Cutting edge: IFN-gamma signaling to macrophages is required for optimal Valpha14i NK T/NK cell cross-talk

Activated NK T cells are known to rapidly stimulate NK cells and, subsequently, CD8(+) T cells and B cells. In this report, we first demonstrate that the downstream effects induced by alpha-galactosylceramide activated NK T cells on NK cells are mainly dependent on IFN-gamma. We found that NK T cell activation of NK cells requires a functional IFN-gamma signaling in macrophages and dendritic cells but not in B cells, NK cells, or NK T cells. NK T cell activation is dendritic cell-dependent whereas NK T cell activation of NK cells is indirect and in part mediated by macrophages. Interestingly, in this context, macrophage participation in the CD1d Ag presentation of alpha-galactosylceramide to NK T cells is not necessary. These data indicate that NK T cell-dependent activation of macrophages is required for optimal NK T cell-induced stimulation of NK cells.     

Impaired NK cell development in an IFN-gamma transgenic mouse: aberrantly expressed IFN-gamma enhances hematopoietic stem cell apoptosis and affects NK cell differentiation

Aberrant expression of IFN-gamma has been demonstrated to cause a wide variety of alterations in cell function and development. Previously we reported that constitutive expression of IFN-gamma in bone marrow (BM) and thymus results in a total absence of B cells and a substantial decrease in the number of hematopoietic progenitor cells. In this study, we demonstrate a severe deficiency of NK1.1(+)CD3(-) cells in this transgenic mouse model. Compared with normal control littermates, we found a pronounced reduction of NK cells in IFN-gamma transgenic mouse spleen and liver despite maintenance of normal function. In addition, we observed a reduced number of BM cells in the IFN-gamma transgenic mouse despite normal expression of hematopoietic growth factors in the BM. Interestingly, these cells were less responsive to stem cell factor (SCF) despite c-kit expression on hematopoietic stem cells (HSCs). We observed that addition of exogenous IFN-gamma inhibited proliferation of HSCs and differentiation of NK precursors from HSCs in normal mice in response to SCF, IL-7, fms-like tyrosine kinase 3 ligand, and IL-15. Furthermore, we found that HSCs express the IFN-gammaRalpha subunit and undergo apoptosis in response to exogenous IFN-gamma. Thus, we have demonstrated the occurrence of a severe deficiency of NK cells and lower numbers of BM cells in an IFN-gamma transgenic mouse model. Furthermore, because exogenous IFN-gamma affects the responsiveness to hematopoietic growth factors such as SCF in vitro, our results indicate that chronic expression of IFN-gamma in vivo leads to widespread immune system defects, including alterations in NK cell differentiation.     

IFN-gamma acts on T cells to induce NK cell mobilization and accumulation in target organs

The mechanism(s) that regulates NK cell mobilization and the significance of this process to NK cell activity are unknown. After Con A-induced hepatitis, NK cells are mobilized from the spleen and bone marrow into the periphery in an IFN-gamma-dependent fashion. Intraperitoneal administration of IFN-gamma stimulates the mobilization of NK cells into the circulation, but not their cell death or proliferation. Increased number of circulating NK cells was coupled with their accumulation in the peritoneum, liver, and tumor-bearing lung tissue. Furthermore, increased number of NK cells in the lung reduced metastasis of Lewis lung carcinoma cells (3LL cell line) resulting in significantly extended NK-dependent survival. Mobilization of NK cells was specific and required the presence of T cells. Moreover, mobilization and migration of spleen NK cells in response to IFN-gamma treatment is dependent on the chemokine receptor CXCR3. Mechanistic insights regarding the role of IFN-gamma in the regulation of NK cell mobilization and their accumulation at sites of tumor metastasis may lead to the development of novel immunotherapy for cancer.     

Inhibition of NF-kappa B activity in T and NK cells results in defective effector cell expansion and production of IFN-gamma required for resistance to Toxoplasma gondii

To define the role of NF-kappa B in the development of T cell responses required for resistance to Toxoplasma gondii, mice in which T cells are transgenic for a degradation-resistant (Delta N) form of I kappa B alpha, an inhibitor of NF-kappa B, were challenged with T. gondii and their response to infection compared with control mice. I kappa B alpha(Delta N)-transgenic (Tg) mice succumbed to T. gondii infection between days 12 and 35, and death was associated with an increased parasite burden compared with wild-type (Wt) controls. Analysis of the responses of infected mice revealed that IL-12 responses were comparable between strains, but Tg mice had a marked reduction in systemic levels of IFN-gamma, the major mediator of resistance to T. gondii. In addition, the infection-induced increase in NK cell activity observed in Wt mice was absent from Tg mice and this correlated with NK cell expression of the transgene. Infection-induced activation of CD4(+) T cells was similar in Wt and Tg mice, but expansion of activated CD4(+)T cells was markedly reduced in the Tg mice. This difference in T cell numbers correlated with a reduced capacity of these cells to proliferate after stimulation and was associated with a major defect in the ability of CD4(+) T cells from infected mice to produce IFN-gamma. Together, these studies reveal that inhibition of NF-kappa B activity in T and NK cells results in defective effector cell expansion and production of IFN-gamma required for resistance to T. gondii.     

Enhancing effect of IFN-gamma on helper T cell activity and IL 2 production

A single injection of young murine immune interferon (IFN-gamma) in young (3 mo) or old (14 to 24 mo) mice 3 days before carrier-priming significantly enhances helper T cell activity of their spleen cells. Maximal enhancement is attained when IFN-gamma is injected once immediately before priming or for 4 consecutive days from the time of priming. Helper activity for anti-TNP antibody response was titrated in vitro by adding graded numbers of spleen cells from HRBC-primed mice of a given age to cultures containing a constant number of spleen cells from 3-mo-old normal mice and TNP-HRBC. When T cell-enriched spleen cells from HRBC-primed young or old mice, uninjected or injected with IFN-gamma, were separated by nylon wool filtration into passed (Thi) and adherent (Th2) cells, the helper activity of both T cell subpopulations was found to be enhanced by IFN-gamma injection. Helper activity of purified Th1 and Th2 cells was also increased by their in vitro preincubation with IFN-gamma. Furthermore, interleukin 2 (IL 2) production by mitogen-activated spleen cells from young and old mice is enhanced by addition of IFN-gamma to cultures. These data altogether indicate that IFN-gamma plays an important role in immunoregulation of helper T cell activity.     

Differential requirement for the SAP-Fyn interaction during NK T cell development and function

The adaptor molecule SAP (signaling lymphocytic activation molecule-associated protein) plays a critical role during NK T (NKT) cell development in humans and mice. In CD4(+) T cells, SAP interacts with the tyrosine kinase Fyn to deliver signals required for TCR-induced Th2-type cytokine production. To determine whether the SAP-dependent signals controlling NKT cell ontogeny rely on its binding to Fyn, we used the OP9-DL1 system to initiate structure function studies of SAP in murine NKT cell development. In cultures containing wild-type (WT) hematopoietic progenitors, we noted the transient emergence of cells that reacted with the NKT cell-specific agonist alpha-galactosyl ceramide and its analog PBS57. Sap(-/-) cells failed to give rise to NKT cells in vitro; however, their development could be rescued by re-expression of WT SAP. Emergence of NKT cells was also restored by a mutant version of SAP (SAP R78A) that cannot bind to Fyn, but with less efficiency than WT SAP. This finding was accentuated in vivo in Sap(R78A) knock-in mice as well as Sap(R78A) competitive bone marrow chimeras, which retained NKT cells but at significantly reduced numbers compared with controls. Unlike Sap(R78A) CD4(+) T cells, which produce reduced levels of IL-4 following TCR ligation, alpha-galactosyl ceramide-stimulated NKT cells from the livers and spleens of Sap(R78A) mice produced Th2 cytokines and activated NK cells in a manner mimicking WT cells. Thus, SAP appears to use differential signaling mechanisms in NKT cells, with optimal ontogeny requiring Fyn binding, while functional responses occur independently of this interaction.     

Restoration of NK T cell development in fyn-mutant mice by a TCR reveals a requirement for Fyn during early NK T cell ontogeny

NK T cells are a unique lymphocyte population that have developmental requirements distinct from conventional T cells. Mice lacking the tyrosine kinase Fyn have 5- to 10-fold fewer mature NK T cells. This study shows that Fyn-deficient mice have decreased numbers of NK1.1(-) NK T cell progenitors as well. 5-Bromo-2'-deoxyuridine-labeling studies indicate that the NK T cells remaining in fyn(-/-) mice exhibit a similar turnover rate as wild-type cells. The fyn(-/-) NK T cells respond to alpha-galactosylceramide, a ligand recognized by NK T cells, and produce cytokines, but have depressed proliferative capacity. Transgenic expression of the NK T cell-specific TCR alpha-chain Valpha14Jalpha18 leads to a complete restoration of NK T cell numbers in fyn(-/-) mice. Together, these results suggest that Fyn may have a role before alpha-chain rearrangement rather than for positive selection or the peripheral upkeep of cell number. NK T cells can activate other lymphoid lineages via cytokine secretion. These secondary responses are impaired in Fyn-deficient mice, but occur normally in fyn mutants expressing the Valpha14Jalpha18 transgene. Because this transgene restores NK T cell numbers, the lack of secondary lymphocyte activation in the fyn-mutant mice is due to the decreased numbers of NK T cells present in the mutant, rather than an intrinsic defect in the ability of the other fyn(-/-) lymphoid populations to respond.     

T cell subsets and IFN-gamma production in resistance to systemic candidosis in immunized mice

In addition to previous evidence for the roles of T cell-dependent immunity and delayed-type hypersensitivity in acquired resistance to systemic candidosis in mice, in the present study we have investigated the relative contributions of L3T4+ and Lyt-2+ lymphocytes in the protective immunity induced by vaccination with low virulence Candida albicans cells. We have also addressed the issue of the mode of Candida Ag recognition by specific T cells leading to cytokine release. Spleen cells from immunized mice produced high levels of IFN-gamma in vitro in response to Candida Ag, and this activity was abolished only by the combined treatment of the responder population with anti-L3T4 and anti-Lyt-2.2 mAb plus C. Positively selected L3T4+ and Lyt-2+ cells also produced IFN-gamma in vitro, provided accessory cells (plastic-adherent and Thy-1- Ia- splenocytes, respectively) were added to the lymphocyte-yeast cell cocultures. The production of IFN-gamma by purified L3T4+ and Lyt-2+ cells was inhibited by addition of the respective anti-class II and anti-class I H-2 antibody to the cultures. In vivo, administration of anti-L3T4, anti-Lyt-2.2 mAb or a combination of both significantly impaired the resistance of immunized mice to challenge with virulent C. albicans, as manifested by increased recovery of the yeast from the mouse kidneys. A similar effect was observed upon neutralization of endogenous IFN-gamma by treatment with rat mAb. These results suggest that both L3T4+ and Lyt-2+ T cells play a role in acquired immunity to systemic C. albicans infection, and that their activity may involve IFN-gamma-mediated stimulation of candidacidal mechanisms.     

IL-21 in synergy with IL-15 or IL-18 enhances IFN-gamma production in human NK and T cells

NK and T cell-derived IFN-gamma is a key cytokine that stimulates innate immune responses and directs adaptive T cell response toward Th1 type. IL-15, IL-18, and IL-21 have significant roles as activators of NK and T cell functions. We have previously shown that IL-15 and IL-21 induce the expression of IFN-gamma, T-bet, IL-12R beta 2, and IL-18R genes both in NK and T cells. Now we have studied the effect of IL-15, IL-18, and IL-21 on IFN-gamma gene expression in more detail in human NK and T cells. IL-15 clearly activated IFN-gamma mRNA expression and protein production in both cell types. IL-18 and IL-21 enhanced IL-15-induced IFN-gamma gene expression. IL-18 or IL-21 alone induced a modest expression of the IFN-gamma gene but a combination of IL-21 and IL-18 efficiently up-regulated IFN-gamma production. We also show that IL-15 activated the binding of STAT1, STAT3, STAT4, and STAT5 to the regulatory sites of the IFN-gamma gene. Similarly, IL-21 induced the binding of STAT1, STAT3, and STAT4 to these elements. IL-15- and IL-21-induced STAT1 and STAT4 activation was verified by immunoprecipitation with anti-phosphotyrosine Abs followed by Western blotting with anti-STAT1 and anti-STAT4 Abs. IL-18 was not able to induce the binding of STATs to IFN-gamma gene regulatory sites. IL-18, however, activated the binding of NF-kappa B to the IFN-gamma promoter NF-kappa B site. Our results suggest that both IL-15 and IL-21 have an important role in activating the NK cell-associated innate immune response.     

Interleukin 2-mediated immune interferon (IFN-gamma) production by human T cells and T cell subsets

Human interleukin 2 (IL 2, or T cell growth factor), which was free of lectin and interferon activity (IFN), induced human peripheral T lymphocytes to produce immune IFN (IFN-gamma). In contrast, non-T cells and macrophages did not produce IFN-gamma in response to IL 2. IL 2 acted directly on unstimulated T cells to induce IFN-gamma production, and also acted in synergy with a suboptimal dose (2 micrograms/ml) of concanavalin A (Con A) to enhance IFN-gamma production. The IFN-gamma-inducing activity of partially purified IL 2 was absorbed along with the IL 2 activity by murine IL 2-dependent CT-6 cell line cells. This further supports the view that IFN-gamma-inducing activity is identical to IL 2. When T cells were separated further into helper/inducer T4+ and suppressor/cytotoxic T8+ subsets by negative selection with monoclonal antibody and complement, both T4+ and T8+-enriched cells produced significant levels of IFN-gamma in response to IL 2. Complete removal of macrophages from purified T lymphocyte populations by treatment of OKM1 plus complement consistently reduced IFN-gamma production in response to IL 2 to a limited degree; readdition of macrophages restored IFN-gamma production by both T cell subsets. This observation that IL 2 contributes to the production of IFN-gamma by human lymphocytes suggests that a cascade of lymphocyte-cell interactions participates in human immune responses.     

Type I IFN substitutes for T cell help during viral infections

Certain virus infections depend on the presence of T cell help for the generation of primary CD8(+) T cell responses. However, the mechanisms that render these particular viral infections T cell help dependent is largely unknown. In this study, we compared CD8(+) T cell responses elicited by lymphocytic choriomeningitis virus infection, as prototype of a T cell help independent infection, with T cell help dependent CD8(+) T cell responses induced by vaccinia virus infection. In this paper, we show that a key parameter decisive for T cell help independence is the ability of an infectious agent to stimulate early and robust production of type I IFN. Experimental provision of type I IFN during VV infection rendered the ensuing CD8(+) T cell response completely T cell help independent. Our results support a model in which type I IFN has to be present during the first 3 d of Ag encounter and has to act directly on the responding CD8(+) T cells to promote their survival and effector differentiation. We show that type I IFN signaling on responding CD8(+) T cells induces profound upregulation of CD25 and increased IL-2 expression; however, neither this nor IL-15 accounts for the type I IFN effects on responding CD8(+) T cells. Thus, type I IFN can effectively replace the requirement of T cell help by directly promoting CD8(+) T cell survival and differentiation independent of the type I IFN-induced cytokines IL-2 and IL-15.     

TL1A synergizes with IL-12 and IL-18 to enhance IFN-gamma production in human T cells and NK cells

TL1A, a recently described TNF-like cytokine that interacts with DR3, costimulates T cells and augments anti-CD3 plus anti-CD28 IFN-gamma production. In the current study we show that TL1A or an agonistic anti-DR3 mAb synergize with IL-12/IL-18 to augment IFN-gamma production in human peripheral blood T cells and NK cells. TL1A also enhanced IFN-gamma production by IL-12/IL-18 stimulated CD56(+) T cells. When expressed as fold change, the synergistic effect of TL1A on cytokine-induced IFN-gamma production was more pronounced on CD4(+) and CD8(+) T cells than on CD56(+) T cells or NK cells. Intracellular cytokine staining showed that TL1A significantly enhanced both the percentage and the mean fluorescence intensity of IFN-gamma-producing T cells in response to IL-12/IL-18. The combination of IL-12 and IL-18 markedly up-regulated DR3 expression in NK cells, whereas it had minimal effect in T cells. Our data suggest that TL1A/DR3 pathway plays an important role in the augmentation of cytokine-induced IFN-gamma production in T cells and that DR3 expression is differentially regulated by IL-12/IL-18 in T cells and NK cells.     

Prostaglandin D2 inhibits the production of IFN-gamma by invariant NK T cells: consequences in the control of B16 melanoma

Invariant NK T (iNKT) cells are a subset of innate/memory lymphocytes that recognize lipid Ags presented by CD1d-expressing APCs such as dendritic cells (DCs). Upon primary stimulation through their TCR, iNKT cells promptly produce large amounts of IFN-gamma and/or IL-4 that play critical roles in the regulation of innate and adaptive immune responses. To date, the role of environmental factors on iNKT cell functions has been poorly investigated. In this study, we addressed the question of whether PGD2, a potent eicosanoid lipid mediator involved in immune responses and inflammation, could be important in DC/iNKT cell cross-talk. We show that PGD2 dramatically reduced the production of IFN-gamma, but not IL-4, by iNKT cells in response to the superagonist alpha-galactosylceramide (alpha-GalCer) both in vitro and in vivo. This effect is mediated by the D prostanoid receptor 1 (DP1) expressed by DCs and iNKT cells and requires protein kinase A activation. We also report that PGD2 and BW245C (a selective DP1 agonist) reduce the protective effects of alpha-GalCer in B16F10-induced melanoma metastasis, an effect that depends on IFN-gamma production by iNKT cells. As a whole, these data reveal novel pathways regulating iNKT cell biologic functions and confirm the immunoregulatory roles of PGD2 on the innate response.     

Coordinated and distinct roles for IFN-alpha beta,IL-12, and IL-15 regulation of NK cell responses to viral infection

NK cell cytotoxicity, IFN-gamma expression, proliferation, and accumulation are rapidly induced after murine CMV infections. Under these conditions, the responses were shown to be elicited in overlapping populations. Nevertheless, there were distinct signaling molecule requirements for induction of functions within the subsets. IL-12/STAT4 was critical for NK cell IFN-gamma expression, whereas IFN-alphabeta/STAT1 were required for induction of cytotoxicity. The accumulation/survival of proliferating NK cells was STAT4-independent but required IFN-alphabeta/STAT1 induction of IL-15. Taken together, the results define the coordinated interactions between the cytokines IFN-alphabeta, IL-12, and IL-15 for activation of protective NK cell responses during viral infections, and emphasize these factors' nonredundant functions under in vivo physiological conditions.     

Paradoxical roles for programmed cell death signaling during viral infection of the central nervous system

Programmed cell death (PCD) is an essential mechanism of antimicrobial defense. Recent work has revealed an unexpected diversity in the types of PCD elicited during infection, as well as defined unique roles for different PCD modalities in shaping the immune response. Here, we review recent work describing unique ways in which PCD signaling operates within the infected central nervous system (CNS). These studies reveal striking complexity in the regulation of PCD signaling by CNS cells, including both protective and pathological outcomes in the control of infection. Studies defining the specialized molecular mechanisms shaping PCD responses in the CNS promise to yield much needed new insights into the pathogenesis of neuroinvasive viral infection, informing future therapeutic development.     

T cell phenotype and intracellular IFN-gamma production in peritoneal exudate cells and gut intraepithelial lymphocytes during acute Toxoplasma gondii infection in mice

Although there are many reports on the splenic (systemic) T cell response after Toxoplasma gondii infection, little information is available regarding the local T cell responses of peritoneal exudate cells (PEC) and gut intraepithelial lymphocytes (IEL) following peroral infection with bradyzoites. Mice were infected with 40 cysts of the 76K strain of T. gondii, and then sacrificed at days 0, 1, 4, 7 and 10 postinfection (PI). The cellular composition and T cell responses of PEC and IEL were analyzed. The total number of PEC and IEL per mouse increased after infection, but the ratio of increase was higher in IEL. Lymphocytes were the major component of both PEC and IEL. The relative percentages of PEC macrophages and neutrophils/eosinophils increased significantly at day 1 and 4 PI, whereas those of IEL did not change significantly. The percentage of PEC NK1.1 and gamma delta T cells peaked at day 4 PI (p < 0.0001), and CD4 and CD8 alpha T cells increased continuously after infection. The percentages of IEL CD8 alpha and gamma delta T cells decreased slightly at first, and then increased. CD4 and NK1.1 T cells of IEL did not change significantly after infection. IFN-gamma-producing PEC NK1.1 T cells increased significantly from day 1 PI, but the other T cell subsets produced IFN-gamma abundantly thereafter. The proportion of IEL IFN-gamma-producing CD8 alpha and gamma delta T cells increased significantly after infection, while IEL NK1.1 T cells had similar IFN-gamma production patterns. Taken together, CD4 T cells were the major phenotype and the important IFN-gamma-producing T cell subsets in PEC after oral infection with T. gondii, whereas CD8 alpha T cells had these roles in IEL. These results suggest that PEC and IEL comprise different cell differentials and T cell responses, and according to infection route these factors may contribute to the different cellular immune responses.     

The roles of IL-10 and TGF-beta in controlling IL-4 and IFN-gamma production during experimental Fasciola hepatica infection

Hosts infected with Fasciola hepatica experience immunosuppression during the acute and chronic phases of the disease. This immunosuppression may allow parasite survival in the face of an ongoing immune response. In bovine hosts early IL-4 and continued IgG1 production is one of the few remaining features of the characteristic type 0/2 helper (Th0/2) response present in the chronic stage of disease. Here we demonstrate elevated levels of parasite-specific, in vitro peripheral blood mononuclear cell (PBMC)-derived transforming growth factor (TGF)-β1 from the early phases of infection and increasing levels of IL-10 as the infection becomes chronic. In vitro neutralisation of these cytokines during culture of PBMCs from experimentally-infected cattle increased IL-4 and IFN-γ production in response to parasite-specific and non-specific stimulation. At 4 weeks p.i. neutralisation of TGF-β results in an increase in parasite driven IL-4, while also having a greater role, compared with IL-10, in influencing specific and non-specific IFN-γ. At 12 weeks p.i. ex vivo parasite driven IL-4 was not restored by inhibiting either IL-10 or TGF-β. However IL-10 influenced both parasite-specific and non-specific IFN-γ production at this time. This highlights the roles of IL-10 and TGF-β in fasciolosis, however the cellular sources of these have yet to be defined. This suggests that suppression of IFN-γ production by parasite molecules occurs during infection and it is possible that the suppression of IFN-γ production may mediate parasite survival in this disease.