Journal: European journal of immunology

Article Title: Activated monocytes augment TRAIL-mediated cytotoxicity by human NK cells through release of IFN-γ.

doi: 10.1002/eji.201242735

Figure Lengend Snippet: Figure 1. ZA upregulates TRAIL expression on NK cells indirectly via monocytes. (A) NK cells were cocultured with irradiated PBMCs for 7 days with or without ZA and stained for the indicated surface and intra- cellular markers and analyzed by flow cytometry. Representative flow cytometry plots from one of three independent experiments with simi- lar results are shown. (B) NK cells were cultured with irradiated PBMCs or monocytes for 7 days in the presence or absence of ZA. Expression of TRAIL was analyzed by flow cytometry on day 7 of culture. Each symbol represents the result of one experiment and the line indicates the mean MFI; data pooled from four independent experiments. (C) Secretion of soluble TRAIL (S-TRAIL) or FasL (S-FasL) from NK cells was analyzed by ELISA. Results are shown as mean ± SEM of data pooled from three experiments. p-values were calculated by paired (between same feeder) or unpaired (between different feeders) two-tailed Student’s t-test.

Article Snippet: NK cell mediated tumor cytotoxicity against J82 (bladder cancer, ATCC), PC3 (prostate cancer, ATCC), MCF7 (breast cancer, ATCC), and EST025 (melanoma, IPD– ESTDAB) was analyzed on day 8–11.

Techniques: Expressing, Irradiation, Staining, Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: European journal of immunology

Article Title: Activated monocytes augment TRAIL-mediated cytotoxicity by human NK cells through release of IFN-γ.

doi: 10.1002/eji.201242735

Figure Lengend Snippet: Figure 2. ZA treatment augments NK-cell cytotoxicity against different tumor cell lines. (A) NK cells were cocultured with irradiated monocytes in the presence or absence of ZA and analyzed for cytotoxicity in a 51Cr release assay against untreated or b-AP15-treated J82 tumor cells. Data are shown as mean + SEM of results pooled from three experiments. p-values were calculated from two-tailed Student’s t-test. (B) The ability of ZA-primed NK cells to kill b-AP15-treated J82 and PC3 tumor cells (left) or untreated MCF7 and EST025 (right) were analyzed in a 51Cr release assay at E:T ratios of 1:1–6:1. NK cells and tumor cells were washed before coculture. Data are shown as mean ± SEM of triplicates. One of three independent experiments with similar results is shown.

Article Snippet: NK cell mediated tumor cytotoxicity against J82 (bladder cancer, ATCC), PC3 (prostate cancer, ATCC), MCF7 (breast cancer, ATCC), and EST025 (melanoma, IPD– ESTDAB) was analyzed on day 8–11.

Techniques: Irradiation, Release Assay, Two Tailed Test

Journal: European journal of immunology

Article Title: Activated monocytes augment TRAIL-mediated cytotoxicity by human NK cells through release of IFN-γ.

doi: 10.1002/eji.201242735

Figure Lengend Snippet: Figure 3. ZA treatment augments TRAIL mediated anti-tumor effects of NK cells. (A) NK cells were cocultured with irradiated PBMCs or mono- cytes and tested for cytotoxicity against b-AP15-treated J82 cells. Data are shown as mean + SEM of results pooled from six NK cell cultures. p-values were calculated by paired (in between same feeder cell groups) or unpaired (between different feeder cell groups) two tailed Student’s t-test. (B) NK cells were cocultured with irradiated monocytes in the presence or absence of ZA, washed and tested for cytotoxicity against b-AP15-treated J82 cells in the presence of neutralizing antibodies against TRAIL, NKG2D, NKp44, FasL, or Concanamycin A. Data are shown as mean + SEM of results pooled from four NK cell cultures. p-values were calculated by paired Student’s t-test. *p < 0.04 between unprimed and ZA-primed NK cells.

Article Snippet: NK cell mediated tumor cytotoxicity against J82 (bladder cancer, ATCC), PC3 (prostate cancer, ATCC), MCF7 (breast cancer, ATCC), and EST025 (melanoma, IPD– ESTDAB) was analyzed on day 8–11.

Techniques: Irradiation, Two Tailed Test

Journal: European journal of immunology

Article Title: Activated monocytes augment TRAIL-mediated cytotoxicity by human NK cells through release of IFN-γ.

doi: 10.1002/eji.201242735

Figure Lengend Snippet: Figure 4. Bisphosphonates and IL-2 induce IFN-γ production in mono- cytes. (A) Purified NK cells were cultured in the lower chamber of 24- well plates in the presence of IL-2 (500 U/mL) and monocytes were added to the upper insert in the presence or absence of ZA for 6 days. TRAIL expression was analyzed by flow cytometry. Data are shown as mean + SEM of results pooled from six cultures. (B) Monocytes (Mono) were cultured in 500 U/mL IL-2 in the presence or absence of ZA for 4 days and production of IFN-γ was analyzed by ELISA. Data are shown as mean + SEM of results pooled from three cultures. p-values were calculated by paired two tailed Student’s t-test. (C) Purified monocytes cultured for 4 days in presence of 10 μM ZA or pamidronate (pam) or left untreated and production of IFN-γ was analyzed by ELISA. Data are shown as mean + SEM of results pooled from two experiments.

Article Snippet: NK cell mediated tumor cytotoxicity against J82 (bladder cancer, ATCC), PC3 (prostate cancer, ATCC), MCF7 (breast cancer, ATCC), and EST025 (melanoma, IPD– ESTDAB) was analyzed on day 8–11.

Techniques: Cell Culture, Expressing, Cytometry, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: European journal of immunology

Article Title: Activated monocytes augment TRAIL-mediated cytotoxicity by human NK cells through release of IFN-γ.

doi: 10.1002/eji.201242735

Figure Lengend Snippet: Figure 6. Adoptive infusion of ZA-primed NK cells reduces tumor pro- gression in tumor bearing mice. SCID/Beige mice were injected with J82 cells and (A) left untreated or (B) were treated with b-AP15 on days 10, 21, 24, and 31 followed by infusion of ZA-primed or unprimed NK cells on days 11, 22, 25, and 31. Tumor size was analyzed by caliper measurement twice weekly. (C) Survival was measured daily. p-values for tumor progression (B) and survival (C) were analyzed by two-way ANOVA and log-rank tests, respectively. Data are shown as mean ± SEM (n = 7) mice per group pooled from two experiments.

Article Snippet: NK cell mediated tumor cytotoxicity against J82 (bladder cancer, ATCC), PC3 (prostate cancer, ATCC), MCF7 (breast cancer, ATCC), and EST025 (melanoma, IPD– ESTDAB) was analyzed on day 8–11.

Techniques: Injection

Journal: European journal of immunology

Article Title: Activated monocytes augment TRAIL-mediated cytotoxicity by human NK cells through release of IFN-γ.

doi: 10.1002/eji.201242735

Figure Lengend Snippet: Figure 5. ZA-treated monocytes augment TRAIL-mediated cytotoxicity of human NK cells through the release of IFN-γ. Purified NK cells were (A) cultured with supernatant from ZA-treated or untreated monocytes or (B) cocultured with ZA-treated or untreated monocytes in the pres- ence or absence of IFN-γ-neutralizing antibodies (α-IFNγ) for 6 days and analyzed for TRAIL expression. Data are shown as mean + SEM of results pooled from six (A) or three (B) experiments. (C) NK cells from (A) were analyzed for cytotoxicity against b-AP15-treated J82. Data are shown as mean + SEM of results pooled from six experiments. (D, E) NK cells were treated with 500 U/mL IL-2 on days 0 and 4 and 500 pg/mL recombinant human IFN-γ (hIFNγ) every other day for 6 days or left untreated and analyzed for (D) TRAIL expression by flow cytometry and (E) cytotoxicity against b-AP15-treated J82 tumor cells by 51Cr-release assay. Data are shown as mean + SEM of results pooled from four experiments. p-values were obtained from paired two tailed Student’s t-test.

Article Snippet: NK cell mediated tumor cytotoxicity against J82 (bladder cancer, ATCC), PC3 (prostate cancer, ATCC), MCF7 (breast cancer, ATCC), and EST025 (melanoma, IPD– ESTDAB) was analyzed on day 8–11.

Techniques: Cell Culture, Expressing, Recombinant, Cytometry, Release Assay, Two Tailed Test