Proliferation of alloreactive T cells induced by mature DCs in patients with CD40 deficiency. (A) Proliferation of alloreactive T cells induced by mature DCs: allogeneic MLR. T cells were purified by negative selection from healthy donor PBMCs and were cultured with LPS + IFN-γ-matured DCs from controls and HIGM3 patients at 3 different ratios. DCs were 0.5%, 3%, and 6% of T cells (see “Patients, materials, and methods”). After 5 days at 37°C, proliferation of alloreactive T cells was assessed by [³H]-thymidine uptake. Results are expressed as mean cpm ± SD of 1 experiment performed in triplicate. ^*Significant difference in the control subject in comparison with patients as assessed by statistical analysis (P < .05). ♦ indicates healthy control; □, HIGM3 patient 1; and ▴, HIGM3 patient 2. (B) Proliferation of alloreactive naive T cells induced by TNF-α, LPS, or LPS+IFN-γ-matured DCs: allogeneic MLR. Naive lymphocytes were purified by negative selection from healthy donor PBMCs and cultured (1.5 × 10⁵ cells per well) with soluble TNF-α (light gray bars), LPS (black bars), or LPS + IFN-γ-matured (dark gray bars) DCs (5 × 10³ cells/well) from control subjects (adult and child) and HIGM3 patient 1, in the presence of 1 ng/mL SEB (see “Patients, materials, and methods”). After 5 days at 37°C, we measured proliferation of alloreactive naive T cells. The formation of a formazan salt was measured using an ELISA reader, as described in “Patients, materials, and methods.” Results are expressed as extinction coefficient at 450 nm (mean ± SD of an experiment performed in triplicate). Adult and child data are from control subjects. These MLR experiments were performed in 2 different laboratories. Data are from 1 of 3 representative, independent experiments. ^*Significant difference in the patient compared with healthy subjects as assessed by statistical analysis (P < .05).