Diterpenes inhibit IL-12 production by DC and enhance Th2 cells polarization

Sugiol and 12-hydroxy-6,7-secoabieta-8,11,13-triene-6,7-dial (Secoferruginol) are diterpenes isolated from the heartwood of Cryptomeria japonica and are pharmacologically active substances. Dendritic cells (DC) have a key influence on the differentiation of na?ve T cells into Th1 or Th2 effector cells. We demonstrate that Sugiol and Secoferruginol activate human DC as documented by phenotypic and functional maturation and altered cytokine production. Human monocytes were exposed to Sugiol or Secoferruginol alone, or in combination with LPS and thereafter co-cultured with na?ve T cells. The expression levels of CD83 on Sugiol-primed DC were enhanced. Sugiol dose-dependently inhibited IL-12p70 production by LPS-primed DC and to a lesser extent, the production of the proinflammatory cytokines. Na?ve T cells co-cultured with Sugiol-primed DC, turned into typical Th2 which produced large quantities of IL-4 and released small amounts of IFN-gamma and reduced Th1 cell polarizing capacity. Sugiol-primed DC induced the development of Th2 cells via the enhanced expression of OX40L and augmented the Th2 cell polarizing capacity of DC via the inhibition of IL-12p70. Similar results were obtained with Secoferruginol. These results suggest that some diterpenes modulate human DC function in a fashion that favors Th2 cell polarization and might have implication in autoimmune diseases.     

Unique ability of activated CD4+ T cells but not rested effectors to migrate to non-lymphoid sites in the absence of inflammation

Recent studies suggest that effector T cells generated by immune responses migrate to multiple non-lymphoid sites, even those without apparent expression of antigen or inflammation. To investigate the ability of distinct CD4(+) T lymphocyte subsets to enter and persist in non-lymphoid, noninflamed compartments, we examined the migration and persistence of na?ve, effector, and rested effector CD4(+) T cells generated in vitro following transfer to nonimmunized adoptive hosts. Th1 and Th2 effectors migrated to both lymphoid and non-lymphoid organs (peritoneum, fat pads, and lung). In contrast, rested effectors and na?ve cells migrated only to lymphoid areas. Adhesion molecule expression, but not chemokine receptor expression, correlated with the ability to enter non-lymphoid sites. Donor cells persisted longer in lymphoid than in non-lymphoid sites. When hosts with na?ve and memory donor cells were challenged with antigen, effectors developed in situ, which also migrated to non-lymphoid sites. Memory cells showed an accelerated shift to non-lymphoid migration, in keeping with memory effector formation. These results suggest that only recently activated effector T cells can disperse to non-lymphoid sites in the absence of antigen and inflammation, and as effectors return to rest, they lose this ability. These data also argue that memory cells in lymphoid sites are longer lived and not in equilibrium with those in non-lymphoid sites.     

Epicubenol and Ferruginol induce DC from human monocytes and differentiate IL-10-producing regulatory T cells in vitro

Epicubenol and 19-hydroxyferruginol (Ferruginol) are sesquiterpenes isolated from the black heartwood of Cryptomeria japonica. Dendritic cells (DC) are specialized antigen-presenting cells that monitor the antigenic environment and activate na?ve T cells. The role of DC is not only to sense danger but also to tolerize the immune system to antigens encountered in the absence of maturation/inflammatory stimuli. In this study, we attempted to investigate the effects of Epicubenol and Ferruginol on the phenotypic and functional maturation of human monocytes-derived DC in vitro. Human monocytes were cultured with GM-CSF and IL-4 for 6 days under standard conditions, followed by another 2 days with Epicubenol or Ferruginol. The expression levels of CD1a, CD83, and HLA-DR as expressed by mean fluorescence intensity (MFI) on Epicubenol-primed DC or Ferruginol-primed DC were enhanced. Allogeneic Epicubenol-primed DC or Ferruginol-primed DC co-cultured with na?ve T cells at 1:5 ratio, secreted IL-10 and TGF-beta, but little IL-4. Moreover, T cells that develop in co-culture of Epicubenol-primed DC or Ferruginol-primed DC and na?ve T cells at 1:5 ratio suppressed the proliferation of autologous T cells at Treg cells: Ttarget cells and this suppression of proliferation was inhibited by anti-IL-10 mAb. The expression of FoxP3 mRNA on T cells that develop in co-culture of Epicubenol-primed DC or Ferruginol-primed DC and na?ve T cells was lower. From these results, Epicubenol and Ferruginol may induce IL-10-producing Treg 1 cells from na?ve T cells by modulating DC function. It seems that Epicubenol and Ferruginol appear to be a target for tolerance after transplantation and in autoimmune diseases.     

Polarization of naive CD4+ T cells toward the Th1 subset by CTLA-4 costimulation

In this study, we examined in vitro the role of CTLA-4 costimulation in the polarization of naive CD4+ T cells toward the Th1 subset. When CTLA-4 costimulation was blocked by the inclusion of anti-CTLA-4 Fab in cultures during priming of naive CD4+ T cells with anti-CD3 in the presence of splenic adherent cells, they were polarized toward the Th2 subset. Conversely, the engagement of CTLA-4 with immobilized anti-CTLA-4 or with CD80-P815 cells polarized naive CD4+ T cells costimulated with anti-CD3 and anti-CD28 toward the Th1 subset. The CTLA-4 costimulation during priming augmented TGF-beta1 mRNA accumulation in naive CD4+ T cells, and the inclusion of anti-TGF-beta in cultures for priming suppressed the effect of CTLA-4 costimulation on the Th1 polarization. The addition of low doses of TGF-beta1 in cultures for priming of naive CD4+ T cells enhanced the production of Th1 cytokines upon secondary stimulation, although Th2 cytokine production was not affected by the doses of TGF-beta1. The CTLA-4 costimulation was also shown to suppress IL-4 production of naive CD4+ T cells upon priming. These results indicate that the costimulation against CTLA-4 drives polarization of naive CD4+ T cells toward the Th1 subset independent of IL-12 through, at least in part, the enhancement of TGF-beta1 production, and it also hampers Th2 subset differentiation by affecting IL-4 production of naive CD4+ T cells.     

Proteome profiling of interleukin-12 treated human T helper cells

Selective activation of T helper subsets 1 (Th1) and 2 (Th2) plays a crucial role in different pathological conditions. Th1 cell response is involved in pathogenesis of autoimmune diseases, such as type II diabetes and multiple sclerosis, and Th2 cell response in pathogenesis of allergy and asthma. Cytokine interleukin-12 (IL-12) is one of the key factors in the differentiation of na?ve CD4(+) T cells into Th1 cells. In this study we used 2-DE and MS to find and identify IL-12 regulated proteins in human CD4(+) T cells. In total, 42 protein spots were found to be differentially expressed following IL-12 stimulation, of which 22 were up- and 20 down-regulated. Among the upregulated proteins there are a multifunctional cytokine macrophage migration inhibitory factor and a known IL-12 target gene Programmed cell death 4. Downregulated proteins include p21-activated kinase 2 and its upstream GTPase Cdc42. Compared to previous reports our analysis provides a new view on the IL-12 induced changes on CD4(+) T cells underscoring the importance of creating and combining the data generated at various levels to build a comprehensive view of a given biological process of the cell.     

Th2 cell-specific cytokine expression and allergen-induced airway inflammation depend on JunB

Na?ve CD4+ T cells differentiate into effector T helper 1 (Th1) or Th2 cells, which are classified by their specific set of cytokines. Here we demonstrate that loss of JunB in in vitro polarized Th2 cells led to a dysregulated expression of the Th2-specific cytokines IL-4 and IL-5. These cells produce IFN-gamma and express T-bet, the key regulator of Th1 cells. In line with the essential role of Th2 cells in the pathogenesis of allergic asthma, mice with JunB-deficient CD4+ T cells exhibited an impaired allergen-induced airway inflammation. This study demonstrates novel functions of JunB in the development of Th2 effector cells, for a normal Th2 cytokine expression pattern and for a complete Th2-dependent immune response in mice.     

TSC1/2 signaling complex is essential for peripheral naïve CD8+ T cell survival and homeostasis in mice

The PI3K-Akt-mTOR pathway plays crucial roles in regulating both innate and adaptive immunity. However, the role of TSC1, a critical negative regulator of mTOR, in peripheral T cell homeostasis remains elusive. With T cell-specific Tsc1 conditional knockout (Tsc1 KO) mice, we found that peripheral na?ve CD8(+) T cells but not CD4(+) T cells were severely reduced. Tsc1 KO na?ve CD8(+) T cells showed profound survival defect in an adoptive transfer model and in culture with either stimulation of IL-7 or IL-15, despite comparable CD122 and CD127 expression between control and KO CD8(+) T cells. IL-7 stimulated phosphorylation of Akt(S473) was diminished in Tsc1 KO na?ve CD8(+)T cells due to hyperactive mTOR-mediated feedback suppression on PI3K-AKT signaling. Furthermore, impaired Foxo1/Foxo3a phosphorylation and increased pro-apoptotic Bim expression in Tsc1 KO na?ve CD8(+)T cells were observed upon stimulation of IL-7. Collectively, our study suggests that TSC1 plays an essential role in regulating peripheral na?ve CD8(+) T cell homeostasis, possible via an mTOR-Akt-FoxO-Bim signaling pathway.     

ICOS expression by activated human Th cells is enhanced by IL-12 and IL-23: increased ICOS expression enhances the effector function of both Th1 and Th2 cells

Previous mouse studies have shown that IL-4 increases the expression of ICOS on activated Th cells, resulting in enhanced ICOS expression on Th2 cells. In this study, we show that ICOS expression on human Th cells is not increased by IL-4, but by IL-12 and by IL-23 instead. Consequently, ICOS expression during IL-12-driven Th1 cell polarization was transiently increased compared with the levels on Th0 cells and IL-4-driven Th2 cells. Addition of IL-12 and/or IL-23 during restimulation increased ICOS expression to the same extent on pre-established Th1, Th2, and Th0 cells, indicating that ICOS levels are not stably imposed by prior polarization. In contrast to the findings in the mouse, IL-4 significantly suppressed the ICOS-enhancing effects of IL-12 and IL-23. The functional consequence of variable ICOS levels was shown in coculture experiments with cells expressing the ICOS-ligand B7-related protein 1 (either transfected Chinese hamster ovary cells or autologous dendritic cells). Ligation of ICOS on 2-day-preactivated effector cells increased their cytokine production to an extent proportional to their ICOS expression levels. As the ICOS-enhancing potentials of IL-12 and IL-23 were maintained for several days after stimulation, both on Th1 and Th2 cells, we propose the concept that local regulation of ICOS expression on activated Th cells by IL-12 and/or IL-23 may provide a powerful means to amplify effector T cell responses in peripheral tissues, independently of the polarized state of the Th cells.     

An orally active Th1/Th2 balance modulator, M50367, suppresses Th2 differentiation of naïve Th cell in vitro

We previously reported an orally active anti-allergic agent, M50367, modulated Th1/Th2 balance to down-regulate Th2 response in a murine model of atopic asthma. In this study, we examined the effect of M50354, the active metabolite of M50367, on the differentiation of na?ve Th cells into Th1/Th2 cells. M50354 at 3 microM decreased the generation of Th2 cells by 0.2-fold and increased that of Th1 cells by 1.6-fold from na?ve Th cells primed with antigenic peptide and antigen-presenting cells. Its effect was also seen when na?ve Th cells were primed with anti-T cell receptor and anti-CD28 agonistic antibodies instead of antigen and antigen-presenting cells. M50354 decreased early endogenous IL-4 production in the nai;ve Th cell priming culture without affecting interferon-gamma production and proliferation. In contrast, M50354 had no effect on interferon-gamma and IL-4 production from mature Th1 and Th2 cells. These results suggest that M50354 directly acts on na?ve Th cells to suppress their differentiation into Th2 cells.     

Leishmania: naive human T cells sensitized with promastigote antigen and IL-12 develop into potent Th1 and CD8(+) cytotoxic effectors.

Russo, D. M., Chakrabarti, P., and Higgins, A. Y. 1999. Leishmania: Naive human T cells sensitized with promastigote antigen and IL-12 develop into potent Th1 and CD8(+) cytotoxic effectors. Experimental Parasitology 93, 161-170. The differentiation of naive human T cells into Leishmania-specific Th1 or cytotoxic effector cells was examined by sensitizing T cells in vitro with dead Leishmania antigen in the presence or absence of IFN-gamma or IL-12. These Leishmania-specific T cell lines proliferated and produced cytokines in response to challenge with autologous Leishmania-infected macrophages. Sensitization in the presence of IL-12 or IFN-gamma induced Leishmania-specific human Th1 responses, with IL-12 inducing more potent Th1 responses. However, IL-12-induced Th1 responses were IFN-gamma dependent. T cell lines exhibited Th2 or Th0 phenotypes when primed in the absence of cytokines. Only T cell lines primed in the presence of IL-12 contained high percentages of CD8(+) cells. These cells lysed autologous Leishmania-infected but not uninfected macrophages in an MHC-dependent manner. Thus, this in vitro sensitization system can be used to delineate the conditions for optimally priming human Leishmania-specific effector cells.     

The biology of IL-12: coordinating innate and adaptive immune responses

Cytokines play critical roles in regulating all aspects of immune responses, including lymphoid development, homeostasis, differentiation, tolerance and memory. Interleukin (IL)-12 is especially important because its expression during infection regulates innate responses and determines the type and duration of adaptive immune response. IL-12 induces interferon-gamma (IFN-gamma) production by NK, T cells, dendritic cells (DC), and macrophages. IL-12 also promotes the differentiation of na?ve CD4+ T cells into T helper 1 (Th1) cells that produce IFN-gamma and aid in cell-mediated immunity. As IL-12 is induced by microbial products and regulates the development of adaptive immune cells, IL-12 plays a central role in coordinating innate and adaptive immunity. IL-12 and the recently identified cytokines, IL-23 and IL-27, define a family of related cytokines that induce IFN-gamma production and promote T cell expansion and proliferation.     

Role of CD47 in the induction of human naive T cell anergy

We recently reported that CD47 ligation inhibited IL-2 release by umbilical cord blood mononuclear cells activated in the presence of IL-12, but not IL-4, preventing the induction of IL-12Rbeta(2) expression and the acquisition of Th1, but not the Th2 phenotype. Here we show that in the absence of exogenous cytokine at priming, CD47 ligation of umbilical cord blood mononuclear cells promotes the development of hyporesponsive T cells. Naive cells were treated with CD47 mAb for 3 days, expanded in IL-2 for 9-12 days, and restimulated by CD3 and CD28 coengagement. Effector T cells generated under these conditions were considered to be anergic because they produced a reduced amount of IL-2 at the single-cell level and displayed an impaired capacity 1) to proliferate, 2) to secrete Th1/Th2 cytokines, and 3) to respond to IL-2, IL-4, or IL-12. Moreover, CD47 mAb strongly suppressed IL-2 production and IL-2Ralpha expression in primary cultures and IL-2 response of activated naive T cells. Induction of anergy by CD47 mAb was IL-10 independent, whereas inclusion of IL-2 and IL-4, but not IL-7, at priming fully restored T cell activation. Furthermore, CD28 costimulation prevented induction of anergy. Thus, CD47 may represent a potential target to induce anergy and prevent undesired Th0/Th1 responses such as graft vs host diseases, allograft rejection, or autoimmune diseases.     

Constitutive expression of CIITA directs CD4 T cells to produce Th2 cytokines in the thymus

We generated mice expressing a human type III CIITA transgene (CIITA Tg) under control of the CD4 promoter to study the role of CIITA in CD4 T cell biology. The transgene is expressed in peripheral CD4 and CD8 T cells, as well as in thymocytes. When CD4 T cells were differentiated towards the Th2 lineage, both control and CIITA Tg Th2 cells expressed similar levels of Th2 cytokines. Th1 cells from control and CIITA Tg mice cells produced comparable levels of IFN-gamma. CIITA Tg Th1 cells also expressed IL-4, IL-5, and IL-13 in the absence of Stat6. There was an approximate 10-fold increase in the number of peripheral na?ve CD4 T cells and NK1.1- thymocytes producing IL-4 from CIITA Tg mice compared to control mice. Finally, Th1 cells from irradiated control mice reconstituted with CIITA Tg bone marrow displayed the same cytokine production profiles as Th1 cells from CIITA Tg mice. Together, our data demonstrate that CIITA expression pre-disposes CD4 T cells to produce Th2 type cytokines. Moreover, phenotypic similarities between Th1 cells expressing the CIITA transgene and CIITA deficient Th1 cells suggest that the role of CIITA in cytokine regulation is complex and may reflect both direct and indirect mechanisms of T cell development and differentiation.