The transcription factor T-bet regulates mucosal T cell activation in experimental colitis and Crohn's disease.

The balance between pro and antiinflammatory cytokines secreted by T cells regulates both the initiation and perpetuation of inflammatory bowel diseases (IBD). In particular, the balance between interferon (IFN)-gamma/interleukin (IL)-4 and transforming growth factor (TGF)-beta activity controls chronic intestinal inflammation. However, the molecular pathways that evoke these responses are not well understood. Here, we describe a critical role for the transcription factor T-bet in controlling the mucosal cytokine balance and clinical disease. We studied the expression and function of T-bet in patients with IBD and in mucosal T cells in various T helper (Th)1- and Th2-mediated animal models of chronic intestinal inflammation by taking advantage of mice that lack T-bet and retroviral transduction techniques, respectively. Whereas retroviral transduction of T-bet in CD62L(+) CD4(+) T cells exacerbated colitis in reconstituted SCID mice, T-bet-deficient T cells failed to induce colitis in adoptive transfer experiments suggesting that overexpression of T-bet is essential and sufficient to promote Th1-mediated colitis in vivo. Furthermore, T-bet-deficient CD62L(-) CD4(+) T cells showed enhanced protective functions in Th1-mediated colitis and exhibited increased TGF-beta signaling suggesting that a T-bet driven pathway of T cell activation controls the intestinal balance between IFN-gamma/IL-4 and TGF-beta responses and the development of chronic intestinal inflammation in T cell-mediated colitis. Furthermore, TGF-beta was found to suppress T-bet expression suggesting a reciprocal relationship between TGF-beta and T-bet in mucosal T cells. In summary, our data suggest a key regulatory role of T-bet in the pathogenesis of T cell-mediated colitis. Specific targeting of this pathway may be a promising novel approach for the treatment of patients with Crohn's disease and other autoimmune diseases mediated by Th1 T lymphocytes.

A novel transcription factor, T-bet, directs Th1 lineage commitment.

Naive T helper cells differentiate into two subsets, Th1 and Th2, each with distinct functions and cytokine profiles. Here, we report the isolation of T-bet, a Th1-specific T box transcription factor that controls the expression of the hallmark Th1 cytokine, IFNgamma. T-bet expression correlates with IFNgamma expression in Th1 and NK cells. Ectopic expression of T-bet both transactivates the IFNgamma gene and induces endogenous IFNgamma production. Remarkably, retroviral gene transduction of T-bet into polarized Th2 and Tc2 primary T cells redirects them into Th1 and Tc1 cells, respectively, as evidenced by the simultaneous induction of IFNgamma and repression of IL-4 and IL-5. Thus, T-bet initiates Th1 lineage development from naive Thp cells both by activating Th1 genetic programs and by repressing the opposing Th2 programs.

T helper differentiation proceeds through Stat1-dependent, Stat4-dependent and Stat4-independent phases.

Much of our focus in understanding Th1/Th2 development has been on the signals delivered by IL-12 and IL-4 as final determinants of terminal T cell differentiation. Because extinction of IL-12 signaling in early Th2 development could potentially be important in imprinting a more permanent Th2 phenotype on a population of T cells, we have also examined various parameters regulating the IL-12 signaling pathway. Whereas IL-4 appears to repress functional IL-12 signaling through inhibition of IL-12R beta 2 expression, IFN-gamma in the mouse, and IFN-alpha in the human appear to induce IL-12R beta 2 expression and promote IL-12 responsiveness. We propose that Th1 development can be considered in two stages, capacitance and development. Capacitance would simply involve expression of IL-12R beta 1 and beta 2 subunits, regulated by TCR, IL-4 and IFNs. The second stage, development, we propose is the true IL-12 induced developmental stage, involving expression of Stat4 inducible proteins. In the human, this may also occur via IFN-alpha, which is able to activate Stat4. It is perhaps possible that all of Stat4 actions on Th1 development may be exert directly by Stat4 at the IFN-gamma gene, however we suggest that, more likely, Stat4 may act to induce Th1 development through the induction of other non-cytokine genes, whose stable expression maintains the transcriptional state of a Th1 cell.

Modulation of airway inflammation by passive transfer of allergen-specific Th1 and Th2 cells in a mouse model of asthma.

Although evidence is strong that Th cells play a major role in mediating the airway inflammation observed in asthma, the relative contributions of the Th cell subsets, Th1 and Th2, are unclear. It has been suggested that asthma is driven by Th2 predominant responses in the lung, but other data suggest a role for Th1 cells as well. Here we show by intracellular cytokine staining and flow cytometric analysis that in the murine model of OVA-induced airway inflammation, both Th1 and Th2 cells are recruited to the airways. Th1 cells predominate early in the response and Th2 cells predominate late. We further show that increasing the number of Th1 cells by passive transfer of OVA-specific Th1 cells results in increased inflammation. This effect is observed regardless of whether the T cells are transferred before sensitization or after airway inflammation is already in progress. Transfer of Th1 cells also results in increased recruitment of host T cells of both Th1 and Th2 phenotypes. Passive transfer of Th2 cells results in little change in the inflammatory response. These results demonstrate that Ag-specific Th1 cells are not protective in this model of asthma, but rather may potentiate the inflammatory response.

Regulation of the interleukin (IL)-12R beta 2 subunit expression in developing T helper 1 (Th1) and Th2 cells.

The developmental commitment to a T helper 1 (Th1)- or Th2-type response can significantly influence host immunity to pathogens. Extinction of the IL-12 signaling pathway during early Th2 development provides a mechanism that allows stable phenotype commitment. In this report we demonstrate that extinction of IL-12 signaling in early Th2 cells results from a selective loss of IL-12 receptor (IL-12R) beta 2 subunit expression. To determine the basis for this selective loss, we examined IL-12R beta 2 subunit expression during Th cell development in response to T cell treatment with different cytokines. IL-12R beta 2 is not expressed by naive resting CD4+ T cells, but is induced upon antigen activation through the T cell receptor. Importantly, IL-4 and IFN-gamma were found to significantly modify IL-12 receptor beta 2 expression after T cell activation. IL-4 inhibited IL-12R beta 2 expression leading to the loss of IL-12 signaling, providing an important point of regulation to promote commitment to the Th2 pathway. IFN-gamma treatment of early developing Th2 cells maintained IL-12R beta 2 expression and restored the ability of these cells to functionally respond to IL-12, but did not directly inhibit IL-4 or induce IFN-gamma production. Thus, IFN-gamma may prevent early Th cells from premature commitment to the Th2 pathway. Controlling the expression of the IL-12R beta 2 subunit could be an important therapeutic target for the redirection of ongoing Th cell responses.

Identification of IL-4 promoter elements conferring Th2-restricted expression during T helper cell subset development.

Selective cytokine gene expression by T cell subsets underlies polarization of cellular and humoral immune responses. Our interest has been to define the molecular basis for restricted cytokine expression by Th1 and Th2 cells. IL-4 is selectively expressed by Th2 cells, providing a model for Th2-specific gene expression. To allow for promoter analysis during the process of Th1/Th2 differentiation within a normal cellular context, we have taken a transgenic approach. We generated a series of murine transgenic lines harboring both the DO11.10 alphabeta-TCR transgene and the luciferase gene driven by regions of the IL-4 promoter. The results identify proximal promoter regions that provide significantly Th2-restricted IL-4 gene expression. The IL-4 -741- to +60-bp region allows, on the average, 40-fold higher inducible reporter activity in Th2 cells than in Th1 cells. When trimerized, the region spanning -88 to -61 bp, containing a composite NF-AT/AP-1 site, also confers significant Th2-specific reporter activity. These results suggest that trans-acting factors binding this NF-AT/AP-1 composite site cooperate to allow substantial Th2-selective reporter expression. Finally, because reporter expression is low relative to endogenous IL-4 mRNA in activated Th2 cells, we suggest that additional control elements outside of the IL-4 promoter may be required to enhance overall IL-4 gene activity.

Genes that regulate interleukin-4 expression in T cells.

Interleukin-4 is an immunomodulatory cytokine which plays a central role in the regulation of allergic and atopic immune responses. Significant progress has been made in gaining a detailed understanding of the transcriptional regulation of the interleukin-4 gene. The recent identification and characterization of several key transcription factors has helped to elucidate the molecular mechanisms of T helper cell cytokine gene expression.

Regulation of interleukin-12 signalling during T helper phenotype development.

The experiments described above have allowed us to define the molecular events in IL-12 signalling. Within minutes after IL-12 treatment of responsive cells, Stat1, Stat3, and Stat4 are tyrosine phosphorylated. These molecules form nuclear DNA-binding complexes consisting of homodimeric Stat1 and heterodimeric Stat3-Stat4 complexes. In a murine in vitro phenotype development model, T cells rapidly and selectively lose their capacity to respond to IL-12 upon acquisition of the Th2 phenotype. This hyporesponsiveness is manifested by the inability of IL-12 to induce IFN gamma production in differentiated Th2 cells, as well as the inability of IL-12 to induce the activation of Stat4. Despite the functional defect of IL-12 signalling in Th2 cells, all known components of the IL-12 signal transduction pathway are present. We speculate that in Th2 cells, the second receptor chain may be absent or one of the other components may be modified. Genetic experiments in Balb/c and B10.D2 strains of mice have demonstrated several differences in T helper differentiation in vitro. Stimulation of T cells under neutral conditions results in a bias of Balb/c T cells toward the Th2 extreme and B10 T cells toward the Th1 extreme of cytokine production. Following stimulation under neutral conditions, B10 T cells retain the ability to respond to IL-12 while Balb/c T cells lose IL-12 responsiveness. Mating experiments have demonstrated that the B10 genetic effect is dominant in F1 mice. Analysis of backcrossed animals has suggested that the ability to respond to IL-12 in the secondary stimulation may be controlled by a single dominant B10 gene. The results we describe may have profound implications for allergy. Since allergic responses are largely due to the activation of the Th2 subset of T lymphocytes, a better understanding of T cell phenotype development may reveal multiple targets for therapeutic intervention. First, a better understanding of Th1 phenotype induction in response to IL-12 may allow prevention of in vivo allergic responses using pharmacological tools which bias allergen-specific responses to the Th1 subset. Second, a molecular explantation of why Th2 cells fail to reverse phenotype in response to IL-12 may allow treatment of atopic individuals to remove the disease-promoting T lymphocyte compartment. Finally, a better understanding of the basis for genetic differences in murine T helper cell differentiation may allow us to identify a causative genetic element in humans, yielding better diagnostic and therapeutic methods.

Ligand-induced autoregulation of IFN-gamma receptor beta chain expression in T helper cell subsets.

Interferon gamma (IFN-gamma) responsiveness in certain cells depends on the state of cellular differentiation or activation. Here an in vitro developmental system was used to show that IFN-gamma produced during generation of the CD4+ T helper cell type 1 (TH1) subset extinguishes expression of the IFN-gamma receptor beta subunit, resulting in TH1 cells that are unresponsive to IFN-gamma. This beta chain loss also occurred in IFN-gamma-treated TH2 cells and thus represents a specific response of CD4+ T cells to IFN-gamma rather than a TH1-specific differentiation event. These results define a mechanism of cellular desensitization where a cytokine down-regulates expression of a receptor subunit required primarily for signaling and not ligand binding.

Developmental commitment to the Th2 lineage by extinction of IL-12 signaling.

Developmental-commitment to Th1 or Th2 responses critically influences host susceptibility to particular pathogens. We describe a novel mechanism governing stable commitment to Th2 differentiation. Naive T cells develop strongly polarized Th1 and Th2 profiles by 7 days after activation. However, commitment of these developing cells differs substantially. Although IL-4 reverses early Th1 differentiation, IL-12 cannot reverse early Th2 differentiation. Th1 reversibility results from maintenance of IL-4 signal transduction, whereas Th2 commitment results from rapid loss of IL-12 signaling. The IL-12 signaling defect in Th2 cells results in failure to phosphorylate Jak2, Stat3, and Stat4. Since Th2 cells express the mRNA for the cloned murine IL-12 receptor beta subunit, the signaling defect may involve expression or function of unidentified receptor components. The rapid extinction of IL-12 signaling in Th2 cells provides a demonstration of a mechanism for the stable commitment to a T helper phenotype.

Interleukin 12 signaling in T helper type 1 (Th1) cells involves tyrosine phosphorylation of signal transducer and activator of transcription (Stat)3 and Stat4.

Interleukin 12 (IL-12) initiates the differentiation of naive CD4+ T cells to T helper type 1 (Th1) cells critical for resistance to intracellular pathogens such as Leishmania major. To explore the basis of IL-12 action, we analyzed induction of nuclear factors in Th1 cells. IL-12 selectively induced nuclear DNA-binding complexes that contained Stat3 and Stat4, recently cloned members of the family of signal transducers and activators of transcription (STATs). While Stat3 participates in signaling for several other cytokines, Stat4 was not previously known to participate in the signaling pathway for any natural ligand. The selective activation of Stat4 provides a basis for unique actions of IL-12 on Th1 development. Thus, this study presents the first identification of the early events in IL-12 signaling in T cells and of ligand activation of Stat4.

Identification of cis-acting regulatory elements controlling interleukin-4 gene expression in T cells: roles for NF-Y and NF-ATc.

Activity of the murine interleukin-4 (IL-4) promoter was localized to several cis-acting elements present within the first 300 bp from the transcriptional initiation site. Five repeated elements, P0 to P4, that share the common consensus ATTTTCCNNT were located between -40 and -250, and each was shown to interact with the T-cell-specific factor NF(P). These distinct P sites appear functionally interchangeable and cooperatively confer cyclosporin A-sensitive and ionomycin-inducible promoter activity. NF(P) may be closely related to the cytoplasmic component of NF-AT (nuclear factor of activated T cells), a T-cell-specific factor essential for IL-2 gene transcription, as judged from indistinguishable molecular weights and protease fragmentation patterns of UV-photolabeled factors. Also, we identified an element in the IL-4 promoter with homology to the Y box common to all major histocompatibility complex class II gene promoters. Our data show that the IL-4 promoter Y box -114CTGATTGG-107 significantly enhances overall promoter activity, since point mutations within this element diminish promoter activity by 85%. The factor binding this region is indistinguishable from the cloned nuclear factor NF-Y, as judged from interactions with specific anti-NF-Y monoclonal and polyclonal antibodies. Last, we point out the presence of two sites that share sequence identity to the OAP region of the ARRE-1 site within the IL-2 promoter (K. S. Ullman, W. M. Flanagan, C. A. Edwards, and G. R. Crabtree, Science 254:558-562, 1991). These regions, -85GTGTAATA-78 and -245GTGTAATT-238, reside adjacent to the NF(P) binding sites P1 and P4 and bind a distinct nuclear factor.