Multiscreen maip n45

To assess whether IDO vaccination resulted in measurable T-cell responses in the two long-term patients, we performed indirect IFN- ELISPOT as previously described. Briefly, PBMCs were stimulated once in ex vivo medium +5% HS, 120 U/L interleukin-2 and 15 umol/L IDO5 peptide prior to analysis to extend the sensitivity of the assay. After 7 days in culture, cells were counted and analyzed in IFN-y ELISPOT. Nitrocellular bottomed 96-well plates (MultiScreen MAIP N45; Millipore) were coated with IFN-y capture mAb (Mabtech) overnight. Wells were washed, blocked by X-vivo medium and the effector cells were added in duplicates at different concentrations with or without 5 umol/L of the IDO5 peptide. Plates were incubated overnight and medium was discharged and wells washed prior to addition of biotinylated secondary Ab (Mabtech). Plates were incubated at room temperature (RT) for 2 h, washed and avidin-enzyme conjugate was added to each well. Plates were incubated at RT for 1 h and the enzyme substrate NBT/BCIP (Invitrogen Life Technologies) was added to each well and incubated at RT for 5–10 min. Upon the emergence of dark purple spots, the reaction was terminated by washing with tap water. The spots were counted using the ImmunoSpot Series 2.0 Analyzer (CTL Analyzers).

Enzyme-linked immunospot (ELISPOT) assays were used to quantify IFNγ-releasing survivin-specific T cells in PBMC samples as described previously [6 (link),35 (link)]. Nitrocellulose-bottomed 96-well plates (MultiScreen MAIP N45, Millipore, Schwalbach, Germany) were coated with an anti-IFNγ antibody (1-D1K, Mabtech, Stockholm, Sweden), and nonspecific binding was blocked using AIM-V (Life Technologies, Gaithersburg, MD). Lymphocytes were isolated from heparinized peripheral blood samples of study patients and subsequently incubated overnight at 37°C at different cell concentrations together with T2 cells loaded with HLA-matched survivin epitope-specific peptides used for vaccination. After two washing procedures, the biotinylated detection antibody (7-B6–1-Biotin, Mabtech) was added. Specific binding was visualized using alkaline phosphatase-avidin together with substrate (Life Technologies). The resulting spots were quantified using the AlphaImager System (Alpha Innotech, San Leandro, CA). IFNγ- secretion was considered as a specific response, if the spot count for any of the specific peptides was more than three times the count of background spots (no peptide) in at least two independent experiments. Patients with a detection of a vaccine-induced SSTR at at least one time point (i.e. week 8 or 16 after onset of vaccination) were defined as positive.