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.

Tracking Virus-Specific CD4+ T Cells during and after Acute Hepatitis C Virus Infection

“…Importantly, whereas after spontaneous or treatment induced viral clearance tetramer+ CD4+ T cells levelled off at frequencies between 0.01% and 0.1%, the frequency further declined to very low (<0.01%) or undetectable levels in patients who developed chronic infection. This finding is consistent with a previous study employing an HCV specific HLA-DR4 tetramer that found a significant tetramer+ CD4+ T cell population in long-term recovered patients but not in patients with chronic hepatitis C [8]. Similarly, with the HLA-DR1 tetramer we found maintained levels of tetramer+ CD4+ T cells in three spontaneously recovered patients, one of whom had cleared HCV more than 20 years before, but only low frequencies in patients with chronic hepatitis.…”

“…This is the first HCV specific HLA class II tetramer to be tested and validated using epitope specific CD4+ T cell clones and we could demonstrate that this tetramer possesses similar sensitivity and specificity to HIV or influenza specific HLA class II tetramers characterized in a similar manner [8]–[11]. In addition, we took advantage of the fact that the HCV peptide used for the tetramer synthesis was highly targeted in our HLA-DR1+ study cohort.…”

“…Until recently, direct evaluation of virus-specific CD4+ T cells has not been possible, except in transgenic systems. However, recent advances in HLA class II tetramer technology and improvements in staining techniques have allowed CD4+ T cell populations to be observed ex vivo in a variety of settings, including HCV, influenza and HIV [8]–[11]. These tools are based on the refolding of soluble, biotinylated HLA class II molecules with specific viral peptides in vitro, and tetramerisation around fluorescently labelled streptavidin molecules to allow strong binding of the complex with the T cell receptor of the specific cells present within a population.…”

Prolonged Activation of Virus-Specific CD8+T Cells after Acute B19 Infection

“…CTL responses to HLA-A2 and non-HLA-A2 epitopes showed similar kinetics, frequencies, and phenotypes. Thus, future studies might reasonably track defined epitopes, rather than requiring individual mapping, as is the case in, for example, HCV [17–19]. …”

Adaptive Immunity to the Hepatitis C Virus

“…More recently, HLA class II tetramers were used to visualize HCV-specific CD4+ T cells in the blood of all HCV-infected subjects regardless of infection outcome (Lucas et al , 2007). CD4+ T cell populations remain detectable in the circulation of subjects who resolved the infection but frequencies dropped below the level of detection in those who followed a persistent course (Day et al , 2003; Lucas et al , 2007). This is consistent with comprehensive mapping of CD4+ T cell responses in individuals with chronic versus resolved infections (Day et al , 2002; Schulze zur Wiesch et al , 2005).…”