Optimization and validation of an 8-color intracellular cytokine staining (ICS) assay to quantify antigen-specific T cells induced by vaccination

J Immunol Methods. 2007 May 31;323(1):39-54. doi: 10.1016/j.jim.2007.03.002. Epub 2007 Apr 3.


Candidate HIV-1 vaccines currently being evaluated in clinical trials are designed to elicit HIV-1-specific cellular immunity. Intracellular cytokine staining (ICS) assays allow sensitive, quantitative ex vivo assessments of antigen-specific T cells including immunophenotyping of responding cells and measurement of multiple effector functions. Additionally, the use of banked cryopreserved PBMC samples makes this assay attractive in the setting of large efficacy trials where it is less feasible to perform immunoassays on freshly isolated samples. Here we describe extensive studies to optimize and quantitatively validate the 8-color ICS assay for use in clinical trials of candidate vaccines, which includes measurement of viable IFN-gamma, IL-2, TNF-alpha and IL-4 producing CD4+ and CD8+ T cells. We show that omission of viability dye staining results in an over-estimate of the true antigen-specific T cell response by up to two-fold. After optimization, the 8-color assay was validated for specificity, precision, linearity, limit of quantitation and robustness. The assay has a lower quantitation limit generally below 0.04%, depending on the cytokine subset. Additionally, with appropriate gating, the 8-color assay gives comparable cytokine-positive responses to those observed with the conventional 4-color assay. In conclusion, we provide the first description of a quantitatively validated ICS assay, which permits quantitative and qualitative evaluation of vaccine-induced immunogenicity and analysis of immune correlates of protection.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • AIDS Vaccines / immunology*
  • Acquired Immunodeficiency Syndrome / prevention & control
  • Clinical Trials as Topic
  • Cytokines / analysis*
  • Cytokines / metabolism
  • Cytomegalovirus / immunology
  • Cytomegalovirus Infections / immunology
  • Flow Cytometry
  • HIV-1 / immunology
  • Humans
  • Immunoassay / methods*
  • Intracellular Fluid / chemistry*
  • Intracellular Fluid / immunology
  • Intracellular Fluid / metabolism
  • Sensitivity and Specificity
  • T-Lymphocyte Subsets / immunology
  • T-Lymphocyte Subsets / metabolism*


  • AIDS Vaccines
  • Cytokines