Detection of cardiac myosin heavy chain-α-specific CD4 cells by using MHC class II/IAk 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.

Versatility of using major histocompatibility complex class II dextramers for derivation and characterization of antigen-specific, autoreactive T cell hybridomas

“…Briefly, the α and β constructs of IA s allele along with the peptide of interest was expressed together using baculovirus expression systems in SF9 insect cells (Invitrogen, Carlsbad, CA). Soluble MHC class II monomers of IA s were then purified, concentrated, and biotinylated using biotin ligase (25 μg/10 nmol of substrate; Avidity, Denver, CO) [12, 14, 15]. The biotinylated monomers were assembled to fluorophore conjugated dextran molecules (kindly provided by Immudex, Copenhagen, Denmark) at a molar ratio of 20:1 in 1x Tris HCl 0.05 M, pH 7.2, by incubating in the dark for 30 minutes at room temperature (RT) [12].…”

“…Lymph node cells (LNC) were stimulated with PLP 139-151 (20 μg/ml) at a density of 5×10 6 cells/ml for two days in clone medium (RPMI medium supplemented with 10% fetal bovine serum [FBS], 1 mM sodium pyruvate, 4 mM L-glutamine, 1x each of non-essential amino acids and vitamin mixture, and 100 U/ml penicillin-streptomycin [Lonza, Walkersville, MD]) [14, 15, 17]. After two days, the cultures were supplemented with clone medium containing interleukin (IL)-2 (hereafter called IL-2 medium) and maintained for an additional two days.…”

Direct Staining with Major Histocompatibility Complex Class II Dextramers Permits Detection of Antigen-Specific, Autoreactive CD4 T Cells In Situ

“…Briefly, α and β constructs for each IA allele containing the sequences of the respective peptides were expressed in baculovirus using Sf9 insect cells (Invitrogen, Carlsbad, CA), and the soluble monomers of IA s and IA k were purified [22], [24], [25]. After being concentrated, the soluble MHC class II proteins were biotinylated using biotin protein ligase at an optimized concentration of 25 µg/10 nmol of substrate as recommended by the manufacturer (Avidity, Denver, CO).…”

“…After the single cell suspensions were prepared, the lymph node cells (LNC) were stimulated with the immunizing peptides (PLP 139–151, 20 µg/ml or Myhc 334–352, 50 µg/ml) at a density of 5×10 6 cells/ml for two days, and interleukin (IL)-2 medium was then added [21], [22], [24]. Cells were stained with dextramers on the indicated days in IL-2 medium, pH 7.6, containing 2.5% fetal bovine serum at RT for 2 hours, followed by staining with anti-CD4 (eBioscience, San Diego, CA) [19].…”

Analysis, Isolation, and Activation of Antigen-Specific CD4+ and CD8+ T Cells by Soluble MHC-Peptide Complexes

“…The tetramer staining efficiency also critically depends on CD4 + T cells surface glycosylation and pretreatment of cells with a broadly active neuraminidase (e.g., from Vibrio cholerae ) can increase tetramer staining by nearly 100% (77, 78). Testing on 37 Flu-specific Th1 clones we found increases of tetramer staining upon desialylation in the range of 20–95% (unpublished data).…”