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T cell facs phenotyping

Sija Landman, Marjan Cruijsen, Paulo C M Urbano, Gerwin Huls, Piet E J van Erp, Esther van Rijssen, Irma Joosten, Hans J P M Koenen
Regulatory T cells (Treg) can show plasticity whereby FOXP3 expression, the master transcription factor for Treg suppressor function, is lost and proinflammatory cytokines are produced. Optimal FOXP3 expression strongly depends on hypomethylation of the FOXP3 gene. 5-Azacytidine (Aza) and its derivative 5-aza-2'-deoxycytidine (DAC) are DNA methyltransferase inhibitors (DNMTi) that are therapeutically used in hematological malignancies, which might be an attractive strategy to promote Treg stability. Previous in vitro research primarily focused on Treg induction by DAC from naïve conventional CD4+ T cells (Tconv)...
2018: Journal of Immunology Research
Heike Schmitt, Ulrike Billmeier, Walburga Dieterich, Timo Rath, Sophia Sonnewald, Stephen Reid, Simon Hirschmann, Kai Hildner, Maximilian J Waldner, Jonas Mudter, Arndt Hartmann, Robert Grützmann, Clemens Neufert, Tino Münster, Markus F Neurath, Raja Atreya
OBJECTIVE: Anti-tumour necrosis factor (TNF) antibodies are successfully used for treatment of Crohn's disease. Nevertheless, approximately 40% of patients display failure to anti-TNF therapy. Here, we characterised molecular mechanisms that are associated with endoscopic resistance to anti-TNF therapy. DESIGN: Mucosal and blood cells were isolated from patients with Crohn's disease prior and during anti-TNF therapy. Cytokine profiles, cell surface markers, signalling proteins and cell apoptosis were assessed by microarray, immunohistochemistry, qPCR, ELISA, whole organ cultures and FACS...
May 30, 2018: Gut
Anja Kerstein, Antje Müller, Silke Pitann, Gabriela Riemekasten, Peter Lamprecht
OBJECTIVES: To examine functional features of CD4+CD8+ double-positive T-cells in patients with granulomatosis with polyangiitis (GPA) using phenotypic and transcriptomic analysis. METHODS: Staining of cellular surface marker was performed using freshly collected whole blood. For intracellular cytokine staining freshly collected whole blood was stimulated with phorbol myristate acetate and ionomycin. Multicolor flow cytometric analysis was performed on a FACSCanto II cytometer using FACSDiva software...
March 2018: Clinical and Experimental Rheumatology
Tyler J Wildes, Adam Grippin, Kyle A Dyson, Brandon M Wummer, David J Damiani, Rebecca S Abraham, Catherine Flores, Duane A Mitchell
PURPOSE: Adoptive T cell immunotherapy (ACT) has emerged as a viable therapeutic for peripheral and central nervous system (CNS) tumors. In peripheral cancers, optimal efficacy of ACT is reliant on dendritic cells (DCs) in the tumor microenvironment. However, the CNS is largely devoid of resident migratory DCs to function as antigen-presenting cells during immunotherapy. Herein, we demonstrate that cellular interactions between adoptively-transferred tumor-reactive T cells and bone marrow-derived HSPCs lead to the generation of potent intratumoral DCs within the CNS compartment...
April 30, 2018: Clinical Cancer Research: An Official Journal of the American Association for Cancer Research
Randy F Sweis, Yuanyuan Zha, Lomax Pass, Brian Heiss, Tara Chongsuwat, Jason J Luke, Thomas F Gajewski, Russell Szmulewitz
BACKGROUND: Immunotherapies targeting the PD-1 checkpoint pathway have recently gained regulatory approval in numerous cancer types. With the widespread use of immune checkpoint therapies, varying patterns of responses and immune-related adverse events are being observed. CASE PRESENTATION: In this case, we highlight a patient who developed recurrent, large-volume ascites, while simultaneously having a 49% reduction in peritoneal tumor lesion size by RECIST criteria...
April 4, 2018: Journal for Immunotherapy of Cancer
Mads N Olesen, Josefine R Christiansen, Steen Vang Petersen, Poul Henning Jensen, Wojciech Paslawski, Marina Romero-Ramos, Vanesa Sanchez-Guajardo
We have previously shown that immunological processes in the brain during α-synuclein-induced neurodegeneration vary depending on the presence or absence of cell death. This suggests that the immune system is able to react differently to the different stages of α-synuclein pathology. However, it was unclear whether these immune changes were governed by brain processes or by a direct immune response to α-synuclein modifications. We have herein locally increased the peripheral concentration of α-synuclein or its pathology-associated variants, nitrated or fibrillar, to characterize the modulation of the CD4 T cell pool by α-synuclein and brain microglia in the absence of any α-synuclein brain pathology...
January 2018: Heliyon
Holger Winkels, Erik Ehinger, Melanie Vassallo, Konrad Buscher, Huy Q Dinh, Kouji Kobiyama, Anouk A J Hamers, Clément Cochain, Ehsan Vafadarnejad, Antoine-Emmanuel Saliba, Alma Zernecke, Akula Bala Pramod, Amlan K Ghosh, Nathaly Anto Michel, Natalie Hoppe, Ingo Hilgendorf, Andreas Zirlik, Catherine C Hedrick, Klaus Ley, Dennis Wolf
RATIONALE: Atherosclerosis is a chronic inflammatory disease that is driven by the interplay of pro- and anti-inflammatory leukocytes in the aorta. Yet, the phenotypic and transcriptional diversity of aortic leukocytes is poorly understood. OBJECTIVE: We characterized leukocytes from healthy and atherosclerotic mouse aortas in-depth by single-cell RNA-sequencing and mass cytometry (cytometry by time of flight) to define an atlas of the immune cell landscape in atherosclerosis...
June 8, 2018: Circulation Research
Livius Penter, Kerstin Dietze, Lars Bullinger, Jörg Westermann, Hans-Peter Rahn, Leo Hansmann
FACS index sorting allows the isolation of single cells with retrospective identification of each single cell's high-dimensional immune phenotype. We experimentally determine the error rate of index sorting and combine the technology with T cell receptor sequencing to identify clonal T cell expansion in aplastic anemia bone marrow as an example.
March 14, 2018: European Journal of Immunology
Willemijn J M Janssen, Valeria Grobarova, Jardin Leleux, Lieneke Jongeneel, Marielle van Gijn, Joris M van Montfrans, Marianne Boes
BACKGROUND: Proline-serine-threonine phosphatase interacting protein 1 (PSTPIP1) is a cytosolic adaptor protein involved with T-cell activation, differentiation, and migration. On cognate T-cell contact, PSTPIP1 is recruited to surface-expressed CD2, where it regulates F-actin remodeling. An immune synapse (IS) is thereby rapidly formed, consisting of T-cell receptor clusters surrounded by a ring of adhesion molecules, including CD2. OBJECTIVE: From genetic screening of patients with primary immunodeficiencies, we identified 2 mutations in PSTPIP1, R228C and T274M, which we further characterized in the primary patients' T cells...
February 9, 2018: Journal of Allergy and Clinical Immunology
Marina Conde, Susanne Michen, Ralf Wiedemuth, Barbara Klink, Evelin Schröck, Gabriele Schackert, Achim Temme
BACKGROUND: Survivin, belonging to the inhibitor of apoptosis (IAP) gene family, is abundantly expressed in tumors. It has been hypothesized that Survivin facilitates carcinogenesis by inhibition of apoptosis resulting in improved survival of tumorigenic progeny. Additionally, Survivin plays an essential role during mitosis. Together with its molecular partners Aurora B, Borealin and inner centromere protein it secures bipolar chromosome segregation. However, whether increased Survivin levels contribute to progression of tumors by inducing chromosomal instability remains unclear...
December 28, 2017: BMC Cancer
Du-Chu Chen, You-De Liang, Liang Peng, Yi-Ze Wang, Chun-Zhi Ai, Xin-Xing Zhu, Ya-Wei Yan, Yasmeen Saeed, Bin Yu, Jingying Huang, Yuxin Gao, Jiaqi Liu, Yi-Zhou Jiang, Min Liu, Demeng Chen
Background: Renal cell carcinoma (RCC) is the most common kidney cancer, accounting for approximately 80-90% of all primary kidney cancer. Treatment for patients with advanced RCC remains unsatisfactory. Rare cancer stem cells (CSCs) are proposed to be responsible for failure of current treatment. Methods: OncoLnc was used as a tool for interactively exploring survival correlations. Gene manipulation and expression analysis were carried out using siRNA, RT-PCR and Western blotting...
2017: Cancer Cell International
Lizhen Ma, Dandan Li, Chunxiu Yuan, Xiangqian Zhang, Na Ta, Xiaochao Zhao, Yumei Li, Xingang Feng
BACKGROUND: It is well known that immunization of radiation-attenuated (RA) schistosoma cercariae or schistosomula can induce high levels of protective immunity against schistosoma cercariae reinfection in many animals. Many studies have shown that the Th1 cellular immune response is crucial for the protective effect elicited by RA schistosomula. However, the molecular mechanism of this strong protective immunity remains unclear. METHODS: The expression profiles of Schistosoma japonicum calreticulin (SjCRT) in RA and normal schistosoma-derived cells were investigated by flow cytometry...
November 13, 2017: Parasites & Vectors
Q Hao, X L Chen, L Ma, X Ye, H Wang, T T Wang, Y Hu, Y L Zhao
BACKGROUND AND PURPOSE: The cerebral arteriovenous malformation (cAVM) is a usual and continually unaware reason of heamorrhage and seizure. It contains of feeder arteries, drain veins and abnormal vessel nets. However, pathologic mechanisms of the development of cAVM are unknown. The purpose of this study was to explore a novel protocol to isolate, culture and passage endothelial cells (ECs) from human cAVM lesions. METHODS: We developed a protocol for isolating and growing ECs from eight patients with cAVM...
March 2018: Microvascular Research
Poonam Gaur, Nootan K Shukla, Satya N Das
Recent studies have suggested an important role of T helper 17 (Th17) cells in tumor biology however, their phenotypic and functional aspects are poorly understood in context with oral cancer. We therefore, investigated the various phenotypic and functional markers of Th17 cells elucidating their relevance in oral squamous cell carcinoma (OSCC). Multi-color flow cytometry (FACs) was used to analyze the frequency and different markers of circulating Th17 cells ex vivo in peripheral blood mono-nuclear cells (PBMCs) from 69 OSCC patients and 35 healthy controls...
October 2017: Immunological Investigations
Supriya Khanra, Nibedeeta R Sarraf, Anjan K Das, Syamal Roy, Madhumita Manna
Emergence of resistance to drugs used to treat the Indian Kala-azar patients makes control strategy shattered. In this bleak situation, Miltefosine (MIL) was introduced to treat mainly antimonial unresponsive cases. Within years, resistance to MIL has been reported. While checking the MIL sensitivity of the recent KA clinical isolates (n = 26), we came across one isolate which showed four times more EC50 for MIL than that of MIL-Sensitive (MIL-S) isolates and considered as putative MIL-Resistant (MIL-R)...
September 4, 2017: Scientific Reports
Tripp Leavitt, Michael S Hu, Michael T Longaker
Flow cytometry is a powerful tool in cell biology in that it allows real-time characterization of cellular phenotypes. Additionally, through the process of fluorescence-activated cell sorting (FACS), living cells can be isolated for future in vitro experiments, including single cell analysis. Here we describe the isolation of live fibroblasts from the dermis of the skin in mice using FACS. This method circumvents the need for ex vivo expansion in cell culture, which can alter phenotypic and functional characteristics of cells...
2017: Methods in Molecular Biology
Leo Hansmann, Arnold Han, Livius Penter, Michaela Liedtke, Mark M Davis
Multiple myeloma is characterized by the clonal expansion of malignant plasma cells in the bone marrow. But the phenotypic diversity and the contribution of less predominant B-lineage clones to the biology of this disease have been controversial. Here, we asked whether cells bearing the dominant multiple myeloma immunoglobulin rearrangement occupy phenotypic compartments other than that of plasma cells. To accomplish this, we combined 13-parameter FACS index sorting and t-Stochastic Neighbor Embedding (t-SNE) visualization with high-throughput single-cell immunoglobulin sequencing to track selected B-lineage clones across different stages of human B-cell development...
September 2017: Cancer Immunology Research
Nana H Overgaard, Jazmina L Cruz, Jennifer A Bridge, Hendrik J Nel, Ian H Frazer, Nicole L La Gruta, Antje Blumenthal, Raymond J Steptoe, James W Wells
CD4(+)CD8(+) double-positive (DP), mature, peripheral T cells are readily detectable in a variety of species and tissues. Despite a common association with autoimmune and malignant skin disorders, however, little is understood about their role or function. Herein, we show that DP T cells are readily detectable in the blood, spleen, and peripheral lymph nodes of naïve C57BL/6 mice. DP T cells were also present in Jα18(-/-) and CD1d(-/-) mice, indicating that these cells are not NK-T cells. After skin administration of CASAC adjuvant, but not Quil A adjuvant, both total DP T cells and skin-infiltrating DP T cells increased in number...
September 2017: Journal of Leukocyte Biology
Richard S Ahn, Keyon Taravati, Kevin Lai, Kristina M Lee, Joanne Nititham, Rashmi Gupta, David S Chang, Sarah T Arron, Michael Rosenblum, Wilson Liao
Human skin consists of multiple cell types, including epithelial, immune, and stromal cells. Transcriptomic analyses have previously been performed from bulk skin samples or from epithelial and immune cells expanded in cell culture. However, transcriptomic analysis of bulk skin tends to drown out expression signals from relatively rare cells while cell culture methods may significantly alter cellular phenotypes and gene expression profiles. To identify distinct transcriptomic profiles of multiple cell populations without substantially altering cell phenotypes, we employed a fluorescence activated cell sorting method to isolate keratinocytes, dendritic cells, CD4+ T effector cells, and CD8+ T effector cells from healthy skin samples, followed by RNA-seq of each cell population...
May 2, 2017: Scientific Reports
Christopher A Lamb, John C Mansfield, Gaik W Tew, Deena Gibbons, Anna K Long, Peter Irving, Lauri Diehl, Jeff Eastham-Anderson, Maria B Price, Graeme O'Boyle, David E J Jones, Sharon O'Byrne, Adrian Hayday, Mary E Keir, Jackson G Egen, John A Kirby
Background and Aims: The αEβ7 integrin is crucial for retention of T lymphocytes at mucosal surfaces through its interaction with E-cadherin. Pathogenic or protective functions of these cells during human intestinal inflammation, such as ulcerative colitis [UC], have not previously been defined, with understanding largely derived from animal model data. Defining this phenotype in human samples is important for understanding UC pathogenesis and is of translational importance for therapeutic targeting of αEβ7-E-cadherin interactions...
May 1, 2017: Journal of Crohn's & Colitis
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