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

Abstract

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...

Impact of Allogeneic Hematopoietic Stem Cell Transplantation on the HIV Reservoir and Immune Response in 3 HIV-Infected Individuals

“…Antigen-specific CD4+ T cells were measured using the CD25/CD134 upregulation assay, as previously described [22, 23]. Briefly, PBMC at 1 × 10 6 /ml in Iscove’s Modified Dulbecco’s medium (Life Technologies) containing 10% human AB serum (Sigma) were cultured for 44–48 hours in the presence of (i) no antigen (negative control); (ii) SEB (1μg/mL; Sigma; positive control); (iii) CMV lysate (1/500 dilution; Grade III purified; Meridian) and (iv) HIV Gag peptide pool (123 peptides from HXB2 sequence; each at 2μg/mL; AIDS Reagent Program).…”

Negative modulation of suppressive HIV-specific regulatory T cells by IL-2 adjuvanted therapeutic vaccine

“…We demonstrated that naïve CD45RO - CD25 + CD127 low FoxP3 + cells were the most affected by IL-2 treatment as compared to the total memory counterpart. Importantly, we revealed a significant decrease in CD25 + CD127 low FoxP3 + CD39 + Tregs, which were shown to be highly suppressive [6,34]. More interestingly, when we analysed HIV-specific CD39 + FoxP3 + Tregs, we found that they decreased significantly after IL-2 therapy compared to HIV-specific CD39 - FoxP3 + and CD39 - FoxP3 - effectors.…”

“…We have previously shown that by using CD39 and FoxP3 we were able to delineate two populations of antigen-specific CD4 + CD25 + CD134 + T cells with different origin and function, namely Tregs and Teffs (Fig 6A) and [6,9] and that CD39 + Tregs are potent suppressors of HIV-specific responses in both natural infection and vaccination [9,34]. Accordingly, we confirm here that frequency of CD39 + FoxP3 + CD25 + CD134 + HIV-specific Tregs was positively correlated with VL after TI (r = 0.7 and p = 0.01; Fig 6C) and interestingly IL-2 therapy decreased significantly the frequency of CD39 + FoxP3 + CD25 + CD134 + HIV-specific Tregs, but not CMV-specific Tregs (p = 0.01, Fig 6B).…”

“…Human peripheral thymic-derived naive and effector Tregs are delineated as CD4 + CD25 hi CD127 low FoxP3 + CD45RA + and CD45RA - respectively [2–4], while circulating antigen-specific Tregs, best at regulating targeted immune responses, can be identified by the expression of co-stimulatory molecules such as CD134 (OX40) [5,6] or CD137 (4-1BB) [7,8]. We have recently shown in a therapeutic vaccine study, that vaccinees who displayed lower levels of HIV-specific CD4 + CD134 + CD25 + CD39 + FoxP3 + Tregs showed better responses to the vaccine even though global CD4 + CD25 hi CD127 low FoxP3 + were slightly increased, probably reflecting restoration of CD4 + T-cell compartment [9].…”

New technologies for monitoring human antigen-specific T cells and regulatory T cells by flow-cytometry

“…However, recently, several markers for activated Treg have been described [40][41][42][43]. CD137 seems to be the most promising, since it is selectively expressed by 5-7 hours stimulated Treg [40] and can be combined with CD154 for simultaneous analysis of Tcon and Treg.…”