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

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+CD127lowCD25highCD39+ 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. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim