In summary, our study demonstrates that human DN T cells exert a strong RG7422 molecular weight suppressive activity toward CD4+ and CD8+ T cells. Moreover, we showed that human DN T cells possess a number of important biological features that highly differ from naturally occurring CD4+CD25+ Tregs. First, DN T cells exert their suppressive activity exclusively after preactivation with APCs, whereas CD4+CD25+ Tregs arise in the thymus 23. Second, human DN T cells inhibit early T-cell activation by modulating TCR-signaling,

whereas initial T-cell activation is not suppressed by CD4+CD25+ Tregs 40, 41. Third, the regulatory function of DN T cells cannot be abolished by exogenous IL-2 or CD28 engagement 41, 42. Lastly, in contrast to naturally occurring CD4+CD25+ Tregs, both resting and APC-primed DN T cells do not express Foxp3. Taken together, our results demonstrate that human DN T cells are a new subset of inducible Tregs exerting a very potent suppressive Selleckchem Small molecule library activity toward cellular immune responses. Further understanding of the mechanisms involved in human DN T-cell suppression may have important implications for novel

immunotherapies. T cells were cultured in RPMI-1640 medium (Gibco, Karlsruhe, Germany) plus 10% human AB-serum (PAN Biotech, Aidenbach, Germany). The following recombinant human cytokines were used: 800 U/mL granulocyte-macrophage colony-stimulating factor (GM-CSF; Schering-Plough, Brussels, Belgium), 500 U/mL IL-2 (Proleukin,

Novartis Pharma, Nuernberg, Germany), 500 U/mL IL-4, 5 ng/mL transforming growth factor-β1 (TGF-β; both from Tebu, Offenbach, Germany), 10 ng/mL IL-1β, 1000 U/mL IL-6, 10 ng/mL tumor necrosis factor (TNF) (all from PromoCell, Heidelberg, Germany), and 1 μg/mL prostaglandin E2 (Alexis Biochemicals, Loerrach, Germany). Preparation of TCGF was described previously 43. PBMC were separated by density gradient centrifugation (Biocoll, Biochrom, Berlin, Germany) from leukapheresis products obtained from healthy volunteers. Informed consent was provided according to the Declaration of Helsinki. CD4+, CD8+, and DN T cells were isolated from PBMC via magnetic separation according to the manufacturer’s instructions (Miltenyi Arachidonate 15-lipoxygenase Biotec, Bergisch-Gladbach, Germany). Viability and purity of the T cells were monitored by flow cytometry. CD4+CD25+ Tregs were isolated from PBMC by sorting CD4+CD25+high T cells with a MoFlo cell sorter (Beckman Coulter, Krefeld, Germany). Cells were analyzed for Foxp3 expression and used for functional assays if a purity of >95% Foxp3+ cells could be documented. Naive and memory T cells were isolated from CD4+ T cells by depletion of CD45RO+ or CD45RA+ cells using MicroBeads (Miltenyi Biotec). DC were generated from leukapheresis products as described previously 44.