Induction Of GATA-3 Research Articles

Stage-specific GATA3 induction promotes ILC2 development after lineage commitment

Group 2 innate lymphoid cells (ILC2s) are a subset of innate lymphocytes that produce type 2 cytokines, including IL-4, IL-5, and IL-13. GATA3 is a critical transcription factor for ILC2 development at multiple stages. However, when and how GATA3 is induced to the levels required for ILC2 development remains unclear. Herein, we identify ILC2-specific GATA3-related tandem super-enhancers (G3SE) that induce high GATA3 in ILC2-committed precursors. G3SE-deficient mice exhibit ILC2 deficiency in the bone marrow, lung, liver, and small intestine with minimal impact on other ILC lineages or Th2 cells. Single-cell RNA-sequencing and subsequent flow cytometry analysis show that GATA3 induction mechanism, which is required for entering the ILC2 stage, is lost in IL-17RB+PD-1− late ILC2-committed precursor stage in G3SE-deficient mice. Cnot6l, part of the CCR4-NOT deadenylase complex, is a possible GATA3 target during ILC2 development. Our findings implicate a stage-specific regulatory mechanism for GATA3 expression during ILC2 development.

CARD11-dependent signaling in T cells is critical for the activation of multiple AP-1 transcription factor complexes.

Abstract The CARD11 scaffold enables TCR-induced NF-κB, mTORC1 and JNK signaling. While the requirement of CARD11 for NF-κB stimulation is well known, it remains unclear if/how CARD11 influences other transcription factors (TFs) essential for T cell activation: AP-1 and NFAT. We sought to delineate the importance of CARD11 in activating AP-1- or NFATc1-dependent transcription in human T cells, employing mitogen-activated wild-type (WT) or CARD11 KO Jurkat T cells. RNA-seq analysis revealed significant downregulation of AP-1-mediated transcriptional programs in CARD11 KO cells, with a concomitant increase in NFATc1 expression. Although JNK inhibition in WT cells had a surprisingly minimal impact on AP-1 activation, loss of CARD11 substantially reduced mitogen-induced AP-1 component expression and activity, encompassing all canonical JUN and FOS family members. We further demonstrated that deleterious CARD11 variants derived from CARD11-associated Atopy with Dominant Interference of NF-κB Signaling (CADINS) disease patients also impaired AP-1 function. Conversely, JNK inhibition in WT T cells enhanced NFATc1 activity with concomitant upregulation of the NFAT-dependent TH2 master regulator GATA3, congruent with enhanced GATA3 induction in CADINS patient T cells. Our novel findings indicate that CARD11 governs the activation of AP-1 and NFAT TF complexes originally thought to be induced via CARD11-independent pathways, with implications for human T cell differentiation and function.

Dominant interfering CARD11 variants disrupt JNK signaling in T cells and impaired CARD11-JNK signaling increases expression of GATA3

CARD11 is a critical scaffold protein linking antigen receptor (AgR) engagement to transcriptional programs required for effector lymphocyte survival, proliferation, and differentiation. Prior work established that CARD11 mediates these effects by enabling AgR-induced signaling through NF-kB, mTORC1 and c-Jun N-terminal kinase 2 (JNK2). Although JNK influences helper CD4+ T cell differentiation, mechanisms governing JNK signal regulation and function in human lymphocytes remain incompletely understood. We therefore asked if/how dominant interfering (DI) CARD11 variants impact JNK signaling and JNK-specific transcriptomic changes in T cells. Wild-type (WT) and CARD11 knockout (KO) Jurkat T cells were transfected with plasmids expressing WT or DI CARD11 variants to assess effects on CARD11-dependent JNK signaling. Additionally, RNA sequencing was performed on mitogen-activated WT and CARD11 KO Jurkat T cells +/– JNK inhibition. Our results revealed that CARD11 is required for AgR-dependent activation of JNK1 and JNK2. Furthermore, heterozygous DI CARD11 mutations found in patients with CARD11-associated atopy with dominant interference of NF-kB signaling (CADINS) disease disrupted AgR-dependent JNK1/2 phosphorylation, c-Jun accumulation, and AP-1 transcriptional activity with variable potency, similar to the degree of impaired NF-kB activation. Intriguingly, JNK inhibition in Jurkat and primary CD4+ T cells enhanced AgR-induced expression of GATA3, the master transcriptional regulator of Th2 cell differentiation. These results were recapitulated in effector Tcells generated from CADINS patient PBMC, with greater GATA3 induction observed after mitogen activation relative to healthy controls. Intriguingly, simultaneous reactivation of JNK signaling through a CARD11-independent mechanism blunted AgR-induced GATA3 induction, suggesting JNK activity can directly modulate GATA3 expression. In summation, our novel findings imply that defective CARD11-dependent JNK signaling in CD4+ T cells contributes to enhanced Th2 differentiation and the atopic diathesis observed in CADINS patients, unveiling a new potential therapeutic target for ameliorating severe allergic disease manifestations.

Realization of the T Lineage Program Involves GATA-3 Induction of Bcl11b and Repression of Cdkn2b Expression.

The zinc-finger transcription factor GATA-3 plays a crucial role during early T cell development and also dictates later T cell differentiation outcomes. However, its role and collaboration with the Notch signaling pathway in the induction of T lineage specification and commitment have not been fully elucidated. We show that GATA-3 deficiency in mouse hematopoietic progenitors results in an early block in T cell development despite the presence of Notch signals, with a failure to upregulate Bcl11b expression, leading to a diversion along a myeloid, but not a B cell, lineage fate. GATA-3 deficiency in the presence of Notch signaling results in the apoptosis of early T lineage cells, as seen with inhibition of CDK4/6 (cyclin-dependent kinases 4 and 6) function, and dysregulated cyclin-dependent kinase inhibitor 2b (Cdkn2b) expression. We also show that GATA-3 induces Bcl11b, and together with Bcl11b represses Cdkn2b expression; however, loss of Cdkn2b failed to rescue the developmental block of GATA-3-deficient T cell progenitor. Our findings provide a signaling and transcriptional network by which the T lineage program in response to Notch signals is realized.

Schnurri 3 promotes Th2 cytokine production during the late phase of T-cell antigen stimulation.

Th1 and Th2 polarization is determined by the coordination of numerous factors including the affinity and strength of the antigen‐receptor interaction, predominant cytokine environment, and costimulatory molecules present. Here, we show that Schnurri (SHN) proteins have distinct roles in Th1 and Th2 polarization. SHN2 was previously found to block the induction of GATA3 and Th2 differentiation. We found that, in contrast to SHN2, SHN3 is critical for IL‐4 production and Th2 polarization. Strength of stimulation controls SHN2 and SHN3 expression patterns, where higher doses of antigen receptor stimulation promoted SHN3 expression and IL‐4 production, along with repression of SHN2 expression. SHN3‐deficient T cells showed a substantial defect in IL‐4 production and expression of AP‐1 components, particularly c‐Jun and Jun B. This loss of early IL‐4 production led to reduced GATA3 expression and impaired Th2 differentiation. Together, these findings uncover SHN3 as a novel, critical regulator of Th2 development.

TNF-alpha and Notch signaling regulates the expression of HOXB4 and GATA3 during early T lymphopoiesis.

During the early thymus colonization, Notch signaling activation on hematopoietic progenitor cells (HPCs) drives proliferation and T cell commitment. Although these processes are driven by transcription factors such as HOXB4 and GATA3, there is no evidence that Notch directly regulates their transcription. To evaluate the role of NOTCH and TNF signaling in this process, human CD34+ HPCs were cocultured with OP9-DL1 cells, in the presence or absence of TNF. The use of a Notch signaling inhibitor and a protein synthesis inhibitor allowed us to distinguish primary effects, mediated by direct signaling downstream Notch and TNF, from secondary effects, mediated by de novo synthesized proteins. A low and physiologically relevant concentration of TNF promoted T lymphopoiesis in OP9-DL1 cocultures. TNF positively modulated the expression of both transcripts in a Notch-dependent manner; however, GATA3 induction was mediated by a direct mechanism, while HOXB4 induction was indirect. Induction of both transcripts was repressed by a GSK3β inhibitor, indicating that activation of canonical Wnt signaling inhibits rather than induces their expression. Our study provides novel evidences of the mechanisms integrating Notch and TNF-alpha signaling in the transcriptional induction of GATA3 and HOXB4. This mechanism has direct implications in the control of self-renewal, proliferation, commitment, and T cell differentiation.

The adaptor protein SAP regulates type II NKT-cell development, cytokine production, and cytotoxicity against lymphoma.

CD1d-restricted NKT cells represent a unique lineage of immunoregulatory T cells that are divided into two groups, type I and type II, based on their TCR usage. Because there are no specific tools to identify type II NKT cells, little is known about their developmental requirements and functional regulation. In our previous study, we showed that signaling lymphocytic activation molecule associated protein (SAP) is essential for the development of type II NKT cells. Here, using a type II NKT-cell TCR transgenic mouse model, we demonstrated that CD1d-expressing hematopoietic cells, but not thymic epithelial cells, meditate efficient selection of type II NKT cells. Furthermore, we showed that SAP regulates type II NKT-cell development by controlling early growth response 2 protein and promyelocytic leukemia zinc finger expression. SAP-deficient 24αβ transgenic T cells (24αβ T cells) exhibited an immature phenotype with reduced Th2 cytokine-producing capacity and diminished cytotoxicity to CD1d-expressing lymphoma cells. The impaired IL-4 production by SAP-deficient 24αβ T cells was associated with reduced IFN regulatory factor 4 and GATA-3 induction following TCR stimulation. Collectively, these data suggest that SAP is critical for regulating type II NKT cell responses. Aberrant responses of these T cells may contribute to the immune dysregulation observed in X-linked lymphoproliferative disease caused by mutations in SAP.

LAPCs contribute to the pathogenesis of allergen-induced allergic airway inflammation in mice.

The inflammatory immune response associated with allergic airway inflammation in asthma involves T helper type 2 (Th2) immunity. Given the data that a newly described late activator antigen-presenting cell (LAPC) population promotes Th2 immunity in viral infections, we undertook studies to investigate whether LAPCs have a pathogenic role in allergic airway inflammation. We employed acute ovalbumin (OVA) and house dust mite (HDM) sensitization and challenge models to establish allergic airway inflammation in mice, followed by the analysis of lungs and draining lymph node (DLN) cell infiltrates, immunoglobulin E (IgE) production, and airway hyper-responsiveness (AHR). We tested whether adoptive transfer of LAPCs isolated from mice with established allergic airway inflammation augments the development of sensitization in naïve mice. We provide evidence that in both OVA and HDM mouse models of allergic inflammation, LAPCs accumulate in the lungs and draining lymph nodes (DLNs), concomitant with the onset of lung pathology, allergen-specific IgE production, eosinophilia, and Th2 cytokine production. Adoptive transfer experiments using OVA-activated LAPCs reveal exacerbation of disease pathology with an increase in lung inflammatory cells, eosinophilia, circulating IgE, Th2 cytokine production, and a worsening of AHR. OVA-activated LAPCs preferentially increased GATA-3 induction in naïve CD4(+) T cells. Together, these data suggest an important role for LAPCs in polarizing the Th2 response in mouse models of allergic airway inflammation.

Nucleic acid sensing by T cells initiates Th2 cell differentiation

While T-cell responses are directly modulated by Toll-like receptor (TLR) ligands, the mechanism and physiological function of nucleic acids (NAs)-mediated T cell costimulation remains unclear. Here we show that unlike in innate cells, T-cell costimulation is induced even by non-CpG DNA and by self-DNA, which is released from dead cells and complexes with antimicrobial peptides or histones. Such NA complexes are internalized by T cells and induce costimulatory responses independently of known NA sensors, including TLRs, RIG-I-like receptors (RLRs), inflammasomes and STING-dependent cytosolic DNA sensors. Such NA-mediated costimulation crucially induces Th2 differentiation by suppressing T-bet expression, followed by the induction of GATA-3 and Th2 cytokines. These findings unveil the function of NA sensing by T cells to trigger and amplify allergic inflammation.

CARMA1 Controls Th2 Cell-Specific Cytokine Expression through Regulating JunB and GATA3 Transcription Factors

The scaffold protein CARMA1 is required for the TCR-induced lymphocyte activation. In this study, we show that CARMA1 also plays an essential role in T cell differentiation. We have found that the adoptive transfer of bone marrow cells expressing constitutively active CARMA1 results in lung inflammation, eosinophilia, and elevated levels of IL-4, IL-5, and IL-10 in recipient mice. In contrast, CARMA1-deficient T cells are defective in TCR-induced expression of Th2 cytokines, suggesting that CARMA1 preferentially directs Th2 differentiation. The impaired cytokine production is due to reduced expression of JunB and GATA3 transcription factors. CARMA1 deficiency affects JunB stability resulting in its enhanced ubiquitination and degradation. In contrast, TCR-dependent induction of GATA3 is suppressed at the transcriptional level. We also found that supplementation with IL-4 partially restored GATA3 expression in CARMA1-deficient CD4(+) splenocytes and subsequently production of GATA3-dependent cytokines IL-5 and IL-13. Therefore, our work provides the mechanism by which CARMA1 regulates Th2 cell differentiation.

Requirement of GATA-binding protein 3 for Il13 gene expression in IL-18-stimulated Th1 cells

Recent reports have revealed that CD4(+) T(h) cell subsets have the ability to alter their gene expression pattern in response to extracellular stimuli. We previously highlighted the plasticity of T(h)1 cells by demonstrating that T(h)1 cells gain the capacity to produce IL-3, IL-9, IL-13 and granulocyte macrophage colony-stimulating factor in response to antigen, IL-2 and IL-18, and based on their unique function, we designated these activated T(h)1 cells as 'super T(h)1 cells'. However, the precise molecular mechanism underlying IL-13 production by super T(h)1 cells has not been elucidated. Here, we show that the GATA-binding protein 3 (Gata3) is essentially required for II13 gene expression in super T(h)1 cells. Gata3 is synergistically induced in T-box expressed in T-cells (T-bet)-expressing T(h)1 cells when co-stimulated with anti-CD3, IL-18 and IL-4 through the activation of nuclear factor of activated T cells, nuclear factor kappa-light-chain-enhancer of activated B cells and signal transducer and activator of transcription 6, respectively. However, Gata3 induction is not satisfactory, and additional TCR or anti-CD3 signaling is prerequisite for triggering IL-13 production by Gata3 plus T-bet-expressing T(h)1 cells. These findings suggest that Gata3, which is not originally expressed in T(h)1 cells, alters the cytokine production profile by T(h)1 cells.

GATA3 inhibits lysyl oxidase-mediated metastases of human basal triple-negative breast cancer cells

Discovery of mechanisms that impede the aggressive and metastatic phenotype of human basal triple-negative type breast cancers (BTNBC) could provide novel targets for therapy for this form of breast cancer that has a relatively poor prognosis. Previous studies have demonstrated that the expression of GATA3, the master transcriptional regulator of mammary luminal differentiation, can reduce the tumorigenicity and metastatic propensity of the human BTNBC MDA-MB-231 cell line (MB231), although the mechanism for reduced metastases was not elucidated. We demonstrate through gene expression profiling that GATA3 expression in 231 cells resulted in the dramatic reduction in the expression of Lysyl oxidase (LOX), a metastasis-promoting matrix remodeling protein, in part, through methylation of the LOX promoter. Suppression of LOX expression by GATA3 was further confirmed in the BTNBC Hs578T cell line. Conversely, reduction of GATA3 expression by siRNA in luminal BT474 cells increased LOX expression. Reconstitution of LOX expression in 231-GATA3 cells restored metastatic propensity. A strong inverse association between high LOX and low GATA3 expression was confirmed in a panel of 51 human breast cancer cell lines. Similarly, human breast cancer microarray data demonstrated that high LOX/low GATA3 expression is associated with the BTNBC subtype of breast cancer and poor patient prognosis. Expression of GATA3 reprograms BTNBC to a less aggressive phenotype and inhibits a major mechanism of metastasis through inhibition of LOX. Induction of GATA3 in BTNBC cells or novel approaches that inhibit LOX expression or activity could be important strategies for treating BTNBC.

Negative Regulation of Human Th2 cells by Type I Interferon (140.15)

Abstract Type I interferon (IFN-α/β) is a potent anti-viral cytokine that regulates multiple aspects of innate and adaptive responses to viral infections. As some respiratory viral infections have been linked to the etiology and exacerbation of asthma, we wished to determine the effects of IFN-α/β on driving the development of human Th2 cells. In these studies, we found that IFNα/β inhibited the ability of IL-4 to promote Th2 differentiation and significantly de-stablized cytokine expression from fully polarized Th2 cells. Further, this effect was specific to IFNα/β as neither IL-12 nor IFNγ inhibited IL-4-regulated Th2 development or stability. IFNα/β inhibited Th2 development by blocking the induction of GATA-3, and this effect was found to bypass the induction of GATA3 via IL-4-mediated STAT6 activation. Finally, IFNα/β also potently inhibited Th2 cytokine secretion from fully committed CRTH2+ cells isolated directly from peripheral blood. Thus, we have found a novel function of IFNα/β in the inhibition of Th2 responses that may be useful as a therapeutic approach for the treatment of asthma.

IL-33 Induces Antigen-Specific IL-5+ T Cells and Promotes Allergic-Induced Airway Inflammation Independent of IL-4

Type 2 cytokines (IL-4, IL-5, and IL-13) play a pivotal role in helminthic infection and allergic disorders. CD4(+) T cells which produce type 2 cytokines can be generated via IL-4-dependent and -independent pathways. Although the IL-4-dependent pathway is well documented, factors that drive IL-4-independent Th2 cell differentiation remain obscure. We report here that the new cytokine IL-33, in the presence of Ag, polarizes murine and human naive CD4(+) T cells into a population of T cells which produce mainly IL-5 but not IL-4. This polarization requires IL-1R-related molecule and MyD88 but not IL-4 or STAT6. The IL-33-induced T cell differentiation is also dependent on the phosphorylation of MAPKs and NF-kappaB but not the induction of GATA3 or T-bet. In vivo, ST2(-/-) mice developed attenuated airway inflammation and IL-5 production in a murine model of asthma. Conversely, IL-33 administration induced the IL-5-producing T cells and exacerbated allergen-induced airway inflammation in wild-type as well as IL-4(-/-) mice. Finally, adoptive transfer of IL-33-polarized IL-5(+)IL-4(-)T cells triggered airway inflammation in naive IL-4(-/-) mice. Thus, we demonstrate here that, in the presence of Ag, IL-33 induces IL-5-producing T cells and promotes airway inflammation independent of IL-4.

Increased activation of GATA‐3, IL‐2 and IL‐5 of cord blood mononuclear cells in infants with IgE sensitization

Risk of allergic diseases has been linked to abnormal patterns of fetal immune development, suggesting that priming of the immune system may occur in utero. The aim of the study was to investigate whether the pattern of immune response in cord blood mononuclear cells (CBMC) shows association with allergic diseases and IgE sensitization at 2 yr of age, and to study the effect of maternal probiotic supplementation on CBMC immune responses. CBMC were isolated from 98 neonates in a randomized double-blinded intervention study. CBMC were stimulated with beta-lactoglobulin, and phytohemaglutinin (PHA). Secretion of interferon-gamma (IFN-gamma), interleukin-5 (IL-5), and IL-13 was measured by an ELISA; IL-2, IL-4, and IL-10 by a cytokine bead assay. T-cell polarization-associated IL-4 receptor and IL-12R expressions, and the respective transcription factors GATA-3 and T-bet were analyzed with RT-PCR. The above responses were compared with the development of allergic diseases and IgE sensitization at 2 yr of age, and with the maternal probiotic or placebo supplementation. PHA-stimulated GATA-3 expression and IL-2 secretion in CBMC were higher in IgE-sensitized children at an age of 2 yr than in the non-sensitized, non-allergic children (p = 0.03 and 0.026). PHA-induced expression of GATA-3 correlated with IL-5 (p = 0.003, r = 0.300) and IL-13 (p = 0.007, r = 0.278) secretion of CBMC, and IL-5 secretion of beta-lactoglobulin-stimulated CBMC was higher in IgE-sensitized children at 2 yr of age than in the non-sensitized, non-allergic children (p = 0.013). Probiotic bacteria had no effect on CBMC immune responses. In CBMC-enhanced induction of GATA-3, which activates several Th2 cytokines genes, was a risk factor for IgE sensitization. The immune deviation towards Th2-type immunity developed already in utero and seemed to modulate the pattern of immune response favoring an IgE response to environmental antigens.

Transcriptional regulation of Th2 differentiation

Naive CD4 T cells have a series of fates open to them; differentiation to Th2 cells depends on the concerted action of three transcription factors, GATA-3, STAT5 and Gfi-1, and displays striking positive reinforcement. The earliest events in Th2 differentiation are T cell receptor driven induction of GATA-3 and of IL-2, the latter activating STAT5. GATA-3 and STAT-5, acting together, lead to early transcription of IL-4. Endogenous IL-4, acting through IL-4Rα and STAT6 strikingly upregulate GATA-3 and IL-4, leading to commitment of the cell to high rate IL-4 production and to the Th2 phenotype. GATA-3 and STAT5 target distinct sites within the Il4 gene, resulting in accessibility and thus the two transcription factors work in concert to activate the gene. Gfi-1 enhances the cytokine driven out-growth of Th2 cells and selects cells expressing the highest amounts of GATA-3. Thus, the interaction of the three transcription factors leads both to Th2 fate determination and selective outgrowth of differentiated Th2 cells and are thus responsible for a robust differentiation process resulting in the appearance of CD4 T cells capable of producing IL-4 and its related cytokines.

T-bet regulates Th1 responses through essential effects on GATA-3 function rather than on IFNG gene acetylation and transcription

T helper type 1 (Th1) development is facilitated by interrelated changes in key intracellular factors, particularly signal transducer and activator of transcription (STAT)4, T-bet, and GATA-3. Here we show that CD4+ cells from T-bet−/− mice are skewed toward Th2 differentiation by high endogenous GATA-3 levels but exhibit virtually normal Th1 differentiation provided that GATA-3 levels are regulated at an early stage by anti–interleukin (IL)-4 blockade of IL-4 receptor (R) signaling. In addition, under these conditions, Th1 cells from T-bet−/− mice manifest IFNG promotor accessibility as detected by histone acetylation and deoxyribonuclease I hypersensitivity. In related studies, we show that the negative effect of GATA-3 on Th1 differentiation in T-bet−/− cells arises from its ability to suppress STAT4 levels, because if this is prevented by a STAT4-expressing retrovirus, normal Th1 differentiation is observed. Finally, we show that retroviral T-bet expression in developing and established Th2 cells leads to down-regulation of GATA-3 levels. These findings lead to a model of T cell differentiation that holds that naive T cells tend toward Th2 differentiation through induction of GATA-3 and subsequent down-regulation of STAT4/IL-12Rβ2 chain unless GATA-3 levels or function is regulated by T-bet. Thus, the principal function of T-bet in developing Th1 cells is to negatively regulate GATA-3 rather than to positively regulate the IFNG gene.

Th2-dominated antitumor immunity induced by DNA immunization with the genes coding for a basal core peptide PDTRP and GM-CSF

Our previous study showed that DNA vaccination with a plasmid vector encoding a core peptide of mucin1 (PDTRP) provided modest protection against challenge with tumor cells that expressed mucin1 protein. We report here that a DNA vaccine comprising a modified PDTRP plasmid and GM-CSF coding sequence at the C-terminus induced better protection against tumor challenge. The increased protection was directly correlated with a stronger PDTRP-specific immune response induced by the GM-CSF fusion plasmid. The plasmid encoding GM-CSF and the target PDTRP antigen induced a greater PDTRP-specific Th proliferation, antibodies, and cytotoxicity. Interestingly, the modified plasmid vaccine predominantly enhanced the type 2 immune responses manifested by an increased IgG1 to IgG2a antibody ratio and a greater induction of GATA-3 and IL-4 mRNA than that of T-bet and IFN-gamma mRNA in spleen cells from vaccinated mice. In addition, protection against tumor challenge in vaccinated mice showed that there was no significant change in mice survival after in vivo CD8+CTL depletion, indicating that antitumor immunity augmented by plasmid encoding GM-CSF and target PDTRP gene vaccine was dominated by Th2 immune response.

ICOS-Mediated Costimulation on Th2 Differentiation Is Achieved by the Enhancement of IL-4 Receptor-Mediated Signaling

ICOS is the third member of the CD28 family molecules and plays a critical role in many T cell-dependent immune responses. Although accumulated data suggest that ICOS costimulatory signals play an important role in Th2-mediated immune responses, the molecular basis for this selective differentiation mechanism is largely unknown. To clarify this mechanism, we used DO11.10 TCR transgenic ICOS-/- mice and evaluated the nature of ICOS costimulatory signals during the process of Ag-specific activation and differentiation of naive CD4+ T cells. Results obtained from these experiments demonstrated that Ag stimulation of naive CD4+ T cells in the absence of an ICOS signal resulted in impaired Th2 development. Unlike previous reports, we found that primary IL-4 production by these T cells was intact and that IL-4R sensitivity of these T cells was reduced as evidenced by a profound defect in IL-4-induced Stat6 phosphorylation and the early induction of GATA-3. The fact that ICOS ligation of wild-type T cells significantly enhanced IL-4-induced Stat6 phosphorylation and primary GATA-3 induction, but not IL-4 transcription, of naive CD4+ T cells was consistent with the results obtained from ICOS-/- T cell experiments. These observations led us to propose that the predominant effect of ICOS-mediated costimulation on Th2 differentiation is achieved by the enhancement of IL-4R-mediated signaling.

Regulation of T helper type 2 cell differentiation by murine Schnurri-2

Schnurri (Shn) is a large zinc finger protein implicated in cell growth, signal transduction, and lymphocyte development. Vertebrates possess at least three Shn orthologues (Shn-1, Shn-2, and Shn-3), which appear to act within the bone morphogenetic protein, transforming growth factor β, and activin signaling pathways. However, the physiological functions of the Shn proteins remain largely unknown. In Shn-2–deficient mice, mature peripheral T cells exhibited normal anti–T cell receptor–induced proliferation, although there was dramatic enhancement in the differentiation into T helper type (Th)2 cells and a marginal effect on Th1 cell differentiation. Shn-2–deficient developing Th2 cells showed constitutive activation of nuclear factor κB (NF-κB) and enhanced GATA3 induction. Shn-2 was able to compete with p50 NF-κB for binding to a consensus NF-κB motif and inhibit NF-κB–driven promoter activity. Thus, Shn-2 plays a crucial role in the control of Th2 cell differentiation by regulating NF-κB function.