FcγRIIb expression on monocytes is upregulated during HD preculture. (A) Histograms showing expression of FcγRIIb on monocytes from fresh PBMCs, and taken after 18 and 36 hours of HD preculture, and 36 hours of LD preculture, and on B cells from fresh PBMCs and after HD preculture. (B) Time course of the increase in FcγRIIb expression on monocytes during HD preculture. In contrast, B cells show no change in FcγRIIb expression during HD preculture. Bars show mean and range of 2 donors. Analysis in A and B was using a FACSCantoII. (C) Western blot showing an increase in the expression of the b2 isoform of FcγRIIb during HD preculture. (D) Comparison of FcγRIIb expression on fresh and HD precultured monocytes. (E) Plots showing relationship between FcγRIIb expression on monocytes after HD preculture and proliferation and TNFα release in response to TGN1412; n = 14, analyzed using 2-tailed Spearman rank correlation (Graphpad Prism 6). FcγRIIb expression and proliferation plots from all donors are shown in supplemental Figure 4. Analysis in D and E was using a FACSCalibur flow cytometer.