Multiplexed combinatorial tetramer staining in a mouse model of virus infection

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Use of fluorescently labelled multimers, particularly tetramers of peptide and MHC class I glycoprotein (pMHC-I) complexes, is essential for the analysis of CD8 + T cell immunity in basic research and clinical settings. A recently described combinatorial approach using pMHC-I multimers coupled to a unique combination of distinct fluorochromes has facilitated the simultaneous screening of multiple T cell specificities within a single human blood sample. The present analysis establishes that this multiplexed tetramer staining protocol can also be applied in mouse models of a disease to detect multiple subdominant CD8 + T cell specificities in the presence of prominent immunodominant T cell sets at different stages of infection. We have established a modified protocol that concurrently identified influenza-specific CD8 + T cells at the acute and long-term memory phases of influenza virus infection in B6 mice. Highly dominant (D bNP 366 +CD8 + and D bPA 224 +CD8 +) and subdominant (K bPB1 703 +CD8 +, D bPB1-F2 62 +CD8 + and K bNS2 114 +CD8 +) T cell responses can be detected simultaneously at levels comparable to the conventional tetramer staining with this combinatorial approach. The technique proved particularly useful with aged mice, where we used 5-fold fewer animals, making the detection of multiple T cell specificities more cost-effective and less time-consuming. Overall, our study establishes that this comprehensive concurrent analysis of multiple T cell specificities is of value for analysing mouse models of disease, especially in situations where sample size and/or response magnitude is limiting.