The costimulatory effect of IL-18 on the induction of antigen-specific IFN-gamma production by resting T cells is IL-12 dependent and is mediated by up-regulation of the IL-12 receptor beta2 subunit

IL-18 was originally described as a cytokine which induced IFN-gamma production by established Th1 cells in an IL-12-independent manner. However, subsequent studies demonstrated that exogenous IL-18 in the absence of IL-12 failed to drive Th1 differentiation of naive cells and induced IFN-gamma from established Th1 cells only in combination with IL-12. We have examined the role of endogenous IL-18 in controlling Th1 lineage commitment. When naive TCR-transgenic T cells were stimulated with antigen, anti-IL-18 antibodies resulted in partial inhibition of IFN-gamma production, but did not inhibit Th1 differentiation. To distinguish whether the inhibitory effect of anti-IL-18 antibodies was mediated directly by blocking IFN-gamma production or indirectly by blocking IL-12Rbeta2 up-regulation, naive T cells from IL-12 - / - mice were stimulated with anti-CD3 and IL-18. These cells failed to produce IFN-gamma, but markedly up-regulated IL-12Rbeta2 expression. We propose that the major effect of IL-18 on Th1 development is mediated by up-regulation of IL-12Rbeta2 expression, thereby enhancing IL-12-mediated signaling. The enhancement of IL-12Rbeta2 expression by IL-18 may be particularly important for the differentiation of foreign antigen- or autoantigen-specific Th1 cells when the stimulatory concentration of IL-12 in the microenvironment is just below the threshold required for Th1 development.     

Cytokine regulation of IL-12 receptor beta2 expression: differential effects on human T and NK cells

The biological activities of IL-12 are mediated through a specific, high-affinity receptor composed of IL-12 receptor(R)beta1 and IL-12Rbeta2 subunits that exist primarily on T and NK cells. Remarkably, the expression of IL-12Rbeta2 on CD4(+) T cells in mouse and humans appears to be differentially regulated by IFN-gamma and IFN-alpha, respectively. Using an antibody specific for the human IL-12Rbeta2 subunit, the effect of IFN-gamma, IFN-alpha, IL-12 and IL-2 on the regulation of IL-12R expression and IL-12 responsiveness of human T and NK cells was assessed. The presence of IFN-alpha or IFN-gamma in cultures enhanced IL-12Rbeta2 expression of CD4(+) and CD8(+) T cells. The enhancing effect of IFN-alpha and IFN-gamma was independent of endogenous IL-12. Furthermore, the clearest effects of IFN-alpha and IFN-gamma on IL-12Rbeta2 expression on T cells were seen by abrograting the inhibition induced by the presence of IL-4 in cultures. In contrast to T cells, IFN-alpha and IFN-gamma had little effect on regulating IL-12Rbeta2 expression on human NK cells. Taken together, these data show that there is differential regulation of IL-12Rbeta2 expression by IFN-alpha and IFN-gamma on human T and NK cells.     

GATA-3 suppresses Th1 development by downregulation of Stat4 and not through effects on IL-12Rbeta2 chain or T-bet

To further understand the interaction among GATA-3, Stat4, and T-bet in helper T cell development, we first showed that retroviral expression of GATA-3 in developing Th1 cells suppresses Th1 development through downregulation of Stat4 rather through downregulation of the IL-12Rbeta2 chain. Correspondingly, Stat4 levels are greatly suppressed during physiological Th2 development. Then, using cells doubly infected with GFP- and YFP-expressing retroviruses, we showed that retroviral GATA-3 expression in developing Th1 cells does not block Th1 development in cells coexpressing Stat4 but does so in cells coexpressing T-bet. Finally, we showed that retroviral Stat4 expression could facilitate Th2-->Th1 conversion in cells bearing an IL-12Rbeta2 transgene, even in cells lacking T-bet. These findings reassert that Stat4 signaling is a central element of Th1/Th2 development.     

Positive and negative regulation of IL-12 receptor expression of naive CD4(+) T cells by CD28/CD152 co-stimulation

IL-12 is a critical cytokine for polarizing naive CD4(+) T cells toward Th1 subset. Therefore, it is important to elucidate the mechanism of IL-12R expression of naive CD4(+) T cells. In this report, we present evidence to show that expression of both IL-12Rbeta1 and beta2 mRNA is regulated by signals mediated through CD28 and CD152. Naive CD4(+) T cells stimulated with anti-CD3 alone neither expressed IL-12Rbeta2 mRNA nor bound detectable level of rIL-12, although they expressed a very low level of IL-12Rbeta1 mRNA when stimulated with a high dose of anti-CD3. Expression of IL-12Rbeta1 and beta2 mRNA was induced by the co-ligation of CD3 and CD28, and it was down-regulated by the ligation of CD152. CD28 ligation induced not only IL-12Rbeta1 and beta2 mRNA expression, but also enhanced IFN-gammaR to mediate up-regulation of IL-12R by IFN-gamma.     

Differential regulation of Th1 responses and CD154 expression in human CD4+ T cells by IFN-alpha

Like interleukin (IL)-12, interferon (IFN)-alpha has been shown to play an important role in inducing human Th1 responses. Recent studies have shown that human Th1 responses driven by IL-12 are associated with enhanced expression of CD154. The present study examined the effects of IFN-alpha on CD154 expression in human CD4+ T cells, with special attention to the relationship with Th1 responses. Highly purified CD4+ T cells from healthy donors were stimulated with immobilized anti-CD3 with or without IFN-alpha and IL-12 in the complete absence of accessory cells. IFN-alpha suppressed CD154 protein and mRNA expression in CD4+ T cells at the initial phase of activation with immobilized anti-CD3, but enhanced it in the subsequent maturation phase irrespective of the presence of IL-12. By contrast, IFN-alpha by itself did not enhance IFN-gamma production or mRNA expression in CD4+ T cells in the absence of IL-12 even in the presence of stimulation with anti-CD28, but enhanced it in the presence of IL-12. Accordingly, IFN-alpha enhanced IL-12Rbeta2 mRNA expression in anti-CD3-stimulated CD4+ T cells. Neither IFN-alpha nor IL-12 influenced the stability of CD154 mRNA in anti-CD3-activated CD4+ T cells. These results indicate that IFN-alpha by itself enhances CD154 expression in CD4+ T cells independently of the induction of IFN-gamma mRNA expression. The data also suggest that the optimal induction of human Th1 responses by IFN-alpha might require the presence of IL-12 and that the induction of Th1 responses and CD154 expression in human CD4+ T cells might be regulated through different mechanisms.     

Stat6-independent GATA-3 autoactivation directs IL-4-independent Th2 development and commitment

The initial source of IL-4-inducing Th2 development and the mechanism of stable Th2 commitment remain obscure. We found the reduced level of IL-4 production in Stat6-deficient T cells to be significantly higher than in Th1 controls. Using a novel cell surface affinity matrix technique, we found that IL-4-secreting Stat6-deficient T cells stably expressed GATA-3 and Th2 phenotype. Introducing GATA-3 into Stat6-deficient T cells completely restored Th2 development, inducing c-Maf, Th2-specific DNase I hypersensitive sites in the IL-4 locus, and Th2 cytokine expression. The fact that GATA-3 fully reconstitutes Th2 development in Stat6-deficient T cells indicates it is a master switch in Th2 development. Finally, GATA-3 exerts Stat6-independent autoactivation, creating a feedback pathway stabilizing Th2 commitment.     

IL-12-induced reversal of human Th2 cells is accompanied by full restoration of IL-12 responsiveness and loss of GATA-3 expression

IL-12 is a potent inducer of IFN-gamma production and drives the development of Th1 cells. Human polarized Th2 cells do not express the signaling beta2-subunit of the IL-12R and, therefore, do not signal in response to IL-12. The question was raised as to what extent the loss of the IL-12Rbeta2 chain in Th2 cells has bearing on the stability of the human Th2 phenotype. In the present report, we show that restimulation of human fully polarized Th2 cells in the presence of IL-12 primes for a shift towards Th0/Th1 phenotypes, accompanied by suppression of GATA-3 expression and induction of T-bet expression. These reversed cells are further characterized by a marked IL-12Rbeta2 chain expression and fully restored IL-12-inducible STAT4 activation. The IL-12-induced phenotypic shift proved to be stable as a subsequent restimulation in the presence of IL-4 and in the absence of IL-12 could not undo the accomplished changes. Identical results were obtained with cells from atopic patients, both with polyclonal Th2 cell lines and allergen-specific Th2 cell clones. These findings suggest the possibility of restoring IL-12 responsiveness in established Th2 cells of atopic patients by stimulation in the presence of IL-12, and that IL-12-promoting immunotherapy can be beneficial for Th2-mediated immune disorders, targeting both naive and memory effector T cells.     

Ectopic expression of activated Stat6 induces the expression of Th2-specific cytokines and transcription factors in developing Th1 cells

Stat6 is critical for IL-4-mediated Th2 cell development, but its molecular mechanism remains unclear. Here we constructed Stat6:ER, a Stat6-estrogen receptor fusion protein that can be activated by 4-hydroxy-tamoxifen, independently of IL-4 and endogenous Stat6. Retrovirus-mediated introduction of Stat6:ER into developing Th1 cells induced Th2-specific cytokines and suppressed IFNgamma production in a 4-HT-dependent manner and in the absence of IL-4. It also induced GATA-3 and c-maf expression and downregulated IL-12Rbeta2 chain expression. Its decreased ability to induce the Th2 phenotype with progressing Th1 cell commitment correlated with a decreased induction of GATA-3 and c-maf. This study indicates that Stat6 functions upstream of GATA-3 and c-Maf to induce Th2 development.     

Skewed expression and up-regulation of the IL-12 and IL-18 receptors in resting and activated CD4 T cells from HIV-1-infected patients

IL-12 and IL-18 synergistically induce the production of IFN-gamma by resting and activated T cells. To evaluate whether this induction was affected in HIV-1-infected patients, PBMC or isolated CD4 T cells were cultured with IL-12 plus IL-18, anti-CD3 plus anti-CD28, or PHA for 72 h. Cell samples were labeled daily to assess the levels of IL-12 receptor beta1 (IL-12Rbeta1), IL-12Rbeta2, and IL-18Ralpha. Culture supernatants were analyzed for the presence of Th1- and Th2-related cytokines by ELISA or cytometric bead array and analyzed by flow cytometry. A twofold increase in the percentage of CD4-resting T cells expressing IL-12Rbeta1 and IL-18Ralpha from HIV-1-infected patients was observed when compared with cells from HIV-1-negative donors. Higher IL-12Rbeta1 and IL-18Ralpha expression correlated (r=0.87; P<0.007) to increased production of IFN-gamma by isolated CD4 T cells in the presence of IL-12 and IL-18. Moreover, exogenous IL-12 and IL-18 induced the up-regulation of IL-12Rbeta2 to twice higher in CD4 T cells from HIV-1-positive individuals compared with controls. Conversely, upon activation with anti-CD3 and anti-CD28 antibodies, only 25% of the CD4+ T cells from HIV-1 patients showed an increase in the IL-12beta2 when compared with 50% in healthy controls. Furthermore, the percentage of IL-12Rbeta1-positive cells correlated inversely with the CD4 nadir of patients, suggesting that deregulation of the IL-12 and IL-18 pathways may play a role in the immunopathogenesis of HIV-1 infection.     

Instruction of naive CD4+ T-cell fate to T-bet expression and T helper 1 development: roles of T-cell receptor-mediated signals

Using T-cell receptor (TCR) transgenic mice, we demonstrate that TCR stimulation of naive CD4(+) T cells induces transient T-bet expression, interleukin (IL)-12 receptor beta2 up-regulation, and GATA-3 down-regulation, which leads to T helper (Th)1 differentiation even when the cells are stimulated with peptide-loaded I-A(b)-transfected Chinese hamster ovary cells in the absence of interferon-gamma (IFN-gamma) and IL-12. Sustained IFN-gamma and IL-12 stimulation augments naive T-cell differentiation into Th1 cells. Intriguingly, a significant Th1 response is observed even when T-bet(-/-) naive CD4(+) T cells are stimulated through TCR in the absence of IFN-gamma or IL-12. Stimulation of naive CD4(+) T cells in the absence of IFN-gamma or IL-12 with altered peptide ligand, whose avidity to the TCR is lower than that of original peptide, fails to up-regulate transient T-bet expression, sustains GATA-3 expression, and induces differentiation into Th2 cells. These results support the notion that direct interaction between TCR and peptide-loaded antigen-presenting cells, even in the absence of T-bet expression and costimulatory signals, primarily determine the fate of naive CD4(+) T cells to Th1 cells.     

Corrective effects of interleukin-12 on age-related deficiencies in IFN-gamma production and IL-12Rbeta2 expression in virus-specific CD8+ T cells.

Interleukin-12 receptor beta2 (IL-12Rbeta2) has been shown to be selectively expressed on Th1 T cell subsets, and we have previously shown that influenza-specific CD8+ cytotoxic T lymphocyte (CTL) deficiency in old mice was associated with deficient Th1 (interferon-gamma [IFN-gamma]) cytokine production. This study tested whether IL-12Rbeta2 expression was also deficient in CD8+ CTL from old mice and the effect of IL-12 treatment on these responses. Splenic lymphocytes from influenza-primed old and young BALB/c mice were stimulated with influenza virus in vitro with and without IL-12 and then enriched for CD8+ T cells. IFN-gamma was significantly reduced, whereas IL-4 and IL-12p40 (an antagonist of IL-12 function) were evaluated in old when compared with young mice. This was true for secreted protein measured by ELISA and for mRNA levels quantitated by RT-PCR. IL-12Rbeta2 mRNA expression in CD8+ CTL was also significantly reduced in old mice. IL-12 treatment in vitro caused significant upregulation of IFN-gamma and IL-12Rbeta2 and downregulation of IL-4 in CD8+ T cells from old mice and young mice. The present demonstration of an age-related downregulation in IL-12Rbeta2 expression and our previous data showing reduced IFN-gamma and elevated IL-4 production provide strong evidence that CD8+ CTL deficiency in aging results from a Th1/Th2 cytokine production switch. Agents that increase IL-12Rbeta2 expression and redirect Th2 to Thl immune responses are likely to enhance CD8+ CTL-mediated control of viral infections in aging.     

IL-23 up-regulates IL-10 and induces IL-17 synthesis by polyclonally activated naive T cells in human

Interleukin (IL)-23 is a heterodimeric cytokine of the IL-12 family. Human IL-23 is known to induce interferon (IFN)-gamma production and proliferation in T cells, preferentially in the CD45RO+ memory subset. Yet, its role in the differentiation of human naive T cells remains largely unknown. We investigated the effect of recombinant human (rh)IL-23 on cord blood CD4+ and CD8+ T cells during polyclonal activation. The IL-23 receptor complex was not detectable in resting naive T cells. Nevertheless, both IL-23 receptor subunits, IL-12Rbeta1 and IL-23R, were rapidly induced after activation in both naive CD4+ and CD8+ T cells. In both cell types, rhIL-23 enhanced IFN-gamma production. This effect was demonstrable as early as 2 days after activation, illustrating that a functional IL-23 receptor is rapidly induced in naive T cells upon activation. In naive CD8+ T cells, rhIL-23 specifically induced the secretion of IL-17, a pro-inflammatory cytokine. Moreover, rhIL-23 significantly increased the production of IL-10 in both naive CD4+ and CD8+ T cells. IL-17 and IL-10 levels were not affected by the addition of rhIL-12. We conclude that IL-23 induces a specific cytokine profile, remarkably distinct from IL-12, in activated human naive T cells.     

Stat5 activation plays a critical role in Th2 differentiation

Upon TCR engagement, naive CD4 T cells differentiate toward the Th1 or Th2 phenotype. IL-4, acting through Stat6, plays a major role in Th2 differentiation; IL-2 has also been reported to be essential. Here, we report that retroviral (RV)-mediated expression of a constitutively active Stat5A mutant (STAT5A1*6) can fully restore IL-4 production when naive CD4 T cells are primed in the absence of IL-2. Furthermore, STAT5A1*6 expression causes Th2 differentiation in the absence of IL-4 or in Stat6- or IL-4Ralpha-deficient cells. Infection with STAT5A1*6-NGFR-RV does not enhance GATA-3 expression. STAT5A1*6-NGFR-RV and GATA-3-GFP-RV each render the Il4 gene accessible, but the sites of restriction enzyme accessibility are different. Stat5A binds to HSII and HSIII sites of the Il4 gene. Coinfection with STAT5A1*6-NGFR-RV and GATA-3-GFP-RV results in optimal Th2 priming.