The transcription factor Sox4 is a downstream target of signaling by the cytokine TGF-ß and suppresses T(H)2 differentiation.

Sox4 is a transcription factor that regulates various developmental processes. Here we show that Sox4 was induced by TGF-ß and negatively regulated the transcription factor GATA-3, the master regulator of function of T helper type 2 (T(H)2) cells, by two distinct mechanisms. First, Sox4 bound directly to GATA-3, preventing its binding to GATA-3 consensus DNA sequences. Second, Sox4 bound to the promoter region of the gene encoding interleukin 5 (IL-5), a T(H)2 cytokine, and prevented binding of GATA-3 to this promoter. T(H)2 cell-driven airway inflammation was modulated by alterations in Sox4 expression. Thus, Sox4 acted as a downstream target of TGF-ß to inhibit GATA-3 function, T(H)2 differentiation and T(H)2 cell-mediated inflammation.

Senescent CD8+ T cells acquire NK cell-like innate functions to promote antitumor immunity.

It has been suggested that aging of the immune system (immunosenescence) results in a decline in the acquired immune response, which is associated with an increase in age-related tumorigenesis. T-cell senescence plays a critical role in immunosenescence and is involved in the age-related decline of the immune function, which increases susceptibility to certain cancers. However, it has been shown that CD8+ T cells with the senescent T-cell phenotype acquire an natural killer (NK) cell-like function and are involved in tumor elimination. Therefore, the role of senescent CD8+ T cells in tumor immunity remains to be elucidated. In this study, we investigated the role of senescent CD8+ T cells in tumor immunity. In a murine model of transferred with B16 melanoma, lung metastasis was significantly suppressed in aged mice (age ≥30 weeks) in comparison to young mice (age 6-10 weeks). We evaluated the cytotoxic activity of CD8+ T cells in vitro and found that CD8+ T cells from aged mice activated in vitro exhibited increased cytotoxic activity in comparison to those from young mice. We used Menin-deficient effector T cells as a model for senescent CD8+ T cells and found that cytotoxic activity and the expression of NK receptors were upregulated in Menin-deficient senescent CD8+ T cells. Furthermore, Menin-deficient CD8+ T cells can eliminate tumor cells in an antigen-independent manner. These results suggest that senescent effector CD8+ T cells may contribute to tumor immunity in the elderly by acquiring NK-like innate immune functions, such as antigen-independent cytotoxic activity.

The histone demethylase Utx controls CD8+ T-cell-dependent antitumor immunity via epigenetic regulation of the effector function.

CD8+ T cells play a central role in antitumor immune responses. Epigenetic gene regulation is essential to acquire the effector function of CD8+ T cells. However, the role of Utx, a demethylase of histone H3K27, in antitumor immunity remains unclear. In this study, we examined the roles of Utx in effector CD8+ T-cell differentiation and the antitumor immune response. In a murine tumor-bearing model, an increased tumor size and decreased survival rate were observed in T-cell-specific Utx KO (Utx KO) mice compared with wild-type (WT) mice. The number of CD8+ T cells in tumor-infiltrating lymphocytes (TILs) was significantly decreased in Utx KO mice. We found that the acquisition of effector function was delayed and attenuated in Utx KO CD8+ T cells. RNA sequencing revealed that the expression of effector signature genes was decreased in Utx KO effector CD8+ T cells, while the expression of naïve or memory signature genes was increased. Furthermore, the expression of Cxcr3, which is required for the migration of effector CD8+ T cells to tumor sites, was substantially decreased in Utx KO CD8+ T cells. These findings suggest that Utx promotes CD8+ T-cell-dependent antitumor immune responses partially through epigenetic regulation of the effector function.

The Loss of H3K27 Histone Demethylase Utx in T Cells Aggravates Allergic Contact Dermatitis.

The pathogenesis of allergic contact dermatitis (ACD) requires the activation of Ag-specific T cells, including effector and regulatory T cells. The differentiation and function of these T cells is epigenetically regulated through DNA methylation and histone modifications. However, the roles of altered histone H3K27 methylation in T cells in the development of ACD remain unknown. Two types of histone H3K27 demethylases, Utx and Jmjd3, have been reported in mammals. To determine the role of the histone H3K27 demethylase expression of T cells in the development of ACD, we generated T cell-specific, Utx-deficient (Utx KO) mice or Jmjd3-deficient (Jmjd3 KO) mice. Unlike control mice, Utx KO mice had severer symptoms of ACD, whereas Jmjd3 KO mice showed symptoms identical to those in control mice. In Utx KO mice with ACD, the massive infiltration of myeloid cells, including neutrophils and dendritic cells, has been observed. In addition, the expression of proinflammatory cytokines in CD4+ T cells of the draining lymph nodes (LNs) and in CD8+ T cells of the skin was increased in Utx KO mice, whereas the ratio of Foxp3+ regulatory CD4+ T cells to Foxp3- conventional CD4+ T cells was decreased in both the draining LNs and the skin of Utx KO mice with ACD. Furthermore, Foxp3+ regulatory CD4+ T cells of Utx KO mice with ACD expressed a decreased level of CCR4 (a skin-tropic chemokine receptor) in comparison with control. Thus, in CD4+ T cells, Utx could potentially be involved in the regulation of the pathogenesis of ACD.

Long-term transition of antibody titers in healthcare workers following the first to fourth doses of mRNA COVID-19 vaccine: Comparison of two automated SARS-CoV-2 immunoassays.

Anti-spike receptor binding domain (S-RBD) antibody against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which best correlates with virus-neutralizing antibody is useful for estimating the period of protection and identifying the timing of additional booster doses. Long-term transition of the S-RBD antibody titer and the antibody responses among healthy individuals remain unclear. In the present study, therefore, we monitored the S-RBD antibody titers of 16 healthcare workers every 4 weeks for 76 weeks after vaccination with a fourth dose of mRNA-1273 (Moderna) following three doses of BNT162b2 (Pfizer/BioNTech) using two commercial automated immunoassays (Roche and Abbott). Two antibody responses to the vaccine were similar with an up-down change before and after the second (weeks 3), third (weeks 40) and fourth (week 72) vaccinations, but the titer did not fall below the assay's positivity threshold in any individual. The peak level of the geometric mean titer (GMT) in the Roche assay was highest after the third vaccination, and that in Abbott assay was highest after the fourth vaccination but almost equal to that after the third vaccination. Both the geometric mean fold rise (GMFR) demonstrated by the Roche and Abbott assays were highest after the third vaccination. Antibody titers determined by the Roche and Abbott assays showed a positive strong correlation (correlation coefficient: 0.70 to 0.99), but the ratio (Roche/Abbott) of antibodies demonstrated by both assays increased 0.46- to 8.26-fold between weeks 3 and 76. These findings will be helpful for clinicians when interpreting results for SARS-CoV-2 antibody levels and considering future vaccination strategies.

[A Case of Transcatheter Arterial Chemoembolization and Surgical Resection of Left 7th Rib Metastasis after Surgery for Hepatocellular Carcinoma].

The patient was an 81-year-old man. After a liver posterior segmentectomy for hepatocellular carcinoma, a painful bulge was observed in the left anterior thoracic region during a routine outpatient visit. Elevated tumor markers and contrast- enhanced CT scan revealed a mass with contrast effect in the left 7th rib. Ultrasound-guided biopsy revealed hepatocellular carcinoma metastatic to the left 7th rib. There were no other obvious metastases, and the diagnosis of a single bone metastasis was made. The patient did not request chemotherapy and underwent transcatheter arterial chemoembolization 4 times. The patient did not show any improvement in tumor markers or shrinkage of the tumor, and his quality of life was deteriorated due to increased pain. The patient underwent left chest wall tumor resection and chest wall reconstruction. Postoperative tumor markers were normalized and pain improved markedly. We report a case of postoperative recurrence- free survival for 2 years.

Glucocorticoid imprints a low glucose metabolism onto CD8 T cells and induces the persistent suppression of the immune response.

Glucocorticoids (GCs), immunosuppressive, and anti-inflammatory agents have various effects on T cells. However, the long-term influence of GCs on the T cell-mediated immune response remain to be elucidated. We demonstrated that the administration of GC during the TCR-mediated activation phase induced long-lasting suppression of glycolysis, even after the withdrawal of GC. The acquisition of the effector functions was inhibited, while the expression of PD-1 was increased in CD8 T cells activated in the presence of GC. Furthermore, adoptive transfer experiments revealed that GC-treated CD8 T cells reduced memory T cell formation and anti-tumor activity. These findings reveal that GCs have long-lasting influence on the T cell-mediated immune response via modulation of T cell metabolism.

BATF-Interacting Proteins Dictate Specificity in Th Subset Activity.

The basic leucine zipper (bZIP) transcription factor BATF is expressed in multiple Th subsets and cooperates with other factors to regulate gene transcription. BATF activates lineage-specific cytokines in Th subsets, activating IL-9 in Th9 cells and IL-17 in Th17 cells, but not IL-9 or IL-17 in the reciprocal subset. The mechanism for this restricted activity is unclear. In this report, we define BATF binding partners that contribute to Th subset-specific functions. Although BATF and IRF4 are expressed in greater amounts in Th9 than Th17, increased expression of both factors is not sufficient to induce IL-9 in Th17 cells. BATF also requires heterodimer formation with Jun family members to bind DNA and induce gene expression. Using primary mouse T cell culture, we observed that JunB and c-Jun, but not JunD, promote IL-9 production in Th9 cells. Ectopic expression of BATF with either JunB or c-Jun generates modest, but significant, increases in IL-9 production in Th17 cells, suggesting that the low expression of Jun family members is one factor limiting the ability of BATF to induce IL-9 in Th17 cells. We further identified that Bach2 positively regulates IL-9 production by directly binding to the Il9 gene and by increasing transcription factor expression in Th9 cells. Strikingly, cotransduction of Bach2 and BATF significantly induces IL-9 production in both Th9 and Th17 cells. Taken together, our results reveal that JunB, c-Jun, and Bach2 cooperate with BATF to contribute to the specificity of BATF-dependent cytokine induction in Th subsets.

Histone H3K27 Demethylase Negatively Controls the Memory Formation of Antigen-Stimulated CD8+ T Cells.

Although the methylation status of histone H3K27 plays a critical role in CD4+ T cell differentiation and its function, the role of Utx histone H3K27 demethylase in the CD8+ T cell-dependent immune response remains unclear. We therefore generated T cell-specific Utx flox/flox Cd4-Cre Tg (Utx KO) mice to determine the role of Utx in CD8+ T cells. Wild-type (WT) and Utx KO mice were infected with Listeria monocytogenes expressing OVA to analyze the immune response of Ag-specific CD8+ T cells. There was no significant difference in the number of Ag-specific CD8+ T cells upon primary infection between WT and Utx KO mice. However, Utx deficiency resulted in more Ag-specific CD8+ T cells upon secondary infection. Adoptive transfer of Utx KO CD8+ T cells resulted in a larger number of memory cells in the primary response than in WT. We observed a decreased gene expression of effector-associated transcription factors, including Prdm1 encoding Blimp1, in Utx KO CD8+ T cells. We confirmed that the trimethylation level of histone H3K27 in the Prdm1 gene loci in the Utx KO cells was higher than in the WT cells. The treatment of CD8+ T cells with Utx-cofactor α-ketoglutarate hampered the memory formation, whereas Utx inhibitor GSK-J4 enhanced the memory formation in WT CD8+ T cells. These data suggest that Utx negatively controls the memory formation of Ag-stimulated CD8+ T cells by epigenetically regulating the gene expression. Based on these findings, we identified a critical link between Utx and the differentiation of Ag-stimulated CD8+ T cells.

PEPPI-MS: Polyacrylamide-Gel-Based Prefractionation for Analysis of Intact Proteoforms and Protein Complexes by Mass Spectrometry.

Prefractionation of complex mixtures of proteins derived from biological samples is indispensable for proteome analysis via top-down mass spectrometry (MS). Polyacrylamide gel electrophoresis (PAGE), which enables high-resolution protein separation based on molecular size, is a widely used technique in biochemical experiments and has the potential to be useful in sample fractionation for top-down MS analysis. However, the lack of a means to efficiently recover the separated proteins in-gel has always been a barrier to its use in sample prefractionation. In this study, we present a novel experimental workflow, called Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS ("PEPPI-MS"), which allows top-down MS of PAGE-separated proteins. The optimization of Coomassie brilliant blue staining followed by the passive extraction step in the PEPPI-MS workflow enabled the efficient recovery of proteins, separated on commercial precast gels, from a wide range of molecular weight regions in under 10 min. Two-dimensional separation combining offline PEPPI-MS with online reversed-phase liquid chromatographic separation resulted in identification of over 1000 proteoforms recovered from the target region of the gel (≤50 kDa). Given the widespread availability and relatively low cost of traditional sodium dodecyl sulfate (SDS)-PAGE equipment, the PEPPI-MS workflow will be a powerful prefractionation strategy for top-down proteomics.

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.

Eomesodermin controls interleukin-5 production in memory T helper 2 cells through inhibition of activity of the transcription factor GATA3.

The regulation of memory CD4(+) helper T (Th) cell function, such as polarized cytokine production, remains unclear. Here we show that memory T helper 2 (Th2) cells are divided into four subpopulations by CD62L and CXCR3 expression. All four subpopulations produced interleukin-4 (IL-4) and IL-13, whereas only the CD62L(lo)CXCR3(lo) population produced IL-5 accompanied by increased H3-K4 methylation at the Il5 gene locus. The transcription factor Eomesodermin (encoded by Eomes) was highly expressed in memory Th2 cells, whereas its expression was selectively downregulated in the IL-5-producing cells. Il5 expression was enhanced in Eomes-deficient cells, and Eomesodermin was shown to interact with the transcription factor GATA3, preventing GATA3 binding to the Il5 promoter. Memory Th2 cell-dependent airway inflammation was attenuated in the absence of the CD62L(lo)CXCR3(lo) population but was enhanced by Eomes-deficient memory Th2 cells. Thus, IL-5 production in memory Th2 cells is regulated by Eomesodermin via the inhibition of GATA3 activity.

[Glutamine Regulates the Antitumor Activity of CD8 T Cells].

The cancer immunotherapies based on adoptive T cell therapy(ACT)has been receiving increased attention by improvement of the curative effect. T cells for ACT are harvested from the patient, then activated and expanded in vitro. However, in vitro activated T cells frequently show dysfunction after adoptive transfer, such as the exhaustion and the senescence. The exhausted/senescent T cells reduces the effector functions and fails to eliminate tumor cells. Therefore, the development of the culture method avoiding a T cellexhaustion and senescence. Recent findings revealthe dramatic changes of the metabolic status in T cells during T-cell receptor(TCR)-mediated activation. We recently reported that the activation status of glutaminolysis during TCR-stimulation determines the activated CD8 T cell fate. We considered that the therapeutic effect of ACT will be improved by the modulation of glutaminolysis. We demonstrated that the CD8 T cell exhaustion and/or senescence is prevented and the antitumor activity of adoptively transferred CD8 T cells is reinforced by the glutamine restriction during in vitro culture. The adoptively transferred CD8 T cells cultured under glutamine-restricted conditions shows higher infiltration in the tumor sites than that of CD8 T cells cultured under normal conditions. The expression of inhibitory receptors, such as PD-1 is decreased in tumor-infiltrating CD8 T cells cultured under glutamine-restricted conditions. Furthermore, the restriction of glutamine during CD8 T cell activation in vitro drives memory T cell development after adoptive transfer. The effect of glutamine restriction is antagonized by a-ketoglutarate, a metabolite of glutaminolysis. Thus, our recent findings suggest that the glutamine-restricted culture of CD8 T cells in vitro will improve the efficacy of CD8 T cell-based ACT.

The critical role of Bach2 in regulating type 2 chronic airway inflammation.

Although Bach2 (broad complex-tramtrack-bric a brac and Cap'n'collar homology 2) plays an important role in regulating Th2 cell differentiation and type 2 immune responses, the underlying molecular mechanisms remain unclear. Our current studies demonstrate that Bach2 associates with Batf (basic leucine zipper transcription factor ATF-like) family transcription factors and binds to the regulatory regions of the Th2 cytokine gene loci. The Bach2-Batf complex antagonizes the recruitment of the interferon regulatory factor 4 (Irf4)-containing Batf complex to activator protein 1 (AP-1) motifs in the Th2 cytokine gene locus and suppresses Th2 cytokine production and/or Th2 cell differentiation. The deletion of Batf ameliorated the spontaneous development of type 2 airway inflammation that is found in mice with Bach2 deficiency specifically in T cells. Interestingly, Bach2 regulates Batf and Batf3 expression via two distinct pathways. First, the Bach2-Batf complex directly binds to the Batf and Batf3 gene loci and reduces transcription by interfering with the Batf-Irf4 complex. Second, Bach2 suppresses interleukin 4 (IL-4)-induced augmentation of Batf and Batf3 expression through the regulation of IL-4 production. These findings suggest that IL-4 and Batf family transcription factors form a positive feedback amplification loop to induce Th2 cell differentiation and that Bach2-Batf interactions block the formation of this amplification loop. Furthermore, we found that reductions in Bach2 confer an innate immunological function on CD4 T cells to induce antigen-independent cytokine production. Some Bach2-deficient lung CD4 T cells showed characteristic features similar to pathogenic Th2 cells, including IL-33 receptor expression and IL-33-dependent Th2 cytokine production. These results suggest a critical role for Bach2 in regulating Th2 cell differentiation and the subsequent onset of chronic type 2 inflammation.

Inflammatory responses induce an identity crisis of alveolar macrophages, leading to pulmonary alveolar proteinosis.

Pulmonary alveolar proteinosis (PAP) is a severe respiratory disease characterized by dyspnea caused by accumulation of surfactant protein. Dysfunction of alveolar macrophages (AMs), which regulate the homeostasis of surfactant protein, leads to the development of PAP; for example, in mice lacking BTB and CNC homology 2 (Bach2). However, how Bach2 helps prevent PAP is unknown, and the cell-specific effects of Bach2 are undefined. Using mice lacking Bach2 in specific cell types, we found that the PAP phenotype of Bach2-deficient mice is due to Bach2 deficiency in more than two types of immune cells. Depletion of hyperactivated T cells in Bach2-deficient mice restored normal function of AMs and ameliorated PAP. We also found that, in Bach2-deficient mice, hyperactivated T cells induced gene expression patterns that are specific to other tissue-resident macrophages and dendritic cells. Moreover, Bach2-deficient AMs exhibited a reduction in cell cycle progression. IFN-γ released from T cells induced Bach2 expression in AMs, in which Bach2 then bound to regulatory regions of inflammation-associated genes in myeloid cells. Of note, in AMs, Bach2 restricted aberrant responses to excessive T cell-induced inflammation, whereas, in T cells, Bach2 puts a brake on T cell activation. Moreover, Bach2 stimulated the expression of multiple histone genes in AMs, suggesting a role of Bach2 in proper histone expression. We conclude that Bach2 is critical for the maintenance of AM identity and self-renewal in inflammatory environments. Treatments targeting T cells may offer new therapeutic strategies for managing secondary PAP.

Reinforce the antitumor activity of CD8+ T cells via glutamine restriction.

The antitumor activity of activated CD8+ T cells in the tumor microenvironment seems to be limited due to their being metabolically unfit. This metabolic unfitness is closely associated with T-cell exhaustion and impairment of memory formation, which are barriers to successful antitumor adoptive immunotherapy. We therefore assessed the role of glutamine metabolism in the antitumor activity of CD8+ T cells using a tumor-inoculated mouse model. The adoptive transfer of tumor-specific CD8+ T cells cultured under glutamine-restricted (dGln) conditions or CD8+ T cells treated with specific inhibitors of glutamine metabolism efficiently eliminated tumors and led to better survival of tumor-inoculated mice than with cells cultured under control (Ctrl) conditions. The decreased expression of PD-1 and increased Ki67 positivity among tumor-infiltrating CD8+ T cells cultured under dGln conditions suggested that the inhibition of glutamine metabolism prevents CD8+ T-cell exhaustion in vivo. Furthermore, the transferred CD8+ T cells cultured under dGln conditions expanded more efficiently against secondary OVA stimulation than did CD8+ T cells under Ctrl conditions. We found that the expression of a pro-survival factor and memory T cell-related transcription factors was significantly higher in CD8+ T cells cultured under dGln conditions than in those cultured under Ctrl conditions. Given these findings, our study uncovered an important role of glutamine metabolism in the antitumor activity of CD8+ T cells. The novel adoptive transfer of tumor-specific CD8+ T cells cultured in glutamine-restricted conditions may be a promising approach to improve the efficacy of cell-based adoptive immunotherapy.

E3 ubiquitin ligases SIAH1/2 regulate hypoxia-inducible factor-1 (HIF-1)-mediated Th17 cell differentiation.

IL-17 is known to be a cytokine mainly secreted from Th17 cells, which well associate with autoimmune inflammatory responses. In the generation of Th17 cells, RORc and RORa have pivotal roles in controlling the transcription of Il17. We speculated additional regulation in Il17a transcription and randomly screened a 6344 clone cDNA library to identify specific modulators for Il17a promoter activity. After the screen, the E3 ubiquitin ligases SIAH1 and SIAH2 were investigated further and confirmed to increase Il17a promoter activity in a T-cell line and to promote Th17 development ex vivo. This enhancement was a consequence of enhanced expression of hypoxia-inducible factor-1α (HIF-1α) protein, which is reported to directly regulate expression of Il17a and Rorgt at the transcriptional level. In the absence of HIF-1α, both ubiquitin ligases had little effect on Th17 cell differentiation. These results suggest that the SIAH1 and SIAH2 play a pivotal role to promote Th17 cell differentiation through maintaining the stability of HIF-1α protein.

Menin Plays a Critical Role in the Regulation of the Antigen-Specific CD8+ T Cell Response upon Listeria Infection.

Menin, a tumor suppressor protein, is encoded by the MEN1 gene in humans. Certain germinal mutations of MEN1 induce an autosomal-dominant syndrome that is characterized by concurrent parathyroid adenomas and several other tumor types. Although menin is also expressed in hematopoietic lineages, its role in CD8+ T cells remains unclear. We generated Meninflox/flox CD4-Cre (Menin-KO) mice by crossing Meninflox/flox mice with CD4-Cre transgenic (Tg) mice to determine the role of menin in CD8+ T cells. Wild-type (WT) and Menin-KO mice were infected with Listeria monocytogenes expressing OVA to analyze the immune response of Ag-specific CD8+ T cells. Menin deficiency resulted in an impaired primary immune response by CD8+ T cells. On day 7, there were fewer Menin-KO OVA-specific CD8+ T cells compared with WT cells. Next, we adoptively transferred WT and Menin-KO OT-1 Tg CD8+ T cells into congenic recipient mice and infected them with L. monocytogenes expressing OVA to determine the CD8+ T cell-intrinsic effect. Menin-KO OT-1 Tg CD8+ T cells were outcompeted by the WT cells upon infection. Increased expression of Blimp-1 and T-bet, cell cycle inhibitors, and proapoptotic genes was observed in the Menin-KO OT-1 Tg CD8+ T cells upon infection. These data suggest that menin inhibits differentiation into terminal effectors and positively controls proliferation and survival of Ag-specific CD8+ T cells that are activated upon infection. Collectively, our study uncovered an important role for menin in the immune response of CD8+ T cells to infection.

Gfi1, a transcriptional repressor, inhibits the induction of the T helper type 1 programme in activated CD4 T cells.

A transcriptional repressor Gfi1 promotes T helper type 2 (Th2) cell development and inhibits Th17 and inducible regulatory T-cell differentiation. However, the role of Gfi1 in regulating Th1 cell differentiation and the Th1-type immune response remains to be investigated. We herein demonstrate that Gfi1 inhibits the induction of the Th1 programme in activated CD4 T cells. The activated Gfi1-deficient CD4 T cells spontaneously develop into Th1 cells in an interleukin-12- and interferon-γ-independent manner. The increase of Th1-type immune responses was confirmed in vivo in Gfi1-deficient mice using a murine model of nickel allergy and delayed-type hypersensitivity (DTH). The expression levels of Th1-related transcription factors were found to increase in Gfi1-deficient activated CD4 T cells. Tbx21, Eomes and Runx2 were identified as possible direct targets of Gfi1. Gfi1 binds to the Tbx21, Eomes and Runx2 gene loci and reduces the histone H3K4 methylation levels in part by modulating Lsd1 recruitment. Together, these findings demonstrate a novel regulatory role of Gfi1 in the regulation of the Th1-type immune response.