Intracellular cytokine staining for the characterization and quantitation of antigen-specific T lymphocyte responses
1New England Primate Research Center, Harvard Medical School, Division of Immunology, One Pine Hill Drive, Southborough, MA 01772, USA. marie-cliare_gauduin@hms.harvard.edu
Insights
This study optimized an intracellular cytokine staining assay for improved T cell analysis in non-human primates. The enhanced assay offers higher sensitivity and reproducibility for studying immune responses to pathogens.
Area of Science:
- Immunology
- Cellular Biology
- Virology
Background:
- Standard T cell proliferation assays lack sensitivity and quantitative readouts.
- Flow cytometry enables multiparametric detection of cell surface antigens and intracellular cytokines.
- Existing methods require optimization for precise analysis of antigen-specific T cells.
Purpose of the Study:
- To optimize an intracellular cytokine staining assay for sensitive, quantitative analysis of antigen-specific T cells.
- To improve the study of T cell function in the non-human primate model of AIDS.
- To provide new insights into pathogen-specific immune responses.
Main Methods:
- Optimization of an intracellular cytokine staining assay using flow cytometry.
- Inclusion of cross-linked costimulatory anti-CD28 and anti-CD49d monoclonal antibodies (Mabs).
- Fine-tuning of antigen concentration and duration of antigenic stimulation.
Main Results:
- The optimized assay achieved high sensitivity and minimal background staining.
- Addition of costimulatory Mabs enhanced the frequency of cytokine-secreting CD4(+) T cells by up to 3-fold.
- The assay allows for single-cell level delineation of antigen-specific T cells.
Conclusions:
- The optimized intracellular cytokine staining assay provides a sensitive and reproducible method for T cell analysis.
- This assay enables detailed phenotypic analysis of low-frequency antigen-specific T cells.
- The improved method offers new insights into pathogen-specific immune responses in non-human primates.
Abstract:
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.
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