Journal: Oncology Letters

Article Title: Antitumor effects of NK cells expanded by activation pre‑processing of autologous feeder cells before irradiation in colorectal cancer

doi: 10.3892/ol.2023.13818

Figure Lengend Snippet: Combination of anti-CD3 mAb, IFN-r, and IL-2 with radiation further promotes the expression levels of activating ligands in human PBMCs. PBMCs were cultured with or without anti-CD3 mAb, IFN-r, and IL-2 and then irradiated with 25 Gy. The cells were cultured for 0, 24, 48, or 72 h. CD48, CD112, CD155, and natural killer group 2D ligands (MICA, MICB, ULBP-1, ULBP-2/5/6, and ULBP-3) were analyzed by flow cytometry. (A, C, and E) Ratios of MFIs obtained from irradiated PBMCs and activated/irradiated PBMCs. Relative expression ratios were calculated by dividing the MFI of irradiated PBMCs (24, 48, and 72 h) or activated/irradiated PBMCs (24, 48, and 72 h) by that of untreated PBMCs (0 h). (B, D, and F) Representative flow cytometry histograms of each donor [white, untreated PBMCs (0 h); bright gray, irradiated PBMCs (48 h); gray, activated/irradiated PBMCs (48 h); dark gray, irradiated PBMCs (72 h); black, activated/irradiated PBMCs (72 h)]. The data are presented as the mean ± SD of 3 donors. Statistical significance was determined using paired and unpaired Student's t-test. # P<0.05, ## P<0.005, ### P<0.0005 [untreated PBMCs (0 h) vs. 24, 48, or 72 h irradiated or activated/irradiated PBMCs]. $ P<0.05, $$ P<0.005, $$$ P<0.0005 (24, 48, and 72 h irradiated PBMCs vs. 24, 48, and 72 h activated/irradiated PBMCs). mAb, monoclonal antibody; r, recombinant; PBMCs, peripheral blood mononuclear cells; MFI, median fluorescence intensity; MIC, MHC class I polypeptide-related sequence; ULBP, UL16 binding protein.

Article Snippet: The cells were irradiated with or without a dose of 25 Gy in a blood irradiator (Eckert & Ziegler), and cultured for 0, 24, 48, and 72 h. The cells were incubated with antibodies against human CD48-fluorescein isothiocyanate (FITC; 1:50; BD Pharmigen; BD Biosciences; cat. no. 555759), CD112-phycoerythrin (PE; 1:50; BD Pharmigen; BD Biosciences; cat. no. 551057), CD155-PE (1:50; R&D Systems, Inc.; cat. no. FAB25301P) and NKG2D ligands [including MHC class I polypeptide-related sequence A (MICA)-PE (1:50; R&D Systems Inc.; cat. no. FAB1300P), MHC class I polypeptide-related sequence B (MICB)-PE (1:50; R&D Systems Inc.; cat. no. FAB1599P), UL16 binding protein (ULBP)-1-PE (1:50; R&D Systems Inc.; cat. no. FAB1380P), ULBP-2/5/6-PE (1:50; R&D Systems Inc.; cat. no. FAB1298P) and ULBP-3-PE (1:50; R&D Systems Inc.; cat. no. FAB1517P)] for 20 min in the dark at room temperature.

Techniques: Expressing, Cell Culture, Irradiation, Flow Cytometry, Recombinant, Fluorescence, Sequencing, Binding Assay