0. The Fer-1 acquisition and analysis gates for PBLs (5 × 104) were determined by characteristic forward and side-scatter properties of lymphocytes.

Furthermore, analysis gates were restricted to the CD3+CD4+ T-cell subsets. CD45RA+Foxp3low Tregs (I), CD45RA-Foxp3high Tregs (II), and Foxp3lowCD45RA- T cells (III) were determined as previously described [14]. Cells expressing surface and intracellular markers were acquired and analyzed on a logarithmic scale from FL1 to FL9. Surface and intracellular staining To determine the frequency of three distinct Treg subsets, both cell surface and intracellular staining was performed. Briefly, mAbs against surface markers CD3, CD4, CD25, and CD45RA were added to the cell suspension (1 × 107 cells/100 μl) and incubated on ice for 30 minutes in the dark. After washing twice, cells were fixed and permeabilized on ice with fixation/permeabilization buffer (eBioscience, San Diego, CA, USA) for 1 hour in the dark. Cells were then washed twice and incubated with intracellular mAbs for 1 hour at room temperature in the dark. After

intracellular staining, cells were washed twice and examined by multicolor flow cytometry. Appropriate isotype Ab controls were included for each sample. Cell culture RPMI 1640 TPCA-1 in vivo medium supplemented with 10% fetal bovine serum, see more 100 IU/ml penicillin, and 100 mg/ml streptomycin (Sigma, St. Louis, MO) was used for T cell culture. In vitro suppression assay of three distinct Treg subsets Stained cells (mAbs against CD3, CD4, CD25, and CD45RA) at a concentration of 5 × 107 cells/100 μl were sorted using a FACS cell sorter (BD Influx, BD Biosciences). Three Treg Fluorouracil subsets were prepared as live cells as previously described [14]; i.e., Foxp3lowCD45RA+ (I), Foxp3highCD45RA- (II), and Foxp3lowCD45RA- cells (III) were prepared by sorting as CD25++CD45RA+, CD25+++CD45RA-, and CD25++CD45RA-CD4+ T cells, respectively. For HNSCC patients, Additional file 1: Figure S1 demonstrates that the degree of CD25 expression in CD45RA+CD25++ Tregs,

CD45RA-CD25+++ Tregs, and CD45RA-CD25++CD4+ T cells are proportional to Foxp3 expression in CD45RA+Foxp3low Tregs, CD45RA-Foxp3high Tregs, and CD45RA-Foxp3low CD4+ T cells, respectively. After sorting, 1 × 104 responder cells (CD25-CD45RA+CD4+ T cells) were labeled with 1 μM carboxyfluorescein diacetate succinimidyl ester (CFSE) (eBioscience, San Diego, CA, USA) and co-cultured with unlabeled CD25++CD45RA+, CD25+++CD45RA-, or CD25++CD45RA- CD4+ T cells and assessed for their suppressive activities. Soluble anti-CD28 (2 μg/ml) and plate-bound anti-CD3 (0.5 μg/ml) was used to activate T cells in 96-well round-bottom plates, and cells harvested and analyzed by flow cytometry after 86 h of co-culture. All CFSE data were analyzed using the ModFit software provided by Verity Software House (Topsham, USA).