A novel dendritic cell-based direct ex vivo assay for detection and enumeration of circulating antigen-specific human T cells
Although a variety of assays have been used to examine T cell responses in vitro, standardized ex vivo detection of antigen-specific CD4+ T cells from human circulatory PBMCs remains constrained by low-dimensional characterization outputs and the need for polyclonal, mitogen-induced expansion methods to generate detectable response signals. To overcome these limitations, we developed a novel methodology utilizing antigen-pulsed autologous human dendritic target cells in a rapid and sensitive assay to accurately enumerate antigen-specific CD4+ T cell precursor frequency by multiparametric flow cytometry. With this approach, we demonstrate the ability to reproducibly quantitate poly-functional T cell responses following both primary and recall antigenic stimulation. Furthermore, this approach enables more comprehensive phenotypic profiling of circulating antigen-specific CD4+ T cells, providing valuable insights into the pre-existing polarization of antigen-specific T cells in humans. Combined, this approach permits sensitive and detailed ex vivo detection of antigen-specific CD4+ T cells delivering an important tool for advancing vaccine, immune-oncology and other therapeutic studies.
KeywordsHuman T cells Dendritic cells Cytokine Ex vivo In vitro Antigen-specific Circulation Cell-based bioassays
Enzyme-linked immunosorbent assay
Polymerase chain reaction
Peripheral blood mononuclear cells
T helper cell
Fluzone® (influenza) vaccine
Tuberculosis recombinant fusion protein (H4)
We thank Drs. A. Byers, C. Dao, and J. Moser for critical discussion and review of this manuscript. This research was supported fully by Sanofi Pasteur and did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Compliance with ethical standards
Conflict of interest
The authors have no conflict of interest.
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