We studied here the long-term maintenance of distinct populations of T helper type 1 (TH1)-lineage cells in vivo and found that effector TH1 cells, defined by their secretion of interferon-γ (IFN-γ), are short-lived and do not efficiently develop into long-term memory TH1 cells. In contrast, a population of activated TH1-lineage cells that did not secrete IFN-γ after primary antigenic stimulation persistedfor several months in vivo and developed the capacity to secrete IFN-γ upon subsequent stimulation.These data suggest that a linear differentiation pathway, as defined by the transition from IFN-γ-producing to resting memory cells, is relatively limited in vivo and support a revised model for TH1memory differentiation.

The biological activities of IL-12 are mediated through a specific, high-affinity receptor composed of IL-12 receptor(R) β1 and IL-12Rβ2 subunits that exist primarily on T and NK cells. Remarkably, the expression of IL-12Rβ2 on CD4+ T cells in mouse and humans appears to be differentially regulated by IFN- γ and IFN-α, respectively. Using an antibody specific for the human IL-12Rβ2 subunit, the effect of IFN- γ, IFN- α, 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- α or IFN- γ in cultures enhanced IL-12Rβ2 expression of CD4+ and CD8+ T cells. The enhancing effect of IFN- α and IFN- γ was independent of endogenous IL-12. Furthermore, the clearest effects of IFN- α and IFN- γ on IL-12Rβ2 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-α and IFN-γ had little effect on regulating IL-12Rβ2 expression on human NK cells. Taken together, these data show that there is differential regulation of IL-12Rβ2 expression by IFN- α and IFN- γ on human T and NK cells.

Cholera toxin (CT) is a potent mucosal vaccine adjuvant, which has been shown to induce T helper cell type 2 (Th2) responses in systemic and mucosal tissues. We report that CT inhibits the production of interleukin (IL)-12, a major Th2 counterregulatory cytokine. IL-12 p70 production by stimulated human monocytes was inhibited by CT in a dose-dependent manner. This suppression occurred at the level of gene transcription, was maximal at low concentrations of CT, and was dependent on the A subunit of the toxin, since purified CT B subunit had minimal effect. CT also inhibited the production of IL-12 p70 by monocyte-derived dendritic cells, as well as the production of tumor necrosis factor a, but not IL-10, IL-6, or transforming growth factor (TGF)-β1, by stimulated monocytes. The effects of CT were not due to autocrine production of IL-10, TGFβ1, or prostaglandin E 2. CT inhibited the production of IFNg by anti-CD3-stimulated human peripheral blood mononuclear cell, due in part to suppression of IL-12 production, but also to the inhibition of expression of the β1 and β2 chains of the IL-12 receptor on T cells. In vivo, mice given CT before systemic challenge with ipopolysaccharide had markedly reduced serum levels of IL-12 p40 and interferon γ. These data demonstrate two novel mechanisms by which CT can inhibit Th1 immune responses, and help explain the ability of mucosally administered CT to enhance Th2-dependent immune responses.

In this study we demonstrated that CD4 T cells from STAT4/mice exhibit reduced IL-12R expression and poor IL-12R signaling function. This raised the question of whether activated STAT4 participates in Th1 cell development mainly through its effects on IL-12 signaling. In a first approach to this question we determined the capacity of CD4 T cells from STAT4 /bearing an IL-12R 2 chain transgene (and thus capable of normal IL-12R expression and signaling) to undergo Th1 differentiation when stimulated by Con A and APCs. We found that such cells were still unable to exhibit IL-12-mediated IFN-production. In a second approach to this question, we created Th2 cell lines (D10 cells) transfected with STAT4-expressing plasmids with varioustyrosine3phenylalanine mutations and CD4 T cell lines from IL-12 2/mice infected with retroviruses expressing similarly STAT4 mutations that nevertheless express surface IL-12R 2 chains. We then showed that constructs that were unable to support STAT4 tyrosine phosphorylation (in D10 cells) as a result of mutation were also incapable of supporting IL-12-induced IFN-production (in IL-12R 2/cells). Thus, by two complementary approaches we demonstrated that activated STAT4 has an essential downstream role in Th1 cell differentiation that is independent of its role in the support of IL-12R 2 chain signaling. This implies that STAT4 is an essential element in the early events of Th1 differentiation.

Cytokines regulate lymphocyte development and differentiation, but precisely how they control these processes is still poorly understood. By using microarray technology to detect cytokineinduced genes, we identified a cDNA encoding Cybr, which was increased markedly in cells incubated with IL-2 and IL-12. The mRNA was most abundant in hematopoietic cells and tissues. The predicted amino acid sequence is similar to that of GRP-1-associated protein (GRASP), a recently identified retinoic acid-induced cytohesin-binding protein. Physical interaction, dependent on the coiled-coil domains of Cybr and cytohesin-1, was demonstrated by coimmunoprecipitation of the overexpressed proteins from 293T cells. Cytohesin-1, in addition to its role in cell adhesion, is a guanine nucleotide-exchange protein activator of ARF GTPases. Acceleration of guanosine 5 -O-(thiotriphosphate) binding to ARF by cytohesin-1 in vitro was enhanced by Cybr. Because the binding protein modified activation of ADP ribosylation factor by cytohesin-1, we designate this cytokine-inducible protein Cybr (cytohesin binder and regulator).

CpG oligodeoxynucleotides (ODN) have potent effects on innate and adaptive cellular immune responses. In this report, the ability of CpG ODN to confer long-term immunity and protection when used as a vaccine adjuvant with a clinical grade of leishmanial antigen, autoclaved Leishmania major (ALM), or a recombinant leishmanial protein was studied. In two different mouse models of L. major infection, vaccination with ALM plus CpG ODN was able to control infection and markedly reduce lesion development in susceptible BALB/c and resistant C57BL/6 (B6) mice, respectively, up to 12wk after immunization. Moreover, B6 mice immunized with ALM plus CpG ODNs were still protected against infectious challenge even 6 mo after vaccination. In terms of immune correlates of protection, ALM plus CpG ODN-vaccinated mice displayed L. major-specific T helper cell 1 and CD8 responses. In addition, complete protection was markedly abrogated in mice depleted of CD8+ T cells at the time of vaccination. Similarly, mice vaccinated with a recombinant leishmanial protein plus CpG ODN also had long-term protection that was dependent on CD8+ T cells in vivo. Together, these data demonstrate that CpG ODN, when used as a vaccine adjuvant with either a recombinant protein or heat-killed leishmanial antigen, can induce long-term protection against an intracellular infection in a CD8-dependent manner.

Two subunits of the IL-12 receptor (IL-12R), IL-12Rβ1 and IL-12Rβ2, have been identified and cloned. Previous studies demonstrated that the IL-12Rβ1 subunit was required for mouse T and NK cells to respond to IL-12 in vivo. To investigate the role of IL-12Rβ2 in IL-12 signaling, we have generated IL-12Rβ2-deficient (IL-12Rβ22/2) mice by targeted mutation in embryonic stem (ES) cells. Although Con A-activated splenocytes from IL-12Rβ22/2 mice still bind IL-12 with both high and low affinity, no IL-12-induced biological functions can be detected. Con A-activated splenocytes of IL-12Rβ22/2 mice failed to produce IFN-g or proliferate in response to IL-12 stimulation. NK lytic activity of IL-12Rβ22/2 splenocytes was not induced when incubated with IL-12. IL-12Rβ22/2 splenocytes were deficient in IFN-g secretion when stimulated with either Con A or anti-CD3 mAβ in vitro. Furthermore, IL-12Rβ22/2 mice were deficient in vivo in their ability to produce IFN-g following endotoxin administration and to generate a type 1 cytokine response. IL-12-mediated signal transduction was also defective as measured by phosphorylation of STAT4. These results demonstrate that although mouse IL-12Rβ1 is the subunit primarily responsible for binding IL-12, IL- 12Rβ2 plays an essential role in mediating the biological functions of IL-12 in mice.

IFN-γ exerts multiple biological activities in the modulation of immune responses by the induction of transcription factors. Onetranscriptional factor of the IFN regulatory factor family found to be critical in regulating IL-12-dependent IFN-γ production invivo following infectious challenge has been designated IFN consensus sequence-binding protein (ICSBP). In this study, the roleof ICSBP in regulating type 1 responses to T cell-specific stimulation in vitro was assessed. Total splenocytes from ICSBP2/2 micestimulated with soluble anti-CD3 were markedly impaired in the production of IFN-γ compared with similarly stimulated cells from ICSBP1/1 mice. Consistent with the decrease in IFN-γ production, splenocytes from ICSBP2/2 mice stimulated with anti-CD3 in the presence or absence of IFN-γ or a soluble CD40 ligand agonist failed to produce IL-12 p40 and IL-12 p70 protein; however, the deficient production of IFN-γ from ICSBP2/2 mice could be restored by the addition of anti-CD28 Ab in an IL-12-independent manner. In contrast to the previous data, production of IFN-γ from naive CD41/LECAM-1high cells of ICSBP2/2 mice that had been primed in vitro with anti-CD3 was similar to or greater than that of ICSBP1/1 controls. In addition, the presence of IFN-γ in priming cultures enhanced both priming for IFN-γ and IL-12 responsiveness from ICSBP2/2 CD41 T cells. Overall, these results provide evidence that ICSBP is differentially required for the ability of IFN-γ to regulate type 1 cytokine responses from APCs and CD41 T cells.

Regulation of the factors governing IL-12R expression and IL-12 responsiveness has been shown to be important in the generation and stability of Th1- and Th2-type responses. In this regard, cytokines have been shown to have a prominent role in regulating IL-12R expression. In this study, the role that PGE2 and dexamethasone (DXM) have in regulating IL-12R expression was evaluated. Addition of PGE2 or DXM to human PBMCs stimulated with immobilized anti-CD3 plus IL-12 inhibited the production of IFN-g in a dose-responsive manner. Moreover, PBMCs stimulated with immobilized anti-CD3 in the presence of PGE2 or DXM for 3 days, washed extensively, and restimulated in the presence of IL-12 still did not produce IFN-g. This lack of IL-12 responsiveness from cells cultured in either PGE2 or DXM was correlated with diminished surface expression of IL-12Rb1, IL-12Rb2 mRNA expression, and IL-12 binding. Finally, the PGE2- and DXM-mediated inhibition of IL-12R expression was not affected significantly by addition of neutralizing Abs against either IL-4, IL-10, or TGF-b. By contrast, addition of dibutyryl cAMP, 8-bromoadenosine 3: 5 cAMP (8-Br-cAMP), or cholera toxin substantially reduced IL-12R expression, suggesting that PGE2 may be mediating its effects through enhancement of cAMP.

Over the past few years, major advances in several areas of immunology have provided a foundation for the rational design of vaccines against diseases requiring cellular immunity. Among these advances are the cellular mechanisms by which DNA vaccines can sustain long-term humoral and cellular immunity.