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.

Preferential loss of IL-2-secreting CD4+ T helper cells in chronic HCV infection

“…26 Here, a set of three tetramers containing NS3/4-derived peptides and a single class II molecule (DRB1*0401) were used to study small groups of PCRϩ and PCRϪ individuals. While tetramer-positive cells were detectable in those with resolved infection, no responses were detectable in four individuals with persistent viremia.…”

“…In previous studies, we have observed that HCV-specific memory populations are typically CD62L-and CCR7-high (i.e., they have features of "central" memory T cells). 26 While examination of phenotype is theoretically possible using flow cytometricbased cytokine assays, even short-term stimulation can markedly modify the surface expression of CD62L and CCR7 (data not shown), making them unsuitable for this purpose. Thus, we have little current data on the status of the antigen-specific T cells in PCRϩ patients.…”

“…This formula has been previously adopted for the calculation of the percentage of cytokine-secreting or tetramer-positive cells following magnetic enrichment (Miltenyi Biotec). 25,26 CD8 and CD25 Depletion. CD8ϩ and CD25ϩ cell depletions were performed using magnetic bead separation (Dynal, Oslo, Norway) as per the manufacturer's instructions.…”

Ultra‐sensitive class I tetramer analysis reveals previously undetectable populations of antiviral CD8+ T cells

“…Melan-Aspecific cells as shown here), both tissue compartmentalization and self tolerance effects will serve to reduce the frequencies of these populations in the blood, so that ultra-sensitive techniques such as that shown here might be necessary. Finally, the technique can be readily applied to the study of other lymphocyte subsets present at low frequencies as e.g., antigen-specific CD4 + T helper cells by using MHC class II tetramers [19] and NKT cells by using CD1d tetramers, as recently published [20].…”

Recent advances in tumour antigen‐specific therapy: In vivo veritas

“…100 While reliable protocols exist for the synthesis of pMHC class I multimers, pMHC class II multimers are more difficult to produce and are less promising for direct ex vivo detection of antigenspecific CD4 ϩ T cells, due to the low signal to background noise ratio obtained. However, for viral systems it has been reported that pMHC class II multimers can reveal Ag-specific CD4 ϩ T cells in nonpreselected PBMC, 101,102 and differentiate between CD4 ϩ Tcell clones with high and low TCR avidities. 103 While multimers reveal overall numbers of specific T cells, they can be combined with other assays in order to obtain insights into the phenotypic and functional properties of multimer-binding T cells.…”