Journal: eLife

Article Title: iTAP, a novel iRhom interactor, controls TNF secretion by policing the stability of iRhom/TACE

doi: 10.7554/eLife.35032

Figure Lengend Snippet: ( A ). iTAP was knocked out in L929, RAW 264.7 and HEK 293ET cells using CRISPR. Lysates were immunoblotted with anti-iTAP antibodies. A small black arrowhead indicates iTAP protein whereas a non-specific band (white asterisk) serves as a loading control. ( B ). Glycoproteins from lysates isolated from the cells in ( A ) were enriched using concanavalin A-sepharose (conA) and TACE levels were assessed by western blot. Here and throughout, the immature form of TACE is indicated by a white arrow, whereas, the mature form is denoted by a black arrow. iRhom double KO MEFs were used as a reference and the transferrin receptor (TfR) as a loading control. Lower panels: densitometry in HEK 293ET. Left hand panel: Levels of immature TACE normalized to TfR. Right hand panel: levels of mature TACE as a relative proportion of immature TACE in WT and iTAP KO HEK 293ET. ( C,D ). Validation of mature and immature TACE detection in panels of WT versus iRhom2 DKO ( C ) or iTAP KO ( D ) cells, by deglycosylation. ConA enriched lysates from the cell lines in ( A ) were treated with endoglycosidase H (Endo-H; H; which cleaves ER-resident glycans only) and PNGase F (F; which cleaves both ER and post-ER glycans). Here and throughout: the immature TACE is indicated with white arrowheads; the black arrowhead denotes both glycosylated mature TACE and deglycosylated immature TACE respectively (which have similar electrophoretic mobility), whereas red arrowheads denote the fully deglycosylated, mature, TACE polypeptide. ( E ). iTAP expression restores the presence of mature TACE in iTAP KO cells. Lysates from WT or iTAP KO HEK 293ET stably expressing empty vector (-, EV) or human iTAP (+) were screened for mature TACE. Actin was used as a loading control. Middle and lower panels: densitometric analysis indicates that iTAP expression increases the levels of mature TACE but does not affect the levels of immature TACE. Middle panel: levels of mature TACE as a relative proportion of immature TACE in WT and KO upon iTAP or EV expression in WT and iTAP KO HEK 293ET clones. Lower panel: Levels of immature TACE after normalization to actin. ( F ). iTAP KO cells lack mature cell surface TACE. Left hand panel: RAW 264.7 WT or iTAP KO were surface-biotinylated in vivo and lysates were enriched for biotinylated proteins with neutravidin resin. Probing for TfR was used as a cell surface positive control protein whereas anti-p97 probing demonstrates that intracellular proteins were not labeled. Right hand panel: Cell surface biotinylated proteins were deglycosylated using Endo-H ( H ) or PNGase F ( F ). ConA enriched lysates were run as mobility controls, for immature and mature TACE. Blots were probed for TACE and for TfR as a control protein. ( G ). Loss of iTAP has no impact on the mature species of other ADAM metalloproteases. HEK 293ET WT or KO cells were transfected with the indicated panel of V5-tagged ADAMs. The lysates were deglycosylated as described above and Tubulin serves as a loading control. Throughout: Data are presented as mean ± standard deviation and represent three independent experiments. *=p ≤ 0.05, **=p ≤ 0.01, ***=p ≤ 0.001 and n.s. = non significant. 10.7554/eLife.35032.016 Figure 4—source data 1. iTAP KO cells are depleted in mature TACE levels. Densitometric analyses of mature/immature TACE levels. 10.7554/eLife.35032.017 Figure 4—source data 2. iTAP expression restores the presence of mature TACE in iTAP KO cells. Densitometric analyses of mature/immature TACE levels.

Article Snippet: Commercial assay or kit , Concanavalin A Agarose; conA , G-biosciences , 786–216 , .

Techniques: CRISPR, Control, Isolation, Western Blot, Biomarker Discovery, Expressing, Stable Transfection, Plasmid Preparation, Clone Assay, In Vivo, Positive Control, Labeling, Transfection, Standard Deviation

Journal: eLife

Article Title: iTAP, a novel iRhom interactor, controls TNF secretion by policing the stability of iRhom/TACE

doi: 10.7554/eLife.35032

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , Concanavalin A Agarose; conA , G-biosciences , 786–216 , .

Techniques: Generated, CRISPR, Isolation, Immunoprecipitation, Magnetic Beads, Negative Control, Transfection, Construct, Plasmid Preparation, Recombinant, Mutagenesis, Luciferase, Sequencing, TA Cloning, Enzyme-linked Immunosorbent Assay, Software

Journal: Frontiers in Immunology

Article Title: Activation of ADAM17 by IL-15 Limits Human NK Cell Proliferation

doi: 10.3389/fimmu.2021.711621

Figure Lengend Snippet: Description of the commercial antibodies used in this study.

Article Snippet: In vivo grade isotype control, mouse IgG1 , CB5 , C0005 , Crown Bioscience.

Techniques: In Vivo

Journal: Frontiers in Immunology

Article Title: Activation of ADAM17 by IL-15 Limits Human NK Cell Proliferation

doi: 10.3389/fimmu.2021.711621

Figure Lengend Snippet: ADAM17 blockade enhances human NK cell proliferation by IL-15 in vivo. (A) Animal treatment schema. NSG mice were treated as described in the Methods. (B) Enriched NK cells were infused in the presence or absence of rhIL-15 (5μg), as indicated. Mouse peripheral blood was collected and the number of human CD45 + CD56 + CD3 − NK cells were enumerated by flow cytometry and are shown as cells/μl. Data are mean ± SD (n = 3 to 5 mice per group). (C) Additional mice were administered enriched NK cells from a separate donor plus rhIL-15 (5μg) ± MEDI3622 (10 mg/kg). Data are mean ± SD (n = 4 mice per group). **p < 0.01; ****p < 0.0001. (D) The experiment was performed as described in panel (C) NK cells were obtained from six separate donors (panels 1-6). Mice were also treated with rhIL-15 in the presence or absence of a human IgG1 control mAb (panel 7). Mouse peripheral blood was collected at day 21 following NK cell adoptive transfer and human CD45 + CD56 + CD3 − NK cells were enumerated by flow cytometry. Group data was tested for normality (Kolmogorov-Smirnov test) and the differences in means were calculated by comparing means ± SD using an unpaired two-sided Student’s t-test. n = 3 to 7 mice per group. *p < 0.05; **p < 0.01; ****p < 0.0001; ns, not significant.

Article Snippet: In vivo grade isotype control, mouse IgG1 , CB5 , C0005 , Crown Bioscience.

Techniques: In Vivo, Flow Cytometry, Adoptive Transfer Assay

Journal: Frontiers in Immunology

Article Title: Activation of ADAM17 by IL-15 Limits Human NK Cell Proliferation

doi: 10.3389/fimmu.2021.711621

Figure Lengend Snippet: ADAM17 blockade enhances human NK cell proliferation by IL-15 in vitro . (A) Enriched NK cells were labeled with CellTrace Violet dye and placed in culture for 7 days with rhIL-15 (10ng/ml) and/or MEDI3622 (5μg/ml) and/or control human IgG1 (5μg/ml), as indicated. Cells were then harvested and examined for CellTrace dye dilution by flow cytometry. Data are representative of 3 independent experiments using leukocytes from separate donors. An expansion index was calculated as described in the Methods and is the fold expansion of the overall culture for each condition based on dye dilution. Data are means ± SD of three independent experiments using separate donors. Statistical significance is indicated as **p < 0.01. Statistics were calculated using one-way ANOVA. (B) Human PBMCs were labeled with CellTrace Violet dye and placed in culture for 7 days with rhIL-15 (10ng/ml), MEDI3622 (5μg/ml), control human IgG1 (5μg/ml), and/or DREG200 (5μg/ml). Cells were then harvested and examined for CellTrace dye dilution by flow cytometry. Data are representative of 3 independent experiments using leukocytes from separate donors.

Article Snippet: In vivo grade isotype control, mouse IgG1 , CB5 , C0005 , Crown Bioscience.

Techniques: In Vitro, Labeling, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Activation of ADAM17 by IL-15 Limits Human NK Cell Proliferation

doi: 10.3389/fimmu.2021.711621

Figure Lengend Snippet: CD62L expression during NK cell proliferation and its role in their expansion in vivo . (A) NK cells were placed in culture for 7 days in the presence of rhIL-15 (10ng/ml) alone or in the presence of MEDI3622 (5 μg/ml) or an isotype-matched negative control mAb (IgG, 5 μg/ml). CD62L levels were determined by flow cytometry. The histograms show representative data and the bar graph shows mean ± SD of 3 independent experiments using leukocytes from separate donors. *p < 0.05. The y-axis on the bar graph indicates mean fluorescence intensity (MFI). Statistics were calculated as described in Figure 1 . (B) Mice were administered enriched NK cells and rhIL-15 (5μg) in the presence or absence of MEDI3622 (10 mg/kg). After 3 weeks, mouse peripheral blood was collected, and relative CD62L expression levels were determined on human CD45 + CD56 + CD3 − NK cells by flow cytometry. Two separate experiments are shown using NK cells from different donors. The y-axis on the bar graphs indicates MFI. Data are mean ± SD (n = 5 mice per group). **p < 0.01. (C) The experiment was performed as described in panel B and various cell surface markers were evaluated. Data are representative of two separate experiments using NK cells from different donors. Data are means ± SD (n = 5 mice per group). *p < 0.05; ns = not significant. Data was analyzed by using unpaired two-tailed student’s t-test. (D) Mice were administered NK cells and rhIL-15 (5μg) in the presence or absence of DREG200 (10mg/kg) (left panel), DREG200 and/or MEDI3622 (10mg/kg) (middle panel), or mouse IgG isotype-matched mAb (10mg/kg) as a control for DREG200 (right panel). The number of NK cells in the peripheral blood were enumerated by flow cytometry and are shown as cells/μl or as percent change normalized to NK cells plus rhIL-15. Data are means ± SD (n = 4 to 5 per group). ****p < 0.0001; ns, not significant. Data was analyzed by using unpaired two-tailed student’s t-test.

Article Snippet: In vivo grade isotype control, mouse IgG1 , CB5 , C0005 , Crown Bioscience.

Techniques: Expressing, In Vivo, Negative Control, Flow Cytometry, Fluorescence, Two Tailed Test