Physiologic ligand binding of K562 stably expressing WT and βTD mutant CD11b/CD18. (A) Cell surface expression of CD11b/CD18 on K562 cells using flow cytometry. K562 cells were immunostained with the primary heterodimer-specific mAb IB4 (thick lines, unshaded area) or isotype-matched control mAb MOPC-173 (gray shaded area), washed, developed with FITC-rat anti–mouse IgG2a, and analyzed counting 10 000 events. Median fluorescence intensity (MFI) for mAb IB4 staining is shown in each panel. (B) Histograms showing the relative binding of iC3b to WT and mutant CD11b/CD18 in buffer containing 5 mM EDTA, 1 mM each of Ca²⁺ and Mg²⁺, or 1 mM Mn²⁺ expressed as the number of cells showing rosettes (more than 3 EiC3b bound per K562 cell) as a percentage of total number of cells counted in a rosetting assay. Each histogram represents mean ± SD of triplicate determinations from a representative experiment (1 of 3 performed). No binding was observed in EDTA and with mock-transfected cells. The methods are detailed in “Materials and methods.” (C) A titration ligand binding curve showing ligand binding (% rosetting) of an increasing number of EiC3b to a constant number of WT or NGTD mutant CD11b/CD18-expressing K562 cells in buffer containing 1 mM each of Ca²⁺ and Mg²⁺ or 1 mM Mn²⁺ (as labeled). Each data point represents mean ± SD of triplicate determinations. Not shown are rosetting results from mock-transfected cells (0 rosettes under all 3 buffer conditions). The graph shows that NGTD-expressing cells have a higher percentage of rosetting in physiologic buffer cells under all conditions tested as compared with WT cells. The difference is greatest when ligand is limiting (at EiC3b/K562 cell ratio of about 18). No difference is seen in mutant integrin binding to iC3b in the presence of Ca²⁺+Mg²⁺ or Mn²⁺ at all ratios used.