Intracellular cytokine staining for the characterization and quantitation of antigen-specific T lymphocyte responses

Standard proliferation assays used for analysis of T cell function have significant shortcomings, including limited sensitivity, lack of quantitative readouts, and considerable variability. Recently, flow cytometric methods have been developed to allow multiparametric detection of cell surface antig...

Full description

Saved in:
Bibliographic Details
Published in:Methods (San Diego, Calif.) Calif.), 2006-04, Vol.38 (4), p.263-273
Main Author: Gauduin, Marie-Claire
Format: Article
Language:English
Subjects:
Antibodies, Monoclonal - chemistry
Antigen-specific activation
Antigens - chemistry
Brefeldin A - pharmacology
CD28 Antigens - biosynthesis
CD4 + and CD8 + T cell responses
CD4-Positive T-Lymphocytes - metabolism
CD8-Positive T-Lymphocytes - metabolism
Cell Proliferation
Cell Separation
Cryopreservation
Cytokines - metabolism
Flow Cytometry
Humans
Immune System
Integrin alpha4 - biosynthesis
Intracellular cytokine staining
Lymphocyte Activation
Models, Statistical
SIV/macaque model
T-Lymphocytes - immunology
T-Lymphocytes - metabolism
Temperature
Time Factors
Citations: Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Standard proliferation assays used for analysis of T cell function have significant shortcomings, including limited sensitivity, lack of quantitative readouts, and considerable variability. Recently, flow cytometric methods have been developed to allow multiparametric detection of cell surface antigens and intracellular cytokine expression in response to polyclonal stimuli and antigen. We have optimized an intracellular cytokine staining assay in the non-human primate model of AIDS, which allows us to identify antigen-specific T lymphocytes at the single cell level with high sensitivity, while reducing background staining to a minimum. Central to our optimized protocol is the addition of cross-linked costimulatory anti-CD28 and anti-CD49d Mabs, a modification that results in up to 3-fold enhancement of the frequency of cytokine-secreting CD4 + T cells following superantigen or antigen-specific stimulation. Optimization of the antigen concentration and duration of antigenic stimulation resulted in a convenient and highly reproducible assay, which permits delineation of antigen-specific cells at the single cell level, thereby providing new insights into pathogen-specific immune responses and allowing detailed phenotypic analysis of extremely low frequency events.
ISSN:1046-2023
1095-9130
DOI:10.1016/j.ymeth.2005.12.004