Techniques to improve the direct ex vivo detection of low frequency antigen‐specific CD8+ T cells with peptide‐major histocompatibility complex class I tetramers

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Abstract
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. © 2008 International Society for Advancement of Cytometry