Ultrasensitive detection and phenotyping of CD4+ T cells with optimized HLA class II tetramer staining

J Immunol. 2005 Nov 15;175(10):6334-43. doi: 10.4049/jimmunol.175.10.6334.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

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

Substances

  • Epitopes
  • HIV Core Protein p24
  • HLA-DR1 Antigen
  • HLA-DR4 Antigen
  • Hemagglutinin Glycoproteins, Influenza Virus
  • Hemagglutinins, Viral
  • Peptide Fragments
  • Phosphoproteins
  • Tetanus Toxoid
  • Viral Matrix Proteins
  • cytomegalovirus matrix protein 65kDa
  • influenza hemagglutinin (307-319)
  • tetanus toxoid (830-843)