Diagnosis of ADHD using virtual reality and artificial intelligence: an exploratory study of clinical applications.

Introduction: Diagnosis of Attention Deficit/Hyperactivity Disorder (ADHD) is based on clinical evaluation of symptoms by a psychiatrist, referencing results of psychological tests. When diagnosing ADHD, the child's behavior and functionality in real-life situations are critical components. However, direct observation by a clinician is often not feasible in practice. Therefore, such information is typically gathered from primary caregivers or teachers, which can introduce subjective elements. To overcome these limitations, we developed AttnKare-D, an innovative digital diagnostic tool that could analyze children's behavioral data in Virtual Reality using Artificial Intelligence. The purpose of this study was to explore the utility and safety of AttnKare-D for clinical application. Method: A total of 21 children aged between 6 and 12 years were recruited for this study. Among them, 15 were children diagnosed with ADHD, 5 were part of a normal control group, and 1 child was excluded due to withdrawal of consent. Psychological assessments, including K-WISC, Conners CPT, K-ARS, and K-CBCL, were conducted for participants and their primary caregivers. Diagnoses of ADHD were confirmed by child and adolescent psychiatrists based on comprehensive face-to-face evaluations and results of psychological assessments. Participants underwent VR diagnostic assessment by performing various cognitive and behavioral tasks in a VR environment. Collected data were analyzed using an AI model to assess ADHD diagnosis and the severity of symptoms. Results: AttnKare-D demonstrated diagnostic performance with an AUC of 0.893 when compared to diagnoses made by child and adolescent psychiatrist, showing a sensitivity of 0.8 and a specificity of 1.0 at a cut-off score of 18.44. AttnKare-D scores showed a high correlation with K-ARS scores rated by parents and experts, although the correlation was relatively low for inattention scores. Conclusion: Results of this study suggest that AttnKare-D can be a useful tool for diagnosing ADHD in children. This approach has potential to overcome limitations of current diagnostic methods, enhancing the accuracy and objectivity of ADHD diagnoses. This study lays the groundwork for further improvement and research on diagnostic tools integrating VR and AI technologies. For future clinical applications, it is necessary to conduct clinical trials involving a sufficient number of participants to ensure reliable use.

Recruitment of CTCF to the SIRT1 promoter after Oxidative Stress mediates Cardioprotective Transcription.

Because most DNA-binding transcription factors (dbTFs), including the architectural regulator CTCF, bind RNA and exhibit di-/multimerization, a central conundrum is whether these distinct properties are regulated post-transcriptionally to modulate transcriptional programs. Here, investigating stress-dependent activation of SIRT1, encoding an evolutionarily-conserved protein deacetylase, we show that induced phosphorylation of CTCF acts as a rheostat to permit CTCF occupancy of low-affinity promoter DNA sites to precisely the levels necessary. This CTCF recruitment to the SIRT1 promoter is eliciting a cardioprotective cardiomyocyte transcriptional activation program and provides resilience against the stress of the beating heart in vivo . Mice harboring a mutation in the conserved low-affinity CTCF promoter binding site exhibit an altered, cardiomyocyte-specific transcriptional program and a systolic heart failure phenotype. This transcriptional role for CTCF reveals that a covalent dbTF modification regulating signal-dependent transcription serves as a previously unsuspected component of the oxidative stress response.

Enhancer-promoter specificity in gene transcription: molecular mechanisms and disease associations.

Although often located at a distance from their target gene promoters, enhancers are the primary genomic determinants of temporal and spatial transcriptional specificity in metazoans. Since the discovery of the first enhancer element in simian virus 40, there has been substantial interest in unraveling the mechanism(s) by which enhancers communicate with their partner promoters to ensure proper gene expression. These research efforts have benefited considerably from the application of increasingly sophisticated sequencing- and imaging-based approaches in conjunction with innovative (epi)genome-editing technologies; however, despite various proposed models, the principles of enhancer-promoter interaction have still not been fully elucidated. In this review, we provide an overview of recent progress in the eukaryotic gene transcription field pertaining to enhancer-promoter specificity. A better understanding of the mechanistic basis of lineage- and context-dependent enhancer-promoter engagement, along with the continued identification of functional enhancers, will provide key insights into the spatiotemporal control of gene expression that can reveal therapeutic opportunities for a range of enhancer-related diseases.

Transcriptional and Epigenetic Regulation of Context-Dependent Plasticity in T-Helper Lineages.

Th cell lineage determination and functional specialization are tightly linked to the activation of lineage-determining transcription factors (TFs) that bind cis-regulatory elements. These lineage-determining TFs act in concert with multiple layers of transcriptional regulators to alter the epigenetic landscape, including DNA methylation, histone modification and three-dimensional chromosome architecture, in order to facilitate the specific Th gene expression programs that allow for phenotypic diversification. Accumulating evidence indicates that Th cell differentiation is not as rigid as classically held; rather, extensive phenotypic plasticity is an inherent feature of T cell lineages. Recent studies have begun to uncover the epigenetic programs that mechanistically govern T cell subset specification and immunological memory. Advances in next generation sequencing technologies have allowed global transcriptomic and epigenomic interrogation of CD4+ Th cells that extends previous findings focusing on individual loci. In this review, we provide an overview of recent genome-wide insights into the transcriptional and epigenetic regulation of CD4+ T cell-mediated adaptive immunity and discuss the implications for disease as well as immunotherapies.

Direct observation of surface charge and stiffness of human metaphase chromosomes.

Metaphase chromosomes in which both polynucleotides and proteins are condensed with hierarchies are closely related to life phenomena such as cell division, cancer development, and cellular senescence. Nevertheless, their nature is rarely revealed, owing to their structural complexity and technical limitations in analytical methods. In this study, we used surface potential and nanomechanics mapping technology based on atomic force microscopy to measure the surface charge and intrinsic stiffness of metaphase chromosomes. We found that extra materials covering the chromosomes after the extraction process were positively charged. With the covering materials, the chromosomes were positively charged (ca. 44.9 ± 16.48 mV) and showed uniform stiffness (ca. 6.23 ± 1.98 MPa). In contrast, after getting rid of the extra materials through treatment with RNase and protease, the chromosomes were strongly negatively charged (ca. -197.4 ± 77.87 mV) and showed relatively non-uniform and augmented stiffness (ca. 36.87 ± 17.56 MPa). The results suggested undulating but compact coordination of condensed chromosomes. Additionally, excessive treatment with RNase and protease could destroy the chromosomal structure, providing an exceptional opportunity for multiscale stiffness mapping of polynucleotides, nucleosomes, chromatin fibers, and chromosomes in a single image. Our approach offers a new horizon in terms of an analytical technique for studying chromosome-related diseases.

Dynamics of Viral and Host 3D Genome Structure upon Infection.

Eukaryotic chromatin is highly organized in the 3D nuclear space and dynamically regulated in response to environmental stimuli. This genomic organization is arranged in a hierarchical fashion to support various cellular functions, including transcriptional regulation of gene expression. Like other host cellular mechanisms, viral pathogens utilize and modulate host chromatin architecture and its regulatory machinery to control features of their life cycle, such as lytic versus latent status. Combined with previous research focusing on individual loci, recent global genomic studies employing conformational assays coupled with high-throughput sequencing technology have informed models for host and, in some cases, viral 3D chromosomal structure re-organization during infection and the contribution of these alterations to virus-mediated diseases. Here, we review recent discoveries and progress in host and viral chromatin structural dynamics during infection, focusing on a subset of DNA (human herpesviruses and HPV) as well as RNA (HIV, influenza virus and SARS-CoV-2) viruses. An understanding of how host and viral genomic structure affect gene expression in both contexts and ultimately viral pathogenesis can facilitate the development of novel therapeutic strategies.

Long-distance association of topological boundaries through nuclear condensates.

The eukaryotic genome is partitioned into distinct topological domains separated by boundary elements. Emerging data support the concept that several well-established nuclear compartments are ribonucleoprotein condensates assembled through the physical process of phase separation. Here, based on our demonstration that chemical disruption of nuclear condensate assembly weakens the insulation properties of a specific subset (∼20%) of topologically associated domain (TAD) boundaries, we report that the disrupted boundaries are characterized by a high level of transcription and striking spatial clustering. These topological boundary regions tend to be spatially associated, even interchromosomally, segregate with nuclear speckles, and harbor a specific subset of "housekeeping" genes widely expressed in diverse cell types. These observations reveal a previously unappreciated mode of genome organization mediated by conserved boundary elements harboring highly and widely expressed transcription units and associated transcriptional condensates.

Association Between Tic Aggravation and Methylphenidate in Youth With Attention-Deficit/Hyperactivity Disorder.

OBJECTIVE: This study aimed to determine the tic aggravation event rate and cumulative incidence rate in the use of methylphenidate (MPH) treatment in attention-deficit/hyperactivity disorder (ADHD) and the factors that influence tic aggravation. METHODS: We conducted a retrospective chart review of children and adolescents aged between 6 to 15 years, who were diagnosed with ADHD from January 2017 to December 2019. A total of 121 subjects were included. The MPH dosage, psychiatric family history, comorbidity and past history of tics were assessed through chart review and the psychological examinations data were included. Collected data were analyzed using Cox regression and Kaplan-Meier survival analysis. RESULTS: Tic aggravation event rates without a past history of tics were 2.9% with MPH treatment in ADHD. Past history of tics, total MPH dosage and age were the factors associated with tic aggravation ([HR 21.46, p<0.001], [HR 0.94, p=0.023], [HR 0.79, p=0.021] for each). Cumulative incidence of tic aggravation was different between groups with or without past tic history. When treated with MPH, all tic aggravation appeared within approximately eight months but for subjects with a past history of tic, aggravation showed within approximately six months (p<0.001). CONCLUSION: Tic aggravation event rate was significantly low especially in the group without a past history of tics with the use of MPH in ADHD. However, a thorough assessment of past history of tics, and close monitoring during the first six-eight months of treatment with MPH is needed to avert a potential worsening of tics.

Enhancer release and retargeting activates disease-susceptibility genes.

The functional engagement between an enhancer and its target promoter ensures precise gene transcription1. Understanding the basis of promoter choice by enhancers has important implications for health and disease. Here we report that functional loss of a preferred promoter can release its partner enhancer to loop to and activate an alternative promoter (or alternative promoters) in the neighbourhood. We refer to this target-switching process as 'enhancer release and retargeting'. Genetic deletion, motif perturbation or mutation, and dCas9-mediated CTCF tethering reveal that promoter choice by an enhancer can be determined by the binding of CTCF at promoters, in a cohesin-dependent manner-consistent with a model of 'enhancer scanning' inside the contact domain. Promoter-associated CTCF shows a lower affinity than that at chromatin domain boundaries and often lacks a preferred motif orientation or a partnering CTCF at the cognate enhancer, suggesting properties distinct from boundary CTCF. Analyses of cancer mutations, data from the GTEx project and risk loci from genome-wide association studies, together with a focused CRISPR interference screen, reveal that enhancer release and retargeting represents an overlooked mechanism that underlies the activation of disease-susceptibility genes, as exemplified by a risk locus for Parkinson's disease (NUCKS1-RAB7L1) and three loci associated with cancer (CLPTM1L-TERT, ZCCHC7-PAX5 and PVT1-MYC).

Effects of equine-assisted activities on attention and quality of life in children with cerebral palsy in a randomized trial: examining the comorbidity with attention-deficit/hyperactivity disorder.

BACKGROUND: Attention problems and decreased quality of life are frequently accompanied in Cerebral Palsy (CP), which can negatively affect rehabilitation of physical disability. However, the majority of affected children remain untreated in the aspects of attention or psychosocial factors. Equine-Assisted Activities and Therapies (EAAT) use horse as a therapeutic modality including grooming as well as mounted riding activities in which patients exercise and experience mounted stimulation. It is known to help improve attention in children with ADHD, so that it can be an exercise therapy that is expected to improvement of attention as well as rehabilitating effects in CP patients. EAA may be a promising strategy to address the unmet need for CP patients. This study aims to investigate the efficacy of EAA for children with CP, those with both CP and ADHD and confirm the comorbidity between CP and ADHD. METHODS: Forty-six children with cerebral palsy participated in this study. For the exercise group, they participated in a 40-min session twice a week for a 16-week period, while the control group engaged in daily life without any special treatments. Each children individually were assessed on attention and psychological wellbeing at baseline and post-treatment. Comorbidity were identified based on the Diagnostic and Statistical Manual of Mental Disorder 5th edition (DSM-5) and confirmed by Korean Kiddie-Schedule for Affective Disorders and Schizophrenia Present and Lifetime Version (K-SADS-PL). RESULTS: Perseveration rated using the Conner's Performance Test (CPT) showed a significant decrease only in the exercise group (p < .024). However, no significant improvement in children's quality of life was observed after EAA program compared with control group. Among the total participants, fifteen children (31.91%) were diagnosed with ADHD. When conducting an additional analysis with the subsample of CP patients diagnosed with ADHD, the d', commission error and perseveration showed a significant decrease only in the exercise group. Children with CP and ADHD reported an improvement in quality of life both in exercise and control group, but only in the exercise group social functioning exhibited a significant difference. CONCLUSION: The positive effects of the EAA on attention and quality of life were confirmed. Children with CP in the exercise group were more capable to sustain their attention longer. Those with CP and ADHD showed an increase in attention and perceived to have better social skills after receiving 16 weeks of EAA compared to those in the control group. Considering high comorbidity of CP and ADHD, it seems that the EAA program could be the better alternative treatment for CP with attentional problem. The results of this study will contribute to growing evidence for the efficacy of EAA in children especially with CP and ADHD. TRIAL REGISTRATION: This trial was registered on ClinicalTrials.gov ( NCT03870893 ). Registered 26 July 2017.

Global epigenomic analysis of KSHV-infected primary effusion lymphoma identifies functional MYC superenhancers and enhancer RNAs.

Enhancers play indispensable roles in cell proliferation and survival through spatiotemporally regulating gene transcription. Active enhancers and superenhancers often produce noncoding enhancer RNAs (eRNAs) that precisely control RNA polymerase II activity. Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncogenic gamma-2 herpesvirus that causes Kaposi's sarcoma and primary effusion lymphoma (PEL). It is well characterized that KSHV utilizes host epigenetic machineries to control the switch between two lifecycles, latency and lytic replication. However, how KSHV impacts host epigenome at different stages of viral lifecycle is not well understood. Using global run-on sequencing (GRO-seq) and chromatin-immunoprecipitation sequencing (ChIP-seq), we profiled the dynamics of host transcriptional regulatory elements during latency and lytic replication of KSHV-infected PEL cells. This revealed that a number of critical host genes for KSHV latency, including MYC proto-oncogene, were under the control of superenhancers whose activities were globally repressed upon viral reactivation. The eRNA-expressing MYC superenhancers were located downstream of the MYC gene in KSHV-infected PELs and played a key role in MYC expression. RNAi-mediated depletion or dCas9-KRAB CRISPR inhibition of eRNA expression significantly reduced MYC mRNA level in PELs, as did the treatment of an epigenomic drug that globally blocks superenhancer function. Finally, while cellular IRF4 acted upon eRNA expression and superenhancer function for MYC expression during latency, KSHV viral IRF4 repressed cellular IRF4 expression, decreasing MYC expression and thereby, facilitating lytic replication. These results indicate that KSHV acts as an epigenomic driver that modifies host epigenomic status upon reactivation by effectively regulating host enhancer function.

Super-Enhancer Redistribution as a Mechanism of Broad Gene Dysregulation in Repeatedly Drug-Treated Cancer Cells.

Cisplatin is an antineoplastic drug administered at suboptimal and intermittent doses to avoid life-threatening effects. Although this regimen shortly improves symptoms in the short term, it also leads to more malignant disease in the long term. We describe a multilayered analysis ranging from chromatin to translation-integrating chromatin immunoprecipitation sequencing (ChIP-seq), global run-on sequencing (GRO-seq), RNA sequencing (RNA-seq), and ribosome profiling-to understand how cisplatin confers (pre)malignant features by using a well-established ovarian cancer model of cisplatin exposure. This approach allows us to segregate the human transcriptome into gene modules representing distinct regulatory principles and to characterize that the most cisplatin-disrupted modules are associated with underlying events of super-enhancer plasticity. These events arise when cancer cells initiate without ultimately ending the program of drug-stimulated death. Using a PageRank-based algorithm, we predict super-enhancer regulator ISL1 as a driver of this plasticity and validate this prediction by using CRISPR/dCas9-KRAB inhibition (CRISPRi) and CRISPR/dCas9-VP64 activation (CRISPRa) tools. Together, we propose that cisplatin reprograms cancer cells when inducing them to undergo near-to-death experiences.

Resting-state alpha and gamma activity in affective disorder with ADHD symptoms: Comparison between bipolar disorder and major depressive disorder.

Although comorbid attention deficit/hyperactivity disorder (ADHD) symptoms are very common in mood disorder, its neurophysiological correlates have not been explored. This study aimed to examine clinical and neurophysiological correlates of ADHD symptoms in major depressive disorder (MDD) and bipolar disorder (BP). A total of 67 subjects with mood disorder, current depressive episode (38 subjects with MDD and 29 subjects with BP depression) were included in the analysis. Resting quantitative electroencephalography (qEEG) recordings were collected under eyes closed condition. ADHD symptoms, depression, anxiety, and lifetime hypomania were evaluated using self-report questionnaires. In MDD, ADHD symptoms did not show significant associations with anxiety and depression. In BP, ADHD symptoms showed significant associations with depression, anxiety and lifetime hypomania. Significant correlations with Adult ADHD self-report scales (ASRS) inattention score and total score were detected in left and right frontal alpha powers in MDD while significant correlation with ASRS hyperactivity score and ASRS total score were detected in right frontal gamma power in BP. Linear regression analyses revealed that left and right frontal alpha powers, depression and lifetime hypomania showed significant association with ASRS inattention score and ASRS total score in MDD. In BP, linear regression analysis showed ASRS hyperactivity score was associated with lifetime hypomania and the right frontal gamma power. MDD and BP showed different correlation patterns between frontal qEEG measures and ADHD symptoms. This might be associated with distinct neurobiological underpinnings of co-occurring ADHD symptoms in MDD and BP.

Phase separation of ligand-activated enhancers licenses cooperative chromosomal enhancer assembly.

A crucial feature of differentiated cells is the rapid activation of enhancer-driven transcriptional programs in response to signals. The potential contributions of physicochemical properties of enhancer assembly in signaling events remain poorly understood. Here we report that in human breast cancer cells, the acute 17ß-estradiol-dependent activation of functional enhancers requires assembly of an enhancer RNA-dependent ribonucleoprotein (eRNP) complex exhibiting properties of phase-separated condensates. Unexpectedly, while acute ligand-dependent assembly of eRNPs resulted in enhancer activation sensitive to chemical disruption of phase separation, chronically activated enhancers proved resistant to such disruption, with progressive maturation of eRNPs to a more gel-like state. Acute, but not chronic, stimulation resulted in ligand-induced, condensin-dependent changes in spatial chromatin conformation based on homotypic enhancer association, resulting in cooperative enhancer-activation events. Thus, distinct physicochemical properties of eRNP condensates on enhancers serve as determinants of rapid ligand-dependent alterations in chromosomal architecture and cooperative enhancer activation.

Dismissal of RNA Polymerase II Underlies a Large Ligand-Induced Enhancer Decommissioning Program.

Nuclear receptors induce both transcriptional activation and repression programs responsible for development, homeostasis, and disease. Here, we report a previously overlooked enhancer decommissioning strategy underlying a large estrogen receptor alpha (ERα)-dependent transcriptional repression program. The unexpected signature for this E2-induced program resides in indirect recruitment of ERα to a large cohort of pioneer factor basally active FOXA1-bound enhancers that lack cognate ERα DNA-binding elements. Surprisingly, these basally active estrogen-repressed (BAER) enhancers are decommissioned by ERα-dependent recruitment of the histone demethylase KDM2A, functioning independently of its demethylase activity. Rather, KDM2A tethers the E3 ubiquitin-protein ligase NEDD4 to ubiquitylate/dismiss Pol II to abrogate eRNA transcription, with consequent target gene downregulation. Thus, our data reveal that Pol II ubiquitylation/dismissal may serve as a potentially broad strategy utilized by indirectly bound nuclear receptors to abrogate large programs of pioneer factor-mediated, eRNA-producing enhancers.

Enhanced production of enveloped viruses in BST-2-deficient cell lines.

Despite all the advantages that cell-cultured influenza vaccines have over egg-based influenza vaccines, the inferior productivity of cell-culture systems is a major drawback that must be addressed. BST-2 (tetherin) is a host restriction factor which inhibits budding-out of various enveloped viruses from infected host cells. We developed BST-2-deficient MDCK and Vero cell lines to increase influenza virus release in cell culture. BST-2 gene knock-out resulted in increased release of viral particles into the culture medium, by at least 2-fold and up to 50-fold compared to release from wild-type counterpart cells depending on cell line and virus type. The effect was not influenza virus/MDCK/Vero-specific, but was also present in a broad range of host cells and virus families; we observed similar results in murine, human, canine, and monkey cell lines with viruses including MHV-68 (Herpesviridae), influenza A virus (Orthomyxoviridae), porcine epidemic diarrhea virus (Coronaviridae), and vaccinia virus (Poxviridae). Our results suggest that the elimination of BST-2 expression in virus-producing cell lines can enhance the production of viral vaccines. Biotechnol. Bioeng.2017;114: 2289-2297. © 2017 Wiley Periodicals, Inc.

Truncating mutation in the autophagy gene UVRAG confers oncogenic properties and chemosensitivity in colorectal cancers.

Autophagy-related factors are implicated in metabolic adaptation and cancer metastasis. However, the role of autophagy factors in cancer progression and their effect in treatment response remain largely elusive. Recent studies have shown that UVRAG, a key autophagic tumour suppressor, is mutated in common human cancers. Here we demonstrate that the cancer-related UVRAG frameshift (FS), which does not result in a null mutation, is expressed as a truncated UVRAG(FS) in colorectal cancer (CRC) with microsatellite instability (MSI), and promotes tumorigenesis. UVRAG(FS) abrogates the normal functions of UVRAG, including autophagy, in a dominant-negative manner. Furthermore, expression of UVRAG(FS) can trigger CRC metastatic spread through Rac1 activation and epithelial-to-mesenchymal transition, independently of autophagy. Interestingly, UVRAG(FS) expression renders cells more sensitive to standard chemotherapy regimen due to a DNA repair defect. These results identify UVRAG as a new MSI target gene and provide a mechanism for UVRAG participation in CRC pathogenesis and treatment response.

Condensin I and II Complexes License Full Estrogen Receptor α-Dependent Enhancer Activation.

Enhancers instruct spatio-temporally specific gene expression in a manner tightly linked to higher-order chromatin architecture. Critical chromatin architectural regulators condensin I and condensin II play non-redundant roles controlling mitotic chromosomes. But the chromosomal locations of condensins and their functional roles in interphase are poorly understood. Here we report that both condensin complexes exhibit an unexpected, dramatic estrogen-induced recruitment to estrogen receptor α (ER-α)-bound eRNA(+) active enhancers in interphase breast cancer cells, exhibiting non-canonical interaction with ER-α via its DNA-binding domain (DBD). Condensins positively regulate ligand-dependent enhancer activation at least in part by recruiting an E3 ubiquitin ligase, HECTD1, to modulate the binding of enhancer-associated coactivators/corepressors, including p300 and RIP140, permitting full eRNA transcription, formation of enhancer:promoter looping, and the resultant coding gene activation. Collectively, our results reveal an important, unanticipated transcriptional role of interphase condensins in modulating estrogen-regulated enhancer activation and coding gene transcriptional program.

LRP8-Reelin-Regulated Neuronal Enhancer Signature Underlying Learning and Memory Formation.

One of the exceptional properties of the brain is its ability to acquire new knowledge through learning and to store that information through memory. The epigenetic mechanisms linking changes in neuronal transcriptional programs to behavioral plasticity remain largely unknown. Here, we identify the epigenetic signature of the neuronal enhancers required for transcriptional regulation of synaptic plasticity genes during memory formation, linking this to Reelin signaling. The binding of Reelin to its receptor, LRP8, triggers activation of this cohort of LRP8-Reelin-regulated neuronal (LRN) enhancers that serve as the ultimate convergence point of a novel synapse-to-nucleus pathway. Reelin simultaneously regulates NMDA-receptor transmission, which reciprocally permits the required γ-secretase-dependent cleavage of LRP8, revealing an unprecedented role for its intracellular domain in the regulation of synaptically generated signals. These results uncover an in vivo enhancer code serving as a critical molecular component of cognition and relevant to psychiatric disorders linked to defects in Reelin signaling.

The transcription factor KLF2 restrains CD4⁺ T follicular helper cell differentiation.

T follicular helper (Tfh) cells are essential for efficient B cell responses, yet the factors that regulate differentiation of this CD4(+) T cell subset are incompletely understood. Here we found that the KLF2 transcription factor serves to restrain Tfh cell generation. Induced KLF2 deficiency in activated CD4(+) T cells led to increased Tfh cell generation and B cell priming, whereas KLF2 overexpression prevented Tfh cell production. KLF2 promotes expression of the trafficking receptor S1PR1, and S1PR1 downregulation is essential for efficient Tfh cell production. However, KLF2 also induced expression of the transcription factor Blimp-1, which repressed transcription factor Bcl-6 and thereby impaired Tfh cell differentiation. Furthermore, KLF2 induced expression of the transcription factors T-bet and GATA3 and enhanced Th1 differentiation. Hence, our data indicate KLF2 is pivotal for coordinating CD4(+) T cell differentiation through two distinct and complementary mechanisms: via control of T cell localization and by regulation of lineage-defining transcription factors.