Fig 1: Tetravalency and FcγRIIB engagement synergize for optimal stimulation of human CD27.A hCD27+ Jurkat NF-κB GFP reporter cells were co-cultured with either WT CHO or hFcγRIIB+ CHO cells in the presence of the indicated antibodies. NF-κB activation was quantitated by GFP expression using flow cytometry. GFP production from Jurkat cells after co-culture with WT CHO cells (above) or hFcγRIIB+ CHO cells (below) and the specified antibodies; hIgG1 (left), hIgG1 NA (center), or hIgG1 V11 (right). Data points are the mean ± SEM (n = 3) from 3 independent experiments. Statistical significance was determined by one-way ANOVA (Tetra anti-hCD27 versus anti-CD27 IgG), with significance values indicated in the figure. B Human CFSE-labeled PBMCs were stimulated with sub-optimal anti-CD3 and the indicated antibodies, hIgG1 (left), hIgG1 NA (center), or hIgG1 V11 (right). CD8+ (above) and CD4+ (below) T cell proliferation was assessed by CFSE dilution, with CFSE low cells identified by comparison to cells that were cultured in the absence of any stimulation. Each data point is the mean ± SEM (n = 5) from 5 independent experiments across 5 different donors. Statistical significance between bivalent anti-CD27 and bivalent isotype control (colored numbers) and tetravalent anti-CD27 and tetravalent isotype control (black numbers) was determined by one-way ANOVA, with significance values indicated in the figure. Source data are provided as a Source data file.
Fig 2: Tetravalent anti-hCD27 hIgG1 V11 induces greater cluster formation, with FcγRIIB engagement polarizing hCD27 clusters and reducing internalization.A, B Jurkat cells expressing hCD27-GFP+ fusion protein were stimulated with the specified antibodies at the indicated concentrations, before PFA fixation and confocal imaging. A Representative images at 100 nM (white corresponds to hCD27-GFP fluorescence; clustered areas of the signal are indicated by green arrows) and B quantification of the number of clusters per cell section, performed using ImageJ. Data shown are mean ± SEM of CD27 clusters per cell section from 2 independent experiments (n = 54 cells). Statistical significance was determined by one-way ANOVA, with significance values indicated in the figure. C, D hCD27-GFP+ Jurkat cells were stimulated with 1 nM tetra anti-hCD27 hIgG1 V11 and either WT CHO cells or hFcγRIIB+ CHO cells for the specified time points before PFA fixation and confocal imaging using Oxford Nanoimager. C Representative images of hCD27-GFP+ Jurkat cells with WT CHO (upper panels) or hFcγRIIB+ CHO (lower panels) for the specified time points. Dashed blue and green lines (determined by brightfield and confocal images) indicate the plasma membrane of CHO and Jurkat cells, respectively. D Quantification of intracellular GFP MFI of individual cell sections of hCD27-GFP+ Jurkat cells after stimulation. Data are the mean ± SEM from 2 independent experiments (n = 24 cells). Statistical significance was determined by one-way ANOVA, with significance values indicated in the figure. E, F hCD27+ Jurkat NF-κB GFP reporter cells were stimulated with the indicated concentrations of either bivalent anti-hCD27 hIgG1 V11 (left panel) or Tetra anti-hCD27 hIgG1 V11 (right panel) in the presence of WT CHO cells (WT CHO), WT CHO cells and a goat anti-hFc f(ab)2 (WT CHO + Xlink) or hFcγRIIB+ CHO cells (hFcγRIIB+ CHO). GFP reporter production was determined by flow cytometry and presented as E mean fluorescence intensity (MFI) of GFP+ cells and F percentage of GFP+ cells. Data points are the mean ± SEM (n = 3) from 3 independent experiments. Statistical significance between hFcγRIIB+ CHO and WT CHO + Xlink was determined by one-way ANOVA on AUC values, with significance values indicated in the figure. Source data are provided as a Source data file.
Supplier Page from R&D Systems, a Bio-Techne Brand for Recombinant Mouse CD27/TNFRSF7 Fc Chimera Protein, CF