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HLA class I tetramers have revolutionized the study of Ag-specific CD8+ T cell responses. Technical problems and the rarity of Ag-specific CD4+ Th cells have not allowed the potential of HLA class II tetramers to be fully realized. Here, we optimize HLA class II tetramer staining methods through the use of a comprehensive panel of HIV-, influenza-, CMV-, and tetanus toxoid-specific tetramers. We find rapid and efficient staining of DR1- and DR4-restricted CD4+ cell lines and clones and show that TCR internalization is not a requirement for immunological staining. We combine tetramer staining with magnetic bead enrichment to detect rare Ag-specific CD4+ T cells with frequencies as low as 1 in 250,000 (0.0004% of CD4+ cells) in human PBLs analyzed directly ex vivo. This ultrasensitive detection allowed phenotypic analysis of rare CD4+ T lymphocytes that had experienced diverse exposure to Ag during the course of viral infections. These cells would not be detectable with normal flow-cytometric techniques.

Original publication

DOI

10.4049/jimmunol.175.10.6334

Type

Journal article

Journal

J Immunol

Publication Date

15/11/2005

Volume

175

Pages

6334 - 6343

Keywords

Amino Acid Sequence, CD4-Positive T-Lymphocytes, Cell Line, Epitopes, HIV Core Protein p24, HIV Infections, HLA-DR1 Antigen, HLA-DR4 Antigen, Hemagglutinin Glycoproteins, Influenza Virus, Hemagglutinins, Viral, Humans, Immunophenotyping, In Vitro Techniques, Molecular Sequence Data, Peptide Fragments, Phosphoproteins, Protein Structure, Quaternary, Sensitivity and Specificity, Staining and Labeling, Tetanus Toxoid, Viral Matrix Proteins