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

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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Figure 7

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TCR Vβ repertoire of HCV class II tetramer–positive cells. (a) Fresh PBM...
TCR Vβ repertoire of HCV class II tetramer–positive cells. (a) Fresh PBMCs from subject 01-40 were stained with DR4–HCV 1248 tetramer, positively selected by labeling with anti-PE microbeads, and then split into separate tubes for staining with a panel of 16 Vβ antibodies. (b and c) Similarly, short-term stimulated HCV-specific CD4 T cell lines from subjects 01-40 (b) and 98A (c) were stained with DR4–HCV 1248 and subsequently stained with the 16 Vβ antibodies. The Vβ antibodies that positively stained DR4–HCV 1248+ cells are shown for each subject, as well as an example of a negative control Vβ antibody that did not stain the tetramer-positive cells. In subject 98A, the frequency of T cells in bulk PBMCs expressing Vβ5.1 and Vβ12 was 6.6% and 2.2% of CD4 T cells, respectively.