The cells were then washed in cold PBS solution and 1 mL of freshly prepared eBioscience Fix/Perm Buffer was added to each sample before incubating at 4°C for 40 min in the dark. After a second wash, 2% (2 μL) normal rat serum was added and the cells were incubated again at 4°C for 15 min. Anti-human Foxp3-PE was added and incubated at 4°C for 30 min in the dark. In another tube, anti-Foxp3-FITC (eBioscience) and anti-CTLA-4-PE (BD Biosciences) were added at the same time as anti-Foxp3-PE Ab. The appropriate isotype-matched control Abs were used to define positivity. The cells were washed twice with PBS

and fixed in 1% polyformaldehyde. Cells MG-132 research buy were analyzed on a FACSAria (BD Biosciences, San Jose, CA, USA) with FACSDiva software. T-lymphocytes were identified by gating on CD3+ T cells and side scatter, and Tregs were identified as CD25-positive and

Foxp3-positive cells found among p38 MAPK cancer CD4+ T cells within the lymphocyte gate. The absolute number of Treg cells was determined by multiplying the proportion of CD4+CD25+Foxp3+ with the total CD4+ T cell count. CTLA-4 expression within the Tregs was identified as the proportion of CTLA-4 positive cells within the CD4+, CD25+ and Foxp3+ cells. Whole blood samples were incubated with the monoclonal antibody combinations anti-HLA-FITC/anti-CD38-PE/anti-CD8-APC/anti-CD4-APC-Cy7 for 30 min at room temperature. After lysis of red blood cells by FACS lysis buffer (BD Biosciences), cells were washed twice, fixed with 1% polyformaldehyde and analyzed via FACSAria. Lymphocytes were identified by gating on forward scatter and side scatter,

then CD4+ or CD8+ T cells were gated. The percentage of HLA-DR and CD38 expression on CD4+ and CD8+ T cells was determined. PBMC depleted of CD25+ T cells was obtained with MACS CD25 MicroBeads (Miltenyi Biotec, Auburn, CA, USA). Briefly, fresh PBMC were washed twice in PBS-containing 0.5% BSA, resuspended in 80 μL of PBS containing 0.5% BSA and 20 μL of MACS CD25 MicroBeads per 107 total PBMC, and incubated for 25 min at 4–8°C. PBMC were washed twice in PBS-containing 0.5% BSA and applied Dichloromethane dehalogenase to a magnetic column on a MidiMACS separation unit (Miltenyi Biotec). CD25- T cell fractions were collected. PBMC and PBMC depleted of CD25+ T cells were stimulated with one of three treatments: phorbol 12-myristate 13-acetate (20 ng/mL; Sigma-Aldrich, St Louis, MO, USA) and ionomycin (1 μg/mL, Sigma-Aldrich), HIV Gag peptide mix (5 μg/mL; Lianmei, Xian, China), or RPMI 1640. Cells were supplemented with 15% FCS and incubated for 18 hr. Golgiplug (BD BioSciences) was added at a final concentration of 1 μL/106 cells for the last 6 hr of incubation. Cells were washed in PBS and were stained with CD8-APC and CD3-PerCP (BD BioSciences). Following permeabilization in permeabilizing solution (eBioScience), cells were stained with IFN-γ-FITC (BD BioSciences).