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Multicolor Flow Cytometry

Laura C Lighaam, Peter-Paul A Unger, David W Vredevoogd, Dorit Verhoeven, Ellen Vermeulen, Annelies W Turksma, Anja Ten Brinke, Theo Rispens, S Marieke van Ham
Regulatory B cells (Breg) have been described as a specific immunological subsets in several mouse models. Identification of a human counterpart has remained troublesome, because unique plasma membrane markers or a defining transcription factor have not been identified. Consequently, human Bregs are still primarily defined by production of IL-10. In this study, we sought to elucidate if in vitro- induced human IL-10 producing B cells are a dedicated immunological subset. Using deep immune profiling by multicolor flow cytometry and t-SNE analysis, we show that the majority of cells induced to produce IL-10 co-express pro-inflammatory cytokines IL-6 and/or TNFα...
2018: Frontiers in Immunology
Heidi J Zapata, Peter H Van Ness, Stefan Avey, Barbara Siconolfi, Heather G Allore, Sui Tsang, Jean Wilson, Lydia Barakat, Subhasis Mohanty, Albert C Shaw
Both aging and HIV infection are associated with an enhanced pro-inflammatory environment that contributes to impaired immune responses and is mediated in part by innate immune pattern-recognition receptors. MINCLE is a C-type lectin receptor that recognizes trehalose 6,6,' di-mycolate (TDM) or "cord factor," the most abundant glycolipid in Mycobacterium tuberculosis (MTB). Here, we evaluated MINCLE function in monocytes in a cohort of HIV-infected and uninfected young (21-35) and older adults (60 years) via stimulation of PBMCs with TDB (Trehalose-6,6-dibehenate), a synthetic analog of TDM and measurement of cytokine production (IL-10, IL-12, IL-6, TNF-) by multicolor flow cytometry...
September 15, 2018: Journals of Gerontology. Series A, Biological Sciences and Medical Sciences
Fernando J Peña, Jose M Ortiz Rodriguez, María C Gil, Cristina Ortega Ferrusola
Flow cytometry is increasingly used in research and also in clinical andrology. Recent developments in instrumentation, availability of probes and bioinformatics expand the possibilities of flow cytometry well beyond the classical two parametric analyses in use. In this paper, an overview of recent developments in flow cytometry will be presented under the perspective of the authors; aspects such a multicolor assays and computational cytometry will be discussed as well.
September 2018: Reproduction in Domestic Animals, Zuchthygiene
Mikias Negash, Aster Tsegaye, Liya Wassie, Rawleigh Howe
BACKGROUND: Previous studies reported HIV infection alters the distribution and function of γδ T cells and their subsets. γδ T phenotypes in healthy and diseased individuals has received little attention in Ethiopia. We conducted this study to analyze the distribution of γδ T cells, the subsets and levels of expression of activation (CD38), exhaustion or anergy (CD95, PD1), adhesion (N-CAM/CD56 and CD103), among HIV and TB infected patients. METHOD: The distributions of total γδ T cells, Vδ1 and Vδ2 T cells subsets were analyzed in clinical samples collected from asymptomatic HIV, pulmonary TB patients and apparently healthy controls...
September 15, 2018: BMC Infectious Diseases
Michael Gock, Christina S Mullins, Christine Harnack, Friedrich Prall, Robert Ramer, Anja Göder, Oliver H Krämer, Ernst Klar, Michael Linnebacher
AIM: To establish cell line and patient-derived xenograft (PDX) models for neuroendocrine carcinomas (NEC) which is highly desirable for gaining insight into tumor development as well as preclinical research including biomarker testing and drug response prediction. METHODS: Cell line establishment was conducted from direct in vitro culturing of colonic NEC tissue (HROC57). A PDX could also successfully be established from vitally frozen tumor samples. Morphological features, invasive and migratory behavior of the HROC57 cells as well as expression of neuroendocrine markers were vastly analyzed...
September 7, 2018: World Journal of Gastroenterology: WJG
Luke Stanisce, Etty Sims, Cheryl Hou, Yekaterina Koshkareva, John P Gaughan, Igor Kuzin, Andrea Bottaro
OBJECTIVE: The goal of this study was to gain a better understanding of the potential functional specialization of palatine and pharyngeal tonsils, by comparing their cellular composition in paired specimens from a large cohort of adenotonsillectomy patients. DESIGN: Resident B cell, T cell, dendritic cell, and stromal cell subsets were characterized using multicolor flow cytometry in palatine and pharyngeal tonsil specimens from 27 patients, age 2-34 years. RESULTS: Paired comparisons showed highly significant intra-individual differences in resident cell subsets of palatine and pharyngeal tonsils...
September 1, 2018: Archives of Oral Biology
Fabien Pitoiset, Lydie Cassard, Karim El Soufi, Lisa Boselli, Jonathan Grivel, Alexandra Roux, David Klatzmann, Nathalie Chaput, Michelle Rosenzwajg
Multicolor flow cytometry is a technology of choice for phenotyping of immune cells, and it can be used routinely for the follow up of patients in clinical trials. But it is challenging to define combinations of conjugated antibodies that efficiently allow the detailed analysis of major immune cell subsets and the identification of rare cell populations. In a collaborative work among the Immunology, Immunopathology, Immunotherapy (I3 ) laboratory, and the laboratory of immunomonitoring in oncology (L.I.O), we developed and validated 12 different 10-color flow cytometry panels that allow the deep immunophenotyping of cells from whole blood for the follow up of autoimmune and cancer patients...
August 2018: Cytometry. Part A: the Journal of the International Society for Analytical Cytology
Tajana Juranovic, Gary Harvey, Jennie Johnson, Deng Zhang, Milton J Plata, Oscar C Estalilla, Tomislav M Jelic
BACKGROUND Multi-parameter (multicolor) flow cytometric study of the bone marrow aspirate is a very useful tool for diagnosis of plasma cell dyscrasia and for evaluation of post-therapy bone marrow for minimal residual disease. CASE REPORT We present a case of a 50-year-old man with multiple myeloma, whose plasma cells on a bone marrow aspirate flow cytometric study showed atypical placement on a light-scatter dot plot, both on forward and side scatter. The bone marrow aspirate sample was 33 hours and 11 minutes old, and the light-scatter dot plot demonstrated that plasma cells, detected by their expression of CD138, CD38, and CD56, occupied an area otherwise characteristic for dead cells and cell detritus...
August 27, 2018: American Journal of Case Reports
Coline Couillault, Claire Germain, Bertrand Dubois, Hélène Kaplon
Follicular helper T (Tfh) cells are major components of the humoral immune response due to their pivotal role in germinal center formation and antibody affinity maturation following B-cell isotype switching. This CD4+ T-cell subtype is mainly found in the B-cell zone of secondary lymphoid organs as well as in tertiary lymphoid structures (TLS), which are highly organized structures composed of T and B cells, occasionally found at the invasive margin in the tumor microenvironment.We describe here how to perform immunofluorescence staining of tumor tissue sections and multicolor flow cytometry on tumor cell suspensions to identify and visualize these TLS-associated Tfh cells within the tumor microenvironment of various human cancers...
2018: Methods in Molecular Biology
Carolyn G J Moonen, Sven T Alders, Hetty J Bontkes, Ton Schoenmaker, Elena A Nicu, Bruno G Loos, Teun J de Vries
Periodontitis, a chronic inflammatory disease of the periodontium, is characterized by osteoclast-mediated alveolar bone destruction. Gingival fibroblasts (GFs) present in the bone-lining mucosa have the capacity to activate the formation of osteoclasts, but little is known about which local immune cells (co-)mediate this process. The aim of this study was to investigate the cellular interactions of GFs with immune cells, including the contribution of GFs to osteoclast formation and their possible role in the proliferation of these immune cells...
2018: Frontiers in Immunology
Kieu-Suong Le, Patricia Amé-Thomas, Karin Tarte, Françoise Gondois-Rey, Samuel Granjeaud, Florence Orlanducci, Etienne D Foucher, Florence Broussais, Reda Bouabdallah, Thierry Fest, Dominique Leroux, Sapna Yadavilli, Patrick A Mayes, Luc Xerri, Daniel Olive
A better characterization of T-cell subsets in the microenvironment of classical Hodgkin lymphoma (cHL) would help to develop immunotherapies. Using multicolor flow cytometry, we identified in 6 of 43 cHL tissue samples a previously unrecognized subset of CD8 T cells coexpressing CXCR5 and inducible T-cell costimulator (ICOS) molecules (CD8CXCR5+ICOS+ ). These cells shared phenotypic features with follicular helper T (TFH ) cells including low CCR7 expression together with high expression of B-cell lymphoma-6, programmed cell death 1, B and T lymphocyte attenuator, CD200, and OX40...
August 14, 2018: Blood Advances
Nikunj Gevariya, Marjorie Besançon, Karine Robitaille, Valérie Picard, Lamoussa Diabaté, Anwar Alesawi, Pierre Julien, Yves Fradet, Alain Bergeron, Vincent Fradet
BACKGROUND: Several lines of evidence suggest effects of dietary fat on prostate cancer (PCa) development and progression. Targeting omega (ω)-3:ω6 fatty acids (FA) ratio could be beneficial against PCa by favorably modulating inflammation. Here, we studied the effects of ω3- and ω6-enriched diets on prostate tumor growth and inflammatory response in androgen-deprived and non-deprived conditions. METHODS: Immune-competent eugonadal and castrated C57BL/6 mice were injected with TRAMP-C2 prostate tumor cells and daily fed with ω3- or ω6-enriched diet...
August 2, 2018: Prostate
Aras Toker, Linh T Nguyen, Simone C Stone, S Y Cindy Yang, Sarah Rachel Katz, Patricia A Shaw, Blaise A Clarke, Danny Ghazarian, Ayman Al-Habeeb, Alexandra Easson, Wey L Leong, David R McCready, Michael Reedijk, Cynthia J Guidos, Trevor J Pugh, Marcus Q Bernardini, Pamela S Ohashi
Purpose: Regulatory T (Treg) cells expressing the transcription factor FOXP3 are essential for the maintenance of immunologic self-tolerance but play a detrimental role in most cancers due to their ability to suppress antitumor immunity. The phenotype of human circulating Treg cells has been extensively studied, but less is known about tumor-infiltrating Treg cells. We studied the phenotype and function of tumor-infiltrating Treg cells in ovarian cancer and melanoma to identify potential Treg cell-associated molecules that can be targeted by tumor immunotherapies...
July 31, 2018: Clinical Cancer Research: An Official Journal of the American Association for Cancer Research
Andrzej Eljaszewicz, Lukasz Bolkun, Kamil Grubczak, Malgorzata Rusak, Tomasz Wasiluk, Milena Dabrowska, Piotr Radziwon, Wojciech Marlicz, Karol Kamiński, Janusz Kloczko, Marcin Moniuszko
Background: Acute lymphoblastic leukemia (ALL) is a malignant disease of lymphoid progenitor cells. ALL chemotherapy is associated with numerous side effects including neutropenia that is routinely prevented by the administration of growth factors such as granulocyte colony-stimulating factor (G-CSF). To date, the effects of G-CSF treatment on the level of mobilization of different stem and progenitor cells in ALL patients subjected to clinically effective chemotherapy have not been fully elucidated...
2018: Stem Cells International
Rita Folcarelli, Selma van Staveren, Roel Bouman, Bart Hilvering, Gerjen H Tinnevelt, Geert Postma, Oscar F van den Brink, Lutgarde M C Buydens, Nienke Vrisekoop, Leo Koenderman, Jeroen J Jansen
Multicolor Flow Cytometry (MFC)-based gating allows the selection of cellular (pheno)types based on their unique marker expression. Current manual gating practice is highly subjective and may remove relevant information to preclude discovery of cell populations with specific co-expression of multiple markers. Only multivariate approaches can extract such aspects of cell variability from multi-dimensional MFC data. We describe the novel method ECLIPSE (Elimination of Cells Lying in Patterns Similar to Endogeneity) to identify and characterize aberrant cells present in individuals out of homeostasis...
July 19, 2018: Scientific Reports
Kimberly A Muczynski, Nicolae Leca, Arthur E Anderson, Niamh Kieran, Susan K Anderson
Introduction: Current processing of renal biopsy samples provides limited information about immune mechanisms causing kidney injury and disease activity. We used flow cytometry with transplanted kidney biopsy samples to provide more information on the immune status of the kidney. Methods: To enhance the information available from a biopsy, we developed a technique for reducing a fraction of a renal biopsy sample to single cells for multicolor flow cytometry and quantitation of secreted cytokines present within the biopsy sample...
July 2018: KI Reports
Janet Retseck, Alexis Nasr, Yan Lin, Huang Lin, Prateek Mendiratta, Lisa H Butterfield, Ahmad A Tarhini
BACKGROUND: We previously reported early on-treatment significant modulation in circulating regulatory T cell (Treg), myeloid derived suppressor cells (MDSC) and antigen-specific type I CD4+ and CD8+ T cells that correlated with clinical outcome in regionally advanced melanoma patients treated with neoadjuvant ipilimumab. Here, we investigated the long term immunologic impact of CTLA4 blockade. METHODS: Patients were treated with ipilimumab given at 10 mg/kg IV every 3 weeks for 2 doses bracketing surgery...
July 4, 2018: Journal of Translational Medicine
Kathryn D Tuttle, Laurent Gapin
Natural killer T (NKT) cells are a subset of αβ T cells that recognize lipid antigens presented by the nonclassical MHC molecule CD1d. Although numerically small, these cells have been shown to play an important role in the regulation of multiple immune responses, including microbial infection, autoimmunity, and cancer. Even in the steady state, cytokine production by NKT cells influences the basal status and function of other immune cells, including dendritic cells and CD8 T cells. To fully understand their biology and harness them in the clinic, it is imperative to dissect the molecular mechanisms involved in the acquisition of their functionality...
2018: Methods in Molecular Biology
Jiaxiong Tan, Shaohua Chen, Jingying Huang, Youchun Chen, Lijian Yang, Chunli Wang, Jun Zhong, Yuhong Lu, Liang Wang, Kanger Zhu, Yangqiu Li
AIM: The immunosuppressive microenvironment plays a crucial role in T-cell immunodeficiency in multiple myeloma (MM). Overexpression of T-cell immunosuppressive receptors, including programmed death-1 (PD-1) and T-cell immunoglobulin and mucin-domain-containing-3 (Tim-3), may be related to tumor immunosuppression and poor prognosis, and the malignant bone marrow (BM) microenvironment may contribute to such immunosuppression. The purpose of this study was to analyze the distribution of PD-1+ and/or Tim-3+ T cells in different T-cell subset in patients with MM...
June 25, 2018: Asia-Pacific Journal of Clinical Oncology
Elia D Tait Wojno, Celine A Beamer
Innate lymphoid cells (ILCs) comprise a family of innate immune cells that orchestrate mucosal immune responses: initiating, sustaining, and even curbing immune responses. ILCs are relatively rare (≤1% of lymphocytes in mucosal tissues), lack classical cell-surface markers, and can be divided into three subsets (type 1-3 ILCs) based on differences in cytokine production, phenotype, and developmental pathway. Because ILCs can only be identified by combinations of cell-surface markers and cytokine production, multicolor flow cytometry is the most reliable method to purify, characterize, and assess the functionality of ILCs...
2018: Methods in Molecular Biology
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