Intracellular Cytokine Staining

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Kunle Odunsi - One of the best experts on this subject based on the ideXlab platform.

Junko Matsuzaki - One of the best experts on this subject based on the ideXlab platform.

Stephen C De Rosa - One of the best experts on this subject based on the ideXlab platform.

  • Corrigendum: OMIP‐056: Evaluation of Human Conventional T Cells, Donor‐Unrestricted T Cells, and NK Cells Including Memory Phenotype by Intracellular Cytokine Staining
    Cytometry. Part A : the journal of the International Society for Analytical Cytology, 2020
    Co-Authors: One Dintwe, Shamiska Rohith, Katharine V Schwedhelm, M Juliana Mcelrath, Erica Andersen-nissen, Stephen C De Rosa
    Abstract:

    A 26-color Staining panel was developed to profile human antigen-specific T cells in an Intracellular Cytokine Staining (ICS) assay using peptide pools to various antigens of interest. In addition to multiple functional markers, the panel includes differentiation/activation markers and markers to assess γδ, mucosal-associated invariant T, and NK T cells as well as conventional NK cells. Panel optimization was performed using previously cryopreserved PBMC from healthy adults, and then, expression of key functional markers in the panel was cross-validated against a validated ICS assay used in the HIV Vaccine Trials Network (HVTN). The panel is currently being used to evaluate the responses to tuberculosis and malaria vaccine candidates in volunteers from different geographic areas. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

  • corrigendum omip 056 evaluation of human conventional t cells donor unrestricted t cells and nk cells including memory phenotype by Intracellular Cytokine Staining
    Cytometry Part A, 2019
    Co-Authors: One Dintwe, Shamiska Rohith, Katharine V Schwedhelm, Stephen C De Rosa, Juliana M Mcelrath, Erica Andersennissen
    Abstract:

    A 26-color Staining panel was developed to profile human antigen-specific T cells in an Intracellular Cytokine Staining (ICS) assay using peptide pools to various antigens of interest. In addition to multiple functional markers, the panel includes differentiation/activation markers and markers to assess γδ, mucosal-associated invariant T, and NK T cells as well as conventional NK cells. Panel optimization was performed using previously cryopreserved PBMC from healthy adults, and then, expression of key functional markers in the panel was cross-validated against a validated ICS assay used in the HIV Vaccine Trials Network (HVTN). The panel is currently being used to evaluate the responses to tuberculosis and malaria vaccine candidates in volunteers from different geographic areas. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

  • OMIP-056: Evaluation of Human Conventional T Cells, Donor-Unrestricted T Cells, and NK Cells Including Memory Phenotype by Intracellular Cytokine Staining.
    Cytometry. Part A : the journal of the International Society for Analytical Cytology, 2019
    Co-Authors: One Dintwe, Shamiska Rohith, Katharine V Schwedhelm, M Juliana Mcelrath, Erica Andersen-nissen, Stephen C De Rosa
    Abstract:

    A 26-color Staining panel was developed to profile human antigen-specific T cells in an Intracellular Cytokine Staining (ICS) assay using peptide pools to various antigens of interest. In addition to multiple functional markers, the panel includes differentiation/activation markers and markers to assess γδ, mucosal-associated invariant T, and NK T cells as well as conventional NK cells. Panel optimization was performed using previously cryopreserved PBMC from healthy adults, and then, expression of key functional markers in the panel was cross-validated against a validated ICS assay used in the HIV Vaccine Trials Network (HVTN). The panel is currently being used to evaluate the responses to tuberculosis and malaria vaccine candidates in volunteers from different geographic areas. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

  • OMIP-025: Evaluation of human T- and NK-cell responses including memory and follicular helper phenotype by Intracellular Cytokine Staining
    Cytometry. Part A : the journal of the International Society for Analytical Cytology, 2014
    Co-Authors: Gemma Moncunill, M Juliana Mcelrath, Carlota Dobaño, Stephen C De Rosa
    Abstract:

    This panel was developed to assess antigen-specific T cells using peptide pools to various antigens of interest, although other types of antigens such as recombinant proteins or whole pathogens could be considered using different stimulation times. In addition to multiple functional markers, the panel includes differentiation markers and markers to assess follicular helper T cells and NK cells (Table 1). It was optimized using cryopreserved peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus (HIV) uninfected and HIV infected adults with known cytomegalovirus (CMV) responses and it underwent assay qualification. The panel is being used to evaluate the responses to HIV and malaria vaccine candidates in adults and children from different geographic areas.

  • Development of an automated analysis system for data from flow cytometric Intracellular Cytokine Staining assays from clinical vaccine trials
    Cytometry. Part A : the journal of the International Society for Analytical Cytology, 2008
    Co-Authors: Nicholas Shulman, M Juliana Mcelrath, Matthew Bellew, George Snelling, Donald K. Carter, Yunda Huang, Steven G. Self, Stephen C De Rosa
    Abstract:

    Intracellular Cytokine Staining (ICS) by multiparameter flow cytometry is one of the primary methods for determining T-cell immunogenicity in HIV-1 clinical vaccine trials. Data analysis requires considerable expertise and time. The amount of data is quickly increasing as more and larger trials are performed, and thus there is a critical need for high-throughput methods of data analysis. A web-based flow cytometric analysis system, LabKey Flow, was developed for the analyses of data from standardized ICS assays. Using a gating template created manually in commercially available flow cytometric analysis software, the system automatically compensates and analyzes all data sets. Quality control queries were designed to identify potentially incorrect sample collections. Comparison of the semiautomated analysis performed by LabKey Flow and the manual analysis performed using FlowJo software demonstrated excellent concordance (concordance correlation coefficient > 0.990). Manual inspection of the analyses performed by LabKey Flow for eight-color ICS data files from several clinical vaccine trials indicated that template gates can appropriately be used for most data sets. Thus, the semiautomated LabKey Flow analysis system can accurately analyze large ICS data files. Routine use of the system does not require specialized expertise. This high-throughput analysis will provide great utility for rapid evaluation of complex multiparameter flow cytometric measurements collected from large clinical trials. © 2008 International Society for Advancement of Cytometry

Holden T. Maecker - One of the best experts on this subject based on the ideXlab platform.

  • Intracellular Cytokine Staining on PBMCs Using CyTOFTM Mass Cytometry
    Bio-protocol, 2015
    Co-Authors: Dongxia Lin, Sheena Gupta, Holden T. Maecker
    Abstract:

    In this protocol, we use a CyTOF™ mass cytometry to collect single-cell data on a large number of Cytokines/chemokines as well as cell-surface proteins that characterize T cells and other immune cells. The current selected mass window in AW 103-203 includes the lanthanides used for most antibody labeling, along with iridium and rhodium for DNA intercalators. The output data are in the format as .txt and .fcs files, which is compatible with many analysis programs. This protocol could be adapted to include tetramers into the Staining panel, but we have not optimized for that purpose. The principal steps of Intracellular Cytokine Staining are as follows: First, cells are activated for a few hours using either a specific peptide or a non-specific activation cocktail. An inhibitor of protein transport (e.g. Brefeldin A) is added to retain the Cytokines within the cell. Next, EDTA is added to remove adherent cells from the activation vessel. After washing, antibodies to cell surface markers are added to the cells. The cells are then fixed in paraformaldehyde and permeabilized. We use a gentle detergent, saponin, as the permealization buffer because it is less destructive to surface and Intracellular epitopes compared to harsh detergents or methanol. After permeabilization, the metal-conjugated anti-Cytokine antibodies are added into the cell suspension. The stained cells are then sequentially introduced into the mass cytometry for signal intensity analysis.

  • Intracellular Cytokine Staining (ICS) on Human Lymphocytes or Peripheral Blood Mononuclear Cells (PBMCs)
    BIO-PROTOCOL, 2015
    Co-Authors: Sheena Gupta, Holden T. Maecker
    Abstract:

    [Abstract] Production of Cytokines plays an important role in the immune response. Cytokines are involved in many different pathways including the induction of many anti-viral proteins by IFN gamma, the induction of T cell proliferation by IL-2 and the inhibition of viral gene expression and replication by TNF alpha. Cytokines are not preformed factors but are rapidly produced and secreted in response to cellular activation. Intracellular Cytokine detection by flow cytometry has emerged as the premier technique for studying Cytokine production at the single-cell level. It detects the production and accumulation of Cytokines within the endoplasmic reticulum after cell stimulation, allowing direct TH1 versus TH2 determination. It can also be used in combination with other flow cytometry protocols for immunophenotyping using cell surface markers or with MHC multimers to detect an antigen specific response, making it an extremely flexible and versatile method. This capability, combined with the high throughput nature of the instrumentation, gives Intracellular Cytokine Staining an enormous advantage over existing single-cell techniques such as ELISPOT, limiting dilution, and T cell cloning. The principle steps of Intracellular Cytokine Staining is as follows: 1. Cells are activated for a few hours using either a specific peptide or a non-specific activation cocktail; 2. An inhibitor of protein transport (e.g. Brefeldin A) is added to retain the Cytokines within the cell; 3. Next, EDTA is added to remove adherent cells from the activation vessel; 4. After washing, antibodies to cell surface markers can be added to the cells; 5. The cells are then fixed in paraformaldehyde and permeabilized; 6. The anti-Cytokine antibody is added and the cells can be analyzed by flow cytometer.

  • Multiparameter Phenotyping of Human PBMCs Using Mass Cytometry.
    Methods in molecular biology (Clifton N.J.), 2015
    Co-Authors: Michael D. Leipold, Evan W. Newell, Holden T. Maecker
    Abstract:

    The standard for single-cell analysis of phenotype and function in recent decades has been fluorescence flow cytometry. Mass cytometry is a newer technology that uses heavy metal ions, rather than fluorochromes, as labels for probes such as antibodies. The binding of these ion-labeled probes to cells is quantitated by mass spectrometry. This greatly increases the number of phenotypic and functional markers that can be probed simultaneously. Here, we review topics that must be considered when adapting existing flow cytometry panels to mass cytometry analysis. We present a protocol and representative panels for surface phenotyping and Intracellular Cytokine Staining (ICS) assays.

  • a harmonized approach to Intracellular Cytokine Staining gating results from an international multiconsortia proficiency panel conducted by the cancer immunotherapy consortium cic cri
    Cytometry Part A, 2013
    Co-Authors: Holden T. Maecker, Cedrik M. Britten, Maria Jaimes, Leah Price, Lisa K Mcneil, Kunle Odunsi, Junko Matsuzaki, Janet Staats
    Abstract:

    Previous results from two proficiency panels of Intracellular Cytokine Staining (ICS) from the Cancer Immunotherapy Consortium and panels from the National Institute of Allergy and Infectious Disease and the Association for Cancer Immunotherapy highlight the variability across laboratories in reported % CD8+ or % CD4+ Cytokine-positive cells. One of the main causes of interassay variability in flow cytometry-based assays is due to differences in gating strategies between laboratories, which may prohibit the generation of robust results within single centers and across institutions. To study how gating strategies affect the variation in reported results, a gating panel was organized where all participants analyzed the same set of Flow Cytometry Standard (FCS) files from a four-color ICS assay using their own gating protocol (Phase I) and a gating protocol drafted by consensus from the organizers of the panel (Phase II). Focusing on analysis removed donor, assay, and instrument variation, enabling us to quantify the variability caused by gating alone. One hundred ten participating laboratories applied 110 different gating approaches. This led to high variability in the reported percentage of Cytokine-positive cells and consequently in response detection in Phase I. However, variability was dramatically reduced when all laboratories used the same gating strategy (Phase II). Proximity of the Cytokine gate to the negative population most impacted true-positive and false-positive response detection. Recommendations are provided for the (1) placement of the Cytokine-positive gate, (2) identification of CD4+ CD8+ double-positive T cells, (3) placement of lymphocyte gate, (4) inclusion of dim cells, (5) gate uniformity, and 6) proper adjustment of the biexponential scaling.

  • Multiparameter Intracellular Cytokine Staining.
    Methods in molecular biology (Clifton N.J.), 2010
    Co-Authors: Patricia Lovelace, Holden T. Maecker
    Abstract:

    Intracellular Cytokine Staining is a popular method for visualizing cellular responses, most often T-cell responses to antigenic or mitogenic stimulation. It can be coupled with Staining for other functional markers, such as upregulation of CD107 or CD154, as well as phenotypic markers that define specific cellular subsets, e.g., effector and memory T-cell compartments, NK cells, or monocytes. Recent advances in multicolor flow cytometry instrumentation and software have allowed the routine combination of 12 or more markers, creating some technical and analytical challenges along the way, and exposing a need for standardization in the field. Here, we will review best practices for antibody panel design and procedural variables for multicolor Intracellular Cytokine Staining, and present an optimized protocol with variations designed for use with specific markers and sample types.

Helen Mcshane - One of the best experts on this subject based on the ideXlab platform.

  • Assay optimisation and technology transfer for multi-site immuno-monitoring in vaccine trials.
    PloS one, 2017
    Co-Authors: Steven G. Smith, Iman Satti, Hazel M. Dockrell, Stephanie A. Harris, Donna Bryan, K. Barry Walker, Helen Mcshane
    Abstract:

    Cellular immunological assays are important tools for the monitoring of responses to T-cell-inducing vaccine candidates. As these bioassays are often technically complex and require considerable experience, careful technology transfer between laboratories is critical if high quality, reproducible data that allows comparison between sites, is to be generated. The aim of this study, funded by the European Union Framework Program 7-funded TRANSVAC project, was to optimise Standard Operating Procedures and the technology transfer process to maximise the reproducibility of three bioassays for interferon-gamma responses: enzyme-linked immunosorbent assay (ELISA), ex-vivo enzyme-linked immunospot and Intracellular Cytokine Staining. We found that the initial variability in results generated across three different laboratories reduced following a combination of Standard Operating Procedure harmonisation and the undertaking of side-by-side training sessions in which assay operators performed each assay in the presence of an assay 'lead' operator. Mean inter-site coefficients of variance reduced following this training session when compared with the pre-training values, most notably for the ELISA assay. There was a trend for increased inter-site variability at lower response magnitudes for the ELISA and Intracellular Cytokine Staining assays. In conclusion, we recommend that on-site operator training is an essential component of the assay technology transfer process and combined with harmonised Standard Operating Procedures will improve the quality, reproducibility and comparability of data produced across different laboratories. These data may be helpful in ongoing discussions of the potential risk/benefit of centralised immunological assay strategies for large clinical trials versus decentralised units.

  • Intracellular Cytokine Staining and Flow Cytometry: Considerations for Application in Clinical Trials of Novel Tuberculosis Vaccines.
    PloS one, 2015
    Co-Authors: Steven G. Smith, Helen A. Fletcher, Kaatje Smits, Simone A. Joosten, Krista E. Van Meijgaarden, Iman Satti, Nadia Caccamo, Francesco Dieli, Françoise Mascart, Helen Mcshane
    Abstract:

    Intracellular Cytokine Staining combined with flow cytometry is one of a number of assays designed to assess T-cell immune responses. It has the specific advantage of enabling the simultaneous assessment of multiple phenotypic, differentiation and functional parameters pertaining to responding T-cells, most notably, the expression of multiple effector Cytokines. These attributes make the technique particularly suitable for the assessment of T-cell immune responses induced by novel tuberculosis vaccines in clinical trials. However, depending upon the particular nature of a given vaccine and trial setting, there are approaches that may be taken at different stages of the assay that are more suitable than other alternatives. In this paper, the Tuberculosis Vaccine Initiative (TBVI) TB Biomarker Working group reports on efforts to assess the conditions that will determine when particular assay approaches should be employed. We have found that choices relating to the use of fresh whole blood or peripheral blood mononuclear cells (PBMC) and frozen PBMC; use of serum-containing or serum-free medium; length of stimulation period and use of co-stimulatory antibodies can all affect the sensitivity of Intracellular Cytokine assays. In the case of sample material, frozen PBMC, despite some loss of sensitivity, may be more advantageous for batch analysis. We also recommend that for multi-site studies, common antibody panels, gating strategies and analysis approaches should be employed for better comparability.

  • Mycobacterium tuberculosis PPD-induced immune biomarkers measurable in vitro following BCG vaccination of UK adolescents by multiplex bead array and Intracellular Cytokine Staining
    BMC immunology, 2010
    Co-Authors: Steven G. Smith, Helen Mcshane, Maeve K. Lalor, Patricia Gorak-stolinska, Rose Blitz, Natalie E. R. Beveridge, Andrew Worth, Hazel M. Dockrell
    Abstract:

    Background: The vaccine efficacy reported following Mycobacterium bovis Bacillus Calmette Guerin (BCG) administration to UK adolescents is 77% and defining the cellular immune response in this group can inform us as to the nature of effective immunity against tuberculosis. The aim of this study was to identify which Cytokines and lymphocyte populations characterise the peripheral blood cellular immune response following BCG vaccination. Results: Diluted blood from before and after vaccination was stimulated with Mycobacterium tuberculosis purified protein derivative for 6 days, after which soluble biomarkers in supernatants were assayed by multiplex bead array. Ten out of twenty biomarkers measured were significantly increased (p < 0.0025) 1 month after BCG vaccination when compared to paired samples (n = 12) taken prior to vaccination (IFNγ, TNFα, IL-1α, IL-2, IL-6, IL-10, IL-17, GM-CSF, MIP1α, IP-10). All of these remained detectable by multiplex bead array in samples taken 12 months after BCG vaccination of a partially overlapping adolescent group (n = 12). Intracellular Cytokine Staining after 24 hour Mycobacterium tuberculosis purified protein derivative stimulation of PBMC samples from the 12 month group revealed that IFNγ expression was detectable in CD4 and CD8 T-cells and natural killer cells. Polyfunctional flow cytometry analysis demonstrated that cells expressing IFNγ alone formed the majority in each subpopulation of cells. Only in CD4 T-cells and NK cells were there a notable proportion of responding cells of a different phenotype and these were single positive, TNFα producers. No significant expression of the Cytokines IL-2, IL-17 or IL-10 was seen in any population of cells. Conclusions: The broad array of biomarker responses detected by multiplex bead array suggests that BCG vaccination is capable, in this setting, of inducing a complex immune phenotype. Although polyfunctional T-cells have been proposed to play a role in protective immunity, they were not present in vaccinated adolescents who, based on earlier epidemiological studies, should have developed protection against pulmonary tuberculosis. This may be due to the later sampling time point available for testing or on the kinetics of the assays used.

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