Direct visualization of antigen-specific T cells using peptide-MHC-class I tetrameric complexes Norbert Meidenbauer, a Thomas K. Hoffmann, b and Albert D. Donnenberg c, * a Department of Hematology and Oncology, University of Regensburg, D-93053 Regensburg, Germany b Department of Otorhinolaryngology, University of D€ usseldorf, D-40225 D€ usseldorf, Germany c Hillman Cancer Research Center L2.42c, University of Pittsburgh Cancer Institute, 5117 Centre Avenue, Pittsburgh, PA 15213-2582, USA Accepted 7 April 2003 Abstract Peptide-major histocompatibility complexes (MHC) class I tetrameric complexes (‘‘tetramers’’) are proving invaluable as fluo- rescent reagents for enumeration, characterization, and isolation of peptide-specific T cells and have afforded many advantages over previous techniques, particularly the ability to directly quantify and phenotype antigen-specific T cells with minimal in vitro ma- nipulation. Recently, various technical peculiarities of tetramers have led to modifications within this important immunoassay. The current manuscript details potential pitfalls and provides guidance for interpretation of results. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: T cells; MHC-tetramer complexes; CTL; Immune monitoring 1. Introduction 1.1. A new tool for detection of antigen-specific T cells Analysis of antigen-specific and major histocompat- ibility complexes (MHC)-restricted T-lymphocytes spe- cific for a known antigen is laborious and often unidimensional. Techniques for functional and quanti- tative measurements of antigen-specific T cells include limiting dilution assays (LDA), enzyme linked immu- nosorbent assay on a single cell level (ELISPOT) and intracellular cytokine staining. These assays have several disadvantages. In vitro stimulation needed for LDA or intracellular cytokine staining may not accurately reflect the in vivo situation. ELISPOT assays can be performed without in vitro stimulation, but do not allow further characterization of the responding T cells. In contrast, the use of tetramers promises a more exact enumeration, combined with phenotypic and functional analysis of antigen-specific T cells without prior in vitro stimulation [1]. In combination with fluorescence activated cell sorting, it can be used to isolate and expand peptide- specific T cells present at low frequency in vivo [2]. The tetramer is composed of the HLA heavy chain, b2-microglobulin, the nominal peptide, and streptavi- dine. HLA heavy chain and the b2-microglobulin are expressed, for example, in Escherichia coli, whereas the HLA heavy chain is modified and contains a biotinyla- tion site as a substrate for the enzyme BirA. After puri- fying the recombinant molecules the nominal peptide is added, yielding approximately 20–30% of properly as- sembled MHC-peptide monomers. Enzymatic biotiny- lation is induced and streptavidine is added, which connects the monomers via its four biotin binding sites to form a tetrameric complex. Streptavidine is commonly marked by a fluorescent dye, such as phycoerythrin (PE) or allophycocyanine (APC). This fluorescent complex binds to MHC-class I positive T cells expressing the specific T-cell receptor (TCR) for the nominal peptide of the tetramer, thus allowing flowcytrometric analysis. Initial 2-color studies using tetramer reagents in conjunction with anti-CD8 antibody suffered from sev- eral sources of artifact, which impeded optimal use of this powerful technique. This paper describes a Methods 31 (2003) 160–171 www.elsevier.com/locate/ymeth * Corresponding author. Fax: +412-623-7778. E-mail address: donnenbergad@msx.upmc.edu (A.D. Donnen- berg). 1046-2023/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S1046-2023(03)00126-9