IL-2-independent activation and proliferation in human T cells induced by CD28.

Although the role of CD28 in T cell costimulation is firmly established, the mechanisms by which it exerts its costimulatory actions are less clear. In many circumstances it is difficult to distinguish the effects of CD28 from subsequent actions of cytokines, such as IL-2, on T cell proliferation. Here, we report a model of CD28 costimulation using PMA plus the natural ligand CD80 that resulted in very limited stimulation of IL-2, as evidenced by both cytokine production and IL-2 promoter stimulation. Promoter assays revealed CD28-dependent effects on both NF-kappaB and AP-1, but not on NF-AT or the intact IL-2 promoter. In addition, T cell proliferation was completely resistant to the actions of the immunosuppressant cyclosporin A (CsA). Moreover T cell proliferation was unaffected by the addition of blocking Abs to both IL-2 and the IL-2 receptor, demonstrating that this form of costimulation by CD28 was independent of IL-2. We also investigated the effects of stimulating T cell blasts with CD80 alone and found that there was a limited requirement for IL-2 in this system. We conclude that CD28 costimulation can cause substantial T cell proliferation in the absence of IL-2, which is driven by a soluble factor independent of NF-AT transactivation.     

A prominent role for Sp1 during lipopolysaccharide-mediated induction of the IL-10 promoter in macrophages

IL-10 is an antiinflammatory cytokine secreted by activated macrophages and Th2 cells. IL-10 secretion promotes the down-regulation of proinflammatory cytokine synthesis and the development of Th2 responses. In macrophages, proinflammatory cytokines appear to be induced by similar mechanisms, but the IL-10 induction mechanisms have not been examined. We have analyzed the murine IL-10 promoter in the RAW264.7 macrophage line activated with LPS. A comprehensive mutant analysis revealed only one element upstream of the core promoter that was essential for promoter induction. A refined mutant analysis localized this element to nucleotides -89 to -78, and gel shift experiments revealed that it represents a nonconsensus binding site for Sp1. The functional relevance of Sp1 was supported by the high affinity of the interaction, the close correlation between the nucleotides required for Sp1 binding and promoter function, and the ability of an Sp1 consensus sequence to substitute for the -89/-78 promoter sequence. Evidence that Sp1 may be a target of signaling pathways involved in IL-10 induction was provided by the exclusive requirement for the Sp1 binding site, by the ability of the Sp1 site to confer induction to a heterologous promoter, and by the delineation of an Sp1 domain that can mediate induction. No relevant contribution from Rel, C/EBP (CCAAT/enhancer-binding protein), or AP-1 binding sites, which regulate most proinflammatory cytokine promoters, was observed. Together, these results demonstrate that IL-10 gene regulation is distinct from the regulation of proinflammatory cytokine genes, and suggest that Sp1 may be a central mediator of IL-10 induction.     

Signals from CD28 induce stable epigenetic modification of the IL-2 promoter

CD28 costimulation controls multiple aspects of T cell function, including the expression of proinflammatory cytokine genes. One of these genes encodes IL-2, a growth factor that influences T cell proliferation, survival, and differentiation. Antigenic signaling in the absence of CD28 costimulation leads to anergy, a mechanism of tolerance that renders CD4+ T cells unable to produce IL-2. The molecular mechanisms by which CD28 costimulatory signals induce gene expression are not fully understood. In eukaryotic cells, the expression of many genes is influenced by their physical structure at the level of DNA methylation and local chromatin remodeling. To address whether these epigenetic mechanisms are operative during CD28-dependent gene expression in CD4+ T cells, we compared cytosine methylation and chromatin structure at the IL-2 locus in fully activated CD4+ effector T cells and CD4+ T cells rendered anergic by TCR ligation in the absence of CD28 costimulation. Costimulation through CD28 led to marked, stable histone acetylation and loss of cytosine methylation at the IL-2 promoter/enhancer. This was accompanied by extensive remodeling of the chromatin in this region to a structure highly accessible to DNA binding proteins. Conversely, TCR activation in the absence of CD28 costimulation was not sufficient to promote histone acetylation or cytosine demethylation, and the IL-2 promoter/enhancer in anergic cells remained completely inaccessible. These data suggest that CD28 may function through epigenetic mechanisms to promote CD4+ T cell responses.     

Cutting edge: stromal cell-derived factor-1 is a costimulator for CD4+ T cell activation

Stromal cell-derived factor (SDF)-1 is a chemoattractant for T cells, precursor B cells, monocytes, and neutrophils. SDF-1alpha was also found to up-regulate expression of early activation markers (CD69, CD25, and CD154) by anti-CD3-activated CD4+ T cells. In addition, SDF-1alpha costimulated proliferation of CD4+ T cells and production of IL-2, IFN-gamma, IL-4, and IL-10. Stimulation with SDF-1alpha alone did not induce activation marker expression, proliferation, or cytokine production by the CD4+ T cells. SDF-1alpha-mediated costimulation was blocked by anti-CXC chemokine receptor-4 mAb. RANTES also increased activation marker expression by anti-CD3-stimulated peripheral CD4+ T cells, but less effectively than SDF-1alpha did, and did not up-regulate IL-2 production and proliferation. These results indicate that SDF-1 and CXC chemokine receptor-4 interactions not only play a role in T cell migration but also provide potent costimulatory signals to Ag-stimulated T cells.     

Mice lacking endogenous IL-10-producing regulatory B cells develop exacerbated disease and present with an increased frequency of Th1/Th17 but a decrease in regulatory T cells

IL-10-producing B cells, also known as regulatory B cells (Bregs), play a key role in controlling autoimmunity. In this study, we report that chimeric mice specifically lacking IL-10-producing B cells (IL-10(-/-)B cell) developed an exacerbated arthritis compared with chimeric wild-type (WT) B cell mice. A significant decrease in the absolute numbers of Foxp3 regulatory T cells (Tregs), in their expression level of Foxp3, and a marked increase in inflammatory Th1 and Th17 cells were detected in IL-10(-/-) B cell mice compared with WT B cell mice. Reconstitution of arthritic B cell deficient (μMT) mice with different B cell subsets revealed that the ability to modulate Treg frequencies in vivo is exclusively restricted to transitional 2 marginal zone precursor Bregs. Moreover, transfer of WT transitional 2 marginal zone precursor Bregs to arthritic IL-10(-/-) mice increased Foxp3(+) Tregs and reduced Th1 and Th17 cell frequencies to levels measured in arthritic WT mice and inhibited inflammation. In vitro, IL-10(+/+) B cells established longer contact times with arthritogenic CD4(+)CD25(-) T cells compared with IL-10(-/-) B cells in response to Ag stimulation, and using the same culture conditions, we observed upregulation of Foxp3 on CD4(+) T cells. Thus, IL-10-producing B cells restrain inflammation by promoting differentiation of immunoregulatory over proinflammatory T cells.     

IL-10 inhibits human T cell proliferation and IL-2 production.

Human IL-10 has been reported previously to inhibit the secretion of IFN-gamma in PBMC. In this study, we have found that human IL-10 inhibits T cell proliferation to either mitogen or anti-CD3 mAb in the presence of accessory cells. Inhibited T cell growth by IL-10 was associated with reduced production of IFN-gamma and IL-2. Studies of T cell subset inhibition by human IL-10 showed that CD4+, CD8+, CD45RA high, and CD45RA low cells are all growth inhibited to a similar degree. Dose response experiments demonstrated that IL-10 inhibits secretion of IFN-gamma more readily than T cell proliferation to mitogen. In addition, IL-2 and IL-4 added exogenously to IL-10 suppressed T cell cultures reversed completely the inhibition of T cell proliferation, but had little or no effect on inhibition of IFN-gamma production. Thus, in addition to its previously reported biologic properties, IL-10 inhibits human T cell proliferation and IL-2 production in response to mitogen. Inhibition of IFN-gamma production by IL-10 appears to be independent of the cytokine effect of IL-2 production.     

Foxp3 inhibits RORgammat-mediated IL-17A mRNA transcription through direct interaction with RORgammat

The cytokine, transforming growth factor-beta1 (TGF-beta1), converts naive T cells into regulatory T cells that prevent autoimmunity. However, in the presence of interleukin (IL)-6, TGF-beta1 has also been found to promote differentiation into IL-17-producing helper T (Th17) cells that are deeply involved in autoimmunity and inflammation. However, it has not been clarified how TGF-beta1 and IL-6 determine such a distinct fate. Here we found that a master regulator for Th17, retinoic acid-related orphan receptor gammat (RORgammat), was rapidly induced by TGF-beta1 regardless of the presence of IL-6. IL-6 reduced Foxp3 expression, and overexpression of Foxp3 in a T cell line resulted in a strong reduction of IL-17A expression. We have characterized the IL-17A promoter and found that RORgammat binding is sufficient for activation of the minimum promoter in the HEK 293T cells. RORgammat-mediated IL-17A promoter activation was suppressed by forced expression of Foxp3. Foxp3 directly interacted with RORgammat through exon 2 region of Foxp3. The exon 2 region and forkhead (FKH) domain of Foxp3 were necessary for the suppression of RORgammat-mediated IL-17A promoter activation. We propose that induction of Foxp3 is the mechanism for the suppression of Th17 and polarization into inducible Treg.