The majority of the genome is noncoding and was thought to be nonfunctional. However, it is now appreciated that transcriptional control of protein coding genes resides within these noncoding regions. Thousands of genes encoding long intergenic noncoding RNAs (lincRNAs) have been recently identified throughout the genome, which positively or negatively regulate transcription of neighboring target genes. Both TMEVPG1 and its mouse ortholog encode lincRNAs and are positioned near the IFN-γ gene (IFNG). In this study, we show that transcription of both mouse and human TMEVPG1 genes is Th1 selective and dependent on Stat4 and T-bet, transcription factors that drive the Th1 differentiation program. Ifng expression is partially restored in Stat4-/-Tbx21-/- cells through coexpression of T-bet and Tmevpg1, and Tmevpg1 expression contributes to, but alone is not sufficient to, drive Th1-dependent Ifng expression. Our results suggest that TMEVPG1 belongs to the general class of lincRNAs that positively regulate gene transcription.

Positive regulatory factors induced by IL-12/STAT4 and IL-4/STAT6 signaling during T cell development contribute to polarized patterns of cytokine expression manifested by differentiated Th cells. These two critical and antagonistic signaling pathways are under negative feedback regulation by a multimember family of intracellular proteins called suppressor of cytokine signaling (SOCS). However, it is not known whether these negative regulatory factors also modulate Th1/Th2 lineage commitment and maintenance. We show here that CD4(+) naive T cells constitutively express low levels of SOCS1, SOCS2, and SOCS3 mRNAs. These mRNAs and their proteins increase significantly in nonpolarized Th cells after activation by TCR signaling. We further show that differentiation into Th1 or Th2 phenotype is accompanied by preferential expression of distinct SOCS mRNA transcripts and proteins. SOCS1 expression is 5-fold higher in Th1 than in Th2 cells, whereas Th2 cells contain 23-fold higher levels of SOCS3. We also demonstrate that IL-12-induced STAT4 activation is inhibited in Th2 cells that express high levels of SOCS3 whereas IL-4/STAT6 signaling is constitutively activated in Th2 cells, but not Th1 cells, with high SOCS1 expression. These results suggest that mutually exclusive use of STAT4 and STAT6 signaling pathways by differentiated Th cells may derive in part, from SOCS3- or SOCS1-mediated repression of IL-12/STAT4- or IL-4/STAT6 signaling in Th2 and Th1 cells, respectively. Given the strong correlation between distinct patterns of SOCS expression and differentiation into the Th1 or Th2 phenotype, SOCS1 and SOCS3 proteins are therefore Th lineage markers that can serve as therapeutic targets for immune modulation therapy.

The development of Schistosoma mansoni ova-induced granulomas is regulated by cytokines secreted by distinct Th cell subsets. To determine the importance of Th1 and Th2 cells in granuloma formation, we have studied the immune response to S. mansoni ova in Stat4- and Stat6-deficient mice, which lack Th1 and Th2 cells, respectively. Lymphocytes from both naive and infected Stat6-deficient mice produced minimal levels of Th2 cell cytokines and Ag-specific IgG1 and IgE, but showed enhanced production of IFN-gamma and Ag-specific IgG2a and IgG2b following schistosome egg injection. This shift away from a Th2 cell-mediated immune response was coupled with the development of pulmonary and hepatic granulomas that were greatly decreased in size compared with those in control littermates. Hepatic granulomas in Stat6-deficient mice were composed of predominantly mononuclear cells with very sparse appearance of eosinophils, and their diminished size was accompanied by decreased amounts of liver hydroxyproline content as a measure of collagen deposition. In contrast, lymphocytes from infected Stat4-deficient mice produced Th2 cell cytokines in amounts comparable to those produced by control littermates, but low levels of IFN-gamma. While infected Stat4-deficient mice developed pulmonary granulomas following schistosome egg injection that were modestly impaired in size, the granuloma size and amount of collagen deposition in the liver were equivalent to those seen in control littermates. These studies demonstrate that Th2 cells are required for the full development of the granulomas and tissue-destructive fibrotic pathology associated with the immune response to S. mansoni ova.

STAT (signal transducers and activators of transcription) proteins are activated in response to a large number of cytokines, growth factors, and hormones. Upon activation following the binding of ligands to their receptors, STAT proteins dimerize, translocate to the nucleus, and bind to the promoters of specific target genes. To date, seven mammalian members of the STAT family have been identified. Although some cytokines and growth factors can activate multiple STAT proteins, some STATs are activated with considerable specificity. The physiological role of each individual STAT protein is now being examined through the study of "knockout" mice, harboring a null allele for the particular gene. STAT1-deficient mice exhibit a selective signaling defect in response to interferons. STAT4 and STAT6 are essential for Thl-and Th2-responses, respectively. STAT5a-deficient mice exhibit defective mammary gland development. A study of STAT5b-deficient mice indicates that STAT5b mediates the sexually dimorphic effects of growth hormone in the liver. STAT5a and 5b also play different biological roles in the immune system. STAT3-deficient mice die during early embryogenesis, but the role of STAT3 in adult tissues can be assessed by utilizing the CreloxP recombination system to ablate the gene later in life. Analyses of tissue-specific STAT3-deficient mice indicate that STAT3 plays a crucial role in a variety of biological functions, including cell growth, suppression of apoptosis, and cell motility.

For the identification of susceptibility loci for primary biliary cirrhosis (PBC), a genome-wide association study (GWAS) was performed in 963 Japanese individuals (487 PBC cases and 476 healthy controls) and in a subsequent replication study that included 1,402 other Japanese individuals (787 cases and 615 controls). In addition to the most significant susceptibility region, human leukocyte antigen (HLA), we identified two significant susceptibility loci, TNFSF15 (rs4979462) and POU2AF1 (rs4938534) (combined odds ratio [OR] = 1.56, p = 2.84 × 10(-14) for rs4979462, and combined OR = 1.39, p = 2.38 × 10(-8) for rs4938534). Among 21 non-HLA susceptibility loci for PBC identified in GWASs of individuals of European descent, three loci (IL7R, IKZF3, and CD80) showed significant associations (combined p = 3.66 × 10(-8), 3.66 × 10(-9), and 3.04 × 10(-9), respectively) and STAT4 and NFKB1 loci showed suggestive association with PBC (combined p = 1.11 × 10(-6) and 1.42 × 10(-7), respectively) in the Japanese population. These observations indicated the existence of ethnic differences in genetic susceptibility loci to PBC and the importance of TNF signaling and B cell differentiation for the development of PBC in individuals of European descent and Japanese individuals.

NK and T cell-derived IFN-gamma is a key cytokine that stimulates innate immune responses and directs adaptive T cell response toward Th1 type. IL-15, IL-18, and IL-21 have significant roles as activators of NK and T cell functions. We have previously shown that IL-15 and IL-21 induce the expression of IFN-gamma, T-bet, IL-12R beta 2, and IL-18R genes both in NK and T cells. Now we have studied the effect of IL-15, IL-18, and IL-21 on IFN-gamma gene expression in more detail in human NK and T cells. IL-15 clearly activated IFN-gamma mRNA expression and protein production in both cell types. IL-18 and IL-21 enhanced IL-15-induced IFN-gamma gene expression. IL-18 or IL-21 alone induced a modest expression of the IFN-gamma gene but a combination of IL-21 and IL-18 efficiently up-regulated IFN-gamma production. We also show that IL-15 activated the binding of STAT1, STAT3, STAT4, and STAT5 to the regulatory sites of the IFN-gamma gene. Similarly, IL-21 induced the binding of STAT1, STAT3, and STAT4 to these elements. IL-15- and IL-21-induced STAT1 and STAT4 activation was verified by immunoprecipitation with anti-phosphotyrosine Abs followed by Western blotting with anti-STAT1 and anti-STAT4 Abs. IL-18 was not able to induce the binding of STATs to IFN-gamma gene regulatory sites. IL-18, however, activated the binding of NF-kappa B to the IFN-gamma promoter NF-kappa B site. Our results suggest that both IL-15 and IL-21 have an important role in activating the NK cell-associated innate immune response.

Experimental autoimmune encephalomyelitis (EAE) is mediated by myelin-specific CD4(+) T cells secreting Th1 cytokines, while recovery from disease is associated with expression of Th2 cytokines. Investigations into the role of individual cytokines in disease induction have yielded contradictory results. Here we used animals with targeted deletion of the STAT4 or STAT6 genes to determine the role of these signaling molecules in EAE. The STAT4 pathway controls the differentiation of cells into a Th1 phenotype, while the STAT6 pathway controls the differentiation of cells into a Th2 phenotype. We found that mice deficient in STAT4 are resistant to the induction of EAE, with minimal inflammatory infiltrates in the central nervous system. In contrast, STAT6-deficient mice, which have a predominantly Th1 phenotype, experience a more severe clinical course of EAE as compared with wild-type or STAT4 knockout mice. In addition, adoptive transfer studies confirm the regulatory functions of a Th2 environment in vivo. These novel data indicate that STAT4 and STAT6 genes play a critical role in regulating the autoimmune response in EAE.

Our understanding of the pathophysiology of multiple sclerosis (MS) has evolved significantly over the past two decades as the fields of immunology and neurobiology provide new avenues of exploration into the cause and mechanism of the disease. It has been known for decades that T cells have different cytokine phenotypes, yet the cytokine phenotype of pathogenic T cells in MS is still an area of debate. In EAE, it appears that IFNγ and IL-17, produced by Th1 and Th17 cells respectively, are not the critical factor that determines T cell encephalitogenicity. However, there are molecules such as IL-23, T-bet and STAT4, that appear to be critical, yet it is unclear whether all these molecules contribute to a common, yet undefined pathway, or act in a synergistic manner which culminates in encephalitogenicity has still to be determined. Therefore, the focus of research on effector T cells in MS should focus on pathways upstream of the cytokines that define Th1 and Th17 cells, since downstream products, such as IFNγ and IL-17, probably are not critical determinants of whether an effector T cells is capable of trafficking to the CNS and inducing inflammatory demyelination.

The best-characterized type 1 interferon (IFN) signaling pathway depends on signal transducer and activator of transcription 1 (STAT1) and STAT2. The cytokines can, however, conditionally activate all STATs. Regulation of their access to particular signaling pathways is poorly understood. STAT4 is important for IFN-gamma induction, and NK cells are major producers of this cytokine. We report that NK cells have high basal STAT4 levels and sensitivity to type 1 IFN-mediated STAT4 activation for IFN-gamma production. Increases in STAT1, driven during viral infection by either type 1 IFN or IFN-gamma, are associated with decreased STAT4 access. Both STAT1 and STAT2 are important for antiviral defense, but STAT1 has a unique role in protecting against sustained NK cell IFN-gamma production and resulting disease. The regulation occurs with an NK cell type 1 IFN receptor switch from a STAT4 to a STAT1 association. Thus, a fundamental characteristic of NK cells is high STAT4 bound to the type 1 IFN receptor. The conditions of infection result in STAT1 induction with displacement of STAT4. These studies elucidate the critical role of STAT4 levels in predisposing selection of specific signaling pathways, define the biological importance of regulation within particular cell lineages, and provide mechanistic insights for how this is accomplished in vivo.

Systemic lupus erythematosus (SLE) is the prototype autoimmune disease where genes regulated by type I interferon (IFN) are over-expressed and contribute to the disease pathogenesis. Because signal transducer and activator of transcription 4 (STAT4) plays a key role in the type I IFN receptor signaling, we performed a candidate gene study of a comprehensive set of single nucleotide polymorphism (SNPs) in STAT4 in Swedish patients with SLE. We found that 10 out of 53 analyzed SNPs in STAT4 were associated with SLE, with the strongest signal of association (P = 7.1 x 10(-8)) for two perfectly linked SNPs rs10181656 and rs7582694. The risk alleles of these 10 SNPs form a common risk haplotype for SLE (P = 1.7 x 10(-5)). According to conditional logistic regression analysis the SNP rs10181656 or rs7582694 accounts for all of the observed association signal. By quantitative analysis of the allelic expression of STAT4 we found that the risk allele of STAT4 was over-expressed in primary human cells of mesenchymal origin, but not in B-cells, and that the risk allele of STAT4 was over-expressed (P = 8.4 x 10(-5)) in cells carrying the risk haplotype for SLE compared with cells with a non-risk haplotype. The risk allele of the SNP rs7582694 in STAT4 correlated to production of anti-dsDNA (double-stranded DNA) antibodies and displayed a multiplicatively increased, 1.82-fold risk of SLE with two independent risk alleles of the IRF5 (interferon regulatory factor 5) gene.

Rheumatoid arthritis (RA) is one of the most common chronic inflammatory syndromes. As such, RA is often considered the prototype disease for defining both the molecular and pathological basis of immune-mediated chronic inflammatory disease, and for validating targeted therapies. The immunogenetics of RA suggest a key role for aberrant pathways of T-cell activation in the initiation and/or perpetuation of disease. In the T-cell activation process, CD4+ T-cells are engaged by antigenic peptide fragments in a complex with HLA class II molecules, in addition to co-stimulatory molecules, such as CD80/CD86, expressed on the surface of professional antigen presenting cells. The strongest evidence supporting a role for CD4+ T cells in disease pathogenesis is the association between RA and HLA-DRB1; however, the functional role of this association has yet to be defined. Susceptibility to RA may also be linked with several RA-associated allelic variants of genes, especially PTPN22, but also CTLA4, IL2RA, IL-2RB, STAT4, PTPN2 and PADI4, many of which encode molecules directly implicated in pathways of T-cell activation.The presence of inflammatory infiltrates, such as follicular structures, in the synovial membrane provides compelling evidence of ongoing immune reactions in moderate to severe RA. These structures likely play a key role in T cell - B cell cooperation and the local generation of specific autoantibodies; as such, chronically activated synovial T cells represent key cellular targets for therapy. Evidence also supports a role for T-helper (Th) cells, Th17 cells, and impaired CD4+CD25(hi) regulatory T cell (Treg) function in the pathogenesis of RA. In addition to discussing a range of issues regarding T-cell activation in RA, this review describes how therapeutic modulation of T-cell function, as opposed to profound immunosuppression or immunodepletion, has been associated with better disease outcomes in clinical trials. Ultimately, elucidation of the distinct effects of co-stimulation modulation with abatacept on T cells should provide key insights into understanding how to restore immune homeostasis in patients with RA.

As a means of developing therapies that target the pathogenic T cells in multiple sclerosis (MS) without compromising the immune system or eliciting systemic side effects, we investigated the use of T-bet-specific antisense oligonucleotides and small interfering RNAs (siRNA) to silence T-bet expression in autoreactive encephalitogenic T cells and evaluated the biological consequences of this suppression in experimental autoimmune encephalomyelitis, a model for MS. The T-bet-specific AS oligonucleotide and siRNA suppressed T-bet expression, IFNgamma production, and STAT1 levels during antigen-specific T cell differentiation. In vitro suppression of T-bet during differentiation of myelin-specific T cells and in vivo administration of a T-bet-specific antisense oligonucleotide or siRNA inhibited disease. T-bet was shown to bind the IFNgamma and STAT1 promoters, but did not regulate the IL-12/STAT4 pathway. Since T-bet regulates IFNgamma production in CD4(+) T cells, but to a lesser extent in most other IFNgamma-producing cells, T-bet may be a target for therapeutics for Th1-mediated diseases.

Systemic lupus erythematosus (SLE) is a genetically complex disease with heterogeneous clinical manifestations. A polymorphism in the STAT4 gene has recently been established as a risk factor for SLE, but the relationship with specific SLE subphenotypes has not been studied. We studied 137 SNPs in the STAT4 region genotyped in 4 independent SLE case series (total n = 1398) and 2560 healthy controls, along with clinical data for the cases. Using conditional testing, we confirmed the most significant STAT4 haplotype for SLE risk. We then studied a SNP marking this haplotype for association with specific SLE subphenotypes, including autoantibody production, nephritis, arthritis, mucocutaneous manifestations, and age at diagnosis. To prevent possible type-I errors from population stratification, we reanalyzed the data using a subset of subjects determined to be most homogeneous based on principal components analysis of genome-wide data. We confirmed that four SNPs in very high LD (r(2) = 0.94 to 0.99) were most strongly associated with SLE, and there was no compelling evidence for additional SLE risk loci in the STAT4 region. SNP rs7574865 marking this haplotype had a minor allele frequency (MAF) = 31.1% in SLE cases compared with 22.5% in controls (OR = 1.56, p = 10(-16)). This SNP was more strongly associated with SLE characterized by double-stranded DNA autoantibodies (MAF = 35.1%, OR = 1.86, p<10(-19)), nephritis (MAF = 34.3%, OR = 1.80, p<10(-11)), and age at diagnosis<30 years (MAF = 33.8%, OR = 1.77, p<10(-13)). An association with severe nephritis was even more striking (MAF = 39.2%, OR = 2.35, p<10(-4) in the homogeneous subset of subjects). In contrast, STAT4 was less strongly associated with oral ulcers, a manifestation associated with milder disease. We conclude that this common polymorphism of STAT4 contributes to the phenotypic heterogeneity of SLE, predisposing specifically to more severe disease.

Although much promising data that interleukin (IL)-12 could be a powerful therapeutic agent against cancer were reported in animal models, its excessive toxicity has become a problem for its clinical application. IL-27 is a novel IL-12 family member that plays a role in the early regulation of T helper cell 1 initiation, including induction of T-bet and IL-12 receptor beta 2 expression. In the present study, we have evaluated the antitumor activity of IL-27 against a murine tumor model of colon carcinoma C26. C26 cells, which were transduced with the single-chain IL-27 cDNA and became secreting IL-27 (C26-IL-27), exhibited minimal tumor growth in vivo, and all of the mice inoculated with these cells survived healthily with complete tumor remission. Inoculation of mice with C26-IL-27 induced enhanced IFN-gamma production and cytotoxic T-lymphocyte activity against C26 tumor in spleen cells. Recovered mice from the inoculation showed a tumor-specific protective immunity to the following challenge with parental C26 tumor. The antitumor activity of IL-27 was almost diminished in nude mice, and depletion of CD8(+) T cells and neutralization of IFN-gamma in immunocompetent mice reduced greatly the antitumor activity. Moreover, the antitumor activity was abolished in T-bet-deficient mice, whereas it was observed unexpectedly in mice deficient of signal transducer and activator of transcription (STAT) 4. These results suggest that IL-27 has potent abilities to induce tumor-specific antitumor activity and protective immunity and that the antitumor activity is mediated mainly through CD8(+) T cells, IFN-gamma, and T-bet but not through STAT4.

The conditions leading to the activation/differentiation of T-helper (Th) cells dedicated for B-cell antibody production are still poorly characterized. We now demonstrate that interleukin-6 (IL-6) promotes the differentiation of naive T lymphocytes into helper cells able to promote B-cell activation and antibody secretion. IL-6-driven acquisition of B-cell help capacity requires expression of the signal transducer and activator of transcription 3 (STAT3), but not STAT4 or STAT6 transcription factors, suggesting that the ability to provide help to B cells is not restricted to a well-defined Th1 or Th2 effector population. T cell-specific STAT3-deficient mice displayed reduced humoral responses in vivo that could not be related to an altered expansion of CXCR5-expressing helper T cells. IL-6 was shown to promote IL-21 secretion, a cytokine that was similarly found to promote the differentiation of naive T cells into potent B-cell helper cells. Collectively, these data indicate that the ability to provide B-cell help is regulated by IL-6/IL-21 through STAT3 activation, independently of Th1, Th2, Th17, or follicular helper T cell (T(FH)) differentiation.

We use an integrated approach to understand breast cancer heterogeneity by modeling mRNA, copy number alterations, microRNAs, and methylation in a pathway context utilizing the pathway recognition algorithm using data integration on genomic models (PARADIGM). We demonstrate that combining mRNA expression and DNA copy number classified the patients in groups that provide the best predictive value with respect to prognosis and identified key molecular and stromal signatures. A chronic inflammatory signature, which promotes the development and/or progression of various epithelial tumors, is uniformly present in all breast cancers. We further demonstrate that within the adaptive immune lineage, the strongest predictor of good outcome is the acquisition of a gene signature that favors a high T-helper 1 (Th1)/cytotoxic T-lymphocyte response at the expense of Th2-driven humoral immunity. Patients who have breast cancer with a basal HER2-negative molecular profile (PDGM2) are characterized by high expression of protumorigenic Th2/humoral-related genes (24-38%) and a low Th1/Th2 ratio. The luminal molecular subtypes are again differentiated by low or high FOXM1 and ERBB4 signaling. We show that the interleukin signaling profiles observed in invasive cancers are absent or weakly expressed in healthy tissue but already prominent in ductal carcinoma in situ, together with ECM and cell-cell adhesion regulating pathways. The most prominent difference between low and high mammographic density in healthy breast tissue by PARADIGM was that of STAT4 signaling. In conclusion, by means of a pathway-based modeling methodology (PARADIGM) integrating different layers of molecular data from whole-tumor samples, we demonstrate that we can stratify immune signatures that predict patient survival.

The transcription factor GATA3 is crucial for the differentiation of naive CD4(+) T cells into T helper 2 (Th2) cells. Here, we show that deletion of Gata3 allowed the appearance of interferon-gamma (IFN-gamma)-producing cells in the absence of interleukin-12 (IL-12) and IFN-gamma. Such IFN-gamma production was transcription factor T-bet independent. Another T-box-containing transcription factor Eomes, but not T-bet, was induced both in GATA3-deficient CD4(+) T cells differentiated under Th2 cell conditions and in Th2 cells with enforced Runx3 expression, contributing to IFN-gamma production. GATA3 overexpression blocked Runx3-mediated Eomes induction and IFN-gamma production, and GATA3 protein physically interacted with Runx3 protein. Furthermore, we found that Runx3 directly bound to multiple regulatory elements of the Ifng gene and that blocking Runx3 function in either Th1 or GATA3-deficient "Th2" cells results in diminished IFN-gamma production by these cells. Thus, the Runx3-mediated pathway, actively suppressed by GATA3, induces IFN-gamma production in a STAT4- and T-bet-independent manner.

Understanding the developmental mechanisms of follicular helper T cells (TFH cells) in humans is relevant to the clinic. However, the factors that drive the differentiation of human CD4+ helper T cells into TFH cells remain largely undefined. Here we found that transforming growth factor-β (TGF-β) provided critical additional signals for the transcription factors STAT3 and STAT4 to promote initial TFH differentiation in humans. This mechanism did not appear to be shared by mouse helper T cells. Developing human TFH cells that expressed the transcriptional repressor Bcl-6 also expressed RORγt, a transcription factor typically expressed by the TH17 subset of helper T cells. Our study documents a mechanism by which TFH cells and TH17 cells emerge together in inflammatory environments in humans, as is often observed in many human autoimmune diseases.

STAT proteins are a family of latent cytoplasmic transcription factors that are activated by tyrosine phosphorylation in response to a variety of cytokines, growth factors, and hormones. Once activated, STAT proteins translocate into the nucleus and help coordinate gene transcription. One striking feature of STAT signaling is its rapid and transient activation and deactivation cycle, although the molecular mechanisms responsible for this remain poorly understood. Here, we report on a nuclear protein that contains both PDZ and LIM domains and that interacts with activated STAT4 molecules. We show that SLIM is an ubiquitin E3 ligase that acts on STAT proteins to cause their proteosome-mediated degradation and enhance their dephosphorylation. Overexpression of SLIM leads to impaired STAT1 and STAT4 activity due to reduced STAT protein levels, while SLIM-deficiency results in increased STAT expression and thus enhanced IFNgamma production by Th1 cells. These studies suggest that SLIM is a novel ubiquitin E3 ligase whose targets include STAT proteins.

TGF-beta can be a potent suppressor of lymphocyte effector cell functions and can mediate these effects via distinct molecular pathways. The role of TGF-beta in regulating CD16-mediated NK cell IFN-gamma production and antibody-dependent cellular cytotoxicity (ADCC) is unclear, as are the signaling pathways that may be utilized. Treatment of primary human NK cells with TGF-beta inhibited IFN-gamma production induced by CD16 activation with or without IL-12 or IL-2, and it did so without affecting the phosphorylation/activation of MAP kinases ERK and p38, as well as STAT4. TGF-beta treatment induced SMAD3 phosphorylation, and ectopic overexpression of SMAD3 resulted in a significant decrease in IFN-gamma gene expression following CD16 activation with or without IL-12 or IL-2. Likewise, NK cells obtained from smad3(-/-) mice produced more IFN-gamma in response to CD16 activation plus IL-12 when compared with NK cells obtained from wild-type mice. Coactivation of human NK cells via CD16 and IL-12 induced expression of T-BET, the positive regulator of IFN-gamma, and T-BET was suppressed by TGF-beta and by SMAD3 overexpression. An extended treatment of primary NK cells with TGF-beta was required to inhibit ADCC, and it did so by inhibiting granzyme A and granzyme B expression. This effect was accentuated in cells overexpressing SMAD3. Collectively, our results indicate that TGF-beta inhibits CD16-mediated human NK cell IFN-gamma production and ADCC, and these effects are mediated via SMAD3.

Interferon-alpha (IFN-alpha) is a pleiotropic cytokine that has antiviral, antiproliferative, and immunoregulatory functions. There is increasing evidence that IFN-alpha has an important role in T-cell biology. We have analyzed the expression of IL-2Ralpha, c-myc, and pim-1 genes in anti-CD3-activated human T lymphocytes. The induction of these genes is associated with interleukin-2 (IL-2)-induced T-cell proliferation. Treatment of T lymphocytes with IFN-alpha, IL-2, IL-12, and IL-15 upregulated IL-2Ralpha, c-myc, and pim-1 gene expression. IFN-alpha also sensitized T cells to IL-2-induced proliferation, further suggesting that IFN-alpha may be involved in the regulation of T-cell mitogenesis. When we analyzed the nature of STAT proteins capable of binding to IL-2Ralpha, pim-1, and IRF-1 GAS elements after cytokine stimulation, we observed IFN-alpha-induced binding of STAT1, STAT3, and STAT4, but not STAT5 to all of these elements. Yet, IFN-alpha was able to activate binding of STAT5 to the high-affinity IFP53 GAS site. IFN-alpha enhanced tyrosine phosphorylation of STAT1, STAT3, STAT4, STAT5a, and STAT5b. IL-12 induced STAT4 and IL-2 and IL-15 induced STAT5 binding to the GAS elements. Taken together, our results suggest that IFN-alpha, IL-2, IL-12, and IL-15 have overlapping activities on human T cells. These findings thus emphasize the importance of IFN-alpha as a T-cell regulatory cytokine.

Signal transducer and activator of transcription 4 (STAT4) is a central mediator in generating inflammation during protective immune responses and immune-mediated diseases. In the 8 yr since their first description, STAT4-deficient mice have defined the role of STAT4 in a variety of in vivo model systems. Despite the extensive study and use of these mice, the exact role of STAT4 in vivo is still unclear. In this review, I focus on describing the phenotypes of STAT4-deficient immune responses to pathogens and in diseases. Comparing the effects of STAT4 deficiency among numerous model systems will further enhance the development of a systemic model of STAT4 function in vivo.

Th17 cells reactive to the enteric microbiota are central to the pathogenesis of certain types of inflammatory bowel disease. However, Th17 cells display substantial developmental plasticity, such that some progeny of Th17 cell precursors retain a predominantly IL-17A(+) phenotype, whereas others extinguish IL-17 expression and acquire expression of IFN-γ, giving rise to "Th1-like" cells. It remains unclear what role these subsets play in inflammatory bowel disease. Using a Th17 transfer model of colitis, we found that IFN-γ-deficient Th17 cells retained an IL-17A(+) phenotype and were unable to induce colitis in recipients. Development of disease required the transition of a subset of Th17 precursors to Th1-like cells and was contingent on the expression of both Stat4 and T-bet, but not the IL-12 or IFN-γ receptors. Moreover, Th17 cells could provide "help" for the development of pathogenic Th1 cells from naïve precursors. These results indicate that Th17 cells are potent mediators of colitis pathogenesis by dual mechanisms: by directly transitioning to Th1-like cells and by supporting the development of classic Th1 cells.

Allergic asthma is characterized by airway inflammation initiated by adaptive immune responses to aeroallergens. Recent data suggest that severe asthma may be a different form of asthma rather than an increase in asthma symptoms and that innate immune responses to LPS can modulate adaptive immune responses to allergens. In this study, we evaluated the hypothesis that airway exposure to different doses of LPS induces different form of asthma. Our study showed that neutrophilic inflammation and IFN-gamma expression were higher in induced sputum from severe asthma patients than from mild to moderate asthmatics. Animal experiments indicated that allergen sensitization with low-dose LPS (0.1 microg) induced type 2 asthma phenotypes, i.e., airway hyperresponsiveness, eosinophilic inflammation, and allergen-specific IgE up-regulation. In contrast, allergen sensitization with high-dose LPS (10 microg) induced asthma phenotypes, i.e., airway hyperresponsiveness and noneosinophilic inflammation that were not developed in IFN-gamma-deficient mice, but unaffected in the absence of IL-4. During the allergen sensitization period, TNF-alpha expression was found to be enhanced by both low- and high-dose LPS, whereas IL-12 expression was only enhanced by high-dose LPS. Interestingly, the asthma phenotypes induced by low-dose LPS, but not by high-dose LPS, were completely inhibited in TNF-alpha receptor-deficient mice, whereas the asthma phenotypes induced by high-dose LPS were abolished in the homozygous null mutation of the STAT4 gene. These findings suggest that airway exposure levels of LPS induces different forms of asthma that are type 1 and type 2 asthma phenotypes by high and low LPS levels, respectively.