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Effect of dendritic cells on flow cytometric quantification of cytomegalovirus-specific, interferon-gamma-producing T
C C Fleischer1, C Solling, M S Petersen
1Department of Medical Microbiology and Immunology, University of Aarhus, Aarhus C, Denmark.
Insights
Antigen-pulsed dendritic cells (DCs) significantly enhance the detection of antigen-specific T cells in frozen blood samples. This method restores the sensitivity of intracellular cytokine detection assays, crucial for studying T-cell responses.
Area of Science:
- Immunology
- Cellular Biology
- Medical Diagnostics
Background:
- Flow cytometry is vital for quantifying antigen-specific T-cell responses by measuring intracellular cytokines.
- Low frequencies of antigen-specific T cells necessitate improved signal-to-noise ratios for accurate detection.
Purpose of the Study:
- To evaluate the efficacy of antigen-pulsed dendritic cells (DCs) in boosting the detection of antigen-specific, interferon-gamma (IFN-gamma)-producing CD4+ T cells.
- To compare the sensitivity of this method in fresh versus cryopreserved peripheral blood mononuclear cell (MNC) samples.
Main Methods:
- Utilized Cytomegalovirus (CMV) lysate as the antigen in a 10-hour assay.
- Compared intracellular cytokine detection in fresh MNCs versus reconstituted cryopreserved MNCs.
- Assessed the impact of adding CMV lysate-pulsed DCs to both sample types.
Main Results:
- Cryopreserved MNC samples showed a 23-86% lower response to CMV lysate compared to fresh samples.
- Antigen-pulsed DCs did not improve sensitivity in fresh peripheral blood samples.
- CMV lysate-pulsed DCs significantly increased IFN-gamma-producing cells in cryopreserved MNCs, matching fresh sample levels.
Conclusions:
- Antigen-pulsed DCs are effective in restoring the sensitivity of intracellular cytokine detection assays for cryopreserved samples.
- This approach offers a viable method for analyzing antigen-specific T-cell responses in stored blood samples.
- Optimizing T-cell detection in frozen samples is critical for immunological research and diagnostics.
Abstract:
Flow cytometric measurement of intracellular cytokines in T cells exposed to antigen is a widely used method for quantification of an antigen-specific T-cell response. As the frequency of antigen-specific T cells is often very low, any improvement in signal to noise ratio is of great importance. Thus, in this study, the ability of antigen-pulsed dendritic cells (DCs) to increase the number of antigen-specific, interferon-gamma (IFN-gamma)-producing CD4+ T cells measurable both in fresh peripheral blood and in reconstituted frozen blood mononuclear cell (MNC) samples was evaluated. Cytomegalovirus (CMV) was used as antigen in a 10 h assay, using cells from both CMV-seropositive and -seronegative donors. When reconstituted frozen samples were analysed, the general response towards CMV lysate in CMV-seropositive donors was 23-86% lower compared to the corresponding fresh blood samples. Antigen-pulsed DCs could not improve the sensitivity of the intracellular cytokine-detection assay when fresh peripheral blood samples were used. Interestingly, however, the addition of CMV lysate-pulsed DCs to cryopreserved MNC samples substantially increased the frequency of specifically induced IFN-gamma-producing cells to a level comparable to the frequency found in the corresponding fresh blood samples.
