Multiplexed combinatorial tetramer staining in a mouse model of virus infection

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^bNP₃₆₆ ⁺CD8⁺ and D^bPA₂₂₄ ⁺CD8⁺) and subdominant (K^bPB1₇₀₃ ⁺CD8⁺, D^bPB1-F2₆₂ ⁺CD8⁺ and K^bNS2₁₁₄ ⁺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.