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Detection of intracellular antigen-specific cytokines in human T cell populations
E G Kallas1, D C Gibbons, H Soucier
1Infectious Diseases Unit, University of Rochester, Rochester, NY 14642, USA.
Insights
Detecting T cell cytokine responses after vaccination is challenging. This study developed an antigenic expansion method using flow cytometry to enhance detection of interferon-gamma (IFN-γ) and IL-4 production.
Area of Science:
- Immunology
- Vaccinology
Background:
- Assessing antigen-specific T lymphocyte cytokine responses post-vaccination is difficult due to the low frequency of responder cells.
- Existing methods struggle to reliably detect these crucial immune responses.
Purpose of the Study:
- To develop and validate a method for detecting antigen-specific T lymphocyte cytokine production.
- To analyze the profile of intracellular cytokines, specifically interferon-gamma (IFN-γ) and IL-4, after antigenic expansion.
Main Methods:
- Peripheral blood mononuclear cells (PBMCs) were stimulated with antigens and expanded with interleukin-2 (IL-2).
- Intracellular cytokine staining via flow cytometry was performed after restimulation to quantify IFN-γ and IL-4.
- Phenotypic analysis identified cytokine-producing cells, focusing on CD45RO+ memory cells and CD4/CD8 populations.
Main Results:
- Antigenic expansion significantly increased detectable IFN-γ production (6%-20% for influenza), with minimal IL-4 detected.
- Memory cells (CD45RO+) were the primary source of both IFN-γ and IL-4.
- IFN-γ was produced by both CD4+ and CD8+ T cell subsets.
- The method successfully measured antigen-specific IFN-γ increases in a human immunodeficiency virus type 1 (HIV-1) vaccine trial, correlating with other immune markers.
Conclusions:
- Antigenic expansion followed by intracellular cytokine staining is an effective method for detecting low-frequency antigen-specific T cell responses.
- This technique provides valuable insights into vaccine-induced cellular immunity and can be applied in clinical trials.
- The findings highlight the role of memory T cells in producing key cytokines like IFN-γ post-vaccination.
Abstract:
Determination of antigen-specific cytokine responses of T lymphocytes after vaccination is made difficult by the low frequency of responder cells. In order to detect these responses, the profile of intracellular cytokines was analyzed using flow cytometry after antigenic expansion. Peripheral blood mononuclear cells were stimulated with antigens for 5 days, further expanded with interleukin (IL)-2, and then restimulated on day 10. Cytokine production was detected by intracellular staining with monoclonal antibodies after saponin-based permeabilization. Influenza expansion resulted in specific interferon-gamma (IFN-gamma) production of 6%-20%, with less IL-4 production (0%-2%). Tetanus toxoid resulted in even greater production. IL-4 and IFN-gamma were produced mainly by memory cells of the CD45RO+ phenotype. IFN-gamma production was contributed by both CD4 and CD8 populations. These methods were then applied to a clinical trial of a candidate human immunodeficiency virus type 1 vaccine. Antigen-specific increases in IFN-gamma were measured, which corresponded to antibody production, lymphoproliferation, and skin testing.
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