Stimulation Of Naive CD4 Research Articles

David John Williams

<h3>Objective:</h3> Our objective was to determine whether altered naive CD4 T-cell biology contributes to development of disease progression in secondary progressive multiple sclerosis (SPMS). <h3>Methods:</h3> We compared the naive CD4...

IκBα Nuclear Export Enables 4-1BB-Induced cRel Activation and IL-2 Production to Promote CD8 T Cell Immunity.

Optimal CD8 T cell immunity is orchestrated by signaling events initiated by TCR recognition of peptide Ag in concert with signals from molecules such as CD28 and 4-1BB. The molecular mechanisms underlying the temporal and spatial signaling dynamics in CD8 T cells remain incompletely understood. In this study, we show that stimulation of naive CD8 T cells with agonistic CD3 and CD28 Abs, mimicking TCR and costimulatory signals, coordinately induces 4-1BB and cRel to enable elevated cytosolic cRel:IκBα complex formation and subsequent 4-1BB-induced IκBα degradation, sustained cRel activation, heightened IL-2 production and T cell expansion. NfkbiaNES/NES CD8 T cells harboring a mutated IκBα nuclear export sequence abnormally accumulate inactive cRel:IκBα complexes in the nucleus following stimulation with agonistic anti-CD3 and anti-CD28 Abs, rendering them resistant to 4-1BB induced signaling and a disrupted chain of events necessary for efficient T cell expansion. Consequently, CD8 T cells in NfkbiaNES/NES mice poorly expand during viral infection, and this can be overcome by exogenous IL-2 administration. Consistent with cell-based data, adoptive transfer experiments demonstrated that the antiviral CD8 T cell defect in NfkbiaNES/NES mice was cell intrinsic. Thus, these results reveal that IκBα, via its unique nuclear export function, enables, rather than inhibits 4-1BB-induced cRel activation and IL-2 production to facilitate optimal CD8 T cell immunity.

The Transcription Factor Runx3 Establishes Chromatin Accessibility of cis-Regulatory Landscapes that Drive Memory Cytotoxic T Lymphocyte Formation

T cell receptor (TCR) stimulation of naive CD8+ Tcells initiates reprogramming of cis-regulatory landscapes that specify effector and memory cytotoxic T lymphocyte (CTL) differentiation. We mapped regions of hyper-accessible chromatin in naive cells during TCR stimulation and discovered that the transcription factor (TF) Runx3 promoted accessibility to memory CTL-specific cis-regulatory regions before the first cell division and was essential for memory CTL differentiation. Runx3 was specifically required for accessibility to regions highly enriched with IRF, bZIP and Prdm1-like TF motifs, upregulation of TFs Irf4 and Blimp1, and activation of fundamental CTL attributes in early effector and memory precursor cells. Runx3 ensured that nascent CTLs differentiated into memory CTLs by preventing high expression of the TF T-bet, slowing effector cell proliferation, and repressing terminal CTL differentiation. Runx3 overexpression enhanced memory CTL differentiation during iterative infections. Thus, Runx3 governs chromatin accessibility during TCR stimulation and enforces the memory CTL developmental program.

Immune system network in allergic diseases during sublingual immunotherapy: The role of oral mucosal tissue

Specific sublingual immunotherapy (SLIT) represents an approach currently available to redirect inappropriate immune response in atopic patients. Since oral mucosal tissue displays high affinity for allergens, it is conceivable that the sublingual administration route might induce immunological tolerance towards allergens involving cells and mediators specific of oral and intestinal mucosa. The presence in oral mucosa of dendritic cells (DCs) which express the high-affinity receptor for immunoglobulin (Ig)E (FceRI) seems to suggest that the generation of T regulatory cells in periphery is orchestrated by a particular subset of DCs. It seems that repeated stimulation of naive CD4 T cells with allogenic immature DCs induce Tr1 cell maturation. Nevertheless, other cells are involved in this process, such as Toll Like Receptors (TLR), Major Histocompatibility Complex I and II (MHC I and II) and costimulatory molecules, such as CD40, CD 80/B7.1 and CD 86/B7.2. An increase of serum IgG4 and IgA, a reduced number of inflammatory cells infiltrating target organs, as well as a reduction of eosinophilic cationic protein and a T cell suppression in the peripheral blood also occur with SLIT. All these molecules orchestrate the immune network within the regional immune system, recreating a favourable environment for the induction of tolerance operated by SLIT.

T cells: Nutrients guide differentiation.

Stimulation of naive CD4+ T cells in glutamine-deprived conditions favours differentiation into regulatory T cells.

Cryptotanshinone induces inhibition of breast tumor growth by cytotoxic CD4+ T cells through the JAK2/STAT4/ perforin pathway.

Cryptotanshinone (CPT), is a quinoid diterpene isolated from the root of the Asian medicinal plant, Salvia miotiorrhiza bunge. Numerous researchers have found that it could work as a potent antitumor agent to inhibit tumor growth in vitro, buith there has been much less emphasis on its in vivo role against breast tumors. Using a mouse tumor model of MCF7 cells, we showed that CPT strongly inhibited MCF7 cell growth in vivo with polarization of immune reactions toward Th1-type responses, stimulation of naive CD4+ T cell proliferation, and also increased IFN-γ and perforin production of CD4+ T cells in response to tumor-activated splenocytes. Furthermore, data revealed that the cytotoxic activity of CD4+ T cells induced by CPT was markedly abrogated by concanamycin A(CMA), a perforin inhibitor, but not IFN-γ Ab. On the other hand, after depletion of CD4+ T cells or blocked perforin with CMA in a tumor-bearing model, CPT could not effectively suppress tumor growth, but this phenomenon could be reversed by injecting naive CD4+ T cells. Thus, our results suggested that CPT mainly inhibited breast tumor growth through inducing cytotoxic CD4+ T cells to secrete perforin. We further found that CPT enhanced perforin production of CD4+ T cells by up-regulating JAK2 and STAT4 phosphorylation. These findings suggest a novel potential therapeutic role for CPT in tumor therapy, and demonstrate that CPT performs its antitumor functions through cytotoxic CD4+ T cells.

Naive CD4 T-cell activation identifies MS patients having rapid transition to progressive MS

Objective:Our objective was to determine whether altered naive CD4 T-cell biology contributes to development of disease progression in secondary progressive multiple sclerosis (SPMS).Methods:We compared the naive CD4 T-cell gene expression profiles of 19 patients with SPMS and 14 healthy controls (HCs) using a whole-genome microarray approach. We analyzed surface protein expression of critical genes by flow cytometry after T-cell receptor (TCR) stimulation of naive CD4 T cells isolated from HCs and patients with SPMS.Results:Hierarchical clustering segregated patients with SPMS into 2 subgroups: SP-1, which had a short duration of relapsing-remitting multiple sclerosis (MS), and SP-2, which had a long duration of relapsing-remitting MS. SP-1 patients upregulated numerous immune genes, including genes within TCR and toll-like receptor (TLR) signaling pathways. SP-2 patients showed immune gene downregulation in comparison with HCs. We identified an SP-1–specific transcriptional signature of 3 genes (TLR4, TLR2, and chemokine receptor 1), and these genes had higher surface protein expression in SP-1 than in SP-2. After TCR stimulation for 48 hours, only SP-1 showed a progressive linear increase in TLR2 and TLR4 protein expression.Conclusions:Differences in naive CD4 T-cell biology, notably of TCR and TLR signaling pathways, identified patients with MS with more rapid conversion to secondary progression, a critical determinant of long-term disability in MS.

Decreased IL-27 Expression in Association with an Increased Th17 Response in Vogt-Koyanagi-Harada Disease

IL-27 has emerged as an important regulator of proinflammatory T-cell responses in animal models. We investigated the pathophysiological role of IL-27 in Vogt-Koyanagi-Harada (VKH) disease. IL-27P28 and EBI3 mRNA expression in peripheral blood mononuclear cells (PBMCs) were assayed by RT-PCR. Cytokines in the serum and supernatants of PBMCs, naïve CD4(+) T cells and DC-T cocultures were assayed by ELISA. Flow cytometry was used to evaluate the frequencies of IL-17-producing CD4(+) T cells. The active VKH patients showed a decreased IL-27P28 mRNA expression in PBMCs and lower IL-27 expression in the serum and supernatants of PBMCs, but higher Th17 cells in PBMCs. EBI3 mRNA expression was not different among the groups tested. Stimulation of naïve CD4(+) T cells under Th17 polarizing conditions showed a higher Th17 cell differentiation in active VKH patients. IL-27 significantly inhibited Th17 cell differentiation. IL-27-treated DCs showed a significant inhibition on Th17 differentiation. There was a significant defect in the Tr1 cell induction as measured by IL-10 in active VKH patients. Treatment with corticosteroids and cyclosporine A (CsA) resolved the intraocular inflammation in association with an upregulation of IL-27 and a downregulation of IL-17. In vitro experiments showed that corticosteroids, but not CsA, significantly upregulated the expression of IL-27. The present study suggests that decreased IL-27 expression may result in a higher Th17 in active VKH patients, which may promote the autoimmune response observed in these patients. Manipulation of IL-27 may offer a novel target for treatment of this disease.

Analysis of the Transcriptional Program of Developing Induced Regulatory T Cells

CD25+ regulatory T cells develop in the thymus (nTregs), but may also be generated in the periphery upon stimulation of naive CD4 T cells under appropriate conditions (iTregs). To gain insight into the mechanisms governing iTreg development, we performed longitudinal transcriptional profiling of CD25+ T cells during their differentiation from uncommitted naive CD4 T cells. Microarray analysis of mRNA from CD25+ iTregs early after stimulation revealed expression of genes involved in cell cycle progression and T cell activation, which largely overlapped with genes expressed in CD25+ effector T cells (Teffs) used as a control. Whereas expression of these genes remained elevated in Teffs, it declined gradually in developing iTregs, resulting in a more quiescent phenotype in mature iTregs. A similar pattern of kinetics was observed for biological processes and for intracellular pathways over-represented within the expressed genes. A maximum dichotomy of transcriptional activity between iTregs and Teffs was reached at late stages of their maturation. Of interest, members of the FoxO and FoxM1 transcription factor family pathways exhibited a reciprocal expression pattern in iTregs and Teffs, suggesting a role of these transcription factors in determining T cell fate.

Stimulation of Naive CD8+ T Cells by a Variant Viral Epitope Induces Activation and Enhanced Apoptosis

Classically, naive T cells recognize a specific peptide-MHC complex resulting in their activation and differentiation. However, it is known that T cells also have the ability to interact productively with variant ligands, indicating a flexibility in TCR Ag recognition. These altered peptide ligands have been shown to trigger responses ranging from complete activation to full inhibition of T cell responses, and thus may play an important role in initiating or sustaining T cell-mediated immunity. We have found that influenza virus-specific CD8(+) TCR transgenic T cells differentially respond to a native (agonist) and variant viral epitope, differing in two amino acids that are thought to alter TCR recognition. In response to stimulation with the agonist epitope, these cells activate, proliferate, and differentiate into effector CTLs. Conversely, stimulation with the variant epitope results in activation, proliferation, and development of effector activity followed by rapid and extensive apoptotic cell death. Stimulation of the T cells with the altered ligand results in an inability to sustain the expression of the prosurvival molecules, Bcl-2 and Bcl-xL. These data suggest that the response to the agonist and variant epitopes may reflect TCR avidity-dependent differential signaling through the TCR, resulting either in activation-dependent T cell proliferative expansion and survival or in the accelerated death of acutely activated differentiating T cells. This process of CD8(+) T cell activation, proliferation, and differentiation followed by rapid cell death may represent a novel mechanism of altered peptide ligand-induced apoptosis programmed by initial Ag receptor engagement.

Induction of Th2 type immunity in a mouse system reveals a novel immunoregulatory role of basophils

AbstractWhile production of cytokines such as IL-12 by activated dendritic cells supports development of Th1 type immunity, a source of early IL-4 that is responsible for Th2 immunity is not well understood. We now show that coculture of basophils could promote a robust Th2 differentiation upon stimulation of naive CD4 T cells primarily via IL-4. Th2 promotion by basophils was also observed even when naive CD4 T cells were stimulated in a Th1-promoting condition or when fully differentiated Th1 phenotype effector CD4 T cells were restimulated. IL-4–deficient basophils failed to induce Th2 differentiation but suppressed Th1 differentiation. It was subsequently revealed that the IL-4–deficient basophils must engage cell-to-cell contact to exert the inhibitory effect on Th1 differentiation. Stimulation of naive CD4 T cells within an in vivo environment of increased basophil generation supported development of Th2 type immunity. Taken together, our results suggest that basophils may provide an important link for the development of Th2 immunity.

Macrophage migration inhibitory factor is essential for allergic asthma but not for Th2 differentiation

Macrophage migration inhibitory factor (MIF) is increased in asthmatic patients and plays a critical role in the pathogenesis of asthma. We show here that mice lacking MIF failed to develop airway hyper-responsiveness (AHR), tissue eosinophilia, and mucus metaplasia. Analysis of the bronchoalveolar fluids revealed a substantial reduction of IL-13, eotaxin and cysteinyl-leukotrienes. The lack of these cardinal features of asthma in MIF(-/-) mice occurs regardless of high concentrations of IL-4 in the lung and OVA-specific IgE in the serum. Antigen-specific lymphocyte proliferation and IL-13 production were similarly increased in the draining lymph nodes of OVA-immunized and challenged MIF(-/-) mice compared to WT, but were reduced in the spleen of MIF(-/-), thus indicating differential roles of MIF in these compartments. Stimulation of naive CD4(+) cells with anti-CD3 antibody demonstrated that MIF(-/-) cells produced increased amounts of IFN-gamma and IL-4 compared to WT CD4(+) cells. Finally, treatment of sensitized BALB/c mice with neutralizing anti-MIF antibody abrogated the development of ARH and airway inflammation without affecting the production of Th2 cytokines or IgE. The present study demonstrates that MIF is required for allergic inflammation, adding important elements to our knowledge of asthma pathogenesis and suggesting that neutralization of MIF might be of therapeutic value in asthma.

Independent roles for IL-2 and GATA-3 in stimulating naive CD4+ T cells to generate a Th2-inducing cytokine environment

T cell receptor (TCR) signaling plays an important role in early interleukin (IL)-4 production by naive CD4+ T cells. This “antigen-stimulated” early IL-4 is sufficient for in vitro Th2 differentiation. Here, we provide evidence that early IL-4 production by naive CD4+ T cells stimulated with cognate peptide requires TCR-induced early GATA-3 expression and IL-2 receptor signaling, both of which are controlled by the degree of activation of extracellular signal-regulated kinase (ERK). Stimulation of naive CD4+ T cells from TCR transgenic mice with low concentrations of peptide-induced IL-2–dependent STAT5 phosphorylation, IL-4-independent early GATA-3 expression, and IL-4 production. Neutralization of IL-2 abolished early IL-4 production without affecting early GATA-3 expression. In addition, naive CD4+ T cells from GATA-3 conditional KO mice failed to produce early IL-4 in response to TCR/CD28 stimulation. Stimulation with high concentrations of peptide abrogated early GATA-3 expression and IL-2–dependent STAT5 phosphorylation, and resulted in the failure to produce early IL-4. This high concentration–mediated suppression of early IL-4 production was reversed by blockade of the ERK pathway. A MEK inhibition rescued early GATA-3 expression and responsiveness to IL-2; these cells were now capable of producing early IL-4 and undergoing subsequent Th2 differentiation.

The Double Identity of WSX-1 (IL-27R) as an Initiator and an Attenuator of Immune Responses

Two heterodimeric cytokines, IL-23 and IL-27, were recently identified, which have structural and functional homology to IL-12. IL-27, composed of p28 plus Epstein Barr Virus-induced gene (EBI)-3, is a member of the IL-12 cytokine family. IL-27 is produced early after activation of antigen-prese nting cells and induces proliferation of naive CD4 + T cells upon antigen recognition. Stimulation of naive CD4 + T cells with IL-27 through WSX-1, a subunit of functional IL-27 receptor complex, initiates the differentiation of CD4 + T cells into Th1 populations through induction of T-bet followed by expression of IL-12Rβ2. IL-27/WSX-1 thus is critical for proper induction of Th1 responses and lack of WSX-1 results in the impaired Th1 responses in mice. Recent studies revealed that L-27/WSX-1 signaling also has an anti-inflamma tory property. In some protozoan infection, various pro-inflammatory cytokines including TNF-α and IL-6, and even IFN-γ, were over produced, causing lethal inflammatory responses in WSX-1 -/- mice. These data revealed that IL-27/WSX-1 has a suppressive role for pro-inflammatory cytokine production and that IL-27/WSX-1 may work to suppress and/or terminate immune responses and inflammation. Taken together, IL-27/WSX-1 thus has double identity as an initiator and as an attenuator of immune responses and inflammation.

"Bystander polarization" of CD4+ T cells: activation with high-dose IL-2 renders naive T cells responsive to IL-12 and/or IL-18 in the absence of TCR ligation.

Responsiveness of CD4+ T cells to the IFN-gamma-inducing cytokines IL-12 and IL-18 is generally thought to be acquired only after stimulation via the TCR. We report herein that stimulation of naive CD4+ T cells with high-dose IL-2 (1000 U/ml) renders these cells responsive to IL-12 and/or IL-18 without a requirement for TCR ligation. Naive CD4+CD62L+ Tcells from normal C57BL/6 mice or from DO11.10/Rag2(-/- )OVA-specific TCR-transgenic mice secreted substantial amounts of IFN-gamma when stimulated concurrently with high-dose IL-2 plus IL-12 or IL-18. mRNA encoding both chains of the IL-12 and the IL-18 receptors was expressed by CD4+ T cells after stimulation with high-dose IL-2. Furthermore, anti-CD3-induced IL-12/IL-18 responsiveness was fully abrogated in the presence of cyclosporin A whereas IL-2-induced IL-12/IL-18 responsiveness was not, reminiscent of the previously reported IL-12+IL-18 innate pathway of T cell activation. Lastly, after stimulation with IL-2+IL-12, naive CD4+ T cells from DO11.10/Rag2(-/- )mice exhibited polarization towards a Th1 phenotype (high IFN-gamma but no IL-4) during secondary stimulation with immobilized anti-CD3. We have coined the term "bystander polarization" to describe this phenomenon and we speculate that bystander polarization of naive CD4+ T cells may occur in vivo during strong antigen-specific immune responses.

Development of Colitis in Signal Transducers and Activators of Transcription 6-Deficient T-Cell Receptor α-Deficient Mice: A Potential Role of Signal Transducers and Activators of Transcription 6-Independent Interleukin-4 Signaling for the Generation of Th2-Biased Pathological CD4 +ββT Cells

Forbidden CD4(+)betabeta T cells, which produce interleukin (IL)-4 predominantly, are a pathological subset in the development of colitis in T-cell receptor alpha chain (TCRalpha)-deficient mice. Stimulation of naive CD4(+) T cells with IL-4 induces Th2 development via the activation of signal transducers and activators of transcription (STAT) 6. In the present study, we had found that IL-4 enhanced the expression of STAT6 in CD4(+)betabeta T cells isolated from TCRalpha(-/-) mice with colitis, suggesting that the IL-4 signal in the CD4(+)betabeta T cells is mediated by STAT6. To further investigate the role of STAT6 in the development of colitis induced by TCRalpha deficiency, we generated double-deficient mice by crossing TCRalpha(-/-) mice and STAT6(-/-) mice. Surprisingly, STAT6 deficiency did not result in decreased severity of colitis in TCRalpha(-/-) mice. STAT6-deficient CD4(+)betabeta T cells produced IL-4 and intraperitoneal injection of anti-IL-4 monoclonal antibody in the nondiseased TCRalpha(-/-) and STAT6 double-deficient mice prevented the colitis formation, thus indicating that the cells differentiated into the Th2 phenotype have the ability to mediate the development of the colitis in the absence of STAT6.

Major histocompatibility complex class II- fetal skin dendritic cells are potent accessory cells of polyclonal T-cell responses.

Whereas dendritic cells (DC) and Langerhans cells (LC) isolated from organs of adult individuals express surface major histocompatibility complex (MHC) class II antigens, DC lines generated from fetal murine skin, while capable of activating naive, allogeneic CD8+ T cells in a MHC class I-restricted fashion, do not exhibit anti-MHC class II surface reactivity and fail to stimulate the proliferation of naive, allogeneic CD4+ T cells. To test whether the CD45+ MHC class I+ CD80+ DC line 80/1 expresses incompetent, or fails to transcribe, MHC class II molecules, we performed biochemical and molecular studies using Western blot and polymerase chain reaction analysis. We found that 80/1 DC express MHC class II molecules neither at the protein nor at the transcriptional level. Ultrastructural examination of these cells revealed the presence of a LC-like morphology with indented nuclei, active cytoplasm, intermediate filaments and dendritic processes. In contrast to adult LC, no LC-specific cytoplasmic organelles (Birbeck granules) were present. Functionally, 80/1 DC in the presence, but not in the absence, of concanavalin A and anti-T-cell receptor monoclonal antibodies stimulated a vigorous proliferative response of naive CD4+ and CD8+ T cells. Furthermore, we found that the anti-CD3-induced stimulation of naive CD4+ and CD8+ T cells was critically dependent on the expression of FcgammaR on 80/1 DC and that the requirement for co-stimulation depends on the intensity of T-cell receptor signalling.

Relative efficiency of microglia, astrocytes, dendritic cells and B cells in naive CD4+ T cell priming and Th1/Th2 cell restimulation

We have compared the efficiency of central nervous system and peripheral antigen-presenting cells (APC) in T cell priming and restimulation. OVA peptide 323 – 339-dependent activation of DO11.10 TCR-transgenic naive CD4+ and polarized Th1 or Th2 cells was assessed in the presence of microglia and astrocytes from the neonatal mouse brain as well as dendritic cells (DC) and B cells purified from adult mouse lymph nodes. DC were the most efficient in inducing naive T cell proliferation, IL-2 secretion and differentiation into Th1 cells, followed by IFN-γ-preactivated microglia, large and small B cells. Astrocytes failed to activate naive T cells. IFN-γ-pretreated microglia were as efficient as DC in the restimulation of Th1 cells, whereas IFN-γ-pretreated astrocytes, large and small B cells were much less efficient. Conversely, Th2 cells were efficiently restimulated by all the APC types examined. During T cell priming, DC secreted more IL-12 than microglia but similar amounts of IL-12 were secreted by the two cell types upon interaction with Th1 cells. The hierarchy of APC established in this study indicates that DC and microglia are the most efficient in the stimulation of naive CD4+ T cells and in the restimulation of Th1 cells, suggesting that activated microglia may effectively contribute to Th1 responses leading to central nervous system inflammation and tissue damage. These potentially pathogenic responses could be counteracted by the high efficiency of astrocytes as well as microglia in restimulating Th2 cells.

Relative efficiency of microglia, astrocytes, dendritic cells and B cells in naive CD4+ T cell priming and Th1/Th2 cell restimulation.

We have compared the efficiency of central nervous system and peripheral antigen-presenting cells (APC) in T cell priming and restimulation. OVA peptide 323 - 339-dependent activation of DO11.10 TCR-transgenic naive CD4+ and polarized Th1 or Th2 cells was assessed in the presence of microglia and astrocytes from the neonatal mouse brain as well as dendritic cells (DC) and B cells purified from adult mouse lymph nodes. DC were the most efficient in inducing naive T cell proliferation, IL-2 secretion and differentiation into Th1 cells, followed by IFN-gamma-preactivated microglia, large and small B cells. Astrocytes failed to activate naive T cells. IFN-gamma-pretreated microglia were as efficient as DC in the restimulation of Th1 cells, whereas IFN-gamma-pretreated astrocytes, large and small B cells were much less efficient. Conversely, Th2 cells were efficiently restimulated by all the APC types examined. During T cell priming, DC secreted more IL-12 than microglia but similar amounts of IL-12 were secreted by the two cell types upon interaction with Th1 cells. The hierarchy of APC established in this study indicates that DC and microglia are the most efficient in the stimulation of naive CD4(+) T cells and in the restimulation of Th1 cells, suggesting that activated microglia may effectively contribute to Th1 responses leading to central nervous system inflammation and tissue damage. These potentially pathogenic responses could be counteracted by the high efficiency of astrocytes as well as microglia in restimulating Th2 cells.

Interleukin-12 Induces Expression of Interferon Regulatory Factor-1 via Signal Transducer and Activator of Transcription-4 in Human T Helper Type 1 Cells

IRF-1-deficient mice show a striking defect in the development of T helper 1 (Th1) cells. In the present report, we investigate the expression of IRF-1 during differentiation of human T helper cells. No significant differences of IRF-1 mRNA expression were found in established Th1 and Th2 cells; however, interleukin 12 (IL-12) induced a strong up-regulation of IRF-1 transcripts in Th1 but not in Th2 cells. We demonstrate that IL-12-induced up-regulation of IRF-1 is mediated by signal transducer and activator of transcription-4, which binds to the interferon (IFN)-gamma-activated sequence present in the promoter of the IRF-1 gene. Strong IL-12-dependent activation of a reporter gene construct containing the IRF-1 IFN-gamma-activated sequence element provides further evidence for the key role of signal transducer and activator of transcription-4 in the IL-12-induced up-regulation of IRF-1 transcripts in T cells. IRF-1 expression was strongly induced after stimulation of naive CD4(+) T cells via the T cell receptor, irrespective of the cytokines present at priming, indicating that this transcription factor does not play a major role in initiating a Th1-specific transcriptional cascade in differentiating helper T cells. However, our finding that IRF-1 is a target gene of IL-12 suggests that some of the IL-12-induced effector functions of Th1 cells may be mediated by IRF-1.