (n = 18) undergoing neoadjuvant chemotherapy, collecting pre- and post-treatment pathology specimens and MRI data. We found that core biopsy samples can be used with acceptable accuracy (r = 0.76) to determine histological parameters representative of the whole tissue region. Analysis of model histology parameters obtained from tumor vasculature measurements, specifically diffusion distance divided by radius of drug source (L/rb) and blood volume fraction (BVF), provides a statistically significant separation of patients obtaining a pathologic complete response (pCR) from those that do not (Student’s t-test; P < 0.05). With this model, it is feasible to evaluate primary breast tumor vasculature biomarkers in a patient specific manner, thereby allowing a precision approach to breast cancer treatment.
Authors
Terisse A. Brocato, Ursa Brown-Glaberman, Zhihui Wang, Reed G. Selwyn, Colin M. Wilson, Edward F. Wyckoff, Lesley C. Lomo, Jennifer L. Saline, Anupama Hooda-Nehra, Renata Pasqualini, Wadih Arap, C. Jeffrey Brinker, Vittorio Cristini
Abstract
Current clinical methods for the evaluation of lymphatic vessel function, crucial for early diagnosis and evaluation of treatment-response of several pathological conditions, in particular of post-surgical lymphedema, are based on complex and mainly qualitative imaging techniques. To address this unmet medical need, we established a simple strategy for the painless and quantitative assessment of cutaneous lymphatic function. We prepared a lymphatic-specific tracer formulation, consisting of the clinically approved near-infrared fluorescent dye, indocyanine green, and the solubilizing surfactant Kolliphor HS15. The tracer is non-invasively delivered to the dermal layer of the skin using MicronJet600TM hollow microneedles, and the fluorescence signal decay at the injection site is measured over time using a custom-made, portable detection device. The decay rate of fluorescence signal in the skin was used as a direct measure of lymphatic vessel drainage function. With this new method, we could quantify impaired lymphatic clearance in transgenic mice lacking dermal lymphatics and distinguish distinct lymphatic clearance patterns in pigs in different body locations and under manual stimulus. Overall, this method has the potential for becoming a non-invasive and quantitative clinical “office-test” for lymphatic function assessment.
Authors
Anna K. Polomska, Steven T. Proulx, Davide Brambilla, Daniel Fehr, Mathias Bonmarin, Simon Brändli, Mirko Meboldt, Christian Steuer, Tsvetina Vasileva, Nils Reinke, Jean-Christophe Leroux, Michael Detmar
Abstract
In utero hypoxia is a major cause of neonatal morbidity and mortality and predisposes to adult cardiovascular disease. No therapies exist to correct fetal hypoxia. In a new ex utero fetal support system, we tested the hypothesis that hypoxemic support of the fetus impairs myocardial development, whereas normoxic support allows normal myocardial development. Preterm fetal lambs were connected via umbilical vessels to a low-resistance oxygenator and placed in a sterile-fluid environment. Control normoxic fetuses received normal fetal oxygenation, and hypoxemic fetuses received subphysiologic oxygenation. Fetuses with normal in utero development served as normal controls. Hypoxemic fetuses exhibited decreased maximum cardiac output in both ventricles, diastolic function, myocyte and myocyte nuclear size, and increased myocardial capillary density versus control normoxic fetuses. There were no differences between control normoxic fetuses in the fetal support system and normal in utero controls. Chronic fetal hypoxemia resulted in significant abnormalities in myocyte architecture and myocardial capillary density as well as systolic and diastolic cardiac function, whereas control fetuses showed no differences. This ex utero fetal support system has potential to become a significant research tool and novel therapy to correct fetal hypoxia.
Authors
Kendall M. Lawrence, Samson Hennessy-Strahs, Patrick E. McGovern, Ali Y. Mejaddam, Avery C. Rossidis, Heron D. Baumgarten, Esha Bansal, Maryann Villeda, Jiancheng Han, Zhongshan Gou, Sheng Zhao, Jack Rychik, William H. Peranteau, Marcus G. Davey, Alan W. Flake, J. William Gaynor, Carlo R. Bartoli
Abstract
The analysis and validation of flow cytometry–based biomarkers in clinical studies are limited by the lack of standardized protocols that are reproducible across multiple centers and suitable for use with either unfractionated blood or cryopreserved PBMCs. Here we report the development of a platform that standardizes a set of flow cytometry panels across multiple centers, with high reproducibility in blood or PBMCs from either healthy subjects or patients 100 days after hematopoietic stem cell transplantation. Inter-center comparisons of replicate samples showed low variation, with interindividual variation exceeding inter-center variation for most populations (coefficients of variability <20% and interclass correlation coefficients >0.75). Exceptions included low-abundance populations defined by markers with indistinct expression boundaries (e.g., plasmablasts, monocyte subsets) or populations defined by markers sensitive to cryopreservation, such as CD62L and CD45RA. Automated gating pipelines were developed and validated on an independent data set, revealing high Spearman’s correlations (rs >0.9) with manual analyses. This workflow, which includes pre-formatted antibody cocktails, standardized protocols for acquisition, and validated automated analysis pipelines, can be readily implemented in multicenter clinical trials. This approach facilitates the collection of robust immune phenotyping data and comparison of data from independent studies.
Authors
Sabine Ivison, Mehrnoush Malek, Rosa V. Garcia, Raewyn Broady, Anne Halpin, Manon Richaud, Rollin F. Brant, Szu-I Wang, Mathieu Goupil, Qingdong Guan, Peter Ashton, Jason Warren, Amr Rajab, Simon Urschel, Deepali Kumar, Mathias Streitz, Birgit Sawitzki, Stephan Schlickeiser, Janetta J. Bijl, Donna A. Wall, Jean-Sebastien Delisle, Lori J. West, Ryan R. Brinkman, Megan K. Levings
Abstract
Asthma is one of the most common immunological diseases and is characterized by airway hyperresponsiveness (AHR), mucus overproduction, and airway eosinophilia. Although mouse models have provided insight into the mechanisms by which type-2 cytokines induce asthmatic airway inflammation, differences between the rodent and human immune systems hamper efforts to improve understanding of human allergic diseases. In this study, we aim to establish a preclinical animal model of asthmatic airway inflammation using humanized IL-3/GM-CSF or IL-3/GM-CSF/IL-5 Tg NOD/Shi-scid-IL2rγnull (NOG) mice and investigate the roles of human type-2 immune responses in the asthmatic mice. Several important characteristics of asthma — such as AHR, goblet cell hyperplasia, T cell infiltration, IL-13 production, and periostin secretion — were induced in IL-3/GM-CSF Tg mice by intratracheally administered human IL-33. In addition to these characteristics, human eosinophilic inflammation was observed in IL-3/GM-CSF/IL-5 Tg mice. The asthmatic mechanisms of the humanized mice were driven by activation of human Th2 and mast cells by IL-33 stimulation. Furthermore, treatment of the humanized mice with an anti–human IL-13 antibody significantly suppressed these characteristics. Therefore, the humanized mice may enhance our understanding of the pathophysiology of allergic disorders and facilitate the preclinical development of new therapeutics for IL-33–mediated type-2 inflammation in asthma.
Authors
Ryoji Ito, Shuichiro Maruoka, Kaori Soda, Ikumi Katano, Kenji Kawai, Mika Yagoto, Asami Hanazawa, Takeshi Takahashi, Tomoyuki Ogura, Motohito Goto, Riichi Takahashi, Shota Toyoshima, Yoshimichi Okayama, Kenji Izuhara, Yasuhiro Gon, Shu Hashimoto, Mamoru Ito, Satoshi Nunomura
Abstract
Despite the initial promise of immunotherapy for CNS disease, multiple recent clinical trials have failed. This may be due in part to characteristically low penetration of antibodies to cerebrospinal fluid (CSF) and brain parenchyma, resulting in poor target engagement. We here utilized transcranial macroscopic imaging to noninvasively evaluate in vivo delivery pathways of CSF fluorescent tracers. Tracers in CSF proved to be distributed through a brain-wide network of periarterial spaces, previously denoted as the glymphatic system. CSF tracer entry was enhanced approximately 3-fold by increasing plasma osmolality without disruption of the blood-brain barrier. Further, plasma hyperosmolality overrode the inhibition of glymphatic transport that characterizes the awake state and reversed glymphatic suppression in a mouse model of Alzheimer’s disease. Plasma hyperosmolality enhanced the delivery of an amyloid-β (Aβ) antibody, obtaining a 5-fold increase in antibody binding to Aβ plaques. Thus, manipulation of glymphatic activity may represent a novel strategy for improving penetration of therapeutic antibodies to the CNS.
Authors
Benjamin A. Plog, Humberto Mestre, Genaro E. Olveda, Amanda M. Sweeney, H. Mark Kenney, Alexander Cove, Kosha Y. Dholakia, Jeffrey Tithof, Thomas D. Nevins, Iben Lundgaard, Ting Du, Douglas H. Kelley, Maiken Nedergaard
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