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.

Interleukin (IL)-6 is produced by professional antigen-presenting cells (APCs) such as B cells, macrophages, and dendritic cells. It has been previously shown that APC-derived IL-6 promotes the differentiation of naive CD4+ T cells into effector T helper type 2 (Th2) cells. Here, we have studied the molecular mechanism for IL-6–mediated Th2 differentiation. During the activation of CD4+ T cells, IL-6 induces the production of IL-4, which promotes the differentiation of these cells into effector Th2 cells. Regulation of IL-4 gene expression by IL-6 is mediated by nuclear factor of activated T cells (NFAT), as inhibition of NFAT prevents IL-6–driven IL-4 production and Th2 differentiation. IL-6 upregulates NFAT transcriptional activity by increasing the levels of NFATc2. The ability of IL-6 to promote Th2 differentiation is impaired in CD4+ T cells that lack NFATc2, demonstrating that NFATc2 is required for regulation of IL-4 gene expression by IL-6. Regulation of NFATc2 expression and NFAT transcriptional activity represents a novel pathway by which IL-6 can modulate gene expression.

The A20 gene product is a novel zinc finger protein originally described as a tumor necrosis factor ␣ (TNF)-inducible early response gene in human umbilical vein endothelial cells (HUVEC). Its described function is to block TNF-induced apoptosis in fibroblasts and B lymphocytes, but more recently it has also been shown to play a role in lymphoid cell maturation. The mechanism of action of A20 is unknown. The aim of our study was to assess the effect of A20 upon endothelial cell activation. By transfecting bovine aortic endothelial cells (BAEC) with A20 as well as reporter constructs consisting of the promoters of genes known to be up-regulated during endothelial cell activation, i.e. E-selectin, interleukin (IL)-8, tissue factor (TF), and inhibitor of nuclear factor B␣ (IB␣), we demonstrate that A20 expression inhibits gene up-regulation associated with TNF, lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), and hydrogen peroxide (H 2 O 2 )-induced endothelial cell (EC) activation. The mechanism of action of A20 is in part, or totally, due to the blockade of nuclear factor B (NF-B), as shown by its ability to suppress the activity of a NF-B reporter. This effect is specific, as A20 does not block a noninducible, constitutively expressed reporter, Rous sarcoma virus-luciferase (RSV-LUC); nor does it block the c-Tat-inducible, NF-B-independent reporter, human immunodeficiency virus-chloramphenicol acetyltransferase (HIV-CAT). How A20 blocks NF-B is unclear, although we demonstrate that it does not affect p65 (RelA)-mediated gene transactivation. The inhibition of endothelial cell activation by A20 is a novel function for A20.

The phenotype of NFATc2−/− c3−/− (double knockout [DKO]) mice implies a disturbed regulation of T cell responses, evidenced by massive lymphadenopathy, splenomegaly, and autoaggressive phenomena. The population of CD4+ CD25+ T cells from DKO mice lacks regulatory capacity, except a small subpopulation that highly expresses glucocorticoid-induced tumor necrosis factor receptor family–related gene (GITR) and CD25. However, neither wild-type nor DKO CD4+ CD25+ regulatory T cells (T reg cells) are able to suppress proliferation of DKO CD4+ CD25− T helper cells. Therefore, combined NFATc2/c3 deficiency is compatible with the development of CD4+ CD25+ T reg cells but renders conventional CD4+ T cells unresponsive to suppression, underlining the importance of NFAT proteins for sustaining T cell homeostasis.