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

Journal: Cancers

Article Title: Chemotherapeutics Used for High-Risk Neuroblastoma Therapy Improve the Efficacy of Anti-GD2 Antibody Dinutuximab Beta in Preclinical Spheroid Models

doi: 10.3390/cancers15030904

Figure Lengend Snippet: Chemotherapy-induced immune-checkpoint ligand-cell surface abundance on tumor cells. 1 × 10 6 tumor cells treated for 24 h under cell culture conditions with either carboplatin (2 µg/mL, open circles), cisplatin (1 µg/mL, open triangles), etoposide (0.5 µg/mL, open squares), vincristine (0.05 µg/mL, open diamonds), or the cyclophosphamide metabolite 4-HPC (1 µg/mL, open hexagons). After 72 h of culturing, LAN-1 (left panel), CHLA-20 (center), and CHLA-136 (right panel) were analyzed for surface abundance of ( A ) PD-L1, ( B ) CD86, ( C ) CD155, and ( D ) Gal-9, using flow cytometry. Data represent at least five biological replicates. Means and SEM are indicated as black lines and error bars, respectively. For statistical analysis, ANOVA with appropriate post hoc test was used; * p < 0.05 vs., ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus untreated control (medium).

Article Snippet: Immune checkpoint ligand expression analysis by tumor cells was performed in analogy to the stress ligand expression analysis detailed above, using the following antibodies: anti-human CD80-BV421 (Biolegend, mouse IgG1,κ, clone 2D10, 1:20), CD86-PerCP-Vio 700 (Miltenyi Biotec, Bergisch Gladbach, REA968, 1:50), CD112-APC (Miltenyi Biotec, REA1195, 1:50), CD155-PE-Vio 770 (Miltenyi Biotec, REA1081, 1:50), Galectin-9-PE (Miltenyi Biotec, REA435, 1:50), and CD274 (PD-L1)-Vio Bright B515 (Miltenyi Biotec, REA1197, 1:50).

Techniques: Cell Culture, Flow Cytometry, Control