Regulation of IFN-gamma signaling is essential for the cytotoxic activity of CD8(+) T cells.

Previous studies have demonstrated that, as naive murine CD4(+) cells differentiate into Th1 cells, they lose expression of the second chain of IFN-gammaR (IFN-gammaR2). Hence, the IFN-gamma-producing subset of Th cells is unresponsive to IFN-gamma. Analysis of IFN-gamma-producing CD8(+) T cells demonstrates that, like Th1 cells, these cells do not express IFN-gammaR2. To define the importance of IFN-gamma signaling for the development of functional CD8(+) T cells, mice either lacking IFN-gammaR2 or overexpressing this protein were examined. While CD8(+) T cell development and function appear normal in IFN-gammaR2(-/-) mice, CD8(+) T cell function in IFN-gammaR2 transgenic is altered. IFN-gammaR2 transgenic CD8(+) T cells are unable to lyse target cells in vitro. However, these cells produce Fas ligand, perforin, and granzyme B, the effector molecules required for killing. Interestingly, TG CD8(+) T cells proliferate normally and produce cytokines, such as IFN-gamma in response to antigenic stimulation. Therefore, although IFN-gamma signaling is not required for the generation of normal cytotoxic T cells, constitutive IFN-gamma signaling can selectively impair the cytotoxic function of CD8(+) T cells.     

The antineoplastic agent bryostatin-1 differentially regulates IFN-gamma receptor subunits in monocytic cells: transcriptional and posttranscriptional control of IFN-gamma R2

Bryostatin-1 (Bryo-1) is a potent ligand and modulator of protein kinase C that exerts antineoplastic and immunomodulatory activities both in vitro and in vivo. We have previously reported that Bryo-1 synergized with IFN-gamma to induce NO synthase and NO by macrophages. To determine whether this effect was associated with changes in levels of IFN-gammaR, we investigated the effects of Bryo-1 on the expression and regulation of IFN-gammaR chains in monocytic cells. Northern blot analysis revealed that Bryo-1 treatment of the human monocytic cell lines MonoMac6 and THP-1 and human monocytes enhanced the expression of IFN-gammaR2 mRNA but did not affect IFN-gammaR1 mRNA expression. Bryo-1 increased IFN-gammaR2 mRNA in a dose-dependent manner as early as 3 h posttreatment. Bryo-1-induced up-regulation of IFN-gammaR2 mRNA levels is not dependent on de novo protein synthesis as shown by cell treatment with the protein-synthesis inhibitor cycloheximide. Bryo-1 treatment increased the IFN-gammaR2 mRNA half-life by 2 h. EMSA analysis from Bryo-1-treated MonoMac6 cells showed an increased nuclear protein binding to the NF-kappaB motif present in the 5' flanking region of the human IFN-gammaR2 promoter that was markedly decreased by pretreatment with the NF-kappaB inhibitor SN50. These results show for the first time that Bryo-1 up-regulates IFN-gammaR2 expression in monocytic cells. Given the pivotal role that IFN-gamma exerts on monocyte activation and in the initiation and outcome of the immune response, the induction of IFN-gammaR2 by Bryo-1 has significant implications in immunomodulation and could overcome some of the immune defects observed in cancer patients.     

Dynamic regulation of IFN-gamma signaling in antigen-specific CD8+ T cells responding to infection

IFN-gamma plays a critical role in the CD8(+) T cell response to infection, but when and if this cytokine directly signals CD8(+) T cells during an immune response is unknown. We show that naive Ag-specific CD8(+) T cells receive IFN-gamma signals within 12 h after in vivo infection with Listeria monocytogenes and then become unresponsive to IFN-gamma throughout the ensuing Ag-driven expansion phase. Ag-specific CD8(+) T cells regain partial IFN-gamma responsiveness throughout the contraction phase, whereas the memory pool exhibits uniform, but reduced, responsiveness that is also modulated during the secondary response. The responsiveness of Ag-specific CD8(+) T cells to IFN-gamma correlated with modulation in the expression of IFN-gammaR2, but not with IFN-gammaR1 or suppressor of cytokine signaling-1. This dynamic regulation suggests that early IFN-gamma signals participate in regulation of the primary CD8(+) T cell response program, but that evading or minimizing IFN-gamma signals during expansion and the memory phase may contribute to appropriate regulation of the CD8(+) T cell response.     

Ligand-independent down-regulation of IFN-gamma receptor 1 following TCR engagement

Activated T lymphocytes modulate the level of many molecules on their cell surface, including cytokine receptors. This regulation of cytokine receptor expression affects the ability of T cells to respond to cytokines and thus influences the outcome of an immune response. The receptor for IFN-gamma, a proinflammatory cytokine, consists of two copies of a ligand binding chain (IFN-gammaR1) as well as two copies of a second chain (IFN-gammaR2) required for signal transduction. The expression of IFN-gammaR2 is down-regulated at the mRNA level on CD4+ T cells when they differentiate into the Th1, but not the Th2, phenotype. This down-regulation has been demonstrated to depend on the ligand, IFN-gamma, which is produced by Th1 but not Th2 T cells. The regulation of the cell-surface expression of IFN-gamma receptors during primary T cell activation has not been reported. Naive and differentiated T lymphocytes express IFN-gammaR1 at the mRNA level and as a cell-surface protein. In this study, we present evidence that cell-surface expression of IFN-gammaR1 is transiently down-regulated on the surface of naive CD4+ T cells shortly after TCR engagement. Furthermore, this down-regulation is not mediated by the ligand, IFN-gamma, but results from TCR engagement and can be inhibited by cyclosporin A.     

In vivo generation of pathogen-specific Th1 cells in the absence of the IFN-gamma receptor

The precise mechanisms that govern the commitment of CD4 T cells to become Th1 or Th2 cells in vivo are incompletely understood. Recent experiments demonstrate colocalization of the IFN-gammaR chains with the TCR during activation of naive CD4 T cells, suggesting that association of these molecules may be involved in determining lineage commitment. To test the role of IFN-gamma and its receptor in the generation of Th1 Ag-specific CD4 T cells, we analyzed mice after infection with Listeria monocytogenes or lymphocytic choriomeningitis virus. In the absence of IFN-gamma, Ag-specific CD4 T cells were generated in response to both these infections. In addition, IFN-gamma-producing (Th1) Ag-specific CD4 T cells were generated in mice lacking the ligand-binding chain of the IFN-gammaR (IFN-gammaR1-/-) or the signaling chain (IFN-gammaR2-/-). There was no increase in the number of IL-4-producing Ag-specific CD4 T cells, nor was there a decrease in the expression of T-bet in the absence of functional IFN-gamma signaling, indicating that the cells were committed Th1 cells. Thus, both chains of the IFN-gammaR are dispensable for the generation of Th1 Ag-specific CD4 T cells after infection in vivo.     

Regulation of interferon-gamma receptor (INF-gammaR) chains: a peculiar way to rule the life and death of human lymphocytes

Interferon-gamma (IFN-gamma) is a lymphokine produced by activated T lymphocytes and NK cells, that plays an important role in host defense mechanisms by exerting pleiotropic activities on a wide range of cell types. Cellular responses to IFN-gamma are mediated by its heterodimeric cell surface receptor (IFN-gammaR), which activates downstream signal transduction cascades, ultimately leading to the regulation of gene expression. Several observations suggest that the signals resulting from the binding of IFN-gamma to its receptor depend on the number of surface receptors transducing the IFN-gamma signal. This review summarizes recent advances in the understanding of the fine regulation of the response of human lymphocytes to IFN-gamma through an interplay between surface expression of IFN-gammaR and a variety of environmental factors that combine to control their fate.     

Nitric oxide inhibits the proliferation of T-helper 1 and 2 lymphocytes without reduction in cytokine secretion

To study the effect of nitric oxide (NO) on the activity of Th subsets, cloned Th1 and Th2 lymphocytes were stimulated in the presence of an NO donor. NO, when present from the start of incubation, inhibited the proliferation of both Th subsets dose-dependently, achieving complete inhibition at a relatively low level. The addition of NO 24 h after the onset of T cell stimulation also resulted in reduced proliferation of both Th subsets, suggesting that NO affects a late process during T cell activation. Stimulation of T cells in the presence of NO did not induce apoptosis at the concentrations that completely inhibited proliferation, although apoptosis became evident at higher NO concentrations. The secretion of several cytokines (i.e., IFN-gamma, IL-4, and IL-5) was slightly upregulated, while IL-2 production was modestly inhibited in the presence of NO. However, exogenous IL-2 did not reverse the NO-induced inhibition of T cell proliferation, nor did additional stimulation with phorbol esters. Finally, expression of IL-2R was modestly decreased in the presence of NO, although TCR expression was not affected. These studies demonstrate that relatively low concentrations of NO induce a strong and specific inhibition of T cell proliferation in both Th subsets, suggesting that local NO production may regulate Th-mediated tissue inflammation.     

Annexin V associates with the IFN-gamma receptor and regulates IFN-gamma signaling

Many of the biological activities of IFN-gamma are mediated through the IFN-gammaR3-linked Jak-Stat1alpha pathway. However, regulation of IFN-gamma signaling is not fully understood, and not all responses to IFN-gamma are Stat1alpha dependent. To identify novel elements involved in IFN-gamma cell regulation, the cytoplasmic domain of the R2 subunit of the human IFN-gammaR was used as bait in a yeast two-hybrid screen of a human monocyte cDNA library. This identified annexin A5 (AxV) as a putative IFN-gammaR binding protein. The interaction was confirmed in pull-down experiments in which a GST-R2 cytoplasmic domain fusion protein was incubated with macrophage lysates. Furthermore, immunoprecipitation using anti-IFN-gammaR2 Abs showed that AxV interacted with IFN-gammaR2 to form a stable complex following incubation of cells with IFN-gamma. In 293T cells with reduced expression of AxV, brought about by small interfering RNA targeting, activation of Jak2 and Stat1alpha in response to IFN-gamma was enhanced. Inhibition of cell proliferation, a hallmark of the IFN-gamma response, also was potentiated in HeLa cells treated with small interfering RNA directed at AxV. Taken together, these results suggest that through an inducible association with the R2 subunit of the IFN-gammaR, AxV modulates cellular responses to IFN-gamma by modulating signaling through the Jak-Stat1 pathway.     

Preassembly and ligand-induced restructuring of the chains of the IFN-γ receptor complex： the roles of Jak kinases, Statl and the receptor chains

We previously demonstrated using noninvasive technologies that the interferon-gamma （IFN-γ） receptor complex is preassembled [ 1 ]. In this report we determined how the receptor complex is preassembled and how the ligand-mediated conformational changes occur. The interaction of Statl with IFN-γR1 results in a conformational change localized to IFN- γR1. Jakl but not Jak2 is required for the two chains of the IFN-γ receptor complex （IFN-γR1 and IFN-γR2） to interact; however, the presence of both Jakl and Jak2 is required to see any ligand-dependant conformational change. Two IFN- γR2 chains interact through species-specific determinants in their extracellular domains. Finally, these determinants also participate in the interaction of IFN-γR2 with IFN-γR1. These results agree with a detailed model of the IFN-γ receptor that requires the receptor chains to be pre-associated constitutively for the receptor to be active.     

Presentation of IFN-gamma to nitric oxide-producing cells: a novel function for mast cells

We report that mast cells can bind and present IFN-gamma in a functionally active form to macrophages. Flow-cytometric analysis revealed that biotinylated IFN-gamma bound equally well to purified peritoneal mast cells from both IFN-gammaR knockout and wild-type mice, indicating a non-IFN-gammaR binding site. Purified peritoneal mast cells, loaded with IFN-gamma for 30 min and washed, were able to induce NO synthesis by peritoneal macrophages. This response required cell contact and expression of IFN-gammaR on the responding macrophages, but not the mast cells. Human HMC-1 mast cells were also able to present IFN-gamma to mouse macrophages. Enzyme treatment of mouse mast cells revealed that binding of IFN-gamma was predominantly to chondroitin sulfate B (dermatan sulfate). Binding of IFN-gamma to dermatan sulfate was confirmed by inhibition ELISA. This study demonstrates for the first time that mast cells can present IFN-gamma to other cells via glycosaminoglycans. Mast cells may act as a reservoir of surface-stored functionally active cytokines.     

Dominant negative MyD88 proteins inhibit interleukin-1beta /interferon-gamma -mediated induction of nuclear factor kappa B-dependent nitrite production and apoptosis in beta cells

Insulin-dependent diabetes mellitus is an autoimmune disease in which pancreatic islet beta cells are destroyed by a combination of immunological and inflammatory mechanisms. In particular, cytokine-induced production of nitric oxide has been shown to correlate with beta cell apoptosis and/or inhibition of insulin secretion. In the present study, we investigated whether the interleukin (IL)-1beta intracellular signal transduction pathway could be blocked by overexpression of dominant negative forms of the IL-1 receptor interacting protein MyD88. We show that overexpression of the Toll domain or the lpr mutant of MyD88 in betaTc-Tet cells decreased nuclear factor kappaB (NF-kappaB) activation upon IL-1beta and IL-1beta/interferon (IFN)-gamma stimulation. Inducible nitric oxide synthase mRNA accumulation and nitrite production, which required the simultaneous presence of IL-1beta and IFN-gamma, were also suppressed by approximately 70%, and these cells were more resistant to cytokine-induced apoptosis as compared with parental cells. The decrease in glucose-stimulated insulin secretion induced by IL-1beta and IFN-gamma was however not prevented. This was because these dysfunctions were induced by IFN-gamma alone, which decreased cellular insulin content and stimulated insulin exocytosis. These results demonstrate that IL-1beta is involved in inducible nitric oxide synthase gene expression and induction of apoptosis in mouse beta cells but does not contribute to impaired glucose-stimulated insulin secretion. Furthermore, our data show that IL-1beta cellular actions can be blocked by expression of MyD88 dominant negative proteins and, finally, that cytokine-induced beta cell secretory dysfunctions are due to the action of IFN-gamma.     

Characterization of a dipeptide motif regulating IFN-gamma receptor 2 plasma membrane accumulation and IFN-gamma responsiveness

The IFN-gammaR complex is composed of two IFN-gammaR1 and two IFN-gammaR2 polypeptide chains. Although IFN-gammaR1 is constitutively expressed on all nucleated cells, IFN-gammaR2 membrane display is selective and tightly regulated. We created a series of fluorescent-tagged IFN-gammaR2 expression constructs to follow the molecule's cell surface expression and intracellular distribution. Truncation of the receptor immediately upstream of Leu-Ile 255-256 (254X) created a receptor devoid of signaling that overaccumulated on the cell surface. In addition, this truncated receptor inhibited wild-type IFN-gammaR2 activity and therefore exerted a dominant negative effect. In-frame deletion (255Delta2) or alanine substitution (LI255-256AA) of these amino acids created mutants that overaccumulated on the plasma membrane, but had enhanced function. Single amino acid substitutions (L255A or I256A) had a more modest effect. In-frame deletions upstream (253Delta2), but not downstream (257Delta2), of Leu-Ile 255-256 also led to overaccumulation. A truncation within the IFN-gammaR2 Jak2 binding site (270X) led to a mutant devoid of function that did not overaccumulate and did not affect wild-type IFN-gammaR2 signaling. We have created a series of novel mutants of IFN-gammaR2 that have facilitated the identification of intracellular domains that control IFN-gammaR2 accumulation and IFN-gamma responsiveness. In contrast to IFN-gammaR1, not only dominant negative, but also dominant gain-of-function, mutations were created through manipulation of IFN-gammaR2 Leu-Ile 255-256. These IFN-gammaR2 mutants will allow fine dissection of the role of IFN-gamma signaling in immunity.