Pe conjugated anti foxp3 clone 259d c7

Following 3 days co-incubation of yeast stimulated DCs and naive T cells, cells were washed using cold CellWash (BD Biosciences, Erembodegem, Belgium) and resuspended in cold Stain Buffer (BD Biosciences, Erembodegem, Belgium). For surface staining, cells were incubated with PE-Cy7-conjugated anti-CD4 (clone SK3) and PerCP-Cy5.5-conjugated anti-CD25 (clone M-A251) or appropriate nonspecific isotype matched controls (all from BD Biosciences, Erembodegem, Belgium) for 30 min on ice protected from light. For intracellular staining, surface-stained cells were fixed and permeabilized using Human Foxp3 Buffer Set (BD Biosciences, Erembodegem, Belgium), incubated with PE-conjugated anti-Foxp3 (clone 259D/C7) or an appropriate nonspecific isotype matched control (both from BD Biosciences, Erembodegem, Belgium), and washed twice before analysis. Stained cells were acquired on an LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA) using FACSDiva software (BD Biosciences, San Jose, CA, USA). Specific gates and quadrants were defined based on background staining of isotype controls. At least 10,000 cells were analyzed for each sample.

Red blood cell-depleted splenocytes from an IL-7 transgenic mouse were incubated with Fc-Block (anti-mouse CD16/CD32, BD) for 20 min on ice, followed by washing in flow cytometry buffer and staining with fluorophore-conjugated anti-CD8a and anti-CD44 antibodies. Stained cells were seeded in a 96-well plate (1 × 10⁶ cells per well) and re-suspended in serially diluted IL-2-Fc variants. Stimulation was allowed for 10 min at 37 °C, followed by washing with flow cytometry buffer, fixation, permeabilization and intracellular staining with AF488-conjugated anti-pSTAT5 (clone 47/Stat5-pY694, BD). The levels of pSTAT5 in the CD8⁺ CD44^high population were determined by flow cytometry. For flow cytometry of human PBMC, the following antibodies were used: Pacific Blue-conjugated anti-CD8 (clone RPA-T8, BD), APC-conjugated anti-CD4 (clone S3.5, Thermo Fisher), PE-conjugated anti-FoxP3 (clone 259D/C7, BD). Stimulation of PBMC was allowed for 10 min at 37 °C in the presence of 1 μg ml⁻¹ (Treg stain) or 10 μg ml⁻¹ (CD8⁺ T-cell stain) IL-2-Fc. Detection of intracellular pSTAT5 was performed as described above. Buffy coats from normal donors were obtained from the Australian Red Cross Blood Service. Informed consent was obtained from all subjects and approval for this study was obtained from the human research ethics committee of the St Vincent's Hospital (Sydney, Australia).