Effects of anti-CD154 mAb on GPIIb/IIIa-induced T-cell responses in bulk PBMC cultures and GPIIb/IIIa-reactive CD4⁺ T-cell lines.

(A) PBMCs from patient ITP14 were stimulated with trypsin-digested GPIIb/IIIa (5 μg/mL) for 7 days in the presence or absence of anti–HLA-DR (1 μg/mL), anti–HLA-DQ (1 μg/mL), anti-CD154 (0.5, 1, and 2 μg/mL), or isotype-matched control mAb (2 μg/mL), and [³H]thymidine incorporation was measured by liquid scintillation counting. The results shown are the means ± SDs of quadruplicate values. Asterisk indicates significant inhibition of T-cell proliferation by the mAb treatment in comparison with the culture with isotype-control mAb. (B) PBMCs from ITP14 were cultured with trypsin-digested GPIIb/IIIa (5 μg/mL) and pokeweed mitogen (1 μg/mL) for 10 days in the presence or absence of anti–HLA-DR (1 μg/mL), anti–HLA-DQ (1 μg/mL), anti-CD154 (0.25, 0.5, and 1 μg/mL), or isotype-matched control mAb (1 μg/mL), and IgG anti-GPIIb/IIIa antibody levels were measured by ELISA. The results shown are the means ± SDs of duplicate values. Asterisks indicate significant inhibition of IgG anti-GPIIb/IIIa antibody production by the mAb treatment compared with the culture with isotype-matched control mAb. (C) GPIIb/IIIa-reactive CD4⁺ T-cell lines SiM4 and WY9 (2 × 10⁵) were repeatedly treated with irradiated autologous lymphoblastoid B-cell line (10⁶), recombinant fragment (IIbα18-252 for SiM4 or IIIa22-262 for WY9; 5 μg/mL), and IL-2 (50 U/mL) in the presence of anti-CD154 or isotype-control mAb (2 μg/mL), and examined for their capacity to proliferate on stimulation with the antigenic recombinant fragment or glutathione S-transferase in quadruplicate cultures. Results are expressed as a stimulation index, which was calculated as the mean counts per minute incorporated into cultures with the antigenic recombinant fragment divided by the mean counts per minute incorporated into the cultures with glutathione S-transferase. A result representative of 3 independent experiments is shown. (D) Lines SiM4 and WY9, which were repeatedly treated with anti-CD154 or isotype-matched control mAb, were examined for their capacity to stimulate autologous B cells to produce IgG anti-GPIIb/IIIa antibodies on stimulation with IIbα18-252 (SiM4) or IIIa22-262 (WY9). Results shown are the means ± SDs of duplicate values. Asterisks indicate significant inhibition of anti-GPIIb/IIIa antibody production by treatment with anti-CD154 mAb compared with the treatment with isotype-control mAb. The IgG anti-GPIIb/IIIa antibody levels in cultures of B cells alone were 0.020 ± 0.005 (SiM4) and 0.085 ± 0.011 (WY9). The results were similar in 3 independent experiments.