T helper type 17 (Th17) cells play critical roles in the pathogenesis of various autoimmune and inflammatory diseases; however, signaling pathways that affect Th17 cell differentiation are not fully understood. Here, we investigated whether focal adhesion kinase (FAK), an integrator of extracellular signals, regulates differentiation of Th17 cells. The findings reveal that Fak deficiency in CD4 T cells significantly reduces Th17 differentiation, while also promoting regulatory T (Treg) cell differentiation, thereby ameliorating symptoms of experimental autoimmune encephalomyelitis (EAE). Mechanistically, Fak deficiency inhibited nuclear translocation of the NF-κB subunit RelA, thereby reducing the binding of RelA to the promoter region of Il17a. Moreover, pharmacological inhibition of FAK with the specific inhibitor PND1186 prevented Th17 differentiation in vitro, and reduced EAE symptoms in vivo. Thus, FAK plays an essential role in Th17 cell differentiation by stimulating NF-κB signaling.

For several decades, a plant-based expression system has been proposed as an alternative platform for the production of biopharmaceuticals including therapeutic monoclonal antibodies (mAbs), but the immunogenicity concerns associated with plant-specific N-glycans attached in plant-based biopharmaceuticals has not been completely solved. To eliminate all plant-specific N-glycan structure, eight genes involved in plant-specific N-glycosylation were mutated in rice (Oryza sativa) using the CRISPR/Cas9 system. The glycoengineered cell lines, PhytoRice (R), contained a predominant GnGn (G0) glycoform. The gene for codon-optimized trastuzumab (TMab) was then introduced into PhytoRice (R) through Agrobacterium co-cultivation. Selected cell lines were suspension cultured, and TMab secreted from cells was purified from the cultured media. The amino acid sequence of the TMab produced by PhytoRice (R) (P-TMab) was identical to that of TMab. The inhibitory effect of P-TMab on the proliferation of the BT-474 cancer cell line was significantly enhanced at concentrations above 1 mu g/mL (****P < 0.0001). P-TMab bound to a Fc gamma RIIIa variant, Fc gamma RIIIa-F158, more than 2.7 times more effectively than TMab. The ADCC efficacy of P-TMab against Jurkat cells was 2.6 times higher than that of TMab in an in vitro ADCC assay. Furthermore, P-TMab demonstrated efficient tumour uptake with less liver uptake compared to TMab in a xenograft assay using the BT-474 mouse model. These results suggest that the glycoengineered PhytoRice (R) could be an alternative platform for mAb production compared to current CHO cells, and P-TMab has a novel and enhanced efficacy compared to TMab.

Regulatory T (Treg) cells play an important role in immune homeostasis by inhibiting cells within the innate and adaptive immune systems; therefore, the stability and immunosuppressive function of Treg cells need to be maintained. In this study, we found that the expression of insulin receptor substrate 1 (IRS1) by Treg cells was lower than that by conventional CD4 T cells. IRS1-overexpressing Treg cells showed the downregulated expression of FOXP3, as well as Treg signature markers CD25 and CTLA4. IRS1-overexpressing Treg cells also showed diminished immunosuppressive functions in an in vitro suppression assay. Moreover, IRS1-overexpressing Treg cells were unable to suppress the pathogenic effects of conventional T cells in a transfer-induced colitis model. IRS1 activated the mTORC1 signaling pathway, a negative regulator of Treg cells. Moreover, IRS1 destabilized Treg cells by upregulating the expression of IFN-γ and Glut1. Thus, IRS1 acts as a negative regulator of Treg cells by downregulating the expression of FOXP3 and disrupting stability.

T helper type 9 (Th9) cells play important roles in immune responses by producing interleukin-9 (IL-9). Several transcription factors are responsible for Th9 cell differentiation; however, transcriptional regulation of Th9 cells is not fully understood. Here, it is shown that Id1 is an essential transcriptional regulator of Th9 cell differentiation. Id1 is induced by IL-4 and TGF-β. Id1-deficient naïve CD4 T cells fail to differentiate into Th9 cells, and overexpression of Id1 induce expression of IL-9. Mass spectrometry analysis reveals that Id1 interacts with Tcf3 and Tcf4 in Th9 cells. In addition, RNA-sequencing, chromatin immunoprecipitation, and transient reporter assay reveal that Tcf3 and Tcf4 bind to the promoter region of the Il9 gene to suppress its expression, and that Id1 inhibits their function, leading to Th9 differentiation. Finally, Id1-deficient Th9 cells ameliorate airway inflammation in an animal model of asthma. Thus, Id1 is a transcription factor that plays an essential role in Th9 cell differentiation by inhibiting Tcf3 and Tcf4.

Th cells, which orchestrate immune responses to various pathogens, differentiate from naïve CD4 T cells into several subsets that stimulate and regulate immune responses against various types of pathogens, as well as a variety of immune-related diseases. Decades of research have revealed that the fate decision processes are controlled by cytokines, cytokine receptor signaling, and master transcription factors that drive the differentiation programs. Since the Th1 and Th2 paradigm was proposed, many subsets have been added to the list. In this review, I will summarize these events, including the fate decision processes, subset functions, transcriptional regulation, metabolic regulation, and plasticity and heterogeneity. I will also introduce current topics of interest.

Regulatory T (Treg) cells maintain immune homeostasis by preventing abnormal or excessive immune responses. Histone deacetylase 6 (HDAC6) regulates expression of Foxp3, and thus, Treg cell differentiation; however, its role in Treg cell differentiation is unclear and somewhat controversial. Here, we investigated the role of HDAC6 in TGF-β-induced murine Treg cells. HDAC6 expression was higher in Treg cells than in other T helper cell subsets. Pharmacological inhibitors of HDAC6 selectively inhibited Treg cell differentiation and suppressive function. A specific HDAC6 inhibitor induced changes in global gene expression by Treg cells. Of these changes, genes related to cell division were prominently affected. In summary, HDAC6 plays an important role in TGF-β-induced murine Treg cell differentiation by regulating cell proliferation.

Naive T cells undergo radical changes during the transition from dormant to hyperactive states upon activation, which necessitates de novo protein production via transcription and translation. However, the mechanism whereby T cells globally promote translation remains largely unknown. Here, we show that on exit from quiescence, T cells upregulate transfer RNA (tRNA) m1A58 'writer' proteins TRMT61A and TRMT6, which confer m1A58 RNA modification on a specific subset of early expressed tRNAs. These m1A-modified early tRNAs enhance translation efficiency, enabling rapid and necessary synthesis of MYC and of a specific group of key functional proteins. The MYC protein then guides the exit of naive T cells from a quiescent state into a proliferative state and promotes rapid T cell expansion after activation. Conditional deletion of the Trmt61a gene in mouse CD4+ T cells causes MYC protein deficiency and cell cycle arrest, disrupts T cell expansion upon cognate antigen stimulation and alleviates colitis in a mouse adoptive transfer colitis model. Our study elucidates for the first time, to our knowledge, the in vivo physiological roles of tRNA-m1A58 modification in T cell-mediated pathogenesis and reveals a new mechanism of tRNA-m1A58-controlled T cell homeostasis and signal-dependent translational control of specific key proteins.

T cells maintain a quiescent state prior to activation. As inappropriate T cell activation can cause disease, T cell quiescence must be preserved. Despite its importance, the mechanisms underlying the "quiescent state" remain elusive. Here, we identify BTG1 and BTG2 (BTG1/2) as factors responsible for T cell quiescence. BTG1/2-deficient T cells show an increased proliferation and spontaneous activation due to a global increase in messenger RNA (mRNA) abundance, which reduces the threshold to activation. BTG1/2 deficiency leads to an increase in polyadenylate tail length, resulting in a greater mRNA half-life. Thus, BTG1/2 promote the deadenylation and degradation of mRNA to secure T cell quiescence. Our study reveals a key mechanism underlying T cell quiescence and suggests that low mRNA abundance is a crucial feature for maintaining quiescence.

Regulatory T (Treg) cells are a CD4 T-cell subset with an important role in immune tolerance; however, the mechanisms underlying Treg cell differentiation and function are incompletely understood. Here, we show that NFIL3/E4BP4, a transcription factor, plays a key role in Treg cell differentiation and function. Microarray analysis showed that Treg cells had lower Nfil3 expression than all other CD4 T-cell subsets. Overexpression of Nfil3 in Treg cells led to diminished expression of Foxp3 and other signature Treg genes, including Il2ra , Icos , Tnfrsf18 , and Ctla4 . Furthermore, Nfil3 -overexpressing Treg cells exhibited impaired immunosuppressive activity in vitro and in vivo. We discovered that NFIL3 directly binds to and negatively regulates the expression of Foxp3 . In addition, bisulfite sequencing revealed that NFIL3 induces methylation at Foxp3 locus regulatory CpG sites, which contributes to the control of Treg cell stability. Together, these data indicate that NFIL3 impairs Treg cell function through the downregulation of Foxp3 expression.

Th9 cells preferentially produce IL-9 and participate in allergic responses and asthma. Differentiation of Th9 cells is induced by IL-4 and TGF-β, and then the cells are amplified by OX40 signals. The transcription factors PU.1, IRF4, and BATF are required for Th9 differentiation. BATF3 is an AP-1 family transcription factor that is highly homologous to BATF; however, its role in Th9 cells is poorly defined. Here, we show that OX40 signaling induced the expression of Batf3 and that its overexpression in the presence or absence of OX40 signaling increased the expression of IL-9 in Th9 cells. BATF3 physically interacted with IRF4 and was bound to the Il9 locus. A transient reporter assay revealed that the BATF3-IRF4 complex induced Il9 promoter activity. BATF3 rescued Il9 expression and restored the capacity to induce the airway inflammation in Batf KO Th9 cells. Thus, BATF3 itself is sufficient for the induction of Th9 cell differentiation and can substitute for BATF during Th9 cell differentiation.

Regulatory T (Treg) cells play an essential role in maintaining immune homeostasis, but the suppressive function of Treg cells can be an obstacle in the treatment of cancer and chronic infectious diseases. Here, we identified the homeobox protein Hhex as a negative regulator of Treg cells. The expression of Hhex was lower in Treg cells than in conventional T (Tconv) cells. Hhex expression was repressed in Treg cells by TGF-β/Smad3 signaling. Retroviral overexpression of Hhex inhibited the differentiation of induced Treg (iTreg) cells and the stability of thymic Treg (tTreg) cells by significantly reducing Foxp3 expression. Moreover, Hhex-overexpressing Treg cells lost their immunosuppressive activity and failed to prevent colitis in a mouse model of inflammatory bowel disease (IBD). Hhex expression was increased; however, Foxp3 expression was decreased in Treg cells in a delayed-type hypersensitivity (DTH) reaction, a type I immune reaction. Hhex directly bound to the promoters of Foxp3 and other Treg signature genes, including Il2ra and Ctla4, and repressed their transactivation. The homeodomain and N-terminal repression domain of Hhex were critical for inhibiting Foxp3 and other Treg signature genes. Thus, Hhex plays an essential role in inhibiting Treg cell differentiation and function via inhibition of Foxp3.

Yin Yang 1 (YY1) is a ubiquitously expressed transcription factor that functions in cooperation with various cofactors to regulate gene expression. In the immune system, YY1 enhances cytokine production and T helper (Th) 2 effector cell differentiation, resulting in the activation of inflammation. However, no studies have reported the role of YY1 in Th17 cell regulation, which is implicated in rheumatoid arthritis (RA). We investigated the expression of YY1 in Th17 cells in vitro and revealed increased levels of YY1 mRNA and protein. To elucidate the function of YY1 pathogenesis in RA, we used a collagen-induced arthritis (CIA) mouse model with YY1 deficiency. Deficiency of YY1 reduced the severity of arthritis and joint destruction. Moreover, Th17 cells were dramatically reduced in YY1-deficient mice. The cytokine interleukin (IL)-17 was decreased in YY1-deficient CD4+ T cells ex vivo and in vivo. Interestingly, the level of signal transducer and activator of transcription 3 (STAT3), tumor necrosis factor-α, IL-17, IL-6, and IL-1β were markedly decreased in YY1-deficient mice with CIA. The cytokine-inducing function of YY1 was more specific to IL-17 than to interferon-γ. YY1 plays a role in Th17 cell differentiation and RA pathogenesis. Our findings suggest that future RA therapies should target the regulatory mechanism involved in Th17 cell differentiation, in which YY1 may cooperate with the STAT3 signaling pathway.

T helper type 17 (Th17) cells and pTreg cells, which share a common precursor cell (the naïve CD4 T cell), require a common tumor growth factor (TGF)-β signal for initial differentiation. However, terminally differentiated cells fulfill opposite functions: Th17 cells cause autoimmunity and inflammation, whereas Treg cells inhibit these phenomena and maintain immune homeostasis. Thus, unraveling the mechanisms that affect the Th17/Treg cell balance is critical if we are to better understand autoimmunity and tolerance. Recent studies have identified many factors that influence this balance; these factors range from signaling pathways triggered by T cell receptors, costimulatory receptors, and cytokines, to various metabolic pathways and the intestinal microbiota. This review article summarizes recent advances in our understanding of the Th17/Treg balance and its implications with respect to autoimmune disease.

T helper 17 (Th17) cells are one of CD4+ T cell subsets that mediate inflammation and autoimmunity. Unlike other subsets, interleukin 2 (IL-2) inhibits differentiation of Th17 cells but the mechanism that regulates Th17 differentiation is poorly understood. Here, we show that phosphatase and tensin homologue (PTEN) plays key role in Th17 cell differentiation by regulating IL-2 expression. We generated Th17-specific Pten knock-out mice (Pten fl/fl Il17a CRE) to investigate intrinsic role of PTEN. Th17-specific Pten deletion inhibited Th17 differentiation by inducing excessive expression of IL-2 in vitro. Over-expressed IL-2 recognized by IL-2 receptor and induced phosphorylation of STAT5, eventually triggered Foxp3 expression in Th17 cells. Next, we used experimental autoimmune encephalomyelitis (EAE) as in vivo model, Th17-specific deficiency of Pten ameliorated symptoms of EAE. Additionally, we used SF1670 which is PTEN-specific inhibitor for therapeutic approach. Treatment of SF1670 to EAE induced mice showed ameliorated symptoms of EAE, therefore PTEN may be a potential therapeutic target for Th17-mediated diseases.

Objectives Hepatocellular carcinoma (HCC) is a common cancer with high rate of recurrence and mortality. Diverse aetiological agents and wide heterogeneity in individual tumours impede effective and personalised treatment. Tonicity-responsive enhancer-binding protein (TonEBP) is a transcriptional cofactor for the expression of proinflammatory genes. Although inflammation is intimately associated with the pathogenesis of HCC, the role of TonEBP is unknown. We aimed to identify function of TonEBP in HCC. Design Tumours with surrounding hepatic tissues were obtained from 296 patients with HCC who received completion resection. TonEBP expression was analysed by quantitative reverse transcription-quantitative real-time PCR (RT-PCR) and immunohfistochemical analyses of tissue microarrays. Mice with TonEBP haplo deficiency, and hepatocyte-specific and myeloid-specific TonEBP deletion were used along with HCC and hepatocyte cell lines. Results TonEBP expression is higher in tumours than in adjacent non-tumour tissues in 92.6% of patients with HCC regardless of aetiology associated. The TonEBP expression in tumours and adjacent non-tumour tissues predicts recurrence, metastasis and death in multivariate analyses. TonEBP drives the expression of cyclo-oxygenase-2 (COX-2) by stimulating the promoter. In mouse models of HCC, three common sites of TonEBP action in response to diverse aetiological agents leading to tumourigenesis and tumour growth were found: cell injury and inflammation, induction by oxidative stress and stimulation of the COX-2 promoter. Conclusions TonEBP is a key component of the common pathway in tumourigenesis and tumour progression of HCC in response to diverse aetiological insults. TonEBP is involved in multiple steps along the pathway, rendering it an attractive therapeutic target as well as a prognostic biomarker.

Regulatory T (Treg) cells are a distinct subset of CD4+ T cells. Instead of triggering adaptive immunity, they suppress immune responses. Small numbers of Treg cells reside within lymphoid organs and peripheral tissues, but their contribution to immune tolerance is so significant that defects in Treg cell function cause catastrophic immune disorders. Since they were first discovered 20 years ago, efforts have been made to understand the differences in developmental processes between Treg cells and conventional T cells that determine the ultimate fate of the overall T-cell population. Transcription factor Foxp3 is crucial for Treg cell differentiation, but it is not the whole story. Owing to recent advances in Treg cell research, we are now on the verge of appreciating the comprehensive mechanisms underlying Treg cell generation. Here, we discuss major discoveries, active study topics and remaining questions regarding Treg cell development.

Rad50 hypersensitive site 6 (RHS6) is a cis-acting element in the Th2 locus control region which is essential for the expression of Th2 cytokine genes. However, molecular mechanisms how it regulates the genes are poorly understood. We found that RHS6 recruits transcription factors GATA3, SATB1, and IRF4 during Th2 cell differentiation. GATA3, SATB1, and IRF4 have a crucial role in regulating Th2 cytokine genes. Knock-out or knock-down of each of GATA3, SATB1, and IRF4 genes significantly decrease the expression of Th2 cytokine genes. Mechanistically RHS6 mediates the transcription factor-induced regulation of Th2 cytokine genes. GATA3, SATB1, and IRF4 physically associates with each other, and forms a protein complex at RHS6 sequence. They directly bind to the many regulatory elements throughout the Th2 cytokine locus, RHS6 dependent manner. Furthermore, deletion of RHS6 caused a dramatic resistance to airway inflammation in ovalbumin-induced allergic inflammation model. Together, RHS6 recruits GATA3, SATB1, and IRF4 coordinating their regulation of Th2 cytokine genes, so is essential for Th2-mediated allergic airway inflammation.