Independent and synergistic effects of interleukin-18 and interleukin-12 in augmenting cytotoxic T lymphocyte responses and IFN-gamma production in aging.

Aged mice exhibit diminished CD8(+) cytotoxic T lymphocyte (CTL) response to influenza virus. Previously, interleukin-12 (IL-12) was shown to partially restore in vitro influenza virus-specific CD8(+) CTL activity and interferon-gamma (IFN-gamma) production in aged mice. The present study investigated IL-18 production and its ability to collaborate with IL-12 to enhance these responses to the levels of young mice. IL-18 protein production and mRNA expression in influenza virus-specific CTL from aged mice were higher than from young mice. In contrast, IL-18 receptor (IL-18R) mRNA expression was significantly reduced in CD8(+) CTL from aged mice. Generation of CTL in the presence of IL-12 alone caused a significant increase in IFN-gamma production in both old and young mice. IL-18 treatment alone significantly increased IFN-gamma in CTL from young but not old mice. However, a combination of IL-18 and IL-12 significantly increased IFN-gamma in both old and young mice. IL-18 and IL-12, either alone or in combination, stimulated significant influenza virus-specific cytotoxicity in both old and young mice, but no significant synergistic effect was observed. These results represent an initial demonstration of downregulated IL-18R expression in aging mice and are consistent with age-related cytotoxic T lymphocyte 1 (Tc1) deficiency. Potentially, IL-18 and IL-12 can augment IFN-gamma production and reverse CD8(+) CTL deficiency in aging, independently or synergistically.     

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 production from murine CD4 and CD8 cells after mannan-MUC1 immunization.

Immunotherapy with oxidized mannan-MUC1 fusion protein (M-FP) leads to a T1 immune response characterized by the generation of cytotoxic T lymphocytes (CTL), few antibodies, secretion of interleukin-2 (IL-2), IL-12, and interferon-gamma and tumor protection. Immunotherapy with reduced M-FP or fusion protein (FP) alone leads to a T2 immune response characterized by the generation of MUC1 antibodies, few CTL, IL-4 secretion, and no tumor protection. In these studies, cytokine production from T cells was measured from cultures containing whole spleens. We now report the cytokine secretion patterns from spleen cells separated into CD4+ and CD8+ T cells obtained from mice immunized with either oxidized M-FP, reduced M-FP or FP, or the simultaneous administration of oxidized M-FP and FP. Immunization with oxidized M-FP led to the secretion of T1 cytokines from CD8+ T cells (IL-2, IFN-gamma, and tumor necrosis factor-alpha [TNF-alpha]) and from CD4+ T cells (IL-2 and IFN-gamma). IL-12 production, presumably from activated macrophages, was observed in CD8+ but not CD4+ cultures. Immunization with either reduced M-FP or FP led to the secretion of predominantly T2 cytokines from CD4+ T cells (IL-4 and IL-10) and IL-2 production in both CD4+ and CD8+ T cell cultures. The simultaneous immunization of both oxidized M-FP and FP led to the production of both T1 and T2 cytokines from CD8+ T cells (IL-2, IFN-gamma, and TNF-alpha) and CD4+ cells (IL-2, IFN-gamma, IL-4, and IL-10) and IL-12 production in CD8+ cultures that is, both types of immune responses could occur together. The results demonstrate that the cellular immune response observed in oxidized M-FP-immunized mice is indeed dependent on the T1 cytokine profile secreted by CD8+ T cells, and the simultaneous production of both T1 and T2 cytokines is not cross-inhibitory.     

Th1 cytokines facilitate CD8-T-cell-mediated early resistance to infection with Mycobacterium tuberculosis in old mice

Numerous immunological defects begin to emerge as an individual ages, the consequence of which is heightened susceptibility to infectious diseases. Despite this decline in immune function, old mice display an early transient resistance to Mycobacterium tuberculosis infection in the lung, which is dependent on CD8 T cells and gamma interferon (IFN-gamma) production. In this study, we investigated the mechanism of resistance by examining the CD8-T-cell phenotype and function in old na?ve and M. tuberculosis-infected mice. Pulmonary CD8 T cells from na?ve old mice expressed cell surface markers of memory in addition to receptors for several Th1 cytokines. Stimulation of lung cells from na?ve old mice with a combination of Th1 cytokines (interleukin-2 [IL-2], IL-12, and IL-18) resulted in nonspecific production of IFN-gamma by memory CD8 T cells. Following aerosol infection with M. tuberculosis, the lungs of old mice contained significantly more IL-12, IL-18, and IFN-gamma than the lungs of young mice contained. Together, these data demonstrate that the increased and early production of Th1 cytokines in the lungs of M. tuberculosis-infected old mice, in combination with CD8 T cells that can nonspecifically produce IFN-gamma, leads to transient control of M. tuberculosis growth in the lungs of old mice. Further characterization of this mechanism should provide essential information regarding the aging immune system and should contribute to the development of novel strategies to decrease the morbidity and mortality of the aging population associated with infectious diseases.     

The influence of dietary protein antigen on Th1/Th2 balance and cellular immunity.

Dietary administration of ovalbumin (OVA) antigen (Ag) into OVA-specific T cell receptor alphabeta-transgenic (TCR-Tg) mice resulted in the induction of activated CD4+ Th cells expressing CD69 early activation Ag. However, the number of CD4+ Th cells rather decreased by dietary administration of OVA antigen. The production of Th1-cytokines such as IFN-gamma and IL-2 markedly reduced in spleen of OVA-fed mice compared to mice fed with normal diet. In sharp contrast, the production of Th2-cytokine, IL-4 greatly increased in spleen of OVA-fed mice though the number of CD4+ T cells decreased to less than 10% of control mouse spleen. The decrease of IFN-gamma production and the increase of IL-4 production by CD4+ T cells was demonstrated at a single cell level by intracellular cytokine staining analysis. Moreover, such a polarized cytokine production pattern was also demonstrated using highly purified CD4+ T cells obtained from mice fed with OVA. In addition to the decrease of Th1-cytokine production, TCR-Tg mice fed with OVA-containing diet showed greatly reduced in vivo generation of NK cells, LAK cells and CTL. These results suggested that dietary protein antigen caused the polarization of Th1/Th2 balance into Th2-dominant immunity and inhibited cellular immunity.     

Interleukin-12 is necessary for the priming of CD4+ T cells required during the elicitation of HIV-1 gp120-specific cytotoxic T-lymphocyte function

The mechanism of the T-cell response and cytokine induction to restrict human immunodeficiency virus 1 (HIV-1) infection is not clear. During early infection, HIV-infected individuals have a high frequency of virus-specific cytotoxic T lymphocytes (CTLs) that effectively reduces the viral load. However, the CTLs are unable to clear the virus at later stages of infection, leading to disease progression. Dysregulation of cytokines like interleukin-12 (IL-12) and interferon-gamma (IFN-gamma) as a result of the interaction of HIV-1-specific T cells with antigen-presenting cells is one of the possible causes of CTL dysfunction. Secretion of IL-12 is reduced with the progression of HIV infection, correlating with impaired CTL function; however, the role of IL-12 in CTL regulation awaits elucidation. Here, we have studied the role of IL-12 in CTL dysfunction by using DNA immunization of wild-type (WT) and IL-12-deficient mice with HIV-1 gp120 complementary DNA. It was observed that the CTL response in IL-12-deficient mice was significantly less than that in WT mice. Our results further demonstrated that coimmunization with IL-12 vector restored the impaired CTL response in IL-12-deficient mice. However, immunization with IL-12 vector failed to rescue the CTL response in IFN-gamma deficient mice, suggesting that the CTL-promoting function of IL-12 is IFN-gamma-mediated. Our data suggest a phase-specific role of IL-12 in the CTL response, specifically in the priming of CD4+ T cells that provide help to CD8+ T cells. Our results also suggest that IL-12 is vital for the priming of antigen-specific T cells and plays an essential role in IFN-gamma induction in T cells.     

IFN-gamma and IL-12 differentially regulate CC-chemokine secretion and CCR5 expression in human T lymphocytes

Interleukin (IL)-12, especially in the presence of neutralizing anti-IL-4 monoclonal antibodies, primed CD45RO(-) T clones for high CCL3/macrophage-inflammatory protein-1alpha (MIP-1alpha) and CCL4/MIP-1beta levels. In CD4(+) and CD8(+) clones from two patients deficient for IL-12Rbeta1 (IL-12Rbeta1(-/-)), production of CCL3/MIP-1alpha and CCL4/MIP-1beta was defective. CD4(+) clones from two patients deficient for interferon-gamma (IFN-gamma) R1 (IFN-gammaR1(-/-)) produced somewhat decreased CCL4/MIP-1beta levels. IL-12 failed to prime CD4(+) or CD8(+) healthy clones for high CCL5/regulated on activation, normal T expressed and secreted (RANTES) production, although its secretion was impaired in CD4(+) clones from IL-12Rbeta1(-/-) and IFN-gammaR1(-/-) patients. CCR5 surface expression was up-regulated in resting peripheral blood mononuclear cells and CD4(+) clones from both kinds of patients, rendering them more susceptible to CCR5-dependent (R5) HIV-1 infection. Neutralization of IFN-gamma increased CCR5 expression and decreased CC-chemokine secretion by CD4(+) clones from healthy and IL-12Rbeta1(-/-) individuals, suggesting an IFN-gamma-dependent control of CCR5 expression. These data provide the first documented analysis of chemokine secretion and chemokine receptor expression on T cells from IL-12 and IFN-gamma receptor-deficient patients and dissect the role of IL-12 and IFN-gamma on inducing inflammatory chemokine secretion and down-regulating CCR5 expression in human T cells.     

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.     

IL-12 receptor deficiency revisited: IL-23-mediated signaling is also impaired in human genetic IL-12 receptor beta1 deficiency

IFN-gamma and IL-12 are crucial cytokines for cell-mediated immunity against intracellular pathogens. We have previously shown that human IL-12Rbeta1-deficiency leads to impaired IL-12 responsiveness and unusual susceptibility to infections due to mycobacteria and salmonellae. IL-23 is a cytokine with functions that partially overlap with those of IL-12. IL-23 consists of IL-12p40 and a novel p19 protein, and binds to a receptor complex comprising IL-12Rbeta1 and IL-23R. Thus, IL-12Rbeta1-deficiency may impair both IL-12- and IL-23 signaling, and both may contribute to the immunological phenotypes. To examine whether IL-12Rbeta1 is essential for IL-23 signaling in human T cells, we have studied IL-23 responsiveness of four IL-12Rbeta1-deficient individuals. Whereas IL-23 promoted IFN-gamma production by CD4(+) and CD8(+) T cells in controls, IL-12Rbeta1-deficient T cells lacked IL-23-induced IFN-gamma secretion, but responded normally to IL-2, IL-4, IL-15 and IL-18. We also show that induction of IFN-gamma production by IL-23 depends upon TCR-ligation and is enhanced by CD28-costimulation. Furthermore, IL-23 cooperates with IL-12 and IL-18 in promoting IFN-gamma production in controls, but not in patients. We conclude that IL-12Rbeta1-deficiency impairs IL-12- and IL-23-dependent signaling in human T cells. The syndrome caused by IL-12Rbeta1-deficiency thus needs to be reinterpreted as resulting from defective IL-12-as well as IL-23-mediated immunity.     

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.     

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.     

IL-27 and IFN-alpha signal via Stat1 and Stat3 and induce T-Bet and IL-12Rbeta2 in naive T cells.

Interleukin-27 (IL-27) supports proliferation of naive CD4(+) T cells and enhances interferon-gamma (IFN-gamma) production by activated T cells and natural killer (NK) cells. We report here that IL-27 induces Stat1 and Stat3 phosphorylation and activation in human and murine cell lines and primary human T cells. IL-27 also induces T-Bet, a Stat1-dependent gene crucial to Th1 cell commitment. Similarly, IFN-alpha activates Stat1 and Stat3 and T-Bet expression in naive T cells. Induction of T-Bet results in upregulation of IL-12Rbeta2 on naive T cells, which is essential for responsiveness to IL-12 and differentiation to a Th1 phenotype. Both IL-27 and IFN-alpha induce expression of IL-12Rbeta2 in T cells. In contrast, IFN-gamma, which activates Stat1 but not Stat3, induces expression of T-Bet but not IL-12Rbeta2 in naive T cells. We propose that IL-27 and IFN-alpha are important for early Th1 commitment and act upstream of IL-12 and IFN-gamma in this pathway.     

Participation of contrasting changes in IL-10 and IL-12 production in the reduction of Th1-predominance by Hachimi-jio-gan in autoimmune MRL/MP-lpr/lpr mice

Hachimi-jio-gan (Chinese name: Ba-Wei-Di-Huang-Wan, HMG), a traditional Japanese herbal medicine, has been used for disorders accompanying aging. Our previous studies suggested that HMG ameliorated Thl-predominant autoimmune diseases in MRL/lpr mice through inhibition of IL-12 production. In the present study, the oral administration of HMG down-regulated phosphorylated STAT4 and up-regulated phosphorylated STAT6 in CD4 T cells. In the T cells, IL-12Rbeta1 and IL-12Rbeta2 mRNA expression levels were suppressed. In antigen-presenting cells (CD45R- MHC class II+ cells) which control Th1 and Th2 immune responses, the total cell number and the percentage of cells expressing co-stimulatory molecules decreased in the HMG-treated group. In addition, the levels of IL-12 and 18 mRNA expression increased and conversely, IL-10 mRNA expression decreased. Further, the production of IL-10 was up-regulated and that of IL-12 was down-regulated by HMG in the presence of anti-CD40 antibody. These results suggest that the opposite effects on IL-10 production and, IL-12 or IL-18 production in antigen-presenting cells of oral administration of HMG are due a decline in IL-12R expression, and consequently, amelioration of MRL/lpr autoimmune diseases occurs through the suppression of Th1 predominance via STAT4/STAT6 signaling.     

An immunomodulatory arabinomannan extracted from Mycobacterium tuberculosis, Z-100, restores the balance of Th1/Th2 cell responses in tumor bearing mice

The regulatory effect of Z-100 on the balance of Th1/Th2 cell responses in BALB/c mice bearing Meth-A fibrosarcoma was investigated. In tumor bearing mice, Th1 cytokine production (IL-2, IFN-gamma) are suppressed and Th2 cytokine production (IL-4, IL-10) are increased, as compared with those of normal mice. The administration of Z-100 (10 mg/kg) to tumor bearing mice restored the balance of Th1/Th2 cell responses from Th2 dominant state to the normal state. This regulatory effect of Z-100 was eliminated by depletion of adherent cells from splenocytes derived from tumor bearing mice, and by the treatment with 2-ClAdo (a macrophage inhibitor). Similarly, this regulatory effect was diminished by the treatment with anti-IL-12 mAb and anti-IFN-gamma mAb. In addition, the IL-12 p40 mRNA expression in splenic adherent cells and IFN-gamma mRNA expression in CD4+ T cells were increased by the administration of Z-100 to tumor bearing mice. These results suggested that Z-100 restored the balance of Th1/Th2 cell responses to the normal one in tumor bearing mice through the activation of macrophages and up-regulation of IL-12 production from macrophages and IFN-gamma production from CD4+ T cells.     

Aberrant regulation of interleukin-12 receptor beta2 chain on type 1 cytokine-stimulated T lymphocytes in type 1 diabetes

An aberrant mitogen-induced polarization of peripheral blood T cells has been associated with type 1 diabetes (T1D). We studied, in T1D, type 1 and 2 cytokine-induced expression of the interleukin-12 receptor beta2 chain (IL-12Rbeta2 chain), which plays a critical role in regulating T-cell polarization. Peripheral blood lymphocytes from children with newly diagnosed T1D (n=10; mean age 10 years), from children with longstanding T1D (n=8; mean age 12.9 years) and from healthy children (n=15; mean age 11.5 years) were stimulated with phytohaemagglutinin (PHA) in a type 1 (IL-12 and anti-IL-4) or a type 2 (IL-4 and anti-IL-12) cytokine environment. Secretion of interferon-gamma (IFN-gamma), IL-5 and IL-13, as detected by enzyme-linked immunosorbent assay (ELISA), and expression of the IL-12Rbeta2 chain on CD4 and CD8 cells by flow cytometry, were analysed. Children with newly diagnosed and longstanding T1D had lower expression levels of the IL-12Rbeta2 chain on IL-12Rbeta2 chain-positive CD4 T cells (for a type 1 or a type 2 cytokine environment: P=0.01 and P=0.002 or P=0.02 and P=0.01, respectively) and on IL-12Rbeta2 chain-positive CD8 T cells (for a type 1 or a type 2 cytokine environment: P=0.007 and P=0.0007 or P=0.003 and P=0.01, respectively) when compared to healthy children. A decreased percentage of IL-12Rbeta2 chain-expressing CD4 T cells (P=0.07 and P=0.03) and CD8 T cells (P=0.004 and P=0.01) and increased secretion of IL-13 (P=0.006 and P=0.04) in a type 1 cytokine environment was seen in both groups of patients. Peripheral blood T cells from patients with both newly diagnosed and longstanding T1D showed poor polarization towards type 1 cells.     

Induction of interleukin-12 production in mouse macrophages by berberine,a benzodioxoloquinolizine alkaloid,deviates CD4+ T cells from a Th2 to a Th1 response

In this study we investigated whether berberine-mediated induction of interleukin-12 (IL-12) production in antigen-presenting cells could regulate a cytokine profile of antigen-primed CD4+ T helper (Th) cells. Pretreatment with berberine induced IL-12 production in both macrophages and dendritic cells, and significantly increased the levels of IL-12 production in lipopolysaccharide-stimulated macrophages and in CD40 ligand-stimulated dendritic cells. Importantly, berberine pretreatment of macrophages increased their ability to induce interferon-gamma (IFN-gamma) and reduced their ability to induce IL-4 in antigen-primed CD4+ T cells. Berberine did not influence the macrophage cell surface expression of the class II major histocompatibility complex molecule, the co-stimulatory molecules CD80 and CD86, and intracellular adhesion molecule-1. Addition of neutralizing anti-IL-12p40 monoclonal antibody to cultures of berberine-pretreated macrophages and CD4+ T cells restored IL-4 production in antigen-primed CD4+ T cells. The in vivo administration of berberine resulted in the enhanced induction of IL-12 production by macrophages when stimulated in vitro with lipopolysaccharide or heat-killed Listeria monocytogenes, leading to the inhibition of the Th type 2 cytokine profile (decreased IL-4 and increased IFN-gamma production) in antigen-primed CD4+ T cells. These findings may point to a possible therapeutic use of berberine or medicinal plants containing berberine in the Th type 2 cell-mediated immune diseases such as allergic diseases.     

Differential requirement of CD28 for IL-12 receptor expression and function in CD4(+) and CD8(+) T cells

IL-12 is a potent inducer of IFN-gamma production and Th1 responses. Co-stimulation mediated by B7 has been shown to synergize with IL-12 for optimal IFN-gamma production and proliferation in vitro. In this study, we examined the requirement of CD28/B7 interactions for optimal induction of IL-12 receptor(R) beta1 and beta2 expression and IFN-gamma. IL-12-induced IFN-gamma production and STAT4 nuclear translocation were markedly reduced in CD28(-/-) splenocytes compared to that of wild-type (WT) splenocytes. Analysis of IL-12R expression revealed that IL-12 induced similar levels of IL-12R beta2 mRNA expression in WT and CD28(-/-) cells. In contrast, IL-12R beta1 expression was impaired in CD28(-/-) cells, indicating that expression of IL-12R beta1 and beta2 is differentially regulated by CD28. CD28(-/-) CD4(+) but not CD8(+) cells exhibited a defect in IL-12Rbeta1 expression that was associated with a marked decrease in IL-12 binding as well as IL-12-induced IFN-gamma production. IL-2 could restore IL-12R expression to CD28(-/-) CD4(+) cells, however, this occurred independently of IL-2-induced proliferation. Thus, these findings identify distinct requirements for CD28 in the capacity of CD4(+) and CD8(+) cells to respond maximally to IL-12.     

Gene gun-mediated delivery of an interleukin-12 expression plasmid protects against infections with the intracellular protozoan parasites Leishmania major and Trypanosoma cruzi in mice

An interleukin-12 (IL-12) expression plasmid was transferred, using a gene gun, to mice infected with Leishmania major or Trypanosoma cruzi. Transfer of the IL-12 gene to susceptible BALB/c mice resulted in regression of lesion size and reduced the number of parasites in draining lymph nodes (LN) at the site of L. major infection. Coincident with these protective effects, the T-helper type (Th) response shifted towards Th1, as evaluated by cytokine production in vitro and L. major-specific antibody responses. Protective effects of the IL-12 gene were also observed in T. cruzi infection. Treatment of BALB/c mice infected with T. cruzi enhanced the production of interferon-gamma (IFN-gamma) by spleen cells, while suppressed production of interleukin-10 (IL-10) compared with control mice. Administration of anti-CD4 or anti-CD8 monoclonal antibody (mAb) abolished the protective immunity against T. cruzi infection, and treatment with the IL-12 gene could not restore the resistance in these mice. Mice depleted of natural killer (NK) cells with anti-asialo GM1 also became susceptible to infection, while the resistance was restored when these mice were treated with the IL-12 gene. Thus, target cells for the treatment appear to be CD4+ and CD8+ T cells, which are ordinarily activated by NK cells. These results suggest that the transfer of cytokine genes using a gene gun is an effective method for investigating the roles of cytokines and gene therapy in infectious diseases.