title: Detection of cardiac myosin heavy chain-α-specific CD4 cells by using MHC class II/IA(k) tetramers in A/J mice.
abstract: A/J mice bearing the H-2 allele IA(k) are highly susceptible to autoimmune myocarditis induced with cardiac myosin heavy chain (Myhc)-α 334-352, whereas B10.A mice carrying a similar allele IA(k) are relatively resistant, suggesting that the generation of Myhc-α-reactive T cell repertoires is influenced by genetic background. To enumerate the precursor frequencies of Myhc-α-specific CD4 T cells, we sought to create IA(k) tetramers for Myhc-α 334-352. Tetramers were created using approaches that involve covalent tethering of individual peptide sequences or exogenous loading of peptides into empty IA(k) molecules by peptide-exchange reaction. Using ribonuclease 43-56 tetramers as controls, we demonstrated that by flow cytometry (FC), Myhc-α 334-352 tetramers specifically bind myosin-reactive T cells. CD4 cells isolated from A/J mice immunized with Myhc-α 334-352 were used to optimize conditions for tetramer staining, and neuraminidase treatment prior to tetramer staining permitted the detection of Myhc-α-specific cells ex vivo. The reagents are useful tools for monitoring the frequency of Myhc-α-reactive CD4 cells and to determine their pathogenic potential at a single cell level by FC.
authors.affiliation: School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583, USA.
authors.forename: Chandirasegaran
authors.initials: C
authors.lastname: Massilamany
authors.forename: Arunakumar
authors.initials: A
authors.lastname: Gangaplara
authors.forename: Nora
authors.initials: N
authors.lastname: Chapman
authors.forename: Noel
authors.initials: N
authors.lastname: Rose
authors.forename: Jay
authors.initials: J
authors.lastname: Reddy
mesh_terms: D000483:Alleles; D000818:Animals; D015153:Blotting, Western; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D005260:Female; D005434:Flow Cytometry; D000949:Histocompatibility Antigens Class II / Q000276:immunology*; D051379:Mice; D008805:Mice, Inbred A; D009205:Myocarditis / Q000235:genetics / Q000276:immunology*; D018995:Myosin Heavy Chains / Q000235:genetics / Q000276:immunology*
chemical_list: D000949:Histocompatibility Antigens Class II; D018995:Myosin Heavy Chains