Research paper Dynamic quantification of MHC class I–peptide presentation to CD8+ T cells via intracellular cytokine staining Ken C. Pang, Joe Q.Z. Wei, Weisan Chen ⁎ T cell Laboratory, Ludwig Institute for Cancer Research, Melbourne Branch, Austin Health, Heidelberg, VIC, 3084, Australia Received 5 July 2005; received in revised form 22 December 2005; accepted 3 January 2006 Available online 20 February 2006 Abstract In order to further our basic understanding of antigen processing and presentation as well as to translate that knowledge into clinically effective vaccines and immunotherapies, having appropriate tools to study MHC class I–peptide presentation is highly desirable. Current methods are based upon HPLC fractionation of extracted peptides, monoclonal Ab, multivalent T cell receptors (TCR), T cell hybridomas, TCR transgenic cells, and T cell lines. However, each of these is associated with problems that make them either difficult to apply generally or too insensitive to adequately quantitate antigen presentation. We have developed a method based upon intracellular cytokine staining (ICS) that dynamically and relatively quantitates MHC class I–peptide presentation to CD8+ T cells in a manner that is both widely applicable and highly sensitive. It is well-suited to assess antigen presentation in its early stages, does not require fixation nor labeling of antigen presenting cells (APC), can be used to examine cross-presentation, and is able to directly employ ex vivo T cells which obviates the need for the development and maintenance of T cell lines and hybridomas. Our method represents a simple yet powerful tool that others interested in studying antigen processing and presentation should find of great practical value. © 2006 Elsevier B.V. All rights reserved. Keywords: CD8+ T cell; Antigen presentation; Kinetics; Intracellular cytokine staining; Brefeldin A 1. Introduction CD8+ T cells are critical effector cells of the adaptive immune system, and can mediate protection against intracellular pathogens and malignant cells. Despite intensive efforts, identifying vaccination strategies to achieve effective CD8+ T cell responses remains an elu- sive goal. The immunogenicity of any vaccine will be ultimately limited by the efficacy of antigen presentation, therefore having appropriate tools to assess the presenta- tion of antigenic peptides on MHC class I molecules in a quantitative manner is highly desirable. Existing methods include the use of HPLC fractionation of extracted pep- tides (Anton et al., 1997), monoclonal Ab (mAb) or multivalent TCR specific for a given MHC–peptide complex (Andersen et al., 1996; O'Herrin et al., 1997; Porgador et al., 1997), T cell hybridomas (Sanderson and Shastri, 1994; Usherwood et al., 1999; Canaday et al., 2003), TCR transgenic cells (Robson et al., 2003), and T cell lines (Chen et al., 2000; Schnurr et al., 2005). How- ever, each of these is problematic, making them either very difficult to apply generally or too insensitive to adequately quantitate antigen presentation. HPLC frac- tionation, for instance, although permitting direct and Journal of Immunological Methods 311 (2006) 12 – 18 www.elsevier.com/locate/jim Abbreviations: APC, antigen-presenting cell; BFA, Brefeldin A; DC, dendritic cell; FCS, fetal calf serum; ICS, intracellular cytokine staining; MHC, major histocompatibility complex. ⁎ Corresponding author. Tel.: +61 3 9496 3700; fax: +61 3 9496 5334. E-mail address: weisan.chen@ludwig.edu.au (W. Chen). 0022-1759/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2006.01.008