title: Reliable generation and use of MHC class II:gamma2aFc multimers for the identification of antigen-specific CD4(+) T cells.
abstract: MHC tetramers have proven to be powerful reagents for the analysis of MHC class I-restricted T cells. However, generating similarly reliable reagents for MHC class II-restricted T cells has been elusive. Here we evaluated the utility of MHC class II:gamma2aFc multimers, which contain the MHC class II extracellular domains, with or without recombinantly attached peptides, dimerized via a fos-jun leucine zipper and attached to the hinge of murine IgG2a. We have successfully generated 24 multimers in either myeloma or Drosophila melanogaster S2 cells, with an average yield of 7 mg/L. 'Empty' MHC class II:gamma2aFc multimers were effectively used in peptide binding assays. Similar versions that contained recombinantly attached peptides stimulated T cells in an antigen-specific, MHC-restricted manner, and identified antigen-specific nai;ve and effector T cells by flow cytometry. Furthermore, we have successfully used these reagents to stain T cells generated following viral infection. Thus, MHC class II:gamma2aFc multimers are robust and reliable reagents for the analysis of MHC class II-restricted T cells.
authors.affiliation: Department of Immunology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105, USA.
authors.forename: Paula Y
authors.initials: PY
authors.lastname: Arnold
authors.forename: Kate M
authors.initials: KM
authors.lastname: Vignali
authors.forename: Timothy B
authors.initials: TB
authors.lastname: Miller
authors.forename: Nicole L
authors.initials: NL
authors.lastname: La Gruta
authors.forename: Linda S
authors.initials: LS
authors.lastname: Cauley
authors.forename: Laura
authors.initials: L
authors.lastname: Haynes
authors.forename: P
authors.initials: P
authors.lastname: Scott Adams
authors.forename: Susan L
authors.initials: SL
authors.lastname: Swain
authors.forename: David L
authors.initials: DL
authors.lastname: Woodland
authors.forename: Dario A A
authors.initials: DA
authors.lastname: Vignali
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D004331:Drosophila melanogaster; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D005260:Female; D005434:Flow Cytometry; D000949:Histocompatibility Antigens Class II / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000276:immunology*; D009980:Influenza A virus / Q000276:immunology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D010957:Plasmids / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology; D029082:Sendai virus / Q000276:immunology; D013047:Specific Pathogen-Free Organisms; D014407:Tumor Cells, Cultured
chemical_list: D018984:Epitopes, T-Lymphocyte; D000949:Histocompatibility Antigens Class II; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins