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).