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A novel dendritic cell-based direct ex vivo assay for detection and enumeration of circulating antigen-specific human T cells

  • Roberto Carrio
  • Ge Zhang
  • Donald R. DrakeIII
  • Brian C. Schanen
Article

Abstract

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.

Keywords

Human T cells Dendritic cells Cytokine Ex vivo In vitro Antigen-specific Circulation Cell-based bioassays 

Abbreviations

DCs

Dendritic cells

ELISA

Enzyme-linked immunosorbent assay

ELISPOT

Enzyme-linked immunospot

PCR

Polymerase chain reaction

PBMC

Peripheral blood mononuclear cells

TH

T helper cell

FLU

Fluzone® (influenza) vaccine

TB

Tuberculosis recombinant fusion protein (H4)

BFA

Brefeldin A

Notes

Acknowledgements

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.

Supplementary material

10616_2018_222_MOESM1_ESM.pdf (244 kb)
Supplementary material 1 (PDF 245 kb)
10616_2018_222_MOESM2_ESM.pdf (39 kb)
Supplementary material 2 (PDF 39 kb)
10616_2018_222_MOESM3_ESM.pdf (149 kb)
Supplementary material 3 (PDF 150 kb)

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Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  • Roberto Carrio
    • 1
  • Ge Zhang
    • 1
  • Donald R. DrakeIII
    • 1
  • Brian C. Schanen
    • 1
  1. 1.Sanofi PasteurOrlandoUSA

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