Blockade of immunosuppressive cytokines restores NK cell antiviral function in chronic hepatitis B virus infection

NK cells are enriched in the liver, constituting around a third of intrahepatic lymphocytes. We have previously demonstrated that they upregulate the death ligand TRAIL in patients with chronic hepatitis B virus infection (CHB), allowing them to kill hepatocytes bearing TRAIL receptors. In this study we investigated whether, in addition to their pathogenic role, NK cells have antiviral potential in CHB. We characterised NK cell subsets and effector function in 64 patients with CHB compared to 31 healthy controls. We found that, in contrast to their upregulated TRAIL expression and maintenance of cytolytic function, NK cells had a markedly impaired capacity to produce IFN-γ in CHB. This functional dichotomy of NK cells could be recapitulated in vitro by exposure to the immunosuppressive cytokine IL-10, which was induced in patients with active CHB. IL-10 selectively suppressed NK cell IFN-γ production without altering cytotoxicity or death ligand expression. Potent antiviral therapy reduced TRAIL-expressing CD56(bright) NK cells, consistent with the reduction in liver inflammation it induced; however, it was not able to normalise IL-10 levels or the capacity of NK cells to produce the antiviral cytokine IFN-γ. Blockade of IL-10 +/- TGF-β restored the capacity of NK cells from both the periphery and liver of patients with CHB to produce IFN-γ, thereby enhancing their non-cytolytic antiviral capacity. In conclusion, NK cells may be driven to a state of partial functional tolerance by the immunosuppressive cytokine environment in CHB. Their defective capacity to produce the antiviral cytokine IFN-γ persists in patients on antiviral therapy but can be corrected in vitro by IL-10+/- TGF-β blockade.     

Negative regulation of Schistosoma japonicum egg-induced liver fibrosis by natural killer cells

The role of natural killer (NK) cells in infection-induced liver fibrosis remains obscure. In this study, we elucidated the effect of NK cells on Schistosoma japonicum (S. japonicum) egg-induced liver fibrosis. Liver fibrosis was induced by infecting C57BL/6 mice with 18-20 cercariae of S. japonicum. Anti-ASGM1 antibody was used to deplete NK cells. Toll-like receptor 3 ligand, polyinosinic-polycytidylic acid (poly I:C) was used to enhance the activation of NK cells. Results showed that NK cells were accumulated and activated after S. japonicum infection, as evidenced by the elevation of CD69 expression and IFN-γ production. Depletion of NK cells markedly enhanced S. japonicum egg-induced liver fibrosis. Administration of poly I:C further activated NK cells to produce IFN-γ and attenuated S. japonicum egg-induced liver fibrosis. The observed protective effect of poly I:C on liver fibrosis was diminished through depletion of NK cells. Disruption of IFN-γ gene enhanced liver fibrosis and partially abolished the suppression of liver fibrosis by poly I:C. Moreover, expression of retinoic acid early inducible 1 (RAE 1), the NKG2D ligand, was detectable at high levels on activated hepatic stellate cells derived from S. japonicum-infected mice, which made them more susceptible to hepatic NK cell killing. In conclusion, our findings suggest that the activated NK cells in the liver after S. japonicum infection negatively regulate egg-induced liver fibrosis via producing IFN-γ, and killing activated stellate cells.     

Synergy between TLR3 and IL-18 promotes IFN-γ dependent TRAIL expression in human liver NK cells

Natural killer (NK) cells are a component of innate immunity against viral infections through their rapid cytotoxic activity and cytokine production. However, intra-hepatic NK cells’ ability to respond to virus is still mostly unknown. Our results show that the synthetic dsRNA polyinosinic–polycytidylic acid (poly I:C), a mimic of a common product of viral infections, activates NK cells directly in the context of cytokines found in the liver, i.e.: poly I:C plus inflammatory cytokines (IL-18, IL-12, and IL-2) induced NK cell IFN-γ production and TRAIL expression, and anti-inflammatory cytokines (TGF-β and IL-10) inhibit NK cell IFN-γ production. Neutralization of IFN-γ blocks poly I:C plus inflammatory cytokines-induced NK cell TRAIL expression, suggesting that IFN-γ is an autocrine differentiation factor for these cells. A better understanding of the intra-hepatic NK cell activation against viral infection may help in the design of therapies and vaccines for the control of viral hepatitis.     

in vitro generation of IFN-γ-producing Listeria-specific T cells is dependent on IFN-γ production by non-NK cells

In vitro 5-day cultures of naive spleen cells with viable Listeria monocytogenes (VLM), but not heat-killed L. monocytogenes, induced CD4⁺ T cells that produced IFN-γ upon secondary antigen stimulation. The VLM-induced Listeria-specific T cells produced IFN-γ but lacked expression of IL-2 and IL-4. To study the role of IFN-γ in the induction of the IFN-γ-producing T cells, we added anti-IFN-γ mAb to the primary culture and analyzed IFN-γ production upon secondary antigen stimulation. Addition of anti-IFN-γ mAb to the culture suppressed generation of IFN-γ-producing CD4⁺ T cells, suggesting that IFN-γ is important in the induction of IFN-γ-producing CD4⁺ T cells. Furthermore, our results showed that depletion of NK cells from spleen cells by anti-asialo GM1 antibody plus complement before culture enhanced induction of IFN-γ-producing CD4⁺ T cells. Although NK cells are known to produce IFN-γ, the results indicate that NK cell-derived IFN-γ may not be important in induction of the Listeria-specific IFN-γ-producing CD4⁺ T cells in the culture system. In addition, we demonstrated that IFN-γ expression was high in CD4⁺ T cells from cultures of spleen cells with VLM at the primary culture level. These results suggest that IFN-γ derived from T cells may enhance production of IFN-γ by CD4⁺ T cells, while NK cells rather suppress the induction of IFN-γ-producing CD4⁺ T cells.     

Loss of NK stimulatory capacity by plasmacytoid and monocyte-derived DC but not myeloid DC in HIV-1 infected patients

Dendritic cells (DC) are potent inducers of natural killer (NK) cells. There are two distinct populations in blood, myeloid (mDC) and plasmacytoid (pDC) but they can also be generated In vitro from monocytes (mdDC). Although it is established that blood DC are lost in HIV-1 infection, the full impact of HIV-1 infection on DC-NK cell interactions remains elusive. We thus investigated the ability of pDC, mDC, and mdDC from viremic and anti-retroviral therapy-treated aviremic HIV-1+ patients to stimulate various NK cell functions. Stimulated pDC and mdDC from HIV-1+ patients showed reduced secretion of IFN-α and IL-12p70 respectively and their capacity to stimulate expression of CD25 and CD69, and IFN-γ secretion in NK cells was also reduced. pDC activation of NK cell degranulation in response to a tumour cell line was severely reduced in HIV-1+ patients but the ability of mDC to activate NK cells was not affected by HIV-1 infection, with the exception of HLA-DR induction. No differences were observed between viremic and aviremic patients indicating that anti-retroviral therapy had minimal effect on restoration on pDC and mdDC-mediated activation of NK cells. Results from this study provide further insight into HIV-1 mediated suppression of innate immune functions.     

IFN-γ-producing CD4+ T cells promote experimental cerebral malaria by modulating CD8+ T cell accumulation within the brain

It is well established that IFN-γ is required for the development of experimental cerebral malaria (ECM) during Plasmodium berghei ANKA infection of C57BL/6 mice. However, the temporal and tissue-specific cellular sources of IFN-γ during P. berghei ANKA infection have not been investigated, and it is not known whether IFN-γ production by a single cell type in isolation can induce cerebral pathology. In this study, using IFN-γ reporter mice, we show that NK cells dominate the IFN-γ response during the early stages of infection in the brain, but not in the spleen, before being replaced by CD4(+) and CD8(+) T cells. Importantly, we demonstrate that IFN-γ-producing CD4(+) T cells, but not innate or CD8(+) T cells, can promote the development of ECM in normally resistant IFN-γ(-/-) mice infected with P. berghei ANKA. Adoptively transferred wild-type CD4(+) T cells accumulate within the spleen, lung, and brain of IFN-γ(-/-) mice and induce ECM through active IFN-γ secretion, which increases the accumulation of endogenous IFN-γ(-/-) CD8(+) T cells within the brain. Depletion of endogenous IFN-γ(-/-) CD8(+) T cells abrogates the ability of wild-type CD4(+) T cells to promote ECM. Finally, we show that IFN-γ production, specifically by CD4(+) T cells, is sufficient to induce expression of CXCL9 and CXCL10 within the brain, providing a mechanistic basis for the enhanced CD8(+) T cell accumulation. To our knowledge, these observations demonstrate, for the first time, the importance of and pathways by which IFN-γ-producing CD4(+) T cells promote the development of ECM during P. berghei ANKA infection.     

Gamma interferon and perforin control the strength, but not the hierarchy, of immunodominance of an antiviral CD8+ T cell response

The two major antiviral effector mechanisms of CD8(+) T cells are thought to be perforin (Prf)-mediated cell lysis and gamma interferon (IFN-γ)-mediated induction of an antiviral state. By affecting the expression of proteins involved in antigen presentation, IFN-γ is also thought to shape the magnitude and specificity of the CD8(+) T cell response. Here we studied the roles of Prf and IFN-γ in shaping the effector and memory CD8(+) T cell responses to vaccinia virus (VACV). IFN-γ deficiency resulted in increased numbers of anti-VACV effector and memory CD8(+) T cells, which were partly dependent on increased virus loads. On the other hand, Prf-deficient mice showed an increase in the number of VACV-specific CD8(+) T cells only in the memory phase. Treatment of the mice with the antiviral drug cidofovir reduced the numbers of effector and memory cells closer to wild-type levels in IFN-γ-deficient mice and reduced the numbers of memory CD8(+) T cells to wild-type levels in Prf-deficient mice. These data suggest that virus loads are the main reason for the increased strength of the CD8 response in IFN-γ- and Prf-deficient mice. Neither Prf deficiency nor IFN-γ deficiency had an effect on the immunodominance hierarchy of five K(b)-restricted CD8(+) T cell determinants either during acute infection or after recovery. Thus, our work shows that CD8(+) T cell immunodominance during VACV infection is not affected by the effects of IFN-γ on the antigen presentation machinery.     

IL-12 enhances the antitumor actions of trastuzumab via NK cell IFN-γ production

The antitumor effects of therapeutic mAbs may depend on immune effector cells that express FcRs for IgG. IL-12 is a cytokine that stimulates IFN-γ production from NK cells and T cells. We hypothesized that coadministration of IL-12 with a murine anti-HER2/neu mAb (4D5) would enhance the FcR-dependent immune mechanisms that contribute to its antitumor activity. Thrice-weekly therapy with IL-12 (1 μg) and 4D5 (1 mg/kg) significantly suppressed the growth of a murine colon adenocarcinoma that was engineered to express human HER2 (CT-26(HER2/neu)) in BALB/c mice compared with the result of therapy with IL-12, 4D5, or PBS alone. Combination therapy was associated with increased circulating levels of IFN-γ, monokine induced by IFN-γ, and RANTES. Experiments with IFN-γ-deficient mice demonstrated that this cytokine was necessary for the observed antitumor effects of therapy with IL-12 plus 4D5. Immune cell depletion experiments showed that NK cells (but not CD4(+) or CD8(+) T cells) mediated the antitumor effects of this treatment combination. Therapy of HER2/neu-positive tumors with trastuzumab plus IL-12 induced tumor necrosis but did not affect tumor proliferation, apoptosis, vascularity, or lymphocyte infiltration. In vitro experiments with CT-26(HER2/neu) tumor cells revealed that IFN-γ induced an intracellular signal but did not inhibit cellular proliferation or induce apoptosis. Taken together, these data suggest that tumor regression in response to trastuzumab plus IL-12 is mediated through NK cell IFN-γ production and provide a rationale for the coadministration of NK cell-activating cytokines with therapeutic mAbs.     

Phagocytosis, a potential mechanism for myeloid-derived suppressor cell regulation of CD8+ T cell function mediated through programmed cell death-1 and programmed cell death-1 ligand interaction

CD8(+) T cells become exhausted, inducing cell surface protein programmed cell death-1 (PD-1) as chronic virus diseases or tumors progress, but underlying mechanisms of this are unclear. We previously showed that M-CSF is important for developing tolerogenic dendritic cells (DCs) from human CD14(+) monocytes. In this article, we identify M-CSF-derived DCs (M-DCs) after stimulation with IL-10 as myeloid-derived suppressor cells with additional tolerogenic activities to CD8(+) T cells. IL-10 increased PD-1 ligand expression on M-DC, and IL-10-stimulated M-DCs (M-DC/IL-10) induced expression of PD-1 on, and apoptosis of, CD8(+) T cells and phagocytosed CD8(+) T cells. Enhanced phagocytic activity of M-DC/IL-10 required IFN-γ, which further increased PD-1 ligand and PD-2 ligand expression on M-DC/IL-10. IFN-γ-stimulated M-DC/IL-10 cells were phenotypically macrophage-like cells with little or no expression of CD86, a costimulatory molecule, but with high expression levels of CD14, CD200R, and CD80. No phagocytic activity was detected with GM-CSF-derived DCs. We propose that phagocytosis by IFN-γ-stimulated M-DC/IL-10 cells, which may be DCs or, alternatively, a unique subset of macrophages, may be a mechanism by which IFN-γ-producing CD8(+) T cells are tolerized after type 1 immune responses to chronic virus or tumor, and that IFN-γ links effector CD8(+) T cells to their phagocytic clearance.     

Sequential Involvement of NK Cells and CD8+ T Cells in Granuloma Formation of Rhodococcus aurantiacus-Infected Mice

We investigated the effect of in vivo administration of antibodies against T-cell subsets and natural killer (NK) cells on endogenous gamma interferon (IFN-γ) production and granuloma formation in Rhodococcus aurantiacus-infected mice. High titers of endogenous IFN-γ were detected in the extracts of the livers and spleens during 24 hr of the infection, reaching the peak at 8 hr, and the IFN-γ production was reduced by in vivo administration of anti-NK 1.1 monoclonal antibody (MAb) or antibody against asialo GM1⁺ cells. Endogenous IFN-γ declined until 2 days of the infection, then reappeared from 1 week and peaked at 3 weeks. Endogenous IFN-γ at 1 and 3 weeks was reduced by in vivo administration of anti-CD8 MAb, but not by anti-CD4 MAb or anti-NK 1.1 MAb. Granulomatous lesions in the livers and spleens began to appear from 1 week of the infection and developed in 3 weeks. In vivo administration of rat anti-IFN-γ MAb reduced the development of granulomas. In addition, granuloma formation was reduced by depletion of NK cells prior to the infection or depletion of CD8⁺ T cells at 1 week of the infection. Based on these findings, it is presumed that the biphasic production of IFN-γ is attributable to NK cells in the early phase of the infection and CD8⁺ T cells in the phase of granuloma formation, and that granuloma formation is regulated by NK cells and CD8⁺ T cells through the secretion of endogenous IFN-γ.     

Effects of interleukin-18 on natural killer cells: costimulation of activation through Fc receptors for immunoglobulin

The antitumor activity of monoclonal antibodies is mediated by effector cells, such as natural killer (NK) cells, that express Fc receptors for immunoglobulin. Efficacy of monoclonal antibodies, including the CD20 antibody rituximab, could be improved by agents that augment the function of NK cells. Interleukin (IL)-18 is an immunostimulatory cytokine that has antitumor activity in preclinical models. The effects of IL-18 on NK cell function mediated through Fcγ receptors were examined. Human NK cells stimulated with immobilized IgG in vitro secreted IFN-γ as expected; such IFN-γ production was partially inhibited by blocking CD16 with monoclonal antibodies. IL-18 augmented IFN-γ production by NK cells stimulated with immobilized IgG or CD16 antibodies. NK cell IFN-γ production in response to immobilized IgG and/or IL-18 was inhibited by chemical inhibitors of Syk and several other kinases involved in CD16 signaling pathways. IL-18 augmented antibody-dependent cellular cytotoxicity (ADCC) of human NK cells against rituximab-coated Raji cells in vitro. IL-18 and rituximab acted synergistically to promote regression of human lymphoma xenografts in SCID mice. Inasmuch as IL-18 costimulates IFN-γ production and ADCC of NK cells activated through Fc receptors in vitro and augments antitumor activity of rituximab in vivo, it is an attractive cytokine to combine with monoclonal antibodies for treatment of human cancer.     

Heterogeneous and tissue-specific regulation of effector T cell responses by IFN-gamma during Plasmodium berghei ANKA infection

IFN-γ and T cells are both required for the development of experimental cerebral malaria during Plasmodium berghei ANKA infection. Surprisingly, however, the role of IFN-γ in shaping the effector CD4(+) and CD8(+) T cell response during this infection has not been examined in detail. To address this, we have compared the effector T cell responses in wild-type and IFN-γ(-/-) mice during P. berghei ANKA infection. The expansion of splenic CD4(+) and CD8(+) T cells during P. berghei ANKA infection was unaffected by the absence of IFN-γ, but the contraction phase of the T cell response was significantly attenuated. Splenic T cell activation and effector function were essentially normal in IFN-γ(-/-) mice; however, the migration to, and accumulation of, effector CD4(+) and CD8(+) T cells in the lung, liver, and brain was altered in IFN-γ(-/-) mice. Interestingly, activation and accumulation of T cells in various nonlymphoid organs was differently affected by lack of IFN-γ, suggesting that IFN-γ influences T cell effector function to varying levels in different anatomical locations. Importantly, control of splenic T cell numbers during P. berghei ANKA infection depended on active IFN-γ-dependent environmental signals--leading to T cell apoptosis--rather than upon intrinsic alterations in T cell programming. To our knowledge, this is the first study to fully investigate the role of IFN-γ in modulating T cell function during P. berghei ANKA infection and reveals that IFN-γ is required for efficient contraction of the pool of activated T cells.     

Chronic hepatitis C viral infection reduces NK cell frequency and suppresses cytokine secretion: Reversion by anti-viral treatment

Impaired activity of NK (natural killer) cells has been proposed as a mechanism contributing to viral persistence and chronic infection in hepatitis C (HCV) infection. We aimed to assess the impact of HCV infection on NK cells regarding frequency, subset distribution, and cytotoxic and cytokine secretion functions, as well as IFN-α and ribavirin therapeutic effects on NK cells. Significant reduction of total NK frequency and the CD56^dim16⁺ subset was observed in chronic HCV patients. IFN-γ expression upon stimulation with K562 was severely suppressed but cytotoxicity measured by CD107a expression was maintained. These adverse effects were reversed after treatment with pegylated IFN-α and ribavirin; however, these skewed functions were not recovered in treatment-resistant patients. Thus, HCV chronic infection severely affects NK functions, except for cytotoxicity. Altered NK cell frequency and cytokine secretion by HCV infection may contribute to impaired cellular immune response and virus persistence.     

Characterization of L1210 S Cells with Low Sensitivity to Mouse Interferon-γ

A mouse leukemic cell line L1210 Sg with a low sensitivity to interferon-γ (IFN-γ) was described. On the nature of the antiviral action and binding of IFN, L1210 Sg cells were compared with L1210m cell line which is sensitive to IFN-γ. For a half reduction of the vesicular stomatitis virus-RNA synthesis, L1210 Sg cells required 500–fold more IFN-γ than L1210m cells did. However, both cell lines were induced to the antiviral state to the same extent with IFN-α or -β. L1210 Sg and L1210m cells were sensitive to the anti-proliferative action of IFN-α and -β, but insensitive to IFN-γ. (2′-5′)Oligoadenylate synthetase was induced in these cell lines by IFN-β, but not by IFN-γ, which suggests that the induction of this synthetase may not be responsible for the antiviral action of IFN-γ. No substantial difference between L1210 Sg and L1210m cells was found in IFN receptors for IFN-γ and IFN-β either in number per cell or in their affinity to corresponding IFN type. However, differences were noted in time course profiles of cell-associated IFN-γ at 37 C: in L1210m cells, a rise-and-decay profile of IFN-γ bound to cells was observed at 37 C, but in L1210 Sg cells, rise and slight decay was observed. On the other hand, a similar rise-and-decay curve of IFN-β bound to these cells was observed. These results indicated that the low sensitivity of L1210 Sg cells to IFN-γ may be due to this slight decay of receptor-bound IFN-γ.     

T cell-, interleukin-12-, and gamma interferon-driven viral clearance in measles virus-infected brain tissue

Genetic studies with immunocompetent mice show the importance of both T cells and gamma interferon (IFN-γ) for survival of a measles virus (MV) challenge; however, the direct role of T cells and IFN-γ within the MV-infected brain has not been addressed. Organotypic brain explants represent a successful ex vivo system to define central nervous system (CNS)-specific mechanisms of leukocyte migration, activation, and MV clearance. Within the heterogeneous, brain-derived, primed leukocyte population which reduced MV RNA levels in brain explants by 60%, CD3 T cells are the active antiviral cells, as purified CD3-positive cells are highly antiviral and CD3-negative leukocytes are unable to reduce the viral load. Neutralization of CCL5 and CXCL10 decreases leukocyte migration to areas of infection by 70%. However, despite chemokines directing the migration of T cells to infected neurons, chemokine neutralization revealed that migration is not required for viral clearance, suggesting a cytokine-mediated antiviral mechanism. In accordance with our hypothesis, the ability of leukocytes to clear the virus is abrogated when explants are treated with anti-IFN-γ neutralizing antibodies. IFN-γ applied to infected slices in the absence of primed leukocytes reduces the viral load by more than 80%; therefore, in brain tissue, IFN-γ is both necessary and sufficient to clear MV. Secretion of IFN-γ is stimulated by interleukin-12 (IL-12) in the brain, as neutralization of IL-12 results in loss of antiviral activity and stimulation of leukocytes with IL-12/IL-18 enhances their immune effector function of viral clearance. MV-primed leukocytes can reduce both West Nile and mouse hepatitis viral RNAs, indicating that cytokine-mediated viral clearance occurs in an antigen-independent manner. The IFN-γ signal is transduced within the brain explant by the Jak/STAT signaling pathway, as inhibition of Jak kinases results in a loss of antiviral activity driven by either brain-derived leukocytes or recombinant IFN-γ. These results reveal that primed T cells directly act to clear MV infection of the brain by using a noncytolytic IL-12- and IFN-γ-dependent mechanism in the CNS and that this mechanism relies upon Jak/STAT signaling.