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

Keoshkerian, Elizabeth; Helbig, Karla J.; Beard, Michael R.; Zaunders, John; Seddiki, Nabila; Kelleher, Anthony D.; Hampartzoumian, Taline; Zekry, Amany; Lloyd, Andrew R.

doi:10.1016/j.jim.2011.10.004

Cited by 29 publications

(26 citation statements)

References 39 publications

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“…This is our experience as well and likely reflects the different physiological milieu from which the cells are derived, with greater cell-to-cell contact and Ag presentation occurring in lymphoid tissue in the period immediately prior to sampling. Further, our original data from PBLs clearly shows that there is no significant proliferation or apoptosis during the 48 h incubation period (6), an observation we have confirmed during the optimization of the assay for a range of stimulating Ags in humans and macaques (4-9) and (Fig. 2).…”

Section: T Cells In Blood"supporting

confidence: 61%

Comment on “A Cytokine-Independent Approach To Identify Antigen-Specific Human Germinal Center T Follicular Helper Cells and Rare Antigen-Specific CD4+ T Cells in Blood”

Cook

Zaunders

Kelleher

2016

The Journal of Immunology

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Section: T Cells In Blood"supporting

confidence: 61%

Comment on “A Cytokine-Independent Approach To Identify Antigen-Specific Human Germinal Center T Follicular Helper Cells and Rare Antigen-Specific CD4+ T Cells in Blood”

Cook

Zaunders

Kelleher

2016

The Journal of Immunology

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“…The 'OX40' assay exhibited concordance with research methods of antigen-specific T cell detection such as carboxyfluorescein succinimidyl ester (CFSE) proliferation studies and intracellular cytokine production [5]. Further work showed the potential of this assay for the 4 detection of latent TB infection in healthy control cohorts and HIV-infected cohorts and also in detection of Hepatitis C infection [6,7].…”

Section: Introductionmentioning

confidence: 72%

Establishment of a healthy human range for the whole blood ‘OX40’ assay for the detection of antigen-specific CD4+ T cells by flow cytometry

et al. 2014

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Background: 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. Methods: Whole heparinised 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 (VZV), Epstein-Barr Virus (EBV), Cytomegalovirus (CMV), Candida albicans and Streptococcus pneumoniae. Results: The 'OX40' assay technique was clinically validated for routine use in an NHS clinical immunology laboratory by analysis of incubation length (40-50 hours), sample transport time (up to 24 hours at room temperature), concordance with serology testing, proliferation and IFN-γ production. In addition, 63 healthy controls (age range 21-78) were tested for responses to generate a healthy control reference range. Conclusions: 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. © 2013 Clinical Cytometry Society.

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“…This assay has since been utilised to measure Ag-specific CD4 + T-cell responses in several clinical settings [20][21][22][23]. Here, we show that differential surface expression of CD39 enables separation of the Ag-specific CD4 + CD25 + CD134 + T-cell response into a CD39 − population and a Treg-cell-enriched CD39 + population.…”

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confidence: 79%

Human antigen‐specific CD4⁺CD25⁺CD134⁺CD39⁺ T cells are enriched for regulatory T cells and comprise a substantial proportion of recall responses

et al. 2014

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Human Ag-specific CD4 + T cells can be detected by their dual expression of CD134 (OX40) and CD25 after a 44 hours stimulation with cognate Ag. We show that surface expression of CD39 on Ag-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, Ag-specific CD25 + CD134 + CD39 + T cells could be expanded in vitro as cell lines and clones, and retained high Forkhead Box Protein 3, CTLA-4 and CD39 expression, suppressive activity and Ag 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 was 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 Ag-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.Keywords: Antigen-specific T cells r CD25 r CD39 r CD134 r FOXP3 r OX40 r Treg cell Additional supporting information may be found in the online version of this article at the publisher's web-site Correspondence: Laura Cook e-mail: lcook@kirby.unsw.edu.au * These authors contributed equally to this work. The current gold standard marker for the study of Treg cells is Forkhead Box Protein 3 (FOXP3), a constitutively expressed nuclear transcription factor, critical for Treg-cell survival and suppressive function [3]. The key limitation of this marker is that viable cells cannot be isolated, as staining for this intracellular protein involves cell permeabilisation. Also, in the context of activation, this marker loses specificity due to transient, activationinduced up-regulation of FOXP3 in human non-Treg cells [4,5]. An alternative, widely used definition of Treg cells is the combined cell surface expression of high levels of CD25 (IL-2Rα) and low levels of CD127 (IL-7Rα) [6]. Whilst >85% of CD127 low CD25 high Treg cells express FOXP3 and are suppressive ex vivo [6], activated Treg cells cannot be isolated with these markers due to CD127 down-regulation, and CD25 up-regulation, on other subsets of CD4 + T cells following activation by cognate .In 2007, it was shown that murine Treg cells co-express the ectoenzymes CD39 and CD73 [10]. These ectonucleotidases work in concert to convert adenosine triphosphate (ATP), adenosine diphosphate and adenosine monophosphate to immunosuppressive adenosine, which appears to play a role in the suppressive repertoire of Treg cells [10]. Apart from contributing to adenosine produ...

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

Cited by 29 publications

References 39 publications

Comment on “A Cytokine-Independent Approach To Identify Antigen-Specific Human Germinal Center T Follicular Helper Cells and Rare Antigen-Specific CD4+ T Cells in Blood”

Comment on “A Cytokine-Independent Approach To Identify Antigen-Specific Human Germinal Center T Follicular Helper Cells and Rare Antigen-Specific CD4+ T Cells in Blood”

Establishment of a healthy human range for the whole blood ‘OX40’ assay for the detection of antigen-specific CD4+ T cells by flow cytometry

Human antigen‐specific CD4⁺CD25⁺CD134⁺CD39⁺ T cells are enriched for regulatory T cells and comprise a substantial proportion of recall responses

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

Cited by 29 publications

References 39 publications

Comment on “A Cytokine-Independent Approach To Identify Antigen-Specific Human Germinal Center T Follicular Helper Cells and Rare Antigen-Specific CD4+ T Cells in Blood”

Comment on “A Cytokine-Independent Approach To Identify Antigen-Specific Human Germinal Center T Follicular Helper Cells and Rare Antigen-Specific CD4+ T Cells in Blood”

Establishment of a healthy human range for the whole blood ‘OX40’ assay for the detection of antigen-specific CD4+ T cells by flow cytometry

Human antigen‐specific CD4+CD25+CD134+CD39+ T cells are enriched for regulatory T cells and comprise a substantial proportion of recall responses

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Human antigen‐specific CD4⁺CD25⁺CD134⁺CD39⁺ T cells are enriched for regulatory T cells and comprise a substantial proportion of recall responses