Journal: Nature Communications

Article Title: Characterizations of a neutralizing antibody broadly reactive to multiple gluten peptide:HLA-DQ2.5 complexes in the context of celiac disease

doi: 10.1038/s41467-023-44083-4

Figure Lengend Snippet: a Antibodies binding to Ba/F3 cells expressing various pHLA-DQ2.5 as determined by flow cytometry. Gating strategy to determine the MFI is shown in Supplementary Fig. . Data are from an assay performed in triplicates ( n = 3). Heat map represents relative mean MFI (%) of antibodies (DONQ52: 0.5 μg/mL, Control Ab: 10 μg/mL) to DQN0139 (10 μg/mL). Numerical data sets are shown in Supplementary Table . b Antibodies binding to non-HLA-DQ2.5, HLA II expressing Ba/F3 cell lines were determined by flow cytometry. Gating strategy to determine the MFI is shown in Supplementary Fig. . Tu39 is anti-HLA-DR, DP, DQ antibody (mouse IgG2a). SPV-L3 is anti-pan HLA-DQ antibody (mouse IgG2a). MOPC-173 is mouse IgG2a isotype antibody. Control Ab is anti-KLH antibody (clone: IC17-SG181) as an isotype control for DONQ52 and DQN0139. Each binding antibody is colored as indicated. Data are from an assay performed in triplicates ( n = 3) and are shown as mean ± SD. Numerical data sets are shown in Supplementary Table . c Antibodies binding to HLA-DQ2.5+ ( n = 8) or HLA-DQ2.5- ( n = 4) human B cells determined by flow cytometry. Relative values of MFI (%) of antibodies (DONQ52 (blue), Control Ab (gray): 10 μg/m) to DQN0139 (10 μg/mL) are shown. Peptide (-) means B cells cultured without the 33mer gliadin peptide, and Peptide (+) means B cells cultured with the 33mer gliadin peptide. Gating strategy to determine the MFI is shown in Supplementary Fig. . Individual numerical data is shown in Supplementary Table . d Representative SPR sensorgram of the DONQ52 for binding to HLA-DQ2.5:33mer gliadin. K D value was derived by 1:1 binding model. e Representative SPR sensorgram of the DONQ52 binding to the 33mer gliadin peptide itself, or 14D5, which is an anti-gliadin antibody (Abcam PLC). f Schematic representation of DONQ52 binding properties.

Article Snippet: Ten μg/mL of DONQ52, control Ab (anti-KLH antibody, clone IC17-SG181), DQN0139, SPV-L3 (anti-pan HLA-DQ antibody, Beckman Coulter), Tu39 (anti-HLA-DR, DP, DQ antibody, BioLegend), or MOPC-173 (mouse IgG2a isotype antibody, BioLegend) was incubated with 1 × 10 5 cells/100 μL/well Ba/F3 cells expressing HLA II in FACS buffer for 30 min at room temperature, followed by antibody detection with ×50 diluted Goat F(ab′)2 anti-Human IgG, Mouse ads-PE (Southern Biotech), or Goat F(ab′)2 anti-Mouse IgG2a, Human ads-PE (Southern Biotech) by flow cytometry (BD LSRFortessa X-20, Becton, Dickinson and Company).

Techniques: Binding Assay, Expressing, Flow Cytometry, Control, Cell Culture, Derivative Assay

Journal: International journal of cancer

Article Title: In vitro elimination of epidermal growth factor receptor-overexpressing cancer cells by CD32A-chimeric receptor T cells in combination with cetuximab or panitumumab

doi: 10.1002/ijc.32663

Figure Lengend Snippet: In vitro elimination of EGFR+ epithelial cancer cells (ECCs) by CD32A131R-chimeric receptor (CR) and CD16158F-CR T cells is associated with EGFR overexpression. (a) EGFR expression levels in the tumor cell lines as assessed by flow cytometry. The cells were incubated with 3 μg/ml of purified anti-EGFR antibody, washed and then stained with a phycoerythrin (PE)-conjugated antimouse IgG (gray filled histograms). The cells incubated with PE-conjugated antimouse IgG were used as a negative control (light gray filled histograms). Mean fluorescence intensity (MFI) is indicated for each cell line. (b) Spearman’s correlation between EGFR expression levels (MFI) in the HCT116, A549, MDA-MB-231 and MDA-MB-468 cell lines and the ECC elimination by CD16158F-CR T cells (left panel) and CD32A131R-CR T cells (right panel) in combination with cetuximab. A regression line is shown in black. Spearman’s rank correlation coefficient (r) was 0.6316 for CD16158F-CR and 0.533 for CD32A131R-CR. This is a cumulative analysis of five experiments separately performed: 116 = HCT116, 231 = MDA-MB-231, 468 = MDA-MB-468 and 549 = A549 cells.

Article Snippet: PE-conjugated goat antimouse IgG (1012-09) was purchased by Southern Biotech (Birmingham, AL).

Techniques: In Vitro, Over Expression, Expressing, Flow Cytometry, Incubation, Purification, Staining, Negative Control, Fluorescence