neutralisation blocking assays  (R&D Systems)

Journal: Leukemia

Article Title: Oncolytic reovirus enhances rituximab-mediated antibody-dependent cellular cytotoxicity against chronic lymphocytic leukaemia

doi: 10.1038/leu.2015.88

Figure Lengend Snippet: Cytotoxicity of reovirus-activated PBMCs against CLL. Healthy donor PBMCs and CLL patient PBMCs were isolated and either left untreated, or treated with 0.1 or 1 PFU/cell reovirus for at least 16 h. ( a ) PBMCs were used in a 4 h 51 Cr release assay against EHEB and MEC-2 CLL cell targets, at different effector:target (E:T) ratios. Line graphs show the mean (±s.e.m.) % lysis for healthy donors ( n =3) and patient samples ( n =6). ( b ) Expression of CD69 (an early activation marker) on CD3 − /CD56 + NK cells was determined. Histogram plots show representative data for CD69 expression±reovirus treatment. Representative data are shown for healthy donors ( n =3) and patient samples ( n =4). ( c ) PBMCs from healthy donors or patient samples (±reovirus) were cocultured with EHEB or MEC-2 cell targets and the expression of CD107a/b (a marker of cytotoxic granule release) on CD3 − CD56 + NK cells was determined. Bar charts show the mean percentage (±s.e.m.) of total NK cells expressing CD107a/b for healthy donors ( n =4) and patient samples ( n =6).

Article Snippet: To neutralise type I IFNs, PBMCs were treated with reovirus for 16 h±neutralising antibodies (NAbs; IFN block; PBL Interferon Source, Piscataway, NJ, USA) or isotype control (R&D Systems).

Techniques: Isolation, Release Assay, Lysis, Expressing, Activation Assay, Marker

Journal: Leukemia

Article Title: Oncolytic reovirus enhances rituximab-mediated antibody-dependent cellular cytotoxicity against chronic lymphocytic leukaemia

doi: 10.1038/leu.2015.88

Figure Lengend Snippet: Mechanism of NK cell activation by reovirus in CLL patient samples. ( a ) PBMCs from CLL patients were treated with reovirus, cell-free supernatant was collected and the secretion of IFNα was determined by ELISA. Bar chart shows mean ( n =11, ±s.e.m.). ( b ) CLL patient PBMCs were either left untreated or treated with 1 PFU/cell reovirus overnight, in the presence or absence of type 1 IFN-blocking antibodies or appropriate isotype controls. Bar charts show the mean ( n =3, ±s.e.m.) NK cell activation as determined by CD69 expression or CD107a/b degranulation against MEC-2 targets. ( c ) PBMCs were treated with replication-competent (live) or UV-inactivated reovirus and (i) the production of IFNα was determined by ELISA 24 h after treatment ( n =3, ±s.em.), (ii) the expression of CD69 on NK cells ( n =4, ±s.e.m.) and (iii) levels of CD107 NK cell degranulation were determined ( n =4, ±s.e.m.). ( d ) CD14 + monocytes were isolated from CLL samples and left overnight to allow CD14 microbeads to dissociate; monocytes were treated with reovirus and IFNα expression was determined by intracellular flow cytometry (representative of n =2). ( e – g ) PBMCs from CLL patient samples were either left untreated or treated with reovirus, ±CD14 + monocyte depletion. ( e ) Intracellular IFNα was determined by flow cytometry (representative o f n =2). ( f ) Levels of IFNα secretion were determined by ELISA ( n =5, ±s.e.m.). ( g ) NK cell activation was determined by CD69 expression or CD107a/b degranulation ( n =6, ±s.e.m.).

Article Snippet: To neutralise type I IFNs, PBMCs were treated with reovirus for 16 h±neutralising antibodies (NAbs; IFN block; PBL Interferon Source, Piscataway, NJ, USA) or isotype control (R&D Systems).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay, Expressing, Isolation, Flow Cytometry

Journal: Leukemia

Article Title: Oncolytic reovirus enhances rituximab-mediated antibody-dependent cellular cytotoxicity against chronic lymphocytic leukaemia

doi: 10.1038/leu.2015.88

Figure Lengend Snippet: Reovirus potentiates rituximab-mediated ADCC. ( a ) Representative histogram plots showing the expression of CD20, and rituximab binding, on EHEB and MEC-2 CLL cells are shown ( n =2–3). ( b ) PBMCs from CLL patient samples were either left untreated or cultured overnight with reovirus. NK cell (CD3 − CD56 + ) CD107a/b degranulation was determined after coculture with rituximab- or isotype control-labelled EHEB or MEC-2 cell targets. Bar charts show the mean percentage of total NK cells expressing CD107a/b ( n ⩾6, ±s.e.m.). ( c ) Four-hour 51 Cr release assays were carried out using patient PBMCs that were either left untreated or activated with reovirus overnight, and cocultured with rituximab- or isotype control-labelled EHEB or MEC-2 cell targets. Line graphs compare the mean % lysis ( n =6± s.e.m.) between 0 and 0.1 PFU/cell. Statistical significance between 0 vs 0.1 PFU/cell for rituximab-labelled targets is shown.

Article Snippet: To neutralise type I IFNs, PBMCs were treated with reovirus for 16 h±neutralising antibodies (NAbs; IFN block; PBL Interferon Source, Piscataway, NJ, USA) or isotype control (R&D Systems).

Techniques: Expressing, Binding Assay, Cell Culture, Control, Lysis

Journal: Leukemia

Article Title: Oncolytic reovirus enhances rituximab-mediated antibody-dependent cellular cytotoxicity against chronic lymphocytic leukaemia

doi: 10.1038/leu.2015.88

Figure Lengend Snippet: Reovirus enhances NK cell ADCC activity against rituximab-labelled autologous CLL cell targets. ( a ) Expression of CD20 and rituximab binding to CLL cells within patient PBMCs. CLL cells were identified as described in and data shown is representative of n =3 patient samples. ( b ) Patient PBMCs/CLL cells were labelled with increasing doses of rituximab or isotype control and cocultured with autologous PBMCs. CD3 − CD56 + NK cell CD107a/b degranulation was determined and scatter plots show the mean percentage of total NK cells expressing CD107a/b ( n =12, ±s.e.m.). ( c ) Patient PBMCs were either left untreated or activated with reovirus overnight and cocultured with rituximab/isotype-labelled autologous CLL cells. Scatter plots show CD3 − CD56 + NK cell CD107a/b degranulation for each individual sample ( n =24), mean (±s.e.m.) and comparison of 0 PFU/cell vs 0.1 and 0 vs 1 PFU/cell. ( d ) Linear regression analysis of reovirus-induced NK cell activation (NK cell degranulation after treatment with 1 PFU/cell reovirus overnight, n =24) and absolute monocyte count (×10 9 /l) demonstrating a significant correlation ( P =0.0023). ( e ) Comparison of CD3−CD56 + NK cell CD107a/b degranulation after coculture with isotype control-, ofatumumab-, rituximab- and GA101-labelled autologous CLL cells, ±reovirus activation of PBMCs, is shown ( n =7).

Article Snippet: To neutralise type I IFNs, PBMCs were treated with reovirus for 16 h±neutralising antibodies (NAbs; IFN block; PBL Interferon Source, Piscataway, NJ, USA) or isotype control (R&D Systems).

Techniques: Activity Assay, Expressing, Binding Assay, Control, Comparison, Activation Assay

Journal: British Journal of Cancer

Article Title: Targeting ErbB3-mediated stromal–epithelial interactions in pancreatic ductal adenocarcinoma

doi: 10.1038/bjc.2011.263

Figure Lengend Snippet: Western blot and proliferation analysis of CAF-conditioned media effects on AsPC-1 cells. Addition of ErbB3 blocking peptide or anti-NRG-1 neutralising antibody partially abrogates the stimulating effect of CAF-CM.

Article Snippet: ErbB3 blocking/receptor neutralising peptide (clone H3.105.5, Millipore, Temecula, CA, USA) was added directly to the medium to achieve a final concentration of 10 μ g ml −1 .

Techniques: Western Blot, Blocking Assay

Journal: British Journal of Cancer

Article Title: Targeting ErbB3-mediated stromal–epithelial interactions in pancreatic ductal adenocarcinoma

doi: 10.1038/bjc.2011.263

Figure Lengend Snippet: NRG-1 β abrogates the inhibitory effect of erlotinib in vitro . ( A ) ErbB3 expression analysis of nine representative pancreatic cell lines. ( B ) Relative proliferation of nine pancreatic cell lines treated with erlotinib with subsequent stimulation with either EGF or NRG-1 β . Proliferation of the erlotinib-treated control cells is set at 1.0 on the y axis.

Article Snippet: ErbB3 blocking/receptor neutralising peptide (clone H3.105.5, Millipore, Temecula, CA, USA) was added directly to the medium to achieve a final concentration of 10 μ g ml −1 .

Techniques: In Vitro, Expressing, Control

Journal: British Journal of Cancer

Article Title: Targeting ErbB3-mediated stromal–epithelial interactions in pancreatic ductal adenocarcinoma

doi: 10.1038/bjc.2011.263

Figure Lengend Snippet: Effects of MM-121 ErbB3 antibody on AsPC-1 cell proliferation and signalling both in vitro and in vivo . ( A ) Western blot analysis of inhibitory effect of erlotinib, MM-121 and their combination following EGF or NRG-1 stimulation. ( B ) Inhibition of AsPC-1 proliferation with erlotinib, MM-121 and their combination. ( C ) MM-121 inhibits AsPC-1 tumour progression in a dose-dependent manner. ( D ) Western blot analysis of AsPC-1 xenografts depicting the inhibition of ErbB3 activation and expression and inhibition of pAKT.

Article Snippet: ErbB3 blocking/receptor neutralising peptide (clone H3.105.5, Millipore, Temecula, CA, USA) was added directly to the medium to achieve a final concentration of 10 μ g ml −1 .

Techniques: In Vitro, In Vivo, Western Blot, Inhibition, Activation Assay, Expressing