CD28 costimulation accelerates IL-4 receptor sensitivity and IL-4-mediated Th2 differentiation.

The development of Th1 and Th2 cells is determined by the type of antigenic stimulation involved in the initial cell activation step. Evidence indicates that costimulatory signals, such as those delivered by CD28, play an important role in Th2 development, but little is known about how CD28 costimulation contributes to Th2 development. In this study, TCR cross-linking was insufficient for Th2 development, while the addition of CD28 costimulation drastically increased Th2 generation through the IL-4-mediated pathway. Th2 generation following CD28 costimulation was not simply explained by the enhancement of IL-4 production in naive T cells. To generate Th2 cells after TCR cross-linking only, it was necessary to add a 20- to 200-fold excess of IL-4 generated after TCR and CD28 stimulation. TCR cross-linking increased the expression level and binding property of the IL-4R, but enhanced the sensitivity to IL-4 only slightly. In contrast, as evidenced by the enhanced phosphorylation of Jak3, the IL-4Ralpha-chain, and STAT6 following IL-4 stimulation, CD28 costimulation increased IL-4R sensitivity without affecting its expression and binding property. This evidence of the enhancement of IL-4R sensitivity increases our understanding of how CD28 costimulation accelerates Th2 development.     

IL-4 induces differentiation and expansion of Th2 cytokine-producing eosinophils

Innate effector cells that produce Th2-type cytokines are critical in Th2 cell-mediated immune responses. However, it is not known how these cells acquire the ability to produce Th2 cytokines. IL-4 is a potent inducer that directs differentiation of naive CD4(+) T cells into CD4(+) Th2 effector cells. To determine whether IL-4 can induce differentiation and expansion of Th2 cytokine-producing innate cells, we used mice whose il-4 gene was replaced by a knock-in green fluorescence protein (gfp) gene. We found that, directly ex vivo, IL-4 increased the number of GFP(+) cells in the airway and the lung tissue in an Ag-specific manner. The majority of GFP(+) cells were eosinophils, suggesting that IL-4 plays a pivotal role in expanding IL-4-producing eosinophils in vivo. IL-4-producing eosinophils showed some unique features compared with IL-4-producing CD4(+) T cells. They exhibited biallelic expression of the il-4 gene when stimulated and were more dominant IL-4- and IL-5-producing cells. Furthermore, we show that IL-4 drove bone marrow progenitor cells to differentiate into Th2 cytokine-producing eosinophils in vitro. These results strongly suggest IL-4 is a potent factor in directing bone marrow progenitor cells to differentiate into Th2 cytokine-producing eosinophils.     

Either IL-2 or IL-12 is sufficient to direct Th1 differentiation by nonobese diabetic T cells

Th cell differentiation from naive precursors is a tightly controlled process; the most critical differentiation factor is the action of the driving cytokine: IL-12 for Th1 development, IL-4 for Th2 development. We found that CD4(+) T cells from nonobese diabetic mice spontaneously differentiate into IFN-gamma-producing Th1 cells in response to polyclonal TCR stimulation in the absence of IL-12 and IFN-gamma. Instead, IL-2 was necessary and sufficient to direct T cell differentiation to the Th1 lineage by nonobese diabetic CD4(+) T cells. Its ability to direct Th1 differentiation of both naive and memory CD4(+) T cells was clearly uncoupled from its ability to stimulate cell division. Autocrine IL-2-driven Th1 differentiation of nonobese diabetic T cells may represent a genetic liability that favors development of IFN-gamma-producing autoreactive T cells.     

IL-27 limits IL-2 production during Th1 differentiation

Although the ability of IL-27 to promote T cell responses is well documented, the anti-inflammatory properties of this cytokine remain poorly understood. The current work demonstrates that during infection with Toxoplasma gondii, IL-27R-deficient mice generate aberrant IL-2 responses that are associated with the development of a lethal inflammatory disease. Because in vivo depletion of IL-2 prolongs the survival of infected IL-27R-/- mice, these data suggest that IL-27 curbs the development of immunopathology by limiting parasite-induced IL-2 production. Consistent with this hypothesis, IL-27R-/- CD4+ T cells produce more IL-2 than wild-type counterparts during in vitro differentiation, and when rIL-27 is introduced, it can suppress the expression of IL-2 mRNA and protein by the latter group. Additionally, these studies reveal that, like IL-27, IL-12 can inhibit IL-2 production, and although each employs distinct mechanisms, they can synergize to enhance the effect. In contrast, this property is not shared by closely related cytokines IL-6 and IL-23. Thus, while traditionally viewed as proinflammatory agents, the present findings establish that IL-27 and IL-12 cooperate to limit the availability of IL-2, a potent T cell growth and survival factor. Moreover, because the current studies demonstrate that both can induce expression of suppressor of cytokine signaling 3, a protein that tempers cytokine receptor signaling, they also suggest that IL-27 and IL-12 share additionally inhibitory properties.     

Suppressive oligodeoxynucleotides inhibit Th1 differentiation by blocking IFN-gamma- and IL-12-mediated signaling

Repetitive TTAGGG motifs present at high frequency in mammalian telomeres can suppress Th1-mediated immune responses. Synthetic oligonucleotides (ODN) containing TTAGGG motifs mimic this activity and have proven effective in the prevention/treatment of certain Th1-dependent autoimmune diseases. This work explores the mechanism by which suppressive ODN block the induction of Th1 immunity. Findings indicate that these ODN inhibit IFN-gamma-induced STAT1 phosphorylation and IL-12-induced STAT3 and STAT4 phosphorylation. As a result, T-bet expression is reduced as is the maturation of naive CD4+ cells into Th1 effectors. These changes indirectly support the generation of Th2-dominated immune responses. Suppressive ODN may thus represent a novel approach to influence the Th1:Th2 balance in vivo.     

IL-4 is a mediator of IL-12p70 induction by human Th2 cells: reversal of polarized Th2 phenotype by dendritic cells

IL-12 is a key inducer of Th1-associated inflammatory responses, protective against intracellular infections and cancer, but also involved in autoimmune tissue destruction. We report that human Th2 cells interacting with monocyte-derived dendritic cells (DC) effectively induce bioactive IL-12p70 and revert to Th0/Th1 phenotype. In contrast, the interaction with B cells preserves polarized Th2 phenotype. The induction of IL-12p70 in Th2 cell-DC cocultures is prevented by IL-4-neutralizing mAb, indicating that IL-4 acts as a Th2 cell-specific cofactor of IL-12p70 induction. Like IFN-gamma, IL-4 strongly enhances the production of bioactive IL-12p70 heterodimer in CD40 ligand-stimulated DC and macrophages and synergizes with IFN-gamma at low concentrations of both cytokines. However, in contrast to IFN-gamma, IL-4 inhibits the CD40 ligand-induced production of inactive IL-12p40 and the production of either form of IL-12 induced by LPS, which may explain the view of IL-4 as an IL-12 inhibitor. The presently described ability of IL-4 to act as a cofactor of Th cell-mediated IL-12p70 induction may allow Th2 cells to support cell-mediated immunity in chronic inflammatory states, including cancer, autoimmunity, and atopic dermatitis.     

Stat6 is necessary and sufficient for IL-4's role in Th2 differentiation and cell expansion

IL-4 plays a critical role in the differentiation of TCR-stimulated naive CD4 T cells to the Th2 phenotype. In response to IL-4, the IL-4R activates a set of phosphotyrosine binding domain-containing proteins, including insulin receptor substrate 1/2, Shc, and IL-4R interacting protein, as well as Stat6. Stat6 has been shown to be required for Th2 differentiation. To determine the roles of the phosphotyrosine binding adaptors in Th2 differentiation, we prepared a retrovirus containing a mutant of the human (h)IL-4R alpha-chain, Y497F, which is unable to recruit these adaptors. The mutant hIL-4Ralpha, as well as the wild-type (WT) hIL-4Ralpha, was introduced into naive CD4 T cells. Upon hIL-4 stimulation, Y497F worked as well as the WT hIL-4Ralpha in driving Th2 differentiation, as measured by Gata3 up-regulation and IL-4 production. Furthermore, IL-4-driven cell expansion was also normal in the cells infected with Y497F, although cells infected with Y497F were not capable of phosphorylating insulin receptor substrate 2. These results suggest that the signal pathway mediated by Y497 is dispensable for both IL-4-driven Th2 differentiation and cell expansion. Both WT and Y497F hIL-4Ralpha lose the ability to drive Th2 differentiation and cell expansion in Stat6-knockout CD4 T cells. A constitutively activated form of Stat6 introduced into CD4 T cells resulted in both Th2 differentiation and enhanced cell expansion. Thus, activated Stat6 is necessary and sufficient to mediate both IL-4-driven Th2 differentiation and cell expansion in CD4 T cells.     

Th2 differentiation is unaffected by Jagged2 expression on dendritic cells

Expression of the Jagged Notch ligands by dendritic cells (DCs) has been suggested to play a role in instructing Th2 responses. Supporting this hypothesis, we found that Jagged2 but not Jagged1 expression, correlates with the ability of DCs to induce Th2 responses. Jagged2 expression is up-regulated in response to the helminth soluble Schistosoma mansoni egg Ag, which conditions DCs to induce Th2 responses, and is markedly down-regulated following exposure to TLR agonists that generally promote Th1 responses. Conversely, Jagged1 expression is markedly induced by TLR ligation. Despite these correlations, suppression of expression of Jagged2 using retrovirally delivered small interfering RNA failed to affect the ability of DCs to induce Th2 cell differentiation either in vitro or in vivo. Moreover, retrovirally induced expression of Jagged2 did not enhance the ability of DCs to induce Th2 cell responses. Our data indicate that Jagged2 expression by DCs is not sufficient or required for Th2 cell differentiation.     

ICOS costimulation requires IL-2 and can be prevented by CTLA-4 engagement

We investigated the relationship between ICOS, CD28, CTLA-4, and IL-2 to gain a better understanding of this family of costimulatory receptors in the immune response. Using magnetic beads coated with anti-CD3 and varying amounts of anti-ICOS and anti-CTLA-4 Abs, we show that CTLA-4 ligation blocks ICOS costimulation. In addition to inhibiting cellular proliferation, CTLA-4 engagement prevented ICOS-costimulated T cells from producing IL-4, IL-10, and IL-13. Both an indirect and direct mechanism of CTLA-4's actions were examined. First, CTLA-4 engagement on resting cells was found to indirectly block ICOS costimulation by interferring with the signals needed to induce ICOS cell surface expression. Second, on preactivated cells that had high levels of ICOS expression, CTLA-4 ligation blocked the ICOS-mediated induction of IL-4, IL-10, and IL-13, suggesting an interference with downstream signaling pathways. The addition of IL-2 not only overcame both mechanisms, but also greatly augmented the level of cellular activation suggesting synergy between ICOS and IL-2 signaling. This cooperation between ICOS and IL-2 signaling was explored further by showing that the minimum level of IL-2 produced by ICOS costimulation was required for T cell proliferation. Finally, exogenous IL-2 was required for sustained growth of ICOS-costimulated T cells. These results indicate that stringent control of ICOS costimulation is maintained initially by CTLA-4 engagement and later by a requirement for exogenous IL-2.     

Expression of functional selectin ligands on Th cells is differentially regulated by IL-12 and IL-4

Immune responses may be qualitatively distinct depending on whether Th1 or Th2 cells predominate at the site of Ag exposure. T cell subset-specific expression of ligands for vascular selectins may underlie the distinct patterns of recruitment of Th1 or Th2 cells to peripheral inflammatory sites. Here we examine the regulation of selectin ligand expression during murine T helper cell differentiation. Large numbers of Th1 cells interacted with E- and P-selectin under defined flow conditions, while few Th2 and no naive T cells interacted. Th1 cells also expressed more fucosyltransferase VII mRNA than naive or Th2 cells. IL-12 induced expression of P-selectin ligands on Ag-activated naive T cells, even in the presence of IL-4, and on established Th2 cells restimulated in the presence of IL-12 and IFN-gamma. In contrast, Ag stimulation alone induced only E-selectin ligand. Interestingly, restimulation of established Th2 cells in the presence of IL-12 and IFN-gamma induced expression of P-selectin ligands but not E-selectin ligands; IFN-gamma alone did not enhance expression of either selectin ligand. In summary, functional P- and E-selectin ligands are expressed on most Th1 cells, few Th2 cells, but not naive T cells. Furthermore, selectin ligand expression is regulated by the cytokine milieu during T cell differentiation. IL-12 induces P-selectin ligand, while IL-4 plays a dominant role in down-regulating E-selectin ligand.     

TGF-β is necessary for induction of IL-23R and Th17 differentiation by IL-6 and IL-23

TGF-β and IL-6 induce Th17 differentiation, and IL-23 is required for expansion and maintenance of Th17 cells. Recently, it was shown that IL-6 up-regulates IL-23R mRNA in naive CD4⁺ T cells and therefore IL-6 and IL-23 synergistically promote Th17 differentiation. However, the molecular mechanism whereby IL-6 and IL-23 induce Th17 differentiation and the relevance to TGF-β remain unknown. Here, we found that IL-6 up-regulated IL-23R mRNA expression, and IL-6 and IL-23 synergistically augmented its protein expression. The combination induced Th17 differentiation, and TGF-β1 further enhanced it. IL-6 augmented endogenous TGF-β1 mRNA expression, whereas the amount of TGF-β produced was not enough to induce Th17 differentiation by IL-6 alone. However, unexpectedly, the up-regulation of IL-23R and induction of Th17 differentiation by IL-6 and IL-23 were almost completely inhibited by anti-TGF-β. These results suggest that the induction of IL-23R and Th17 differentiation by IL-6 and IL-23 is mediated through endogenously produced TGF-β.     

Cutting edge: the development of IL-4-producing B cells (B effector 2 cells) is controlled by IL-4, IL-4 receptor alpha, and Th2 cells

Although IL-4-producing B cells (B effector 2 cells) are found following infection and immunization, the signals regulating IL-4 production by Be2 cells are unknown. We show that culturing naive B cells with Th2 cells induces up-regulation of IL-4 in the B cells with a concomitant down-regulation of T-bet, IL-12Rbeta2, and IFN-gamma. Up-regulation of IL-4 in the Be2 cells is dependent on both T cells and IL-4 as IL-4Ralpha-deficient B cells primed with Th2 cells did not transcribe IL-4, and B cells primed in the presence of IL-4-deficient Th2 cells produced IFN-gamma instead of IL-4. Likewise, the in vivo development of IL-4-expressing B cells in a nematode infection model was dependent on both T cells and IL-4Ralpha-mediated signals. Thus, the differentiation of naive B cells into IL-4-expressing Be2 cells is regulated by a combination of T cell-dependent signals and the cytokine environment and this process is critically dependent upon the IL-4/IL-4R signaling pathway.     

Aluminium hydroxide adjuvant initiates strong antigen-specific Th2 responses in the absence of IL-4- or IL-13-mediated signaling

Previous studies demonstrate that aluminium hydroxide adjuvant (alum) produces increased Th1 responses in IL-4-deficient mice compared with wild-type animals, although the continued production of IL-5 by spleen cells from these mice also indicates that Th2 responses are induced. In the present study, we demonstrate that alum can induce Th2-associated IL-4 and IL-5 production in the absence of IL-4 signaling in mice deficient in either IL-4Ralpha or Stat6. The Th2 responses observed could not be due to IL-13 as IL-13 responses are also impaired in IL-4Ralpha- and Stat6-deficient mice. We also detected higher levels of IL-4 in IL-4Ralpha gene-deficient, though not Stat6-deficient, mice compared with their wild-type counterparts. The increased levels of IL-4 could be explained by the IL-4R being unavailable to neutralize this cytokine in IL-4Ralpha-deficient mice. While levels of IL-5 production in IL-4Ralpha- or Stat6-deficient mice were similar to IL-4-deficient and wild-type mice, other type 2-associated responses, which are largely or wholly IL-4 dependent, such as the production of IgG1 or IgE Abs, were either reduced or absent. We conclude that alum adjuvants can induce IL-4 production and Th2 responses independently of IL-4 or IL-13, negating the requirement for an early source of IL-4 in the Th2 response induced by this adjuvant.     

The production of IFN-gamma by IL-12/IL-18-activated macrophages requires STAT4 signaling and is inhibited by IL-4

Macrophages release IFN-gamma on combined stimulation with IL-12 and IL-18, but the signaling requirements of this process and its regulation by other cytokines are unknown. Here, we demonstrate that STAT4 is indispensable for IL-12/IL-18-induced production of IFN-gamma by mouse peritoneal macrophages. Type 2 NO synthase (NOS2), which we previously found to be a prerequisite for IL-12-induced IFN-gamma production in NK cells, was not required for IFN-gamma production by these macrophages. IL-12 alone already induced the expression of IFN-gamma mRNA, but nuclear translocation of STAT4, the release of IFN-gamma protein, and the subsequent production of NO was strictly dependent on the simultaneous presence of IL-18. NF-kappa B, which mediates IL-18 effects in T cells, was only weakly activated by IL-12 and/or IL-18 in macrophages. Known inhibitors of macrophage functions (e.g., IL-4 and TGF-beta) also suppressed macrophage IFN-gamma production and the subsequent production of NOS2-derived NO. The inhibitory effect of IL-4 was paralleled by nuclear translocation of STAT6, which in EMSAs was able to bind to the same DNA oligonucleotide as STAT4. These results further define the production of IFN-gamma by macrophages and point to a diversity in the signals required for IFN-gamma production by various cell types.     

Th2-induced airway mucus production is dependent on IL-4Ralpha, but not on eosinophils

Mucus hyperproduction in asthma results from airway inflammation and contributes to clinical symptoms, airway obstruction, and mortality. In human asthmatics and in animal models, excess mucus production correlates with airway eosinophilia. We previously described a system in which TCR transgenic CD4 Th2 cells generated in vitro were transferred into recipient mice and activated in the respiratory tract with inhaled Ag. Th2 cells stimulated airway eosinophilia and a marked increase in mucus production, while mice that received Th1 cells exhibited airway inflammation without eosinophilia or mucus. Mucus could be induced by IL-4-/- Th2 cells at comparable levels to mucus induced by IL-4+/+ Th2 cells. In the current studies we dissect further the mechanisms of Th2-induced mucus production. When IL-4-/- Th2 cells are transferred into IL-4Ralpha-/- mice, mucus is not induced, and BAL eosinophilia is absent. These data suggest that in the absence of IL-4, IL-13 may be critical for Th2-induced mucus production and eosinophilia. To determine whether eosinophils are important in mucus production, IL-5-/- Th2 cells were transferred into IL-5-/- recipients. Eosinophilia was abolished, yet mucus staining in the epithelium persisted. These studies show definitively that IL-5, eosinophils, or mast cells are not essential, but signaling through IL-4Ralpha is critically important in Th2 cell stimulation of mucus production.