The A/E-beads were coated with 10?g/mL anti-CD3, 10?g/mL anti-CD28, and 0.5?g/mL anti-CD2 mAbs, and were used at 1:1 cells/beads ratio. TGF–treated V9?V2?T cells by epigenetic modification of the gene. locus in standard murine CD4 T cells stimulated under Treg-inducing conditions, thereby stabilizing the expression of the Treg-specific grasp transcription factor FoxP3 and enhancing the regulatory activity of CD4 T cells23C25. In this study, we confirm that purified human peripheral blood V9?V2?T cells acquire regulatory activity when activated in the presence of TGF-. More importantly, we demonstrate that pVC strongly MRS1177 upregulates and stabilizes FOXP3 protein expression, induces hypomethylation in the TSDR, and increases the suppressive capacity of V9?V2?T cells expanded in the presence of TGF-. Genome-wide methylation analysis identified additional genes regulated by pVC. We discuss the implications of our findings for the context-dependent modulation of human T-cell functions. Materials and Methods All methods and experiments were carried out in accordance with relevant institutional guidelines and regulations. Cell isolation and circulation cytometry Leukocyte concentrates obtained from healthy adult blood donors were provided by the Institute of Transfusion Medicine, UKSH Campus Kiel. Informed consent was obtained from all subjects. This research was performed in accordance with the declaration of Helsinki and was approved by the Ethics Committee of the Medical Faculty of the University or college of Kiel (Reference D 546/16). Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque (Biochrom, Cambridge, UK) density gradient centrifugation. Total T cells as well as V2?T cells were positively isolated by magnetic cell sorting (MACS) following the manufacturers instructions (Miltenyi Biotec, Bergisch-Gladbach, Germany). CD4 T cells were negatively isolated by Rabbit Polyclonal to PPP4R2 MACS technology (CD4 T Cell Isolation Kit II, Miltenyi Biotec) followed by the depletion of CD25+ Treg using Dynabeads (Life Technologies, Carlsbad, CA, USA). After the use of two consecutives MACS columns (in case of positive selection), the purity of each cell type was typically >97%. MRS1177 Cells were stained with fluorochrome-conjugated monoclonal antibodies (mAb) directed against CD3 (clone SK7), CD4 (clone SK3) and Ki-67 (clone Ki-67) from Biolegend (San Diego, CA, USA); CD86 (clone FM95) and PD-1 (clone PD126.96.36.199) from Miltenyi Biotec; GITR (clone FAB689P) from R&D Systems (Minneapolis, USA); TCR (clone 11F2), TCR V2 (clone B6), CD103 (clone Ber-ACT8) and FOXP3 (clone 259D/C7) and its appropriate isotype control from BD Biosciences (Heidelberg, Germany); Tet1 (clone GT1462) and its isotype control from ThermoFisher Scientific (Waldham, MA, USA). For intracellular staining of FOXP3, Ki-67 and Tet1, cells were fixed and permeabilized using the FoxP3 transcription factor staining buffer (eBioscience, Thermofisher Scientific) according to the manufacturers instructions. Cells were acquired on a LSRII Fortessa cytometer (BD Biosciences) and data were analyzed with FlowJo Software (Tree Star, Ashland, OR, USA) Cell culture Magnetically isolated cells were cultured in 96-well round-bottom plates (Nunc; ThermoFisher Scientific) in medium RPMI 1640 supplemented with 2 mM L-glutamine, 1% penicillin/1% streptomycin, 10?mM HEPES and 10% heat-inactivated fetal bovine serum (complete medium) and incubated at 37?C in a humidified atmosphere of 5% CO2 in air flow. For the initial T-cell growth, MACS-purified total (or V2) T cells were stimulated with 300?nM BrHPP (kindly provided by Innate Pharma, Marseille, France) or with Activation/Expander T cell beads (A/E-beads; Miltenyi Biotec). The A/E-beads were coated with 10?g/mL anti-CD3, 10?g/mL anti-CD28, and 0.5?g/mL anti-CD2 mAbs, and were used at 1:1 cells/beads ratio. Cells (50 103/well) were cultured for eight days with 50 IU/mL recombinant human IL-2 (Novartis, Basel, MRS1177 Switzerland), 2?ng/mL TGF- (Peprotech, Hamburg, Germany) in the presence or absence 50?g/mL (173?M) phospho-modified Vitamin MRS1177 C (pVC, cat. number A8960; Sigma Aldrich/Merck, Darmstadt, Germany). To test the stability of FOXP3 expression, T cells were expanded for eight days under different conditions as explained under Results. Thereafter, cells were washed twice, transferred into new 96-well round-bottom plates and cultured in the presence of 50 IU/mL IL-2 and A/E beads (where indicated) but absence of TGF- and pVC. After additional six days, cells were analyzed for FOXP3 expression as explained above. suppression assay For the suppression assay, T cells were first stimulated for 14 days in the presence of IL-2 and TGF- and pVC where indicated. On day 14, the expanded T cells (20 103/well) were co-cultured for five days with magnetically isolated autologous CD25-depleted CD4 responder T cells (20 103/well). The proliferation of CD4 responder T cells and T cells in microculture wells was simultaneously assessed by.
The A/E-beads were coated with 10?g/mL anti-CD3, 10?g/mL anti-CD28, and 0