Journal: The Journal of Experimental Medicine
Article Title: X-linked susceptibility to mycobacteria is caused by mutations in NEMO impairing CD40-dependent IL-12 production
Figure Lengend Snippet: The impact of CD40 and CD40L deficiency on IL-12 and IFN-γ production. IL-12p70, IL-12p40, and IFN-γ production, measured by classical sandwich ELISA, in a mixture of purified monocytes and T cells, as indicated, upon stimulation with PHA from a healthy donor with (A) CD40L- and (B) CD40-deficient PBMCs. MDDCs obtained from two healthy controls (C2 and C3), P2 and P3, after 24 h of incubation alone or with LPS (1 μg/ml) plus IL-1β (10 ng/ml) as a control of CD40-independent activation, cocultured with L-cells transfected with human CD40L (L-cell-hCD40L) and nontransfected L-cells (L-cell): (C) IL-12p70, IL-12p40, TNF-α, and IL-6 production, measured by classical sandwich ELISA, and (D) FACS analysis of cell surface expression of CD40, CD80, and CD86 costimulatory molecules gating on CD1a-positive MDDCs. (E) Intracellular staining of NEMO protein in MDDCs from P2 and P3, and their respective controls (C2 and C3) in the experimental conditions of Fig. 3 . (F) IgE secretion by B cells in vitro, as measured by ELISA, after the activation of PBMCs from 20 healthy controls (C+, •), P2 (□), and a CD40-deficient patient as the negative control (C−,▵), with soluble CD40L in combination with IL-4. The results shown are representative of two independent experiments.
Article Snippet: Whole blood samples were diluted 1/2 in RPMI 1640 (GIBCO BRL) and infected by incubation with live M. bovis BCG (Pasteur substrain), at a multiplicity of infection of 20:1, alone or with recombinant IFN-γ (5,000 IU/ml; Imukin, Boehringer Ingelheim) or recombinant IL-12p70 (20 ng/ml; R & D Systems), LPS (from Salmonella minnesota , 1 μg/ml; Sigma-Aldrich), TNF-α (20 ng/ml; R & D Systems), IL-1β (10 ng/ml; R & D Systems), phorbol 12-myristate 13-acetate (PMA) (10−7 M; Sigma-Aldrich) with ionomycin (10−5 M; Sigma-Aldrich) and supernatants were recovered after 14 and 48 h. Cytokine production was normalized according to the number of PBMCs in the individual tested.
Techniques: Sandwich ELISA, Purification, Incubation, Activation Assay, Transfection, FACS, Expressing, Staining, In Vitro, Enzyme-linked Immunosorbent Assay, Negative Control