title: Labeling antigen-specific CD4(+) T cells with class II MHC oligomers.
abstract: Class I MHC-peptide oligomers (MHC tetramers) have become popular reagents for the detection and characterization of antigen-specific CD8(+) T cells. Class II MHC proteins can be produced by expression in Escherichia coli followed by in vitro folding, or by native expression in insect cells; biotin can be introduced by site-specific chemical modification of cysteine, or by enzymatic modification of a peptide tag; and a variety of fluorescent streptavidin preparations can be used for oligomerization. Here we review methodologies for production of fluorescent oligomers of soluble class II MHC proteins and discuss their use in analysis of antigen-specific CD4(+) T cells. We explore the experimental conditions necessary for efficient staining of CD4(+) T cells using oligomers of class II MHC proteins, and we establish a standard protocol. Finally, we consider complications and challenges associated with these reagents, discuss the interpretation of staining results, and suggest future directions for investigation, in particular the use of MHC oligomers for the study of T cell avidity modulation.
authors.affiliation: Department of Chemistry, MIT, 77 Massachusetts Avenue, Cambridge 02139, USA.
authors.forename: Thomas O
authors.initials: TO
authors.lastname: Cameron
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Norris
authors.forename: Alka
authors.initials: A
authors.lastname: Patel
authors.forename: Corinne
authors.initials: C
authors.lastname: Moulon
authors.forename: Eric S
authors.initials: ES
authors.lastname: Rosenberg
authors.forename: Elizabeth D
authors.initials: ED
authors.lastname: Mellins
authors.forename: Lucy R
authors.initials: LR
authors.lastname: Wedderburn
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Stern
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics; D015798:HLA-DR1 Antigen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000949:Histocompatibility Antigens Class II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D013194:Staining and Labeling
chemical_list: D015798:HLA-DR1 Antigen; D000949:Histocompatibility Antigens Class II; D011994:Recombinant Proteins