Sabine Stoll

T cell immune responses begin within organized lymphoid tissues. The pace, topology, and outcomes of the cellular interactions that underlie these responses have, so far, been inferred from static imaging of sectioned tissue or from studies of cultured cells. Here we report dynamic visualization of antigen-specific T cells interacting with dendritic cells within intact explanted lymph nodes. We observed immunological synapse formation and prolonged interactions between these two cell types, followed by the activation, dissociation, and rapid migration of T cells away from the antigenic stimulus. This high-resolution spatiotemporal analysis provides insight into the nature of cell interactions critical to early immune responses within lymphoid structures.

Naturally occurring regulatory T cells (T reg cells) are a thymus-derived subset of T cells, which are crucial for the maintenance of peripheral tolerance by controlling potentially autoreactive T cells. However, the underlying molecular mechanisms of this strictly cell contact-dependent process are still elusive. Here we show that naturally occurring T reg cells harbor high levels of cyclic adenosine monophosphate (cAMP). This second messenger is known to be a potent inhibitor of proliferation and interleukin 2 synthesis in T cells. Upon coactivation with naturally occurring T reg cells the cAMP content of responder T cells is also strongly increased. Furthermore, we demonstrate that naturally occurring T reg cells and conventional T cells communicate via cell contact-dependent gap junction formation. The suppressive activity of naturally occurring T reg cells is abolished by a cAMP antagonist as well as by a gap junction inhibitor, which blocks the cell contact-dependent transfer of cAMP to responder T cells. Accordingly, our results suggest that cAMP is crucial for naturally occurring T reg cell-mediated suppression and traverses membranes via gap junctions. Hence, naturally occurring T reg cells unexpectedly may control the immune regulatory network by a well-known mechanism based on the intercellular transport of cAMP via gap junctions.

IL-18 is a recently described cytokine that shares biological activities with IL-12 in driving the development of Th1-type T cells. As dendritic cells (DC) are very potent inducers of T cell proliferation and differentiation we wondered whether they utilize IL-18 as a factor driving Th1 development. We demonstrate by Northern blot and reverse transcription-PCR that various subtypes of human and murine DC as well as the DC-line XS contain IL-18 mRNA. When supernatants of either enriched Langerhans cells (LC) or bone marrow-derived DC were analyzed for production of IL-18 protein, IL-18 production was detected in an IL-18-specific ELISA. To assess whether the IL-18 protein released by DC is functional, we performed a sensitive bioassay using the IL-18-dependent stimulation of concanavalin A-stimulated T cells. Both, supernatants from bone marrow-derived DC and enriched LC induced IFN-gamma production in the T cells. This production was partially inhibitable by addition of anti-IL-18 antiserum. In a TCR-transgenic mouse system we further demonstrate that DC-derived IL-18 potentiates IL-12-dependent Th1 development. Using DC derived from IL-12 knockout animals, we show that DC-derived IL-18 by itself is not capable of inducing Th1 cell differentiation. Together the data demonstrate that subtypes of DC are able to release functional IL-18 that is able to induce IFN-gamma production and Th1 differentiation in primed T cells.

Recently, the novel cytokine IL-18 (IFN-gamma-inducing factor) has been described as a growth and differentiation factor for Th1 cells. Epidermal keratinocytes (KC) are known to direct T cell education by production of cytokines. Therefore, expression of IL-18 was sought in KC. Epidermal RNA was analyzed following stimulation with contact sensitizers or controls for IL-18 mRNA expression by semiquantitative reverse transcription-PCR. Constitutive expression of IL-18 mRNA was low in untreated epidermal cells (EC), but early up-regulation of IL-18 mRNA signals was detected following application of a contact allergen in vivo. The peak strength of IL-18 signals was observed within 4 to 6 h following stimulation with an allergen. Application of an irritant (benzalconiumchloride) or solvents did not result in increased signal strength. To determine the cellular origin of IL-18 mRNA in EC, depletion experiments were performed. IL-18 signals were not affected by depletion with anti-CD3 (T cells) or anti-MHC class II mAb-coupled beads identifying KC as a major source of IL-18. These results were confirmed by analysis of mRNA derived from the KC cell line PAM 212. Strong IL-18 signals could be detected by reverse transcription-PCR. To delineate whether IL-18 protein was produced by EC/KC, a sandwich ELISA was used to assay for IL-18 production. Supernatants from allergen-stimulated EC and KC showed production of IL-18 protein. To confirm that IL-18 protein was functional, EC or KC supernatants were tested for their ability to induce IFN-gamma production. Significant amounts of IFN-gamma were detected in supernatants of allergen-treated cells. In aggregate, our data indicate that murine KC are a source of both IL-18 mRNA and functional protein.