The results of an ELISPOT assay with more than 25 spots in the wells without peptides (control wells) were excluded from the analysis. IFN-γ ELISPOT assays were also performed
using PBMC-depleted CD4+ or CD8+ cells to determine what kind AZD3965 solubility dmso of T cell is responsive to the peptides. In the assay using PBMC-depleted CD4+ or CD8+ cells, the number of cells was adjusted to 3 − 105 cells/well after the depletion. Depletion of CD4+ or CD8+ cells was performed using the MACS separation system with CD4 or CD8 MicroBeads (Miltenyi Biotec, Auburn, CA) in accordance with the manufacturer’s instructions. For the detection of myeloid-derived suppressor cells (MDSCs), PBMCs were isolated from 20 randomly selected patients 2-4 weeks after HCC treatment. To determine the frequency of CD14+HLA-DR−/low MDSCs, two-color fluorescence-activated cell sorting analysis was performed buy GDC-0199 using the following antibodies: anti-CD14 and anti–HLA-DR (Becton Dickinson). Flow cytometry was performed using the FACSAria II system (Becton Dickinson). The frequency of CD14+HLA-DR−/low MDSCs was calculated as a percentage of HLA-DR−/low cells in CD14+ cells. Peptide MRP3765-,
AFP357-, AFP403-, and hTERT461-specific tetramers were purchased from Medical Biological Laboratories Co., Ltd. (Nagoya, Japan). PBMCs were stained with anti–CD8-APCAb (Becton Dickinson, Tokyo, Japan), anti–CCR7-FITCAb (eBioscience, Tokyo, Japan), anti–CD45RA-PerCP-Cy5.5Ab (eBioscience, Tokyo,
Japan), and tetramer-PE for 30 minutes at room temperature. Cells were washed, fixed with 0.5% paraformaldehyde/phosphate-buffered saline, and analyzed using the FACSAria II system. Data are expressed as the mean ± SD. The estimated probability of tumor recurrence-free survival was determined using the Kaplan-Meier method. The Mantel-Cox log-rank test was used to compare curves between groups. The prognostic factors for tumor recurrence-free Succinyl-CoA survival were analyzed for statistical significance using the Kaplan-Meier method (univariate) and the Cox proportional hazard model (multivariate). Linear regression lines for the relationship between the frequency of CD14+HLA-DR−/low MDSCs and the number of TAA-specific T cells were calculated using Pearson’s correlation coefficient. A level of P < 0.05 was considered significant. The clinical profiles of the 69 patients analyzed in the present study are shown in Table 1. HCC was histologically classified as well, moderately, and poorly differentiated in 7, 3, and 1 cases, respectively. In the other cases, HCC was diagnosed on the basis of typical CT findings and elevated AFP levels.