Journal: European Journal of Immunology

Article Title: Inosine pranobex enhances human NK cell cytotoxicity by inducing metabolic activation and NKG2D ligand expression

doi: 10.1002/eji.201847948

Figure Lengend Snippet: IP induces dose‐dependent cell surface NKG2D ligand expression. (A) NKG2D ligands are not typically expressed on healthy quiescent cells. Stimuli including malignant transformation, viral infection, and proliferative lymphocyte activation are associated with NKG2D ligand induction. Expression can cause cytotoxicity, cytokine secretion, or costimulation through binding to the activating receptor, NKG2D. (B) HEK293T cells were cultured in 5 mM glucose with 0.25, 1, or 2 mM IP for 48 h, and cell surface expression of MICA (2C10) was measured by flow cytometry. A strong dose‐dependent increase in MICA expression was observed. Isotype controls (dotted histogram), cells cultured in 5 mM glucose only (light grey shaded histogram) or in 25 mM glucose (dark grey shaded histogram) are also shown. (C) Cells were cultured in 5 or 25 mM glucose with IP in biological triplicates and MICA expression was measured by flow cytometry. In 5 mM glucose, IP produced a significant increase in cell surface MICA expression compared to untreated cells. In 25 mM glucose, a significant increase in MICA expression was observed at higher IP concentrations. (D) HEK293T cells, (E) HT1080 cells (human fibrosarcoma), and (F) HeLa cells (human cervical carcinoma) demonstrate dose‐dependent MICA (2C10) expression when cultured with IP. (G) We tested whether IP influenced total cellular MICA levels by staining permeabilized and non‐permeabilized cells in parallel. Permeabilized cells displayed the same dose‐dependent IP‐induced MICA expression as non‐permeabilized cells. (H) We tested for adequate cell permeabilization by measuring the expression of PCNA (14‐9910‐80) in both non‐permeabilized and permeabilized cells by flow cytometry. PCNA was only detected in permeabilized cells and did not increase in an IP‐dependent manner. (I) The effect of IP on the induction of multiple NKG2D ligands including MICB (MAB1599), ULBP1 (MAB1380), ULBP2 (MAB1298), ULBP3 (MAB1517), ULBP4 (6E6), and ULBP5 (6D10) was tested by flow cytometry. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; and ∗∗∗∗ p < 0.0001. Histograms represent mean and 95% confidence interval. The data shown is from a single experiment that contained three biological replicates. Experiments were performed independently three times with consistent results. Means were compared using t ‐tests. MFI, mean fluorescence intensity.

Article Snippet: MICA (2C10, IgG1), ULBP4 (6E6, IgG2B), and ULBP5 (6D10, IgM) were purchased from Santa Cruz (Santa Cruz, CA); MICB (MAB1599, IgG2b), ULBP1 (MAB1380, IgG2a), ULBP2 (MAB1298, IgG2a), and ULBP3 (MAB1517, IgG2a) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Transformation Assay, Infection, Activation Assay, Binding Assay, Cell Culture, Flow Cytometry, Produced, Staining, Fluorescence