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Jugated antibodies for flow cytometry were the following: CD3, CD4, CD
Responding cellsAn IFN- ELIspot assay was performed to quantify the number of TILs responding to PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28404814 1) tumor-associated peptides, as previously described [14,15] or to 2) mRNA transfected autologous DCs. Briefly, nitrocellulose bottomed 96 well plates (Multiscreen MAIP N45, Millipore, Copenhagen, Denmark) have been coated with IFN- capture antibody (1-DIK, Mabtech, Nacka Strand, Sweden). The wells have been washed, blocked with X-VIVO 15 medium and TILs were added in triplicates at distinct cell concentrations. 1) TILs responding to tumor-associated peptides: TILs have been cultured for 4 hours at 37 with five CO2 in air with or with out tumor-associated peptides (final concentration 5M).Jugated antibodies for flow cytometry have been the following: CD3, CD4, CD8, CD27, CD45RO, CCR7, IFN-, TNF-, and CD107a (BD, Br dby, Denmark). The infusion product and PBMCs obtained prior to and following treatment from each and every patient were tested for reactivity against short-term cultured autologous melanoma cell lines (when readily available), generated from the identical specimen utilized for TIL generation by serial passage of adherent cells [14], or against a panel of four to 8 HLA-A matched allogeneic melanoma cell lines. Additionally, infusion items have been also tested for reactivity against a panel of tumor-derived peptides. The following strategies happen to be applied.Intracellular cytokine staining (ICS)A flow cytometry-based assay was performed in order to depict the production of cytokines when cells in the infusion item have been co-cultured with 1) tumor cell lines, as previously described [14] or with 2) mRNA-transfected autologous DCs: 1) Co-culture with tumor cell lines: TILs were cultured for five hours at 37 with 5 CO2 in air in the presence or absence of melanoma cells at an effector/target ratio of 3:1. two) Co-culture with mRNA-transfected autologous DCs: 1 patient (patient 11) had previously been included in a clinical phase I protocol at our center (ClinicalTrials.gov PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27797473 ID: NCT00978913) in which DCs transfected with mRNA encoding p53, survivin or hTERT had been evaluated in individuals with metastatic breast cancer or malignant melanoma (More file 1). TILs from this patient had been cultured for five hours with i) the DC-vaccine, ii) autologous DCs transfected with p53, survivin and hTERT mRNA, iii) autologous DCs transfected with single mRNA and iv) autologous DCs transfected with mock mRNA (i.e. 1675203-84-5 adverse handle) at an effector/target ratio of 10:1. Staphylococcal Enterotoxin B (SEB) (Sigma-Aldrich, 5 g/ml final concentration) was employed as constructive handle. In selected analyses anti-CD107a (BD, 0.03 ug/ml final concentration) was added at the beginning with the incubation. GolgiPlug (Sigma-Aldrich) was added at a dilution of 1:1000 after the very first hour of incubation. Immediately after 4 additional hours cells have been washed twice with PBS, stained with Fixable Viability Dye (Ebiosciences) and with antibodies directed to surface markers. Cells had been washed a single added time, fixed overnight, permeabilized and subsequently stained with antibodies for intracellular cytokines. Cells had been analyzed working with a BD FACSCanto II flow cytometer. A minimum of 1 x 105 TILs or lymphocytes were acquired.
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