2%)-treated autologous CD4- and CD8-depleted PBMCs (2 �� 105) prepulsed with the peptides. The cells were then washed and suspended in 100 ��l of cold RPMI medium and treated with bispecific CD45 and IFN�� mouse antibodies (IFN�� catch reagent) (2 ��l) for 5 min on ice. The cells were then diluted in AIM-V medium (1 ml) buy Galunisertib
and placed on a slow rotating device (Miltenyi Biotec) to allow IFN�� secretion at 37��C in a 5% CO2 atmosphere. After incubation for 45 min, the cells were washed with cold buffer and treated with 7AAD (7-amino-actinomycin D, Becton Dickinson, Mountain View, CA), PE-conjugated anti-IFN�� (detection reagent) and FITC-conjugated anti-CD4 or CD8 mAbs for staining. After incubation for 10 min at 4��C, the cells were washed and analyzed with a FACS Calibur (Becton Dickinson). Dead cells were sorted by 7AAD staining. The data were analyzed with FlowJo software (Tree Star, Ashland, OR). A net population of IFN��-captured CD4 and CD8 T cells of more than 0.1% was considered significant. IHC was performed as described previously.3 E97823 and EMR8-5 (Funakoshi, Tokyo, Japan)24 mAbs were used to analyze NY-ESO-1 and HLA class I expression, respectively. The reaction was evaluated as +++ (>50% stained cells), ++ (25�C50% stained cells), + (5�C25% stained cells) and ? (<5% stained cells). Table 1 shows a list of the ten patients enrolled in the study. They included six patients with esophageal cancer, three with non-small-cell lung cancer and one with gastric cancer <a href="http://www.selleck.cn/products/XL184.html
">Cabozantinib who were refractory to the standard therapy. Expression of NY-ESO-1 and MHC class I in the tumor was confirmed in biopsy or surgical specimens by IHC in all patients upon entry into the study. Nine patients completed the study with six injections of the NY-ESO-1f peptide with Picibanil and Montanide, but patient OS-f01 was withdrawn from the study after five doses of the vaccine because of disease progression. All patients were considered evaluable for toxicity, immunological and clinical responses. Six patients with a prolonged disease course were allowed to continue vaccination after a cycle of six doses of the vaccine. Toxicity was graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events v.3.0.25 As shown in Table 1, six patients showed Grade 1 fever (38�C39��C) that subsided within a few days without any medication. All patients except OY-f04 developed Z-VAD-FMK mw
an injection-site reaction (Grade 1 or 2). TK-f01, TK-f04 and TK-f05 developed a Grade 2 injection-site reaction early after the first vaccination. The reaction appeared 48�C72 hr after injection, and erythema was accompanied by swelling. Grade 2 induration occurred thereafter without retraction. In patient TK-f02, erythema was first observed after the third injection and accompanied induration after the fifth injection (Supporting Information Fig. 1). The induration gradually subsided during the course of the treatment.