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Rapid assessment of antigen induced cytokine expression in memory T cells by flow cytometry
1Becton Dickinson Immunocytometry Systems, San Jose, CA, USA.
Insights
This study introduces a rapid method to analyze single T cell responses to specific antigens in whole blood. This technique allows for precise measurement of immune function in clinical samples, aiding disease research.
Area of Science:
- Immunology
- Cellular Biology
- Biotechnology
Background:
- Traditional T cell analysis methods use bulk cultures, limiting single-cell insights.
- Assessing individual T cell responses in complex cellular environments is crucial for understanding immunity.
- Existing methods lack the ability to analyze single-cell functional and molecular parameters in response to stimuli.
Purpose of the Study:
- To develop and present a novel methodology for rapid assessment of T-cell subset responses.
- To enable analysis of single T cell responses to specific antigens in whole blood.
- To apply these technologies for measuring immune function parameters in disease and evaluating drug effects.
Main Methods:
- Developed rapid methodologies for assessing T-cell subset responses in whole blood.
- Utilized multiparameter flow cytometry for CD69+/cytokine+ expression analysis in T cells.
- Analyzed T cell responses to polyclonal activators and specific antigens (e.g., CMV, mumps) within 6 hours.
Main Results:
- Quantified T cell responses to specific antigens, with frequencies ranging from 0.05% to 5.0% within 6 hours.
- Demonstrated that antigen-specific T cell responses are host-specific and influenced by co-receptor antibodies.
- The modified technique is compatible with rapid analysis of clinical samples.
Conclusions:
- The new methodology allows for rapid, single-cell analysis of T cell immunity in whole blood.
- This approach facilitates direct examination of in vivo drug concentrations' effects on T cell immunity.
- Ongoing studies aim to assess the sensitivity of this assay to various therapeutic modalities.
Abstract:
Methods for analysis of T cell responses to specific antigen have traditionally relied on measurements of proliferation or cytokine expression in bulk cultures of PBMC in long term incubations with putative antigen. These techniques suffer from the drawback that they do not enable analysis of single cell responses in the context of unselected cellular backgrounds. It is increasingly important to not only identify cells on the basis of expression of unique surface antigens but also to determine functional and molecular parameters of individual cells in response to a variety of stimuli. We have recently developed methodologies to rapidly assess T-cell subset responses to polyclonal activators and specific antigen in whole blood. These procedures determine the percentages of activated cells and the identification of leucocyte subsets capable of expressing various cytokines and cell surface antigens. Multiparameter analysis of CD69+ /cytokine + expression in T cells in response to specific antigen (e.g. CMV, mumps) demonstrated a range of frequencies from 0.05% to 5.0% within 6 h. Frequencies of responding T cells were consistent but varied depending upon the antigen. Antigen specific T cell responses were host specific and both positively and negatively regulated by antibodies to co-receptors involved in APC-T cell interactions. These technologies will be discussed in terms of application to problems of measuring immune function parameters in disease. The modified technique described in this report is compatible with simple and rapid analysis of clinical samples and provides a means to directly examine the effects of in vivo drug concentrations on T cell immunity. Studies are in progress to examine the sensitivity of this cellular assay to drugs and other therapeutic modalities in clinical samples.