A novel assay for detection of hepatitis C virus-specific effector CD4(+) T cells via co-expression of CD25 and CD134.
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.
Article: HIV and co-infections.[Show abstract] [Hide abstract]
ABSTRACT: Despite significant reductions in morbidity and mortality secondary to availability of effective combination anti-retroviral therapy (cART), human immunodeficiency virus (HIV) infection still accounts for 1.5 million deaths annually. The majority of deaths occur in sub-Saharan Africa where rates of opportunistic co-infections are disproportionately high. In this review, we discuss the immunopathogenesis of five common infections that cause significant morbidity in HIV-infected patients globally. These include co-infection with Mycobacterium tuberculosis, Cryptococcus neoformans, hepatitis B virus, hepatitis C virus, and Plasmodium falciparum. Specifically, we review the natural history of each co-infection in the setting of HIV, the specific immune defects induced by HIV, the effects of cART on the immune response to the co-infection, the pathogenesis of immune restoration disease (IRD) associated with each infection, and advances in the areas of prevention of each co-infection via vaccination. Finally, we discuss the opportunities and gaps in knowledge for future research.Immunological Reviews 07/2013; 254(1):114-42. · 12.16 Impact Factor
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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
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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