Regulation Of Th2 Cell Differentiation Research Articles

Neuroprotective protein ADNP-dependent histone remodeling complex promotes T helper 2 immune cell differentiation

Type 2 immune responses are critical in tissue homeostasis, anti-helminth immunity, and allergy. T helper 2 (Th2) cells produce interleukin-4 (IL-4), IL-5, and IL-13 from the type 2 gene cluster under regulation by transcription factors (TFs) including GATA3. To better understand transcriptional regulation of Th2 cell differentiation, we performed CRISPR-Cas9 screens targeting 1,131 TFs. We discovered that activity-dependent neuroprotector homeobox protein (ADNP) was indispensable for immune reactions to allergen. Mechanistically, ADNP performed a previously unappreciated role in gene activation, forming a critical bridge in the transition from pioneer TFs to chromatin remodeling by recruiting the helicase CHD4 and ATPase BRG1. Although GATA3 and AP-1 bound the type 2 cytokine locus in the absence of ADNP, they were unable to initiate histone acetylation or DNA accessibility, resulting in highly impaired type 2 cytokine expression. Our results demonstrate an important role for ADNP in promoting immune cell specialization.

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.

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

Abstract Although prior research implicated c-Jun N-terminal kinase (JNK) in helper CD4 +T cell differentiation, mechanisms governing JNK signaling and function in human lymphocytes remain nebulous. CARD11 is a lymphocyte-specific scaffold protein connecting antigen receptor (AgR) engagement to transcriptional programs necessary for effector cell survival, proliferation, and differentiation. In addition to NF-κB, CARD11 is also required for AgR-induced mTORC1 and JNK2 signaling. We sought to define the impact of dominant interfering (DI) CARD11 variants on JNK signaling and elucidate transcriptomic changes governed by CARD11-dependent JNK activation in T cells. We transfected WT and CARD11 knockout (KO) Jurkat T cells with plasmids expressing WT or DI CARD11 variants to assess effects on CARD11-dependent JNK signaling and performed RNA sequencing on mitogen-activated WT and CARD11 KO Jurkat T cells ± JNK inhibition. Our results revealed that AgR-dependent JNK1 andJNK2 activation are CARD11-dependent. Additionally, heterozygous DI CARD11 mutations derived from patients with CARD11-associated atopy with dominant interference of NF-κB signaling (CADINS) disease disrupted AgR-dependent JNK1/2 phosphorylation, c-Jun accumulation, and AP-1 transcriptional activity with variable potency, mirroring the extent of impaired NF-κB 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. Our novel findings suggest that defects in CARD11-dependent JNK signaling in CD4 +T cells may contribute to severe allergic disease noted in CADINS patients, unveiling a new potential therapeutic target. Supported by funding from the Jeffrey Modell Specific Defect Research Program (ALS) and the Henry M. Jackson Foundation for the Advancement of Military Medicine (BMB).

Glycolysis and subsequent mevalonate biosynthesis play an important role in Th2 cell differentiation

Th2 cytokine such as IL-4, IL -5 and IL-13 are important therapeutic targets for Th2-type chronic inflammation. Several biologics targeting Th2 cytokine and its receptors are effective in clinical practice; however, the development of small-molecule compounds that inhibit Th2 cytokine productions is awaited. We found that an inhibitor for pyruvate dehydrogenase kinase (PDHK) suppresses the differentiation of IL-5/IL-13-producing Th2 cells. The expression of the Th2-related transcriptional factors Pparγ was decreased by treatment with inhibitor, whereas Gata3, a master regulator of Th2 cell differentiation, remained unchanged. The oxygen consumption rate was unaffected, whereas the level of farnesylated proteins was decreased by the PDHK inhibitor. Furthermore, the inhibitors for farnesyltransferase and hydroxymethylglutaryl-CoA reductase showed an inhibitory effect similar to that of the PDHK inhibitor. These results suggest that the mevalonate biosynthesis and subsequent protein prenylation may be novel therapeutic target for Th2 cell-dependent immune dysregulation, such as in allergic diseases.

MiR-23~27~24 clusters restrict Th2 immunity and associated immunopathology during airway allergic reaction (HYP2P.335)

Abstract MiRNAs (miRNAs) are important regulators in T cell differentiation and function. Here we show miR-23~27~24 clusters play a pivotal role in controlling type II immunity. Specifically, under Th2 polarizing condition, T cells with overexpression of miR-23~27~24 clusters exhibited reduced IL-4 secretion whereas T cells devoid of miR-23~27~24 clusters produced elevated amounts of IL-4. Further mechanistic studies revealed miR-24 and miR-27 could repress Gata3, a key regulator of Th2 cell differentiation through an indirect and a direct manner, respectively. Finally, by using an OVA-induced asthma model, we have shown that mice with T cell-specific ablation of miR-23~27~24 clusters developed a more severe airway inflammation characterized by increased IL-4 secretion, lung eosinophil infiltration, mucin production and serum IgE levels. Taken together, our studies identify a miRNA family with important biological function particularly in controlling Th2 immunity and suggest that a tight regulation of this miRNA family is required to maintain optimal effector T cell function and to prevent aberrant immune responses.

Genome-Wide Gene Expression Profiling Revealed a Critical Role for GATA3 in the Maintenance of the Th2 Cell Identity

Functionally polarized CD4+ T helper (Th) cells such as Th1, Th2 and Th17 cells are central to the regulation of acquired immunity. However, the molecular mechanisms governing the maintenance of the polarized functions of Th cells remain unclear. GATA3, a master regulator of Th2 cell differentiation, initiates the expressions of Th2 cytokine genes and other Th2-specific genes. GATA3 also plays important roles in maintaining Th2 cell function and in continuous chromatin remodeling of Th2 cytokine gene loci. However, it is unclear whether continuous expression of GATA3 is required to maintain the expression of various other Th2-specific genes. In this report, genome-wide DNA gene expression profiling revealed that GATA3 expression is critical for the expression of a certain set of Th2-specific genes. We demonstrated that GATA3 dependency is reduced for some Th2-specific genes in fully developed Th2 cells compared to that observed in effector Th2 cells, whereas it is unchanged for other genes. Moreover, effects of a loss of GATA3 expression in Th2 cells on the expression of cytokine and cytokine receptor genes were examined in detail. A critical role of GATA3 in the regulation of Th2-specific gene expression is confirmed in in vivo generated antigen-specific memory Th2 cells. Therefore, GATA3 is required for the continuous expression of the majority of Th2-specific genes involved in maintaining the Th2 cell identity.

Genome-Wide Analysis Reveals Unique Regulation of Transcription of Th2-Specific Genes by GATA3

Differentiation of naive CD4 T cells into Th2 cells is accompanied by chromatin remodeling and increased expression of a set of Th2-specific genes, including those encoding Th2 cytokines. IL-4-mediated STAT6 activation induces high levels of transcription of GATA3, a master regulator of Th2 cell differentiation, and enforced expression of GATA3 induces Th2 cytokine expression. However, it remains unclear whether the expression of other Th2-specific genes is induced directly by GATA3. A genome-wide unbiased chromatin immunoprecipitation assay coupled with massive parallel sequencing analysis revealed that GATA3 bound to 1279 genes selectively in Th2 cells, and 101 genes in both Th1 and Th2 cells. Simultaneously, we identified 26 highly Th2-specific STAT6-dependent inducible genes by DNA microarray analysis-based three-step selection processes, and among them 17 genes showed GATA3 binding. We assessed dependency on GATA3 for the transcription of these 26 Th2-specific genes, and 10 genes showed increased transcription in a GATA3-dependent manner, whereas 16 genes showed no significant responses. The transcription of the 16 GATA3-nonresponding genes was clearly increased by the introduction of an active form of STAT6, STAT6VT. Therefore, although GATA3 has been recognized as a master regulator of Th2 cell differentiation, many Th2-specific genes are not regulated by GATA3 itself, but in collaboration with STAT6.

Transcriptional regulation of Th2 cell differentiation

CD4 T helper 2 (Th2) cells have critical functions in immune responses against extracellular parasites and are involved in asthma and other allergic diseases. The differentiation of naïve CD4 T cells into Th2 cells is initiated from T-cell receptor and cytokine-mediated signaling followed by upregulation of GATA3 and activation of signal transducer and activator of transcription 5 (STAT5), two indispensable events for this differentiation process. In this review, regulation of GATA3 expression and STAT5 activation and functions of these two transcription factors in inducing the expression of Th2 cytokines, cytokine receptors as well as epigenetic modification at Th2 cytokine locus are summarized. Furthermore, I present positive and negative regulatory networks important for Th2 cell commitment, selective growth of committed Th2 cells and suppression of alternative lineage fates. Finally, the difference between in vitro and in vivo Th2 differentiation is discussed.

T follicular helper cells differentiate from Th2 cells in response to helminth antigens

The relationship of T follicular helper (TFH) cells to other T helper (Th) subsets is controversial. We find that after helminth infection, or immunization with helminth antigens, reactive lymphoid organs of 4get IL-4/GFP reporter mice contain populations of IL-4/GFP-expressing CD4+ T cells that display the TFH markers CXCR5, PD-1, and ICOS. These TFH cells express the canonical TFH markers BCL6 and IL-21, but also GATA3, the master regulator of Th2 cell differentiation. Consistent with a relationship between Th2 and TFH cells, IL-4 protein production, reported by expression of huCD2 in IL-4 dual reporter (4get/KN2) mice, was a robust marker of TFH cells in LNs responding to helminth antigens. Moreover, the majority of huCD2/IL-4–producing Th cells were found within B cell follicles, consistent with their definition as TFH cells. TFH cell development after immunization failed to occur in mice lacking B cells or CD154. The relationship of TFH cells to the Th2 lineage was confirmed when TFH cells were found to develop from CXCR5− PD-1− IL-4/GFP+ CD4+ T cells after their transfer into naive mice and antigen challenge in vivo.

Cutting Edge: Ikaros Is a Regulator of Th2 Cell Differentiation

Ikaros, a hematopoietic transcription factor, has well defined effects on early lymphocyte development in the bone marrow and thymus. In this study we demonstrate that Ikaros is a positive regulator of Th2 cytokine gene expression in peripheral T cells. CD4+ T cells from naive Ikaros(null) mice cultured under Th2-skewing conditions express the Th1 cytokine IFN-gamma and have reduced IL-4, IL-5, and IL-13 expression. Ikaros directly associates with several Th2 locus regulatory regions in naive CD4+ T cells. The decreased ability to express Th2 cytokines in Ikaros(null)T cells corresponds with histone 3 hypoacetylation across the Th2 cytokine locus as well as decreased GATA3 and cMaf and increased T-bet and STAT1 expression. These data support a model whereby Ikaros directly activates Th2 gene expression by promoting local chromatin accessibility during CD4+ T cell differentiation and also acts indirectly to regulate expression of Th2- and Th1-specific transcription factors.

Gata-3-specific Dnazyme As An Appraoch For Asthma-therapy

RATIONALE: The transcription factor GATA-3 is a key regulator of Th2 cell differentiation. Since Th2 cells play a principal role in the initiation and manintanance of allergic airway inflammation, GATA-3 represents a promissing target for therapeutic intervention.

Regulation of Th2 Cell Differentiation by mel-18, a Mammalian Polycomb Group Gene

Polycomb group ( PcG) gene products regulate homeobox gene expression in Drosophila and vertebrates and also cell cycle progression of immature lymphocytes. In a gene-disrupted mouse for polycomb group gene mel-18, mature peripheral T cells exhibited normal anti-TCR-induced proliferation; however, the production of Th2 cytokines (IL-4, IL-5, and IL-13) was significantly reduced, whereas production of IFNγ was modestly enhanced. Th2 cell differentiation was impaired, and the defect was associated with decreased levels in demethylation of the IL-4 gene. Significantly, reduced GATA3 induction was demonstrated. In vivo antigen-induced IgG1 production and Nippostrongylus brasiliensis-induced eosinophilia were significantly affected, reflecting the deficit in Th2 cell differentiation. Thus, the PcG gene products play a critical role in the control of Th2 cell differentiation and Th2-dependent immune responses.

The Role of α-Galactosylceramide-Activated Vα14 Natural Killer T Cells in the Regulation of Th2 Cell Differentiation

Vα14 natural killer T (NKT) cells produce large amounts of both IL-4 and IFN-γ upon stimulation with a ligand, α-galactosylceramide (α-GalCer), and play a crucial role in various immune responses, including allergic diseases. Interestingly, Vα14 NKT cells are not essential for the induction of IgE responses but rather induce suppression of specific IgE production upon activation. The suppression in the IgE production is not detected either in Vα14 NKT cell-deficient mice or in IFN-γ-deficient mice. Thus, activated Vα14 NKT cells are likely to exert a potent suppressive activity on Th2 cell differentiation and subsequent IgE production by producing a large amount of IFN-γ. In marked contrast, little regulatory effect of IL-4 produced by Vα14 NKT cells on Th2 cell differentiation is suggested.