Production of PF4/heparin-specific antibodies by PBMCs from healthy human adults or by splenic B cells from unmanipulated wild-type mice following in vitro polyclonal stimulation. PBMCs isolated from healthy donors were cultured at concentrations of 1 × 105, 2 × 104, or 4 × 103 cells per well in 24 or 36 wells of a 96-well plate in the presence of CpG. After 6 to 7 days of culture, supernatants were collected and tested for hPF4/heparin-specific IgM production by ELISA. (A) A representative experiment performed with PBMCs from 1 healthy donor. Each dot represents 1 well, and the horizontal lines indicate the mean values. Based on the values obtained from nonstimulated PBMCs, OD values above the cutoff value of 0.3 were considered positive. Data shown are representative of 16 healthy donors. (B) Antigen specificity of the IgMs. Specificity of the IgMs toward hPF4/heparin complexes, hPF4 alone, or hPF4 in the presence of excess heparin was examined. Data shown are from 6 positive wells with 2 × 104 cells in A and are representative of positive wells from 16 healthy donors. (C-D) Splenic B cells isolated from unmanipulated wild-type mice were cultured at concentrations of 5 × 104 or 1 × 104 cells per well in 12 wells of a 96-well plate in the presence of CpG or lipopolysaccharide (LPS). After 4 days of culture, supernatants were collected and tested for mPF4/heparin-specific IgM production by ELISA. (C) A representative CpG stimulation experiment performed with 1 wild-type mouse is shown. Each dot represents 1 well, and the horizontal lines indicate the mean values. Data shown are representative of 3 independent experiments. (D) A representative LPS stimulation experiment performed with 1 wild-type mouse is shown. Each dot represents 1 well, and the horizontal lines indicate the mean values. Data shown are representative of 3 independent experiments with 3 wild-type mice. (E-F) Antigen specificity of the IgMs. Specificity of the IgMs generated by (E) CpG or (F) LPS stimulation toward mPF4/heparin complexes, mPF4 alone, or mPF4 in the presence of excess heparin was examined. Data shown are from 6 positive wells with 5 × 104 B cells stimulated with CpG in C (left) or stimulated with LPS in D (right) and are representative of positive wells from 3 independent experiments with 3 wild-type mice. Statistical analysis was performed with (A,C,D) the Mann-Whitney test for unpaired data and (B,E,F) the Wilcoxon signed-rank test for paired data.
