Signaling through the murine T cell receptor induces IL-17 production in the absence of costimulation, IL-23 or dendritic cells.

IL-17 (IL-17A or CTLA-8) is the founding member of a novel family of inflammatory cytokines, and emerging evidence indicates that it plays a central role in inflammation and autoimmunity. IL-17 is made primarily, if not exclusively by T cells, but relatively little is known about how its expression is regulated. In the present study, we examined the requirements and mechanisms for IL-17 expression in primary mouse lymphocytes. Like many cytokines, IL-17 is induced rapidly in primary T cells after stimulation of the T cell receptor (TCR) through CD3 crossinking. Surprisingly, however, the pattern of regulation of IL-17 is different in mice than in humans, because "costimulation" of T cells through CD28 only mildly enhanced IL-17 expression, whereas levels of IL-2 were dramatically enhanced. Similarly, several other costimulatory molecules such as ICOS, 4-1BB and CD40L exerted only very weak enhancing effects on IL-17 production. In agreement with other reports, IL-23 enhanced CD3-induced IL-17 expression. However, IL-17 production can occur autonomously in T cells, as neither dendritic cells nor IL-23 were necessary for promoting short-term production of IL-17. Finally, to begin to characterize the TCR-mediated signaling pathway(s) required for IL-17 production, we showed that IL-17 expression is sensitive to cyclosporin-A and MAPK inhibitors, suggesting the involvement of the calcineurin/NFAT and MAPK signaling pathways.

1,25-dihydroxyvitamin D(3) ameliorates Th17 autoimmunity via transcriptional modulation of interleukin-17A.

A new class of inflammatory CD4(+) T cells that produce interleukin-17 (IL-17) (termed Th17) has been identified, which plays a critical role in numerous inflammatory conditions and autoimmune diseases. The active form of vitamin D, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], has a direct repressive effect on the expression of IL-17A in both human and mouse T cells. In vivo treatment of mice with ongoing experimental autoimmune encephalomyelitis (EAE; a mouse model of multiple sclerosis) diminishes paralysis and progression of the disease and reduces IL-17A-secreting CD4(+) T cells in the periphery and central nervous system (CNS). The mechanism of 1,25(OH)(2)D(3) repression of IL-17A expression was found to be transcriptional repression, mediated by the vitamin D receptor (VDR). Transcription assays, gel shifting, and chromatin immunoprecipitation (ChIP) assays indicate that the negative effect of 1,25(OH)(2)D(3) on IL-17A involves blocking of nuclear factor for activated T cells (NFAT), recruitment of histone deacetylase (HDAC), sequestration of Runt-related transcription factor 1 (Runx1) by 1,25(OH)(2)D(3)/VDR, and a direct effect of 1,25(OH)(2)D(3) on induction of Foxp3. Our results describe novel mechanisms and new concepts with regard to vitamin D and the immune system and suggest therapeutic targets for the control of autoimmune diseases.

Characterization of B7S3 as a novel negative regulator of T cells.

T cell activation by APCs is regulated by B7-like costimulatory molecules. In this study, we describe a new B7 superfamily member, B7S3, with two differentially spliced isoforms expressed in lymphoid and nonlymphoid tissues. A soluble B7S3-Ig protein bound to professional APC constitutively as well as to activated but not naive T cells. B7S3-Ig treatment greatly inhibited T cell proliferation and IL-2 production. B7S3-Ig also reduced cytokine production by effector T cells. Interestingly, although human genome appears to contain a single-copy B7S3 homolog, the mouse B7S3 gene has 10 relatives within a 2-Mb region constituting a B7S3 gene family. This study identifies B7S3 as a novel negative regulator of T cells, and suggests evolutionarily divergent T cell regulation mechanisms in mammals.

BTNL2, a butyrophilin-like molecule that functions to inhibit T cell activation.

B7 family members regulate T cell activation and tolerance. Although butyrophilin proteins share sequence homology with the B7 molecules, it is unclear whether they have any function in immune responses. In the present study, we characterize an MHC class II gene-linked butyrophilin family member, butyrophilin-like 2 (BTNL2), the mutation of which has been recently associated with the inflammatory autoimmune diseases sarcoidosis and myositis. Mouse BTNL2 is a type I transmembrane protein with two pairs of Ig-like domains separated by a heptad peptide sequence. BTNL2 mRNA is highly expressed in lymphoid tissues as well as in intestine. To characterize the function of BTNL2, we produced a BTNL2-Ig fusion protein. It recognized a putative receptor whose expression on B and T cells was significantly enhanced after activation. BTNL2-Ig inhibited T cell proliferation and TCR activation of NFAT, NF-kappaB, and AP-1 signaling pathways. BTNL2 is thus the first member of the butyrophilin family that regulates T cell activation, which has implications in immune diseases and immunotherapy.

Crucial role for nuclear factor of activated T cells in T cell receptor-mediated regulation of human interleukin-17.

The biological activities of the inflammatory cytokine interleukin (IL)-17 have been widely studied. However, comparatively little is known about how IL-17 expression is controlled. Here, we examined the basis for transcriptional regulation of the human IL-17 gene. IL-17 secretion was induced in peripheral blood mononuclear cells following anti-CD3 cross-linking to activate the T cell receptor (TCR), and costimulatory signaling through CD28 strongly enhanced CD3-induced IL-17 production. To define cis-acting elements important for IL-17 gene regulation, we cloned 1.25 kb of genomic sequence upstream of the transcriptional start site. This putative promoter was active in Jurkat T cells following CD3 and CD28 cross-linking, and its activity was inhibited by cyclosporin A and MAPK inhibitors. The promoter was also active in Hut102 T cells, which we have shown to secrete IL-17 constitutively. Overexpression of nuclear factor of activated T cells (NFAT) or Ras enhanced IL-17 promoter activity, and studies in Jurkat lines deficient in specific TCR signaling pathways provided supporting evidence for a role for NFAT. To delineate the IL-17 minimal promoter, we created a series of 5' truncations and identified a region between -232 and -159 that was sufficient for inducible promoter activity. Interestingly, two NFAT sites were located within this region, which bound to NFATc1 and NFATc2 in nuclear extracts from Hut102 and Jurkat cells. Moreover, mutations of these sites dramatically reduced both specific DNA binding and reporter gene activity, and chromatin immunoprecipitation assays showed occupancy of NFAT at this region in vivo. Together, these data show that NFAT is the crucial sensor of TCR signaling in the IL-17 promoter.

Functional cooperation between interleukin-17 and tumor necrosis factor-alpha is mediated by CCAAT/enhancer-binding protein family members.

Interleukin (IL)-17 is a recently described cytokine involved in the amplification of inflammatory responses and pathologies. A hallmark feature of IL-17 is its ability to induce expression of other cytokines and chemokines. In addition, IL-17 potently synergizes with tumor necrosis factor-alpha (TNFalpha) to up-regulate expression of many target genes, particularly IL-6. Despite the many observations of IL-17 signaling synergy observed to date, little is known about the molecular mechanisms that underlie this phenomenon. In the osteoblastic cell line MC-3T3, we have found that IL-17 and TNFalpha exhibit potent synergy in mediating IL-6 secretion. Here, we show that at least part of the functional cooperation between IL-17 and TNFalpha occurs at the level of IL-6 gene transcription. Both the NF-kappaB and CCAAT/enhancer-binding protein (C/EBP; NF-IL6) sites in the IL-6 promoter are important for cooperative gene expression, but NF-kappaB does not appear to be the direct target of the combined signal. Microarray analysis using the Affymetrix mouse MG-U74v2 chip identified C/EBPdelta as another gene target of combined IL-17- and TNFalpha-induced signaling. Because C/EBP family members are known to control IL-6, we examined whether enhanced C/EBPdelta expression is involved in the cooperative up-regulation of IL-6 by IL-17 and TNFalpha. Accordingly, we show that C/EBPdelta (or the related transcription factor C/EBPbeta) is essential for expression of IL-6. Moreover, overexpression of C/EBPdelta (and, to a lesser extent, C/EBPbeta) could substitute for the IL-17 signal at the level of IL-6 transcription. Thus, C/EBP family members, particularly C/EBPdelta, appear to be important for the functional cooperation between IL-17 and TNFalpha.