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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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Article Immunology Article has an altmetric score of 10

Ex vivo analysis of human memory CD4 T cells specific for hepatitis C virus using MHC class II tetramers

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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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Figure 4

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Ex vivo MHC class II tetramer staining of HCV-specific CD4 T cells. (a) ...
Ex vivo MHC class II tetramer staining of HCV-specific CD4 T cells. (a) PBMC from subject 01-40 with spontaneously resolved HCV viremia were stained with three control tetramers and three HCV tetramers. Cells were positively selected with anti-PE microbeads and analyzed by flow cytometry; the number of CD4+/tetramer+ cells is indicated in the upper right quadrant. An example of FACS analysis before enrichment for tetramer+ cells is shown for control tetramer DR4-CLIP and HCV tetramer DR4–HCV 1248. The frequencies of tetramer-positive cells in the CD4 T cell pool were determined by splitting the sample 9:1 following labeling with anti-PE beads; 90% of the cells were magnetically enriched, while 10% of the cells were analyzed without enrichment to determine the number of CD4 T cells. The frequencies of the tetramer+ cells of total CD4 cells are as follows: DR4–HCV 1248: 8.2 per 100,000 (0.008%); DR4–HCV 1579: 1.5 per 100,000 (0.0015%); DR4–HCV 1770: 2.4 per 100,000 (0.0024%). Representative data are shown from two independent experiments with fresh PBMC from subject 01-40. (b) PBMC from a DRB1*0401 HCV-seronegative control were stained with the following MHC class II tetramers: 3 control tetramers (DR4-CLIP, DR4-gp100, and DR4–annexin II), a pool of three HCV tetramers, and an HA (residues 306–318) tetramer. The number of CD4+/tetramer+ cells following enrichment with anti-PE microbeads is indicated in the upper right quadrant. The frequency of DR4–influenza HA–specific T cells in this subject was 0.9 per 100,000 (0.0009%).

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ISSN: 0021-9738 (print), 1558-8238 (online)

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