RESUMO
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.
Assuntos
Dermatite de Contato/imunologia , Histona Desmetilases/metabolismo , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Pele/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/metabolismo , Histona Desmetilases/genética , Histonas/genética , Humanos , Mediadores da Inflamação/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Camundongos , Camundongos Knockout , Receptores CCR4/metabolismoRESUMO
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.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Transferência Adotiva , Animais , Diferenciação Celular/imunologia , Listeriose/microbiologia , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Fator 1 de Ligação ao Domínio I Regulador Positivo , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Proteínas com Domínio T/genética , Fatores de Transcrição/genéticaRESUMO
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.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Proteínas de Ligação a DNA/genética , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Células Th1/imunologia , Células Th1/metabolismo , Fatores de Transcrição/genética , Animais , Linfócitos T CD4-Positivos/citologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Citocinas/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Histona Desmetilases/antagonistas & inibidores , Histonas/metabolismo , Interferon gama/biossíntese , Metilação , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Ligação Proteica , Proteínas com Domínio T/genética , Células Th1/citologia , Fatores de Transcrição/deficiência , Fatores de Transcrição/metabolismoRESUMO
Infection with certain pathogens induces a shift of the Th subset balance to a Th1 dominant state. This, in turn, results in the suppression of Th2 responses. We focused on the involvement of interferon regulatory factor-1 (IRF-1) in the suppression of Th2 cells during Listeria infection. We found that the inhibition of IL-4 production by Th2 cells is mediated by a soluble factor (LmSN) produced by Listeria-infected antigen-presenting cells. The inhibition is not observed with T cells from Irf1 gene-targeted mice. IRF-1 suppresses transcription of the Il4 gene in Th2 cells. Under the influence of the LmSN, IRF-1 binds to the 3' untranslated region (UTR) region of the Il4 gene and down-regulates Il4 gene transcription. Finally, we identified IL-1α and IL-1ß as the mediator of the LmSN activity. Signaling through IL-1R induces the stabilization and/or nuclear translocation of IRF-1. We propose that IRF-1 functions to induce the T-cell subset shift via a novel mechanism. Under the influence of IL-1, IRF-1 translocates into the nucleus and acts on the 3'UTR region of the Il4 gene, thus inhibiting its transcription in Th2 cells. As a result, the immune system shifts predominantly to a Th1 response during Listeria infection, resulting in effective protection of the host.
Assuntos
Núcleo Celular/metabolismo , Fator Regulador 1 de Interferon/metabolismo , Interleucina-4/metabolismo , Listeria monocytogenes/imunologia , Listeriose/imunologia , Células Th1/imunologia , Células Th2/imunologia , Regiões 3' não Traduzidas/genética , Animais , Células Cultivadas , Regulação para Baixo , Fator Regulador 1 de Interferon/genética , Interleucina-1/metabolismo , Interleucina-4/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transporte Proteico , Receptores de Interleucina-1/metabolismo , Transdução de Sinais , Células Th1/microbiologia , Equilíbrio Th1-Th2 , Células Th2/microbiologiaRESUMO
BACKGROUND: Although the T-cell subset differentiation pathway has been characterized extensively from the view of host gene regulation, the effects of genes of the pathogen on T-cell subset differentiation during infection have yet to be elucidated. Especially, the bacterial genes that are responsible for this shift have not yet been determined. METHODS: Utilizing a single-gene-mutation Listeria panel, we investigated genes involved in the host-pathogen interaction that are required for the initiation of T-cell subset differentiation in the early phase of pathogen infection. RESULTS: We demonstrate that the induction of T helper types 1 and 2 (Th1 and Th2) subsets are separate phenomena and are mediated by distinct Listeria genes. We identified several candidate Listeria genes that appear to be involved in the host-Listeria interaction. Among them, arpJ is the strongest candidate gene for inhibiting Th2 subset induction. Furthermore, the analysis utilizing arpJ-deficient Listeria monocytogenes (Lm) revealed that the tumor necrosis factor (TNF) superfamily (Tnfsf) 9-TNF receptor superfamily (Tnfrsf) 9 interaction inhibits the Th2 response during Lm infection. CONCLUSIONS: arpJ is the candidate gene for inhibiting Th2 T-cell subset induction. The arpJ gene product influences the expression of Tnfsf/Tnfrsf on antigen-presenting cells and inhibits the Th2 T-cell subset differentiation during Listeria infection.
Assuntos
Diferenciação Celular/imunologia , Listeria monocytogenes/genética , Listeriose/imunologia , Linfócitos T Auxiliares-Indutores/fisiologia , Animais , Células Cultivadas , Genes Bacterianos , Interações Hospedeiro-Patógeno , Listeria monocytogenes/imunologia , Listeriose/microbiologia , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Linfócitos T Auxiliares-Indutores/virologiaRESUMO
It is important to know what kind of T-cell populations are involved in various disease states, and to know the state of T-cell functions involving in the disease. When a T cell's antigen receptors (TCR) recognize a specific antigen, the cell transmits a signal by a transduction mechanism within the T cell's cytoplasm. This signal initiates gene transcription essential for differentiation and activation of T cells. In this chapter, we will describe the methods of analyzing the transcribed mRNA and detecting the translated product in order to know the activation state of T cells.
Assuntos
Linfócitos T , Diferenciação Celular , Citoplasma , RNA Mensageiro/genéticaRESUMO
Infection with Listeria induces a dominant shift to the Th1 immune response and inhibits the Th2 response. Papain is frequently utilized in animal models of allergies. Papain administration induces chemotaxis of basophils to regional lymph nodes (LNs) and production of interleukin (IL)-4 by basophils, resulting in a Th2-dominant status and increased IgE production in LNs. In this model, production of immunoglobulin (Ig) E by LN cells is primarily controlled by IL-4 produced by basophils. Based on this model, it was postulated that Listeria monocytogenes (Lm) infection suppresses IgE production by LN cells. Therefore, the effects of Lm infection on a papain-induced mouse model of allergies were investigated. Following s.c. injection of papain, basophils transiently migrated to draining LNs because of the effects of chemokine (C-C) motif ligand (CCL) 24 and secreted IL-4, inducing a Th2 response. Lm infection blocked recruitment of basophils into the popliteal LNs by inhibiting CCL24 production. Papain-induced class switch recombination (CSR) to IgE is inhibited by Lm infection, whereas CSR to IgG1 is not affected by the same treatment. Therefore, the CSR of IgG1 to IgE is basophil-dependent, whereas the CSR of IgM to IgG1 is basophil-independent. Hence, Lm infection suppresses CSR to IgE without affecting CSR to IgG1.
Assuntos
Basófilos/imunologia , Quimiotaxia , Imunoglobulina E/imunologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Linfonodos/imunologia , Alérgenos/imunologia , Animais , Basófilos/fisiologia , Quimiocina CCL24/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Papaína/imunologiaRESUMO
It is important to know what kind of T cell populations is involved in various disease states and to know the state of T cell functions involved in the disease. When T cell antigen receptors (TCR) recognize a specific antigen, the cell transmits a signal by a transduction mechanism within the T cell's cytoplasm. This signal initiates gene transcription essential for differentiation and activation of T cells. In this chapter, we will describe the methods of analyzing the transcribed mRNA and detecting the translated product in order to know the activation state of T cells.
Assuntos
Técnicas Imunológicas/métodos , Linfócitos T/citologia , Biomarcadores/metabolismo , Membrana Celular/metabolismo , Citocinas/metabolismo , Humanos , Ativação Linfocitária/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Baço/citologiaRESUMO
Gfi1 plays an important role in the development and maintenance of many hematopoietic linage cells. However, the impact of Gfi1-deficiency on the iNKT cell differentiation remains unclear. We herein demonstrate a critical role of Gfi1 in regulating the development of iNKT cell subsets. In the thymus of T cell-specific Gfi1-deficient mice, iNKT cells normally developed up to stage 2, while the number of stage 3 NK1.1pos iNKT cells was significantly reduced. Furthermore, CD4pos iNKT cells were selectively reduced in the peripheral organs of T cell-specific Gfi1-deficient mice. The α-GalCer-dependent production of IFN-γand Th2 cytokines, but not IL-17A, was severely reduced in T cell-specific Gfi1-deficient mice. In addition, a reduction of the α-GalCer-induced anti-tumor activity was observed in Gfi1-deficient mice. These findings demonstrate the important role of Gfi1 in regulating the development and function of NKT1- and NKT2-type iNKT cell subsets.
Assuntos
Proteínas de Ligação a DNA/imunologia , Células T Matadoras Naturais/imunologia , Fatores de Transcrição/imunologia , Animais , Antígenos Ly/imunologia , Antígenos CD4/imunologia , Diferenciação Celular , Citocinas/imunologia , Proteínas de Ligação a DNA/genética , Galactosilceramidas/imunologia , Deleção de Genes , Técnicas de Introdução de Genes , Interferon gama/imunologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Camundongos , Subfamília B de Receptores Semelhantes a Lectina de Células NK/imunologia , Células T Matadoras Naturais/citologia , Fatores de Transcrição/genéticaRESUMO
Although Bach2 has an important role in regulating the Th2-type immune response, the underlying molecular mechanisms remain unclear. We herein demonstrate that Bach2 associates with Batf and binds to the regulatory regions of the Th2 cytokine gene loci. The Bach2-Batf complex antagonizes the recruitment of the Batf-Irf4 complex to AP-1 motifs and suppresses Th2 cytokine production. Furthermore, we find that Bach2 regulates the Batf and Batf3 expressions via two distinct pathways. First, Bach2 suppresses the maintenance of the Batf and Batf3 expression through the inhibition of IL-4 production. Second, the Bach2-Batf complex directly binds to the Batf and Batf3 gene loci and reduces transcription by interfering with the Batf-Irf4 complex. These findings suggest that IL-4 and Batf form a positive feedback amplification loop to induce Th2 cell differentiation and the subsequent Th2-type immune response, and Bach2-Batf interactions are required to prevent an excessive Th2 response.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Fatores Reguladores de Interferon/genética , Proteínas Repressoras/genética , Células Th2/imunologia , Animais , Diferenciação Celular/imunologia , Feminino , Regulação da Expressão Gênica , Fatores Reguladores de Interferon/metabolismo , Interleucina-4/biossíntese , Interleucina-4/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Repressoras/metabolismo , Fator de Transcrição STAT6/genética , Transcrição Gênica/genéticaRESUMO
Although CD4 T-cell senescence plays an important role in immunosenescence, the mechanism behind this process remains unclear. Here we show that T cell-specific Menin deficiency results in the premature senescence of CD4 T cells, which is accompanied by the senescence-associated secretory phenotype after antigenic stimulation and dysregulated cytokine production. Menin is required for the expansion and survival of antigen-stimulated CD4 T cells in vivo and acts by targeting Bach2, which is known to regulate immune homeostasis and cytokine production. Menin binds to the Bach2 locus and controls its expression through maintenance of histone acetylation. Menin binding at the Bach2 locus and the Bach2 expression are decreased in the senescent CD4 T cells. These findings reveal a critical role of the Menin-Bach2 pathway in regulating CD4 T-cell senescence and cytokine homeostasis, thus indicating the involvement of this pathway in the inhibition of immunosenescence.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Senescência Celular , Citocinas/imunologia , Proteínas Proto-Oncogênicas/imunologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/deficiência , Fatores de Transcrição de Zíper de Leucina Básica/genética , Feminino , Homeostase , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genéticaRESUMO
Listeria monocytogenes infection induces various types of immune responses. The Lm-induced immunity not only protects the hosts against Lm infection but also has a therapeutic effect on other diseases such as tumors and infectious diseases. In the present study, we sought to identify the cells and molecules that are primarily responsible for the Lm-induced antitumor immune response. We investigated the mechanism of the antitumor immune response induced by Lm infection using melanoma cells and various types of gene-manipulated mice and B16F10 melanoma cells. Melanoma cells were implanted into mice intrasplenically or intraperitoneally. Lm infection of mice remarkably suppressed the growth of transplanted melanoma. The suppression of melanoma growth was due to the augmented NK cytotoxicity. The Lm-induced NK activation against melanoma was type I interferon- and signal transducer and activator of transcription (STAT)1-dependent but independent of IL-12 and IFN-gamma. In contrast to avirulent Listeria innocua and hly(-) Lm failed to induce NK activation, a mutant Lm strain with minimal hemolytic activity and with normal accessibility to cytoplasm-induced NK activation. We demonstrated that the attenuated Lm entrance into the cytoplasm induces the production of type I IFN followed by the activation of NK cells, which is essential for the Lm-induced antitumor response.
Assuntos
Células Matadoras Naturais/imunologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Melanoma/imunologia , Animais , Interferon Tipo I/imunologia , Interferon gama/imunologia , Interleucina-12/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Transcrição STAT1/imunologia , Baço/patologia , Análise de SobrevidaRESUMO
Bacterial infection induces a shift to type 1 CD4 T cell subset in an infected host and this shift is important for protection of the host from disease development. Many researchers think that the shift is antigen-dependent, but we previously demonstrated an initial induction step for CD4 T cell subsets during Listeria monocytogenes (Lm) infection is antigen-independent. Although Listeria is a TLR2 ligand, the immune system of the Lm-infected host responded to the pathogen to induce expression of CD69 but not CD25 on CD4 T cells, CD8 T cells and B cells even in the absence of TLR2 or MyD88. The antigen-independent activation of type 1 CD4 T cells accelerate the clearance of pathogens by activating innate immune cells with type 1 cytokines. Type 1 CD4 T cells and CD8 T cells also collaborate to protect the host from intracellular Lm infection. Since CD8 T cells function mainly as cytotoxic T cells and CD69-positive CD8 T cells increase during Lm-infection, cytotoxic activity of CD8 T cells was evaluated during Lm-infection. Although CD8 T cells were activated to produce IFN-gamma, the cytotoxic function of CD8 T cells in Lymphocytic choriomeningitis virus (LCMV) p14 TCR-transgenic mouse was not augmented by Lm-infection. Therefore, Lm-infection differentially influences on cytokine production and cytotoxicity of CD8 T cells.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citocinas/metabolismo , Citotoxicidade Imunológica , Listeria monocytogenes , Listeriose/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Interferon gama/metabolismo , Lectinas Tipo C , Ativação Linfocitária , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
Pathogenic infections lead to activation of innate immunity followed by induction of a type 1 T cell subset and, therefore, provide a good model to evaluate when T cells commit to type 1 T cells. Here we show a two-step mechanism of T cell subset commitment during pathogenic infection. The first step is mediated by the basal function of macrophage/dendritic cells and is antigen independent. This step modulates the committed precursor frequency of T cell subsets and influences the expression of T-box expressed in T cells (T-bet) and GATA-3 genes. IL-12 and NK cells are not required for this step. The second step requires antigenic stimulation of T cells together with IL-12 or IL-4, and influences on the expression of T-bet and GATA-3. We propose a two-step T cell subset commitment pathway based on these observations. Therefore, pathogenic infections influence functional T cell commitment before T cells encounter nominal antigen.
Assuntos
Listeriose/imunologia , Listeriose/patologia , Modelos Imunológicos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/patologia , Animais , Células Apresentadoras de Antígenos/imunologia , Antígenos , Diferenciação Celular , Quimiocinas/genética , Proteínas de Ligação a DNA/genética , Fator de Transcrição GATA3 , Expressão Gênica , Interferon gama/biossíntese , Interleucina-12/deficiência , Interleucina-4/biossíntese , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Ovalbumina/imunologia , Receptores de Quimiocinas/genética , Proteínas com Domínio T , Transativadores/genética , Fatores de Transcrição/genéticaRESUMO
In this study, we established a method for Listeria monocytogenes(Lm)-mediated gene transfer into mammalian cells to manipulate the immune response of the host during infection by pathogens. We used the Lm-mediated gene transfer method in an in vivo study to manipulate host immune responses against Leishmania major(L. major )-infection. The injection of Lm modulated the susceptible host into a resistant state against L. major-infection. A more efficient protective effect was obtained with the injection of IL-12-cDNA containing Lm, and the protective effect was stronger than that of the resistant strain. The protective mechanism of Lm-injection against L. major-infection observed here appeared to be a result of the activation of the local immune system by the Lm-mediated gene transfer method. The present study is the first demonstration that a gene introduced into a host by Lm works to modulate the murine host immune response against infections in vivo. Since this system strongly induces Th1 responses and suppresses Th2 responses in infected hosts, the system can be used for controlling infectious diseases and for protection against allergic responses in the future.
Assuntos
Técnicas de Transferência de Genes , Leishmaniose Cutânea/imunologia , Listeria monocytogenes/genética , Células Th1/imunologia , Células Th2/imunologia , Animais , Células COS , Células CACO-2 , Chlorocebus aethiops , Citocinas/genética , Citocinas/imunologia , DNA Complementar/genética , Proteínas de Fluorescência Verde , Humanos , Leishmania major , Listeria monocytogenes/imunologia , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transdução GenéticaRESUMO
IL-12 is a heterodimer composed of p40 and p35 and is a key cytokine that functions to protect the host from viral and microbial infections. IL-12 links the innate immune system with the acquired immune system during infection, and induces differentiation of type 1 T cells that play an important role in the eradication of microbes. The induction of the IL-12 p40 gene is regulated by NF-kappaB in the presence of IFN-gamma. IFN-gamma induces IFN regulatory factor-1 (IRF-1), which in turn induces the transcription of the IL-12 p40 gene. However, the IRF-1 binding site in the promoter region of the IL-12 p40 gene has not yet been formally determined. In the present study, we demonstrated that IRF-1 directly binds to the IL-12 p40 gene promoter and identified its binding site. The IRF-1 binding site in the promoter region of the IL-12 p40 gene is shown to be in the -72 to -58 area of the 5'-upstream region. The -63 to -61 position is the critical site within this region for the binding of IRF-1 to the IL-12 p40 gene promoter. While IFN-gamma must be present for IL-12 p40 gene induction, the p35 gene is strongly induced by LPS, even in the absence of IFN-gamma, and therefore the induction of the p35 gene is IRF-1 independent.