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. 2015 Dec 9;4:e10766. doi: 10.7554/eLife.10766

Figure 2. Antibodies recognizing the same segments of CD11b as CyaA block its binding to CR3.

(A) Schematic representation showing the binding segments of a set of mAbs in the CD11b subunit of CR3, which were mapped by flow cytometry. The ICRF 44, 44 and 2LPM19c mAbs recognize the I-domain of CD11b, the major ligand binding site of CR3. The MEM-174, or OKM1, VIM12 and M1/70 mAbs target amino acid segments 342-424 or 614-682 of CD11b, respectively, which are important for CyaA binding. (B) 2x105 CHO-CD11b/CD18 cells were preincubated without or with saturating concentrations of different mAbs and then incubated with 2 µg/ml of CyaA-biotin. Surface-bound CyaA-biotin was labeled with streptavidin-PE and the cells were analyzed by flow cytometry. CyaA binding was expressed as percentage of toxin binding to CHO-CD11b/CD18 cells treated without mAb. Each bar represents the mean value with SD of at least eight determinations from at least three independent experiments. Significant differences between mean values of CyaA binding to mAb-untreated cells and cells treated with different mAbs are indicated (***, p<0.001; ****, p<0.0001; ANOVA). 3.9, CD11c-specific mAb; MEM-48, CD18-specific mAb; IgG1, IgG2a and IgG2b, isotype control mAbs.

DOI: http://dx.doi.org/10.7554/eLife.10766.007

Figure 2.

Figure 2—figure supplement 1. Flow cytometry profiles of anti-CD11b mAbs on CHO cells expressing the integrin CD11b/CD18 and its mutant variants.

Figure 2—figure supplement 1.

2x105 CHO cells stably expressing intact CD11b/CD18, or the mutant variant lacking the I-domain (CD11bΔ164-339/CD18), or two CR3-CR4 chimeras (CD11b342-424c/CD18 and CD11b614-682c/CD18) with a strongly reduced capacity to bind CyaA were left unstained (grey) or were stained with a panel of anti-CD11b mAbs (red). The cells were analyzed for their capacity to bind mAbs by flow cytometry and typical overlay histograms from one representative binding experiment out of five performed are shown. RFI, relative fluorescence intensity. The data confirmed that the ICRF 44, 44 and 2LPM19c mAbs recognize the I-domain of CR3 and revealed that the MEM-174 mAb has an epitope in the segment 342-424 of CD11b and the OKM1, VIM12 and M1/70 mAbs target the segment 614-682 of CD11b.
DOI: http://dx.doi.org/10.7554/eLife.10766.008
Figure 2—figure supplement 2. Anti-CD11b antibodies do not activate CR3.

Figure 2—figure supplement 2.

2x105 CHO-CD11b/CD18 cells were preincubated without or with saturating concentrations of the indicated anti-CD11b mAbs and the CR3 was stained with the integrin activation-reporting anti-CD18 mAb MEM-148. MEM-148 binding was determined by flow cytometry and expressed as percentage of MEM-148 binding to CHO-CD11b/CD18 cells treated without mAb. Each bar represents the mean value with SD of two independent experiments performed in duplicate. Significant differences between mean values of MEM-148 binding to mAb-untreated cells and cells treated with different anti-CD11b mAbs are indicated (***, p<0.001; ANOVA).
DOI: http://dx.doi.org/10.7554/eLife.10766.009