Distinct characteristics of murine STAT4 activation in response to IL-12 and IFN-alpha

The role of type I IFN in Th1 development, STAT4 activation, and IFN-gamma production in murine T cells has remained unresolved despite extensive examination. Initial studies indicated that IFN-alpha induced Th1 development and IFN-gamma production in human, but not murine, T cells, suggesting species-specific differences in signaling. Later studies suggested that IFN-alpha also induced Th1 development in mice, similar to IL-12. More recent studies have questioned whether IFN-alpha actually induces Th1 development even in the human system. In the present study, we compared the capacity of IL-12 and IFN-alpha to induce Th1 differentiation, STAT4 phosphorylation, and IFN-gamma production in murine T cells. First, we show that IFN-alpha, in contrast to IL-12, cannot induce Th1 development. However, in differentiated Th1 cells, IFN-alpha can induce transient, but not sustained, STAT4 phosphorylation and, in synergy with IL-18, can induce transient, but not sustained, IFN-gamma production in Th1 cells, in contrast to the sustained actions of IL-12. Furthermore, loss of STAT1 increases IFN-alpha-induced STAT4 phosphorylation, but does not generate levels of STAT4 activation or IFN-gamma production achieved by IL-12 or convert transient STAT4 activation into a sustained response. Our findings agree with recent observations in human T cells that IFN-alpha-induced STAT4 activation is transient and unable to induce Th1 development, and indicate that IFN-alpha may act similarly in human and murine T cells.     

IL-12, but not IFN-alpha, promotes STAT4 activation and Th1 development in murine CD4+ T cells expressing a chimeric murine/human Stat2 gene

Humans and mice have evolved distinct pathways for Th1 cell development. Although IL-12 promotes CD4(+) Th1 development in both murine and human T cells, IFN-alphabeta drives Th1 development only in human cells. This IFN-alphabeta-dependent pathway is not conserved in the mouse species due in part to a specific mutation within murine Stat2. Restoration of this pathway in murine T cells would provide the opportunity to more closely model specific human disease states that rely on CD4(+) T cell responses to IFN-alphabeta. To this end, the C terminus of murine Stat2, harboring the mutation, was replaced with the corresponding human Stat2 sequence by a knockin targeting strategy within murine embryonic stem cells. Chimeric m/h Stat2 knockin mice were healthy, bred normally, and exhibited a normal lymphoid compartment. Furthermore, the murine/human STAT2 protein was expressed in murine CD4(+) T cells and was activated by murine IFN-alpha signaling. However, the murine/human STAT2 protein was insufficient to restore full IFN-alpha-driven Th1 development as defined by IFN-gamma expression. Furthermore, IL-12, but not IFN-alpha, promoted acute IFN-gamma secretion in collaboration with IL-18 stimulation in both CD4(+) and CD8(+) T cells. The inability of T cells to commit to Th1 development correlated with the lack of STAT4 phosphorylation in response to IFN-alpha. This finding suggests that, although the C terminus of human STAT2 is required for STAT4 recruitment and activation by the human type I IFNAR (IFN-alphabetaR), it is not sufficient to restore this process through the murine IFNAR complex.     

An indispensable role for STAT1 in IL-27-induced T-bet expression but not proliferation of naive CD4+ T cells

IL-27 is a novel IL-12 family member that plays a role in the early regulation of Th1 initiation, induces proliferation of naive CD4+ T cells, and synergizes with IL-12 in IFN-gamma production. It has been recently reported that IL-27 induces T-bet and IL-12Rbeta2 expression through JAK1/STAT1 activation. In the present study, we further investigated the JAK/STAT signaling molecules activated by IL-27 and also the role of STAT1 in IL-27-mediated responses using STAT1-deficient mice. In addition to JAK1 and STAT1, IL-27-activated JAK2, tyrosine kinase-2, and STAT2, -3, and -5 in naive CD4+ T cells. The activation of STAT2 and STAT5, but not of STAT3, was greatly diminished in STAT1-deficient naive CD4+ T cells. Comparable proliferative response to IL-27 was observed between STAT1-deficient and wild-type naive CD4+ T cells. In contrast, IL-27 hardly induced T-bet and subsequent IL-12Rbeta2 expression, and synergistic IFN-gamma production by IL-27 and IL-12 was impaired in STAT1-deficient naive CD4+ T cells. Moreover, IL-27 augmented the expression of MHC class I on naive CD4+ T cells in a STAT1-dependent manner. These results suggest that IL-27 activates JAK1 and -2, tyrosine kinase-2, STAT1, -2, -3, and -5 in naive CD4+ T cells and that STAT1 plays an indispensable role in IL-27-induced T-bet and subsequent IL-12Rbeta2 expression and MHC class I expression as well but not proliferation, while STAT3 presumably plays an important role in IL-27-induced proliferation.     

Depressed IL-12-mediated signal transduction in T cells from patients with Sézary syndrome is associated with the absence of IL-12 receptor beta 2 mRNA and highly reduced levels of STAT4.

Sézary syndrome (SS) is the leukemic phase of cutaneous T cell lymphoma characterized by the proliferation of clonally derived CD4+ T cells that release cytokines of the Th2 T cell phenotype (IL-4, IL-5, IL-10), whereas Th1 T cell cytokines (IL-2, IFN-gamma) are markedly depressed as is expression of IL-12, a pivotal cytokine for Th1 cell differentiation. Normal Th1 cells express both the beta 1 and beta 2 chains of the IL-12 receptor (IL-12R) and tyrosine phosphorylate STAT4 in response to IL-12. Th2 T cells express only the IL-12R beta 1 and thus do not tyrosine phosphorylate STAT4 in response to IL-12. To determine whether SS cells are Th2-like at the level of IL-12 signal transduction, we analyzed RNA from seven patients for the presence of message for the IL-12R beta 1 and beta 2 genes using RNase protection assays and assessed whether IL-12 induced tyrosine-phosphorylation of STAT4 by immunoblotting. In PBL from six of seven SS patients tested, beta 2 message was expressed at low to undetectable levels and its expression could not be stimulated by either IFN-alpha or IFN- gamma, which stimulated beta 2 expression in control PBL. The absence of beta 2 expression is further supportive evidence for the Th2 lineage of SS cells. However, unlike normal Th2 cells, SS cells also showed severely reduced levels of STAT4, suggesting that they have a depressed response to any inducer of the STAT4 signal transduction pathway, including IFN-alpha. This is the first observation linking STAT4 gene expression with a human disease and suggests that dysregulation of STAT4 expression may be significant to the development and/or progression of SS.     

Direct stimulation of T cells by type I IFN enhances the CD8+ T cell response during cross-priming

Type I IFN (IFN-alphabeta), which is produced rapidly in response to infection, plays a key role in innate immunity and also acts as a stimulus for the adaptive immune response. We have investigated how IFN-alphabeta induces cross-priming, comparing CD8+ T cell responses generated against soluble protein Ags in the presence or absence of IFN-alphabeta. Injection of IFN-alpha was found to prolong the proliferation and expansion of Ag-specific CD8+ T cells, which was associated with marked up-regulation of IL-2 and IL-15 receptors on Ag-specific cells and expression of IL-15 in the draining lymph node. Surprisingly, neither IL-2 nor IL-15 was required for IFN-alpha-induced cross-priming. Conversely, expression of the IFN-alphabetaR by T cells was shown to be necessary for effective stimulation of the response by IFN-alpha. The finding that T cells represent direct targets of IFN-alphabeta-mediated stimulation reveals an additional mechanism by which the innate response to infection promotes adaptive immunity.     

Augmentation of effector CD8+ T cell generation with enhanced granzyme B expression by IL-27

IL-27 is a novel IL-12 family member that plays a role in the early regulation of Th1 initiation. We have recently demonstrated that IL-27 has a potent antitumor activity, which is mainly mediated through CD8+ T cells, and also has an adjuvant activity to induce epitope-specific CTL in vivo. In this study, we further investigated the in vitro effect of IL-27 on CD8+ T cells of mouse spleen cells. In a manner similar to CD4+ T cells, IL-27 activated STAT1, -2, -3, -4, and -5, and augmented the expression of T-bet, IL-12Rbeta2, and granzyme B, and slightly that of perforin in naive CD8+ T cells stimulated with anti-CD3. IL-27 induced synergistic IFN-gamma production with IL-12 and proliferation of naive CD8+ T cells. Moreover, IL-27 enhanced proliferation of CD4+ T cell-depleted spleen cells stimulated by allogeneic spleen cells and augmented the generation of CTL. In STAT1-deficient naive CD8+ T cells, IL-27-induced proliferation was not reduced, but synergistic IFN-gamma production with IL-12 was diminished with decreased expression of T-bet, IL-12Rbeta2, granzyme B, and perforin. In T-bet-deficient naive CD8+ T cells, IL-27-induced proliferation was hardly reduced, but synergistic IFN-gamma production with IL-12 was diminished with decreased expression of IL-12Rbeta2, granzyme B, and perforin. However, IL-27 still augmented the generation of CTL from T-bet-deficient CD4+ T cell-depleted spleen cells stimulated by allogeneic spleen cells with increased granzyme B expression. These results suggest that IL-27 directly acts on naive CD8+ T cells in T-bet-dependent and -independent manners and augments generation of CTL with enhanced granzyme B expression.     

IFN-alpha and IL-12 induce IL-18 receptor gene expression in human NK and T cells

IL-18 is a proinflammatory cytokine that enhances innate and specific Th1 immune responses. During microbial infections, IL-18 is produced by activated macrophages. IL-18 exerts its effects in synergy with IFN-alpha or IL-12 to induce IFN-gamma. Here we show that in human NK and T cells IFN-alpha and IL-12 strongly up-regulate mRNA expression of the IL-18R components, accessory protein-like (AcPL) and IL-1R-related protein (IL-1Rrp). In addition, IFN-alpha enhanced the expression of MyD88, an adaptor molecule involved in IL-18 signaling. Pretreatment of T cells with IFN-alpha or IL-12 enhanced IL-18-induced NF-kappaB activation and sensitized the cells to respond to lower concentrations of IL-18. AcPL and IL-1Rrp genes were strongly expressed in T cells polarized with IL-12, whereas in IL-4-polarized cells these genes were expressed at very low levels, indicating that AcPL and IL-1Rrp genes are preferentially expressed in Th1 cells. In conclusion, the results suggest that IFN-alpha and IL-12 enhance innate as well as Th1 immune response by inducing IL-18R expression.     

Primary Murine CD4+ T Cells Fail to Acquire the Ability to Produce Effector Cytokines When Active Ras Is Present during Th1/Th2 Differentiation

Constitutive Ras signaling has been shown to augment IL-2 production, reverse anergy, and functionally replace many aspects of CD28 co-stimulation in CD4⁺ T cells. These data raise the possibility that introduction of active Ras into primary T cells might result in improved functionality in pathologic situations of T cell dysfunction, such as cancer or chronic viral infection. To test the biologic effects of active Ras in primary T cells, CD4⁺ T cells from Coxsackie-Adenovirus Receptor Transgenic mice were transduced with an adenovirus encoding active Ras. As expected, active Ras augmented IL-2 production in naive CD4⁺ T cells. However, when cells were cultured for 4 days under conditions to promote effector cell differentiation, active Ras inhibited the ability of CD4⁺ T cells to acquire a Th1 or Th2 effector cytokine profile. This differentiation defect was not due to deficient STAT4 or STAT6 activation by IL-12 or IL-4, respectively, nor was it associated with deficient induction of T-bet and GATA-3 expression. Impaired effector cytokine production in active Ras-transduced cells was associated with deficient demethylation of the IL-4 gene locus. Our results indicate that, despite augmenting acute activation of naïve T cells, constitutive Ras signaling inhibits the ability of CD4⁺ T cells to properly differentiate into Th1/Th2 effector cytokine-producing cells, in part by interfering with epigenetic modification of effector gene loci. Alternative strategies to potentiate Ras pathway signaling in T cells in a more regulated fashion should be considered as a therapeutic approach to improve immune responses in vivo.     

Suppression of early IL-4 production underlies the failure of CD4 T cells activated by TLR-stimulated dendritic cells to differentiate into Th2 cells

Dendritic cells (DCs) activated through TLRs provide a potent negative signal for Th2 cell development that is independent of positive signals for Th1 cell development such as IL-12 and IFN-gamma. In this study we demonstrate that the ability of TLR-activated DCs to suppress Th2 cell development is Ag dose-independent and unique to DCs that have been activated through TLRs vs by cytokines. We show that TLR-activated DCs inhibit early IL-4 production by CD4 T cells and thus inhibit their ability to subsequently increase GATA-3 expression and commit to the Th2 lineage. This occurs independently of expression of the GATA-3 antagonist T-bet. Although CD4 T cells activated by TLR-activated DCs make IL-2, they are not capable of phosphorylating STAT5 in response to this cytokine. This inhibition of responsiveness to IL-2 appears to underlie the failure to make early IL-4. Our findings suggest that DCs provide instructional signals for T cell differentiation before cytokine-mediated Th cell selection and outgrowth.     

Microenvironment-dependent requirement of STAT4 for the induction of P-selectin ligands and effector cytokines on CD4+ T cells in healthy and parasite-infected mice

T effector cells require selectin ligands to migrate into inflamed regions. In vitro, IL-12 promotes induction of these ligands as well as differentiation of CD4+ T cells into IFN-gamma-producing Th1 but not Th2 cells. STAT4 is strongly involved in these processes. However, the presence of selectin ligands on various T effector cell subsets in vivo points to more complex regulatory pathways. To clarify the role of the IL-12/STAT4 signaling pathway, we analyzed the impact of STAT4 deficiency on the expression of P-selectin ligands (P-lig) on CD4+ T cells in vitro and in vivo, including conditions of infection. In vitro, we found significant expression of P-lig upon activation not only in the presence, but also in the absence, of IL-12, which was independent of STAT4. TGF-beta, an alternative inducer of selectin ligands in human T cells, was not effective in murine CD4+ T cells, suggesting a role of additional signaling pathways. In vivo, a significant impact of STAT4 for the generation of P-lig+CD4+ T cells was observed for cells from peripheral lymph nodes, but not for those from spleen or lung. However, upon infection with the Th2-inducing parasite Nippostrongylus brasiliensis, P-lig expression became dependent on STAT4 signaling. Interestingly, also the frequency of IL-4-producing cells was greatly diminished in absence of STAT4. These data reveal a hitherto unknown contribution of STAT4 to the generation of Th2 cells in parasite infection and suggest that signals inducing inflammation-seeking properties in vivo vary depending on environmental conditions, such as type of organ and infection.     

IL-21 in synergy with IL-15 or IL-18 enhances IFN-gamma production in human NK and T cells

NK and T cell-derived IFN-gamma is a key cytokine that stimulates innate immune responses and directs adaptive T cell response toward Th1 type. IL-15, IL-18, and IL-21 have significant roles as activators of NK and T cell functions. We have previously shown that IL-15 and IL-21 induce the expression of IFN-gamma, T-bet, IL-12R beta 2, and IL-18R genes both in NK and T cells. Now we have studied the effect of IL-15, IL-18, and IL-21 on IFN-gamma gene expression in more detail in human NK and T cells. IL-15 clearly activated IFN-gamma mRNA expression and protein production in both cell types. IL-18 and IL-21 enhanced IL-15-induced IFN-gamma gene expression. IL-18 or IL-21 alone induced a modest expression of the IFN-gamma gene but a combination of IL-21 and IL-18 efficiently up-regulated IFN-gamma production. We also show that IL-15 activated the binding of STAT1, STAT3, STAT4, and STAT5 to the regulatory sites of the IFN-gamma gene. Similarly, IL-21 induced the binding of STAT1, STAT3, and STAT4 to these elements. IL-15- and IL-21-induced STAT1 and STAT4 activation was verified by immunoprecipitation with anti-phosphotyrosine Abs followed by Western blotting with anti-STAT1 and anti-STAT4 Abs. IL-18 was not able to induce the binding of STATs to IFN-gamma gene regulatory sites. IL-18, however, activated the binding of NF-kappa B to the IFN-gamma promoter NF-kappa B site. Our results suggest that both IL-15 and IL-21 have an important role in activating the NK cell-associated innate immune response.     

Modulation of innate and adaptive immune responses by tofacitinib (CP-690,550)

Inhibitors of the JAK family of nonreceptor tyrosine kinases have demonstrated clinical efficacy in rheumatoid arthritis and other inflammatory disorders; however, the precise mechanisms by which JAK inhibition improves inflammatory immune responses remain unclear. In this study, we examined the mode of action of tofacitinib (CP-690,550) on JAK/STAT signaling pathways involved in adaptive and innate immune responses. To determine the extent of inhibition of specific JAK/STAT-dependent pathways, we analyzed cytokine stimulation of mouse and human T cells in vitro. We also investigated the consequences of CP-690,550 treatment on Th cell differentiation of naive murine CD4(+) T cells. CP-690,550 inhibited IL-4-dependent Th2 cell differentiation and interestingly also interfered with Th17 cell differentiation. Expression of IL-23 receptor and the Th17 cytokines IL-17A, IL-17F, and IL-22 were blocked when naive Th cells were stimulated with IL-6 and IL-23. In contrast, IL-17A production was enhanced when Th17 cells were differentiated in the presence of TGF-β. Moreover, CP-690,550 also prevented the activation of STAT1, induction of T-bet, and subsequent generation of Th1 cells. In a model of established arthritis, CP-690,550 rapidly improved disease by inhibiting the production of inflammatory mediators and suppressing STAT1-dependent genes in joint tissue. Furthermore, efficacy in this disease model correlated with the inhibition of both JAK1 and JAK3 signaling pathways. CP-690,550 also modulated innate responses to LPS in vivo through a mechanism likely involving the inhibition of STAT1 signaling. Thus, CP-690,550 may improve autoimmune diseases and prevent transplant rejection by suppressing the differentiation of pathogenic Th1 and Th17 cells as well as innate immune cell signaling.