TY - JOUR
T1 - Detection of cardiac myosin heavy chain-α-specific CD4 cells by using MHC class II/IAk tetramers in A/J mice
AU - Massilamany, Chandirasegaran
AU - Gangaplara, Arunakumar
AU - Chapman, Nora
AU - Rose, Noel
AU - Reddy, Jay
N1 - Funding Information:
Sources of funding: This work was co-funded by the American Heart Association and the Children's Cardiomyopathy Foundation ( SDG2462390204001 ).
PY - 2011/9/30
Y1 - 2011/9/30
N2 - A/J mice bearing the H-2 allele IAk are highly susceptible to autoimmune myocarditis induced with cardiac myosin heavy chain (Myhc)-α 334-352, whereas B10.A mice carrying a similar allele IAk 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 IAk 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 IAk 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.
AB - A/J mice bearing the H-2 allele IAk are highly susceptible to autoimmune myocarditis induced with cardiac myosin heavy chain (Myhc)-α 334-352, whereas B10.A mice carrying a similar allele IAk 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 IAk 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 IAk 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.
KW - Autoimmunity
KW - Cardiac myosin
KW - MHC class II tetramers
KW - Mouse model
KW - Myocarditis
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U2 - 10.1016/j.jim.2011.07.004
DO - 10.1016/j.jim.2011.07.004
M3 - Article
C2 - 21782819
AN - SCOPUS:79961126870
SN - 0022-1759
VL - 372
SP - 107
EP - 118
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
IS - 1-2
ER -