Journal: Frontiers in Immunology

Article Title: Reovirus infection of tumor cells reduces the expression of NKG2D ligands, leading to impaired NK-cell cytotoxicity and functionality

doi: 10.3389/fimmu.2023.1231782

Figure Lengend Snippet: Reovirus infection reduces the expression of NKG2D ligands on the surface of infected cells. (A) FACS staining for NKG2D-ligands (MICA, MICB, ULBP2, ULBP3) and for human CD4 in MNT-1 cell line and in U87-MG cell line 48 hrs post infection. CD4 staining was done on transduced CD4 + cells, NKG2D ligands staining was done on WT cell lines. Grey histogram depicts mock-infected cells, black line depicts reovirus infected cells, red dashed-line depicts the background staining of mock-infected cells, blue dotted-line depicts the background staining of reovirus-infected cells. Shown is 1 representative staining out of 3 that were performed. MOI=100 was used in this experiment. (B) Summary of the geometric mean fluoresce intensity (MFI) of the staining shown in (A) . Grey columns depict mock-infected cells, white columns depict reovirus-infected cells. Shown is a summary of 3 experiments. Two-tailed unpaired Student’s t test was used to determine statistical significance. FDR of 0.01 was applied. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001. Each dot on the plot depicts the result of an independent experiment. Data is presented as mean ± SEM. (C) Intra cellular FACS staining for CRT, ERp57, PVR and GLUT-1 in the MNT-1 cell line and the U87-MG cell line. Grey histogram depicts mock-infected cells, black line depicts reovirus infected cells, red dashed-line depicts the background staining of mock-infected cells, blue dotted-line depicts the background staining of reovirus-infected cells. Shown is 1 representative staining out of 3 that were performed. MOI=100 was used in this experiment. (D) Summary of the MFI of the staining shown in (C) . Grey columns depict mock-infected cells, white columns depict reovirus-infected cells. Shown is a summary of 3 experiments. Two-tailed unpaired Student’s t test was used to determine statistical significance. FDR of 0.01 was applied. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001. Each dot on the plot depicts the result of an independent experiment. Data is presented as mean ± SEM.

Article Snippet: Moreover, although the antibody used in this study to stain for ULBP2 (R&D, MAB1298) was used in numerous studies to follow the expression of ULBP2 ( , , ), the possible altered expression of ULBP5 and ULBP6 should be further analyzed as this antibody was reported by the vendor to also bind ULBP5 and ULBP6.

Techniques: Infection, Expressing, Staining, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Reovirus infection of tumor cells reduces the expression of NKG2D ligands, leading to impaired NK-cell cytotoxicity and functionality

doi: 10.3389/fimmu.2023.1231782

Figure Lengend Snippet: Reovirus infection impairs the binding of NKG2D-Ig to infected cells. (A) The kinetics of down-regulation of NKG2D ligands (MICA, MICB, ULBP2 and ULBP3) in reovirus-infected MNT-1 cells compared to mock-infected MNT-1 cells. The y axis shows fold change of MFI of reovirus-infected cells divided by MFI of mock-infected cells. The x axis shows time post infection (TPI) in hrs. Shown are 3 pooled experiments in each time point. Statistical significance was tested in comparison to time point 0 hrs post infection. MOI=200 was used in these experiments. Two-tailed unpaired Student’s t test was used to determine statistical significance. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001. Data is presented as mean ± SEM. (B) FACS staining with NKG2D-Ig fusion protein after incubation with mock-infected and reovirus-infected MNT-1 cells, 48 hrs post infection. Grey histogram depicts mock-infected cells, black line depicts reovirus infected cells, red dashed-line depicts the background staining of mock-infected cells, blue dotted-line depicts the background staining of reovirus-infected cells. Shown is 1 representative staining out of 3 that were performed. MOI=100 was used in these experiments. (C) Summary of the MFI of the staining with human NKG2D-Ig at 24 hrs post infection and at 48 hrs post infection. The grey columns depict mock-infected cells and the white columns depicts reovirus-infected cells. Shown is a summary of 3 experiments in each time point. MOI=100 was used in these experiments. Two-tailed unpaired Student’s t test was used to determine statistical significance. *p<0.05, **p<0.01, ***p<0.005 ****p<0.0001. Each dot on the plot depicts the result of an independent experiment. Data is presented as mean ± SEM.

Article Snippet: Moreover, although the antibody used in this study to stain for ULBP2 (R&D, MAB1298) was used in numerous studies to follow the expression of ULBP2 ( , , ), the possible altered expression of ULBP5 and ULBP6 should be further analyzed as this antibody was reported by the vendor to also bind ULBP5 and ULBP6.

Techniques: Infection, Binding Assay, Comparison, Two Tailed Test, Staining, Incubation

Journal: Frontiers in Immunology

Article Title: Reovirus infection of tumor cells reduces the expression of NKG2D ligands, leading to impaired NK-cell cytotoxicity and functionality

doi: 10.3389/fimmu.2023.1231782

Figure Lengend Snippet: The Mechanism of NKG2D ligands downregulation in reovirus-infected cells. (A) ELISA analysis of growth media of mock-infected or reovirus-infected cells at 48 hrs post infection. A sample of RKO-MICA*008 cells treated with PI-PLC enzyme was included as a positive control. Shown 1 out of 3 independent experiments. The black column depicts the positive control of PI-PLC treatment, grey columns depict the mock-infected group, the white columns depict reovirus-infected group. MOI=100 was used in these experiments. Two-tailed unpaired Student’s t test was used to determine statistical significance. FDR of 0.01 was applied. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001. Each dot on the plot depicts the result of an independent experiment. Data is presented as mean ± SEM. (B) Analysis of quantitative polymerase chain reaction (qPCR) of the NKG2D ligands MICA, MICB, ULBP2 and ULBP3 normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at 48 hrs post infection. The grey columns depict mock-infected cells, the white columns depict reovirus-infected cells. Shown is a summary of 3 independent experiments. MOI=100 was used in these experiments. Mann-Whitney tests were used to determine statistical significance. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001. Each dot on the plot depicts the result of an independent experiment. Data is presented as mean ± SEM. (C) Expression of NKG2D-ligands (MICA, MICB, ULBP2, ULBP3) in mock-infected or reovirus-infected MNT-1 cells at 48 hrs post infection. Grey columns depict mock-infected cells, white columns depict reovirus-infected cells. The y axis depicts the MFI. The x axis depicts NKG2D-ligands, grouped by treatment type. Mock-infected and reovirus-infected cells, were treated with mock, 100µM of chloroquine (CQ), 10µM of MG132, or a combination of both. Shown is pooled data from 4 independent experiments. MOI=100 was used in these experiments. Two-way ANOVA followed by Tukey’s test were used to determine statistical significance. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001. Each dot on the plot depicts the result of an independent experiment. Data is presented as mean ± SEM.

Article Snippet: Moreover, although the antibody used in this study to stain for ULBP2 (R&D, MAB1298) was used in numerous studies to follow the expression of ULBP2 ( , , ), the possible altered expression of ULBP5 and ULBP6 should be further analyzed as this antibody was reported by the vendor to also bind ULBP5 and ULBP6.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Positive Control, Two Tailed Test, Real-time Polymerase Chain Reaction, MANN-WHITNEY, Expressing

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