Ex vivo analysis of human memory CD4 T cells specific for hepatitis C virus using MHC class II tetramers
Cheryl L. Day, … , Paul Klenerman, Kai W. Wucherpfennig
Cheryl L. Day, … , Paul Klenerman, Kai W. Wucherpfennig
Published September 15, 2003
Citation Information: J Clin Invest. 2003;112(6):831-842. https://doi.org/10.1172/JCI18509.
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Ex vivo analysis of human memory CD4 T cells specific for hepatitis C virus using MHC class II tetramers

Abstract

Containment of hepatitis C virus (HCV) and other chronic human viral infections is associated with persistence of virus-specific CD4 T cells, but ex vivo characterization of circulating CD4 T cells has not been achieved. To further define the phenotype and function of these cells, we developed a novel approach for the generation of tetrameric forms of MHC class II/peptide complexes that is based on the cellular peptide-exchange mechanism. HLA-DR molecules were expressed as precursors with a covalently linked CLIP peptide, which could be efficiently exchanged with viral peptides following linker cleavage. In subjects who spontaneously resolved HCV viremia, but not in those with chronic progressive infection, HCV tetramer–labeled cells could be isolated by magnetic bead capture despite very low frequencies (1:1,200 to 1:111,000) among circulating CD4 T cells. These T cells expressed a set of surface receptors (CCR7+CD45RA–CD27+) indicative of a surveillance function for secondary lymphoid structures and had undergone significant in vivo selection since they utilized a restricted Vβ repertoire. These studies demonstrate a relationship between clinical outcome and the presence of circulating CD4 T cells directed against this virus. Moreover, they show that rare populations of memory CD4 T cells can be studied ex vivo in human diseases.

Authors

Cheryl L. Day, Nilufer P. Seth, Michaela Lucas, Heiner Appel, Laurent Gauthier, Georg M. Lauer, Gregory K. Robbins, Zbigniew M. Szczepiorkowski, Deborah R. Casson, Raymond T. Chung, Shannon Bell, Gillian Harcourt, Bruce D. Walker, Paul Klenerman, Kai W. Wucherpfennig

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Staining of short-term HCV-specific CD4 T cell lines with MHC class II t...
Staining of short-term HCV-specific CD4 T cell lines with MHC class II tetramers. (a) A short-term T cell line was generated from PBMCs by a single round of stimulation with 1 μg/ml of recombinant C200 antigen encoding the HCV NS3 and NS4 proteins. CD4 T cells were stained with PE-labeled MHC class II tetramers, including a control tetramer, DR4–annexin II, as well as three HCV tetramers, DR4–HCV 1248, DR4–HCV 1579, and DR4–HCV 1770. Cells were analyzed by flow cytometry prior to enrichment for PE-tetramer–positive cells; the percentage of CD4+/tetramer+ cells is indicated in the upper right quadrant. (b) Magnetic enrichment of tetramer-positive cells by positive selection of anti-PE–labeled cells. Data are shown for stimulated CD4 T cells from subject 01-40; similar data were obtained with short-term stimulated lines from subjects 99D and 98A. (c) Cells from an HCV NS3/NS4–stimulated line from subject 98A were stained with a pool of three HCV tetramers, DR4–HCV 1248, DR4–HCV 1579, and DR4–HCV 1770. Data are shown gated on CD4 T cells, without enrichment for tetramer-positive cells. Intracellular cytokine staining was performed by stimulation of cells from this same line with a pool of HCV peptides corresponding to the tetramer epitopes. Data are shown gated on CD4 T cells, and the percentage of IFN-γ+CD4+ cells is indicated in the upper right quadrant.