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: 1.82). 01/2012; 375(1-2):148-58. DOI: 10.1016/j.jim.2011.10.004
Source: PubMed


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.

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    • "Zaunders et al has described the methods of detecting antigen specific CD4+ memory T cells using this assay [51]. The assay has also been used to detect Hepatitis-C-specific-CD4 T cells [52]. "
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    ABSTRACT: Restoration of Cytomegalovirus-specific-CD4 T cell (CMV-Sp-CD4) responses partly accounts for the reduction of CMV-disease with antiretroviral-therapy (ART), but CMV-Sp-CD4 may also drive immune activation and immunosenescence. This study characterized the dynamics of CMV-Sp-CD4 after ART initiation and explored associations with CD4 T cell recovery as well as frequency of naïve CD4 T cells at week 96. Fifty HIV-infected, ART-naïve Thai adults with CD4 T cell count ≤350cells/µL and starting ART were evaluated over 96 weeks ( identifier NCT01296373). CMV-Sp-CD4 was detected by co-expression of CD25/CD134 by flow cytometry after CMV-antigen stimulation. All subjects were CMV sero-positive, 4 had quantifiable CMV-DNA (range 2.3-3.9 log10 copies/mL) at baseline but none had clinically apparent CMV-disease. Baseline CMV-Sp-CD4 response was positive in 40 subjects. Those with CD4 T cell count <100cells/µL were less likely to have positive baseline CMV-Sp-CD4 response (P=0.003). Positive baseline CMV-Sp-CD4 response was associated with reduced odds of quantifiable CMV-DNA (P=0.022). Mean CD4 T cell increase at week 96 was 213 cells/µL. This was associated positively with baseline HIV-VL (P=0.001) and negatively with age (P=0.003). The frequency of CMV-Sp-CD4 increased at week 4 (P=0.008), then declined. Those with lower baseline CMV-Sp-CD4 (P=0.009) or CDC category C (P<0.001) had greater increases in CMV-Sp-CD4 at week 4. At week 96, CD4 T cell count was positively (P<0.001) and the frequency of CMV-Sp-CD4 was negatively (P=0.001) associated with the percentage of naïve CD4 T cells. Increases in CMV-Sp-CD4 with ART occurred early and were greater in those with more advanced immunodeficiency. The frequency of CMV-Sp-CD4 was associated with reduced naïve CD4 T cells, a marker associated with immunosenescence.
    PLoS ONE 10/2013; 8(10):e77479. DOI:10.1371/journal.pone.0077479 · 3.23 Impact Factor
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    • "Cultures were incubated at 37°C for 48 hrs in a humidified atmosphere of 5% CO2 in air. Negative control cultures comprised PBMCs mixed with IMDM with 10% AB serum only while SEB was used for positive control cultures [6] [15]. "
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    ABSTRACT: Current research on antigen specific CD4+ T cells indicates that there is functional and phenotypic heterogeneity within these populations, but the extent of this heterogeneity is poorly described. The CD134/CD25 assay allows live isolation of antigen specific cells in vitro for down-stream molecular analysis. Antigen specific CD4+ T cells were examined at the molecular level by lineage specific transcription factor profiling using qualitative multiplex single cell RT-PCR and Lock Nucleic Acid (LNA) probes allowed unbiased amplification and delineation of expression of Tbx21, Gata3, Rorc, Foxp3 and Bcl-6. It overcomes the limitations of previous assays by allowing identification of transcription factor mRNA in single antigen specific cells with high sensitivity (down to 10 femtograms) and specificity. Patterns of responses can be robustly characterized using <200 cells based on exact binomial calculations. These results are reproducible with a CV of ≈6%. The patterns of heterogeneity are stable within an individual antigen specific response but vary between responses to different antigens. Responses to CMV have a Th1 predominant profile (35.6% of responding cells expressing tbx21) whereas responses to Tetanus Toxoid have a Th2 biased profile (22% of responding cells expressing gata3), with unexpectedly high levels of Treg cells found in both populations. Here we describe a methodology that allows live isolation of Ag specific cells and transcription factor profiling at a single cell level to robustly delineate the different CD4+ T cell subsets within this population. This novel method is a powerful tool that can be used to study CD4+ T cell heterogeneity within extremely small populations of cells and where cell numbers are limited.
    PLoS ONE 10/2013; 8(10):e74946. DOI:10.1371/journal.pone.0074946 · 3.23 Impact Factor
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    ABSTRACT: Recent progress in defining genomic, transcriptomic, and proteomic profiles of human pathogens promise to revolutionize vaccine development. Antigen discovery by traditional biochemical approaches is generally piecemeal and time consuming. These are being replaced by technologies that allow proteomes to be mined for reactive antibody and T-cell antigens in a comprehensive and unbiased fashion. Moreover, naturally acquired or vaccine-induced immunity can now be subjected to a systems biology approach to define the antigens than confer protection. In this chapter, high throughput (HT) proteomic screening technology for vaccine antigen discovery will be reviewed. Future challenges for translation of data obtained by HT screening into vaccines will also be discussed.
    Vaccinology, 06/2012: pages 150-167; , ISBN: 9781405185745
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