Techniques to Improve the Direct Ex Vivo Detection of Low Frequency Antigen-Specific CD8+ T Cells with Peptide-Major Histocompatibility Complex Class I Tetramers

The ability to quantify and characterize antigen-specific CD8(+) T cells irrespective of functional readouts using fluorochrome-conjugated peptide-major histocompatibility complex class I (pMHCI) tetramers in conjunction with flow cytometry has transformed our understanding of cellular immune responses over the past decade. In the case of prevalent CD8(+) T cell populations that engage cognate pMHCI tetramers with high avidities, direct ex vivo identification and subsequent data interpretation is relatively straightforward. However, the accurate identification of low frequency antigen-specific CD8(+) T cell populations can be complicated, especially in situations where T cell receptor-mediated tetramer binding occurs at low avidities. Here, we highlight a few simple techniques that can be employed to improve the Visual resolution, and hence the accurate quantification, of tetramer binding CD8(+) T cell populations by flow cytometry. These methodological modifications enhance signal intensity, especially in the case of specific CD8(+) T cell populations that bind cognate antigen with low avidities, minimize background noise, and enable improved discrimination of true pMHCI tetramer binding events from nonspecific uptake. (C) 2008 international Society for Advancement of cytometry