Journal: Haematologica

Article Title: A subset of anti-HLA antibodies induces FcγRIIa-dependent platelet activation

doi: 10.3324/haematol.2018.189365

Figure Lengend Snippet: HLA monoclonal antibodies induce platelet α-granule release. (A) Platelets were matched for HLA type with the specificity of eight HLA monoclonal antibodies (mAbs) directed at different epitopes. Mean fluorescent intensity (MFI) upon staining with anti-human IgG was measured with flow cytometry for the control (buffer only, no HLA antibodies) and 2.5 mg/mL of the HLA mAbs. Right panel: representative flow cytometry plot of 10 mg/mL SN607D8 with a not matching donor and a matching donor. (B,C) CD62P surface expression of platelets incubated with 2.5 mg/mL (B) or 10 mg/mL (C) HLA mAbs compared to control (buffer only). Representative flow cytometry plots of WIM8E5, SN607D8 and SN230G6. (D) VWF release in platelet supernatant upon incubation with HLA mAbs WIM8E5, SN607D8 and SN230G6 measured by enzyme-linked immunosorbent assay. (E) Representative western blot of SPARC release in platelet supernatant upon incubation with HLA mAbs WIM8E5, SN607D8 and SN230G6. Paired t-tests (A, B and C) or paired ANOVA with the Tukey multiple comparison test (D). Each line represents a separate experiment with a separate donor (A, B and C). Mean ± SD (D). * P <0.05, ** P <0.01, *** P <0.005, **** P <0.001.

Article Snippet: Platelet internalization was quantified by imaging flow cytometry (ImageStream ® X Mark II Imaging Flow Cytometer, Merck Millipore, Amsterdam, the Netherlands).

Techniques: Bioprocessing, Staining, Flow Cytometry, Control, Expressing, Incubation, Enzyme-linked Immunosorbent Assay, Western Blot, Comparison

Journal: Haematologica

Article Title: A subset of anti-HLA antibodies induces FcγRIIa-dependent platelet activation

doi: 10.3324/haematol.2018.189365

Figure Lengend Snippet: Integrin α IIb b 3 activation and platelet agglutination are induced by HLA monoclonal antibodies. A) Integrin α IIb b 3 activation, derived from PAC-1 binding, upon incubation with 10 mg/mL WIM8E5, SN607D8 or SN230G6 compared to control (buffer only, no HLA antibodies). Flow cytometry plots are representative of more than eight independent experiments with different donors. (B) Platelet agglutination upon addition of HLA monoclonal antibodies (mAbs), measured by light transmission aggregometry. Mean ± SD of percentage maximum aggregation. Paired t -tests (A) or paired ANOVA with the Tukey multiple comparison test (B). * P <0.05, ** P <0.01, *** P <0.005, **** P <0.001.

Article Snippet: Platelet internalization was quantified by imaging flow cytometry (ImageStream ® X Mark II Imaging Flow Cytometer, Merck Millipore, Amsterdam, the Netherlands).

Techniques: Activation Assay, Agglutination, Bioprocessing, Derivative Assay, Binding Assay, Incubation, Control, Flow Cytometry, Transmission Assay, Comparison

Journal: Haematologica

Article Title: A subset of anti-HLA antibodies induces FcγRIIa-dependent platelet activation

doi: 10.3324/haematol.2018.189365

Figure Lengend Snippet: HLA monoclonal antibodies activate platelets in an intra-platelet-dependent manner. (A) Schematic representation of theoretically possible inter-platelet activation and intra-platelet activation induced by the HLA monoclonal antibody WIM8E5. (B) Platelet donors were selected as either “WIM8E5 matching” or “WIM8E5 nonmatching” and their platelets were stained with calcein-green or calcein-violet, respectively. Platelets from two donors were mixed in a 1:1 ratio. Platelets from a single donor or the mixed platelets were incubated with control (buffer only, no HLA antibodies) or WIM8E5. By gating for calcein-green or calcein-violet, CD62P exposure was determined for “WIM8E5 matching” and “WIM8E5 nonmatching” platelets. (C) Platelets from a “WIM8E5 matching” donor (stained with calcein green) were mixed 1:1 with platelets from a donor with either nonmatching WIM8E5 or matching WIM8E5 (stained with calcein violet). Samples were incubated with control (buffer only, no HLA antibodies), WIM8E5 or PAR1 activating peptide (PAR1 AP). Percentage double-positive events of calcein-green/calcein-violet-stained platelets are given, representing the ability of HLA antibodies to crosslink the HLA molecule with the FcγRIIa in an inter- or intra-platelet manner. Representative flow cytometry plots are shown for a mix of WIM8E5 matching + WIM8E5 nonmatching platelets and platelets from two different donors with both matching HLA typing. Paired ANOVA with the Tukey multiple comparison test. * P <0.05, ** P <0.01, *** P <0.005, **** P <0.001.

Article Snippet: Platelet internalization was quantified by imaging flow cytometry (ImageStream ® X Mark II Imaging Flow Cytometer, Merck Millipore, Amsterdam, the Netherlands).

Techniques: Bioprocessing, Activation Assay, Staining, Incubation, Control, Flow Cytometry, Immunopeptidomics, Comparison

Journal: Haematologica

Article Title: A subset of anti-HLA antibodies induces FcγRIIa-dependent platelet activation

doi: 10.3324/haematol.2018.189365

Figure Lengend Snippet: Phagocytosis of platelets opsonized by HLA monoclonal antibodies and the effect of FcγRIIa-dependent signaling. Platelets were incubated with 10 mg/mL WIM8E5, SN607D8 or SN230G6 in the presence or absence of 5 mM Syk inhibitor IV. Opsonized platelets were incubated for 1 h with monocyte-derived macrophages and internalization was analyzed through the use of imaging flow cytometry. (A–C) Representative images of imaging flow cytometry. BF: bright field; CD61: extracellular platelet staining; PKH: platelet staining; HLA-DR: macrophage staining. (A) Control without Syk inhibitor, (B) WIM8E5 without Syk inhibitor, (C) WIM8E5 with Syk inhibitor. (D) Intracellular platelet (PKH) fluorescence quantifies the amount of platelets taken up by macrophages. Data are given as mean ± SD, * P <0.05, ** P <0.01. Control: buffer only, no HLA antibodies added. MF: macrophage.

Article Snippet: Platelet internalization was quantified by imaging flow cytometry (ImageStream ® X Mark II Imaging Flow Cytometer, Merck Millipore, Amsterdam, the Netherlands).

Techniques: Bioprocessing, Incubation, Derivative Assay, Imaging, Flow Cytometry, Staining, Control, Fluorescence

Journal: Journal of Immunological Methods

Article Title: Direct uptake of Antagomirs and efficient knockdown of miRNA in primary B and T lymphocytes

doi: 10.1016/j.jim.2015.07.006

Figure Lengend Snippet: Antagomirs efficiently enter primary lymphocytes, are localized in the cytoplasm and show low cytotoxicity. Ex vivo isolated (MACS Technology, Miltenyi Biotech, Germany) unstimulated CD4 + T cells and unstimulated CD19 + B cells were treated with fluorescein-coupled non-targeting control Antagomir (Antagomir-scr) or incubated in serum-free medium without addition of Antagomir (Incubation ctrl). a) Cells were treated with concentrations of 0.125 to 2 μM of fluorescein-coupled Antagomir-scr for 2 h in serum-free medium and fluorescence was analyzed by flow cytometry; b) cells were treated with a concentration of 1 μM of fluorescein-coupled Antagomir-scr, washed after 2 h of incubation with PBS, cultured in FCS-containing RPMI medium for 3 days and fluorescence was analyzed by flow cytometry, d0 = cells after 2 h of incubation; c) cytoplasmic localization of fluorescein-coupled Antagomir-scr in live murine CD4 + T cells, murine CD19 + B cells, human CD4 + T cells and human CD19 + B cells was determined by single cell immunofluorescence analysis (Amnis ImageStreamX MKII, Merck Millipore) 24 h after incubation with Antagomirs, BF — bright field, CD4/CD19 — surface staining; d) viability of murine CD4 + Th1 cells and CD19 + B cells after treatment with different concentrations of Antagomir-scr in serum-free medium for 2 h. Viability was determined by propidium iodide staining, analyzed by flow cytometry and normalized to cells that were incubated in serum-free medium without addition of Antagomir.

Article Snippet: Fluorescein-conjugated Antagomirs were used for quantification of Antagomir incorporation and detected by flow cytometry (MACSQuant, Miltenyi Biotec) or image stream cytometry (Amnis ImageStreamX MKII, Merck Millipore).

Techniques: Ex Vivo, Isolation, Control, Incubation, Fluorescence, Flow Cytometry, Concentration Assay, Cell Culture, Immunofluorescence, Staining