title: Multiplexed combinatorial tetramer staining in a mouse model of virus infection.
abstract: 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(b)NP(366)+CD8+ and D(b)PA(224)+CD8+) and subdominant (K(b)PB1(703)+CD8+, D(b)PB1-F2(62)+CD8+ and K(b)NS2(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.
authors.affiliation: Department of Microbiology and Immunology, University of Melbourne, Parkville 3010, Melbourne, Australia.
authors.forename: Tania
authors.initials: T
authors.lastname: Cukalac
authors.forename: Sophie A
authors.initials: SA
authors.lastname: Valkenburg
authors.forename: Nicole L
authors.initials: NL
authors.lastname: La Gruta
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Turner
authors.forename: Peter C
authors.initials: PC
authors.lastname: Doherty
authors.forename: Katherine
authors.initials: K
authors.lastname: Kedzierska
mesh_terms: D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D002469:Cell Separation; D002478:Cells, Cultured; D020650:Combinatorial Chemistry Techniques; D018604:Epitope Mapping; D018984:Epitopes, T-Lymphocyte / Q000276:immunology / Q000378:metabolism*; D005260:Female; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D009980:Influenza A virus / Q000276:immunology* / Q000472:pathogenicity; D051379:Mice; D008810:Mice, Inbred C57BL; D009976:Orthomyxoviridae Infections / Q000175:diagnosis / Q000276:immunology* / Q000473:pathology; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism*; D055503:Protein Multimerization / Q000276:immunology; D014764:Viral Proteins / Q000276:immunology / Q000378:metabolism*
chemical_list: D018984:Epitopes, T-Lymphocyte; D005456:Fluorescent Dyes; D015395:Histocompatibility Antigens Class I; D010446:Peptide Fragments; D014764:Viral Proteins