Optimization and qualification of an 8-color intracellular cytokine staining assay for quantifying T cell responses in rhesus macaques for pre-clinical vaccine studies

Mitzi M. Donaldson, Shing Fen Kao, Leila Eslamizar, Connie Gee, Gerrit Koopman, Michelle Lifton, Joern E. Schmitz, Andrew W. Sylwester, Aaron Wilson, Natalie Hawkins, Steve G. Self, Mario Roederer, Kathryn E. Foulds

Research output: Contribution to journalArticle

20 Scopus citations

Abstract

Vaccination and SIV challenge of macaque species is the best animal model for evaluating candidate HIV vaccines in pre-clinical studies. As such, robust assays optimized for use in nonhuman primates are necessary for reliable ex vivo measurement of immune responses and identification of potential immune correlates of protection. We optimized and qualified an 8-color intracellular cytokine staining assay for the measurement of IFNγ, IL-2, and TNF from viable CD4 and CD8 T cells from cryopreserved rhesus macaque PBMC stimulated with peptides. After optimization, five laboratories tested assay performance using the same reagents and PBMC samples; similar results were obtained despite the use of flow cytometers with different configurations. The 8-color assay was then subjected to a pre-qualification study to quantify specificity and precision. These data were used to set positivity thresholds and to design the qualification protocol. Upon completion of the qualification study, the assay was shown to be highly reproducible with low inter-aliquot, inter-day, and inter-operator variability according to the qualification criteria with an overall variability of 20-40% for each outcome measurement. Thus, the 8-color ICS assay was formally qualified according to the ICH guidelines Q2 (R1) for specificity and precision indicating that it is considered a standardized/robust assay acceptable for use in pre-clinical trial immunogenicity testing.

Original languageEnglish (US)
Pages (from-to)10-21
Number of pages12
JournalJournal of Immunological Methods
Volume386
Issue number1-2
DOIs
StatePublished - Dec 14 2012

Keywords

  • Antigen-specific
  • Flow cytometry
  • Immune assays
  • Memory
  • Phenotype

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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    Donaldson, M. M., Kao, S. F., Eslamizar, L., Gee, C., Koopman, G., Lifton, M., Schmitz, J. E., Sylwester, A. W., Wilson, A., Hawkins, N., Self, S. G., Roederer, M., & Foulds, K. E. (2012). Optimization and qualification of an 8-color intracellular cytokine staining assay for quantifying T cell responses in rhesus macaques for pre-clinical vaccine studies. Journal of Immunological Methods, 386(1-2), 10-21. https://doi.org/10.1016/j.jim.2012.08.011