Article

A novel assay for detection of hepatitis C virus-specific effector CD4(+) T cells via co-expression of CD25 and CD134.

Inflammation and Infection Research Centre, School of Medical Sciences, University of New South Wales, Sydney NSW, 2052, Australia.
Journal of immunological methods (Impact Factor: 2.35). 01/2012; 375(1-2):148-58. DOI: 10.1016/j.jim.2011.10.004
Source: PubMed

ABSTRACT Hepatitis C virus (HCV)-specific CD4(+) effector T cell responses are likely to play a key role in the immunopathogenesis of HCV infection by promoting viral clearance and maintaining control of viraemia. As the precursor frequency of HCV-specific CD4(+) T cells in peripheral blood is low, favoured assay systems such as intracellular cytokine (ICC) or tetramer staining have limited utility for ex vivo analyses. Accordingly, the traditional lymphocyte proliferation assay (LPA) remains the gold standard, despite detecting responses in only a minority of infected subjects. Recently, we reported development and validation of a novel whole blood CD4(+) effector T cell assay based on ex vivo antigen stimulation followed by co-expression of CD25 and CD134 on CD4(+) T cells. Here we report adaptation of this assay to assessment of HCV-specific responses in cryopreserved peripheral blood mononuclear cells using standardised antigens, including peptide pools, viral supernatants and recombinant viral proteins. The assay allowed detection of HCV-specific CD4 responses in donors with both resolved and chronic infection. Responses were highly correlated with those revealed by LPA. Application of this assay will further define the role of CD4(+) T cells in the immunopathogenesis of HCV infection.

0 Bookmarks
 · 
168 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Background & Aims HCV seroprevalence surveys in longstanding injecting drug users (IDUs) reveal a small minority who remain seronegative, with some exhibiting HCV-specific cellular immunity. This study aimed to characterise this immunity, assess associations with risk behaviours and protection against infection. Methods A nested case-control series from a prospective cohort of seronegative IDUs was selected with incident cases (IN; n = 28) matched by demographics and risk behaviour to exposed uninfected (EU) subjects (n = 28). Samples were assayed for natural killer (NK) cell phenotypes and function, HCV-specific IFNγ in ELISpot, and HCV-specific CD4 T effector responses. IL28B and HLA-C/KIR2DL3 genotypes were tested. Results Numbers of activated (CD69+) NK cells in the mature CD56dimCD16+ subset, and cytotoxic (NKp30+) cells in the CD56brightCD16+ subset were higher in the EU subjects (p = 0.040, p = 0.038 respectively). EU subjects had higher frequencies of interferon gamma (IFNγ) producing NK cells, and lower frequencies of CD107a expression (p = 0.003, p = 0.015 respectively). By contrast, the frequency, magnitude, and breadth of HCV-specific CD4 and CD8 T cell responses did not differ, nor did IL28B, HLA-C, or KIR2DL3 allele frequencies. Conclusions Sustained NK cell activation contributes to protection against HCV infection. HCV-specific cellular immunity is prevalent in EU subjects but does not appear to be protective.
    Journal of Hepatology 10/2014; · 9.86 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Clinical investigation of antigen-specific T cells in potentially immunodeficient patients is an important and often challenging aspect of patient diagnostic work up. Methods for detection of microbial exposure to the T-cell compartment exist but are laborious and time consuming. Recently, a whole blood technique involving flow cytometry and detection of CD25 and OX40 (CD134) expression on the surface of activated CD4+ T cells was shown to be accurate and concordant when compared with more traditional methods of antigen-specific T-cell detection. Whole heparinized blood was collected from healthy donors and set up using the "OX40" assay to detect antigen-specific CD4+ T-cell responses to Varicella Zoster Virus, Epstein-Barr Virus (EBV), Cytomegalovirus, Candida albicans, and Streptococcus pneumoniae. The "OX40" assay technique was clinically validated for routine use in an NHS clinical immunology laboratory by analysis of incubation length (40-50 h), sample transport time (up to 24 h at room temperature), concordance with serology testing, proliferation and interferon-gamma production. In addition, 63 healthy controls (age range 21-78) were tested for responses to generate a healthy control reference range. The OX40 assay, as presented in this report, represents an economical, rapid, robust whole blood technique to detect antigen-specific T cells, which is suitable for clinical immunology diagnostic laboratory use. © 2014 International Clinical Cytometry Society.
    Cytometry Part B Clinical Cytometry 05/2014; · 2.23 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Human antigen-specific CD4(+) T cells can be detected by their dual expression of CD134 (OX40) and CD25 after a 44-hour stimulation with cognate antigen. We show that surface expression of CD39 on antigen-specific cells consistently identifies a substantial population of CD4(+) CD25(+) CD134(+) CD39(+) T cells that have a Treg cell-like phenotype and mostly originate from bulk memory CD4(+) CD45RO(+) CD127(low) CD25(high) CD39(+) Treg cells. Viable, antigen-specific CD25(+) CD134(+) CD39(+) T cells could be expanded in vitro as cell lines and clones, and retained high FOXP3, CTLA-4 and CD39 expression, suppressive activity and antigen specificity. We also utilised this combination of cell surface markers to measure HIV-Gag responses in HIV(+) patients before and after anti-retroviral therapy (ART). Interestingly, we found that the percentage of CD39(-) cells within baseline CD4(+) T-cell responses to HIV-Gag were negatively correlated with HIV viral load pre-ART and positively correlated with CD4(+) T-cell recovery over 96 weeks of ART. Collectively, our data show that antigen-specific CD4(+) CD25(+) CD134(+) CD39(+) T cells are highly enriched for Treg cells, form a large component of recall responses and maintain a Treg-cell-like phenotype upon in vitro expansion. Identification and isolation of these cells enables the role of Treg cells in memory responses to be further defined and provides a development pathway for novel therapeutics. This article is protected by copyright. All rights reserved.
    European Journal of Immunology 03/2014; · 4.97 Impact Factor