Neutralization of proinflammatory cytokines does not restore Treg suppression.

<p>A: Production of IFN-α by activated pDC. Supernatants of activated pDC (CpG/IL-3) or cDC (cytokine cocktail) cultures were harvested. Production of IFN-α was investigated by ELISA. Each circle resembles one individual experiment, bar represents mean; n.d.: not detectable, n = 4. B: Neutralization of cytokines in cocultures of Teff, Treg and pDC. Teff plus Treg (1∶1) were stimulated with allogeneic pDC at a ratio of 1∶5 in absence (Ø) or presence of neutralizing antibodies against IFN-α, IL-1β, IL-6 or TNF-α (10 µg/ml each). Proliferation in cocultures (mean ± SD of triplicates) is normalized to untreated cocultures (set to 100%). One representative experiment out of three is shown. C: Addition of supernatants from standard cultures. CD4⁺ Teff and Treg (1∶1) were stimulated with anti-CD3 mAb (0.5 µg/ml) plus irradiated TC-depleted PBMC. Supernatants from these cocultures were collected on day 3 and titrated (1∶2) to pDC-stimulated CD4⁺ Teff, Treg or cocultures. Proliferation was assessed on day 3 by ³H-Tdr-incorporation. Diagrams show mean values (± SD) of three independent experiments. Proliferation of untreated and treated cocultures was normalized to untreated or treated Teff-proliferation (set to 100%).