I?B? augments IL-12- and IL-18-mediated IFN-? production in human NK cells.
ABSTRACT: Interferon-? (IFN-?) production by natural killer (NK) cells and cytotoxic lymphocytes is a key component of innate and adaptive immune responses. Because inhibitor of ?B-? (I?B?), a Toll-like receptor (TLR)/interleukin-1 receptor (IL-1R) inducible transcription factor, regulates IFN-? production in KG-1 cells, we tested I?B?'s role in the classic lymphocyte pathway of IL-12/IL-18-induced IFN-?. Upon stimulation with IL-12/IL-18, monocyte-depleted human peripheral blood lymphocytes expressed the 79-kDa form of I?B? and released IFN-?. CD56(+) NK cells were shown to be the I?B?-producing lymphocyte subpopulation, which also released abundant IFN-? in response to IL-12/IL-18. Importantly, I?B? was undetectable in CD56(-) lymphocytes where IFN-? release was 10-fold lower. In addition, small interfering RNA knockdown of I?B? suppressed IFN-? expression in CD56(+) cells. The association of I?B? with the IFN-? promoter was documented by chromatin immunoprecipitation. IFN-? promoter activity from I?B? overexpression was confirmed by luciferase reporter assay. Finally, I?B? coprecipitated with p65 and p50 NF-?B in NK cells in response to IL-12/IL-18, suggesting that I?B?'s effects on IFN-? promoter activity are coregulated by NF-?B. These results suggest that I?B? functions as an important regulator of IFN-? in human NK cells, further expanding the class of I?B?-modulated genes.
Project description:Major surgery increases the risk for infectious complications due to the development of immunosuppression. CD56 bright NK cells play a key role in the defense against bacterial infections through the release of Interferon (IFN) ? upon stimulation with monocyte-derived Interleukin (IL) 12. We investigated whether invasive visceral surgery interferes with the IFN-? synthesis of human NK cells in response to Staphylococcus aureus. In a prospective pilot study, peripheral blood mononuclear cells (PBMC) were isolated from 53 patients before and 1 to 7 d after elective visceral surgery. The release of IL-12 and IFN-? from PBMC upon exposure to S. aureus in vitro was quantified. The expression of the IL-12 receptor ?1 chain on the surface, the phosphorylation of signal transducer and activator of transcription (STAT) 4, and the synthesis of IFN-? on/in individual CD56 bright NK cells were investigated using flow cytometry. The modulatory effect of IL-12 on the S. aureus-induced IFN-? production in CD56 bright NK cells was analyzed. The IFN-? secretion from purified CD56 bright NK cells was quantified after stimulation with IL-12 and IL-18. After surgery, CD56 bright NK cells among total PBMC were impaired in the release of IFN-? for at least 5 d. Likewise, the IL-12-induced release of IFN-? from purified CD56 bright NK cells was abolished. Upon stimulation with S. aureus, PBMC secreted less IL-12 but supplementation with recombinant IL-12 did not restore the capacity of CD56 bright NK cells to produce IFN-?. CD56 bright NK cells displayed reduced levels of the IL-12R?1 chain whereas the phosphorylation of STAT4, the key transcription factor for the Ifng gene was not diminished. In summary, after invasive visceral surgery, CD56 bright NK cells are impaired in S. aureus-induced IFN-? production and might contribute to the enhanced susceptibility to opportunistic infections.
Project description:Natural killer (NK) cells are lymphocytes that play an important role in the immune response to infection and malignancy. Recent studies in mice have shown that stimulation of NK cells with cytokines or in the context of a viral infection results in memory-like properties. We hypothesized that human NK cells exhibit such memory-like properties with an enhanced recall response after cytokine preactivation. In the present study, we show that human NK cells preactivated briefly with cytokine combinations including IL-12, IL-15, and IL-18 followed by a 7- to 21-day rest have enhanced IFN-γ production after restimulation with IL-12 + IL-15, IL-12 + IL-18, or K562 leukemia cells. This memory-like phenotype was retained in proliferating NK cells. In CD56(dim) NK cells, the memory-like IFN-γ response was correlated with the expression of CD94, NKG2A, NKG2C, and CD69 and a lack of CD57 and KIR. Therefore, human NK cells have functional memory-like properties after cytokine activation, which provides a novel rationale for integrating preactivation with combinations of IL-12, IL-15, and IL-18 into NK cell immunotherapy strategies.
Project description:Natural killer (NK) cells are classically viewed as lymphocytes that provide innate surveillance against virally infected cells and tumour cells through the release of cytolytic mediators and interferon (IFN)-gamma. In humans, blood CD56(dim) NK cells specialize in the lysis of cell targets. In the lymph nodes, CD56(bright) NK cells secrete IFN-gamma cooperating with dendritic cells and T cells in the generation of adaptive responses. Here we report the characterization of a human NK cell subset located in mucosa-associated lymphoid tissues, such as tonsils and Peyer's patches, which is hard-wired to secrete interleukin (IL)-22, IL-26 and leukaemia inhibitory factor. These NK cells, which we refer to as NK-22 cells, are triggered by acute exposure to IL-23. In vitro, NK-22-secreted cytokines stimulate epithelial cells to secrete IL-10, proliferate and express a variety of mitogenic and anti-apoptotic molecules. NK-22 cells are also found in mouse mucosa-associated lymphoid tissues and appear in the small intestine lamina propria during bacterial infection, suggesting that NK-22 cells provide an innate source of IL-22 that may help constrain inflammation and protect mucosal sites.
Project description:Early interferon-gamma (IFN-?) release by innate cells is critical to direct type 1 immune response able to control intracellular pathogens like Trypanosoma cruzi. Although CD56(bright) natural killer (NK) cells are reported to be potent early IFN-? producers, other CD56(+) cells like CD56(dim) NK cells and NK-like T cells have recently been shown to also release IFN-?. We have here studied the contribution of each CD56(+) lymphocyte populations in early IFN-? production in both adults and neonates. On this purpose, we analysed the kinetics of IFN-? production by RT-PCR, ELISA and flow cytometry from 2 h onwards after T. cruzi and IL-15 stimulation and sought for the responding CD56(+) cells. CD56(bright) and CD56(dim) CD16(-) NK cells were the more potent IFN-? early producers in response to IL-15 and parasites in adults and neonates. In both age groups, the majority of IFN-? producing cells were NK cells. However, on the contrary to neonates, CD3(+) CD56(+) NK-like T cells and CD3(+) CD56(-) 'classical' T cells also contributed to early IFN-? production in adults. Altogether, our results support that whereas NK cells responded almost similarly in neonates and adults, cord blood innate CD56(+) and CD56(-) T cells displayed major quantitative and qualitative defects that could contribute to the well-known neonatal immune immaturity.
Project description:I?B?, encoded by Nfibiz, is a nuclear I?B-like protein harboring ankyrin repeats. I?B? has been shown to regulate IL-6 production in macrophages and Th17 development in T cells. However, the role of I?B? in natural killer (NK) cells has not be understood. In the present study, we found that the expression of I?B? was rapidly induced in response to IL-18 in NK cells, but not in T cells. Analysis of Nfkbiz(-/-) mice revealed that I?B? was essential for the production of IFN-? production and cytotoxic activity in NK cells in response to IL-12 and/or IL-18 stimulation. IL-12/IL-18-mediated gene induction was profoundly impaired in Nfkbiz(-/-) NK cells. Whereas the phosphorylation of STAT4 was normally induced by IL-12 stimulation, STAT4 was not recruited to the Ifng gene regions in Nfkbiz(-/-) NK cells. Acetylation of histone 3 K9 on Ifng regions was also abrogated in Nfkbiz(-/-) NK cells. I?B? was recruited on the proximal promoter region of the Ifng gene, and overexpression of I?B? together with IL-12 activated the Ifng promoter. Furthermore, Nfkbiz(-/-) mice were highly susceptible to mouse MCMV infection. Taken together, these results demonstrate that I?B? is essential for the activation of NK cells and antiviral host defense responses.
Project description:Activation of NK cells is a hallmark of infections with intracellular pathogens. We previously showed that the protozoan parasite Leishmania infantum triggered a rapid NK-cell response in mice that required TLR9-positive myeloid DC and IL-12, but no IFN-alpha/beta. Here, we investigated whether IL-15 or IL-18 mediate the activity of IL-12 or function as independent activators of NK cells. In contrast to earlier studies that described IL-15 as crucial for NK-cell priming in response to TLR ligands, the expression of IFN-gamma, FasL, perforin and granzyme B by NK cells in L. infantum-infected mice was completely preserved in the absence of IL-15, whereas the proliferative capacity of NK cells was lower than in WT mice. IFN-gamma secretion, cytotoxicity and FasL expression of NK cells from infected IL-18(-/-) mice were significantly reduced compared with controls, but, unlike IL-12, IL-18 was not essential for NK-cell effector functions. Part of the NK-cell-stimulatory effect of IL-12 was dependent on IL-18. We conclude that IL-15 is not functioning as a universal NK-cell priming signal and that IL-18 contributes to the NK-cell response in visceral leishmaniasis. The cytokine requirements for NK-cell activation appear to differ contingent upon the infectious pathogen.
Project description:Background: Antibody-dependent cellular cytotoxicity (ADCC), which mainly mediated by natural killer (NK) cells, may play a critical role in human immunodeficiency virus type-1 (HIV-1) disease progression. However, the potential mechanisms that affecting NK-mediated ADCC response are still not well-elucidated. Methods: Antigen-antibody complex model of Ab-opsonized P815 cells was adopted to induce a typical non-specific ADCC response. The capacities of HIV-1 specific NK-ADCC were measured by using the combination model of gp120 protein and plasma of HIV-1 elite controllers. The levels of plasma cytokine were measured by ELISA. Anti-IL-2 blocking antibody was used to analyze the impact of activated CD56+ T cells on NK-ADCC response. Results: IL-2, IL-15, IFN-?, and IFN-? could effectively enhance the non-specific and HIV-1-specific NK-ADCC responses. Compared with healthy controls, HIV-1-infected patients showed decreased plasma IL-2 levels, while no differences of plasma IFN-?, IL-15, and IFN-? were presented. IL-2 production was detected from CD56+ T cells activated through antibody-dependent manner. The capability of NK-ADCC could be weakened by blocking IL-2 secretion from activated CD56+ T cells. Although no difference of frequencies of CD56+ T cells was found between HIV-1-infected patients and healthy controls, deficient IL-2 secretion from activated CD56+ T were found in chronic HIV-1 infection. Conclusions: The impaired ability of activated CD56+ T cells to secreting IL-2 might contribute to the attenuated NK cell-mediated ADCC function in HIV-1 infection.
Project description:In mouse models of chronic inflammatory diseases, Natural Killer (NK) cells can play an immunoregulatory role by eliminating chronically activated leukocytes. Indirect evidence suggests that NK cells may also be immunoregulatory in humans. Two subsets of human NK cells can be phenotypically distinguished as CD16(+)CD56(dim) and CD16(dim/-)CD56(bright). An expansion in the CD56(bright) NK cell subset has been associated with clinical responses to therapy in various autoimmune diseases, suggesting an immunoregulatory role for this subset in vivo. Here we compared the regulation of activated human CD4(+) T cells by CD56(dim) and CD56(bright) autologous NK cells in vitro. Both subsets efficiently killed activated, but not resting, CD4(+) T cells. The activating receptor NKG2D, as well as the integrin LFA-1 and the TRAIL pathway, played important roles in this process. Degranulation by NK cells towards activated CD4(+) T cells was enhanced by IL-2, IL-15, IL-12+IL-18 and IFN-?. Interestingly, IL-7 and IL-21 stimulated degranulation by CD56(bright) NK cells but not by CD56(dim) NK cells. NK cell killing of activated CD4(+) T cells was suppressed by HLA-E on CD4(+) T cells, as blocking the interaction between HLA-E and the inhibitory CD94/NKG2A NK cell receptor enhanced NK cell degranulation. This study provides new insight into CD56(dim) and CD56(bright) NK cell-mediated elimination of activated autologous CD4(+) T cells, which potentially may provide an opportunity for therapeutic treatment of chronic inflammation.
Project description:A subset of CD3(neg)CD56(neg)CD16? Natural Killer (NK) cells is highly expanded during chronic HIV-1 infection. The role of this subset in HIV-1 pathogenesis remains unclear. The lack of NK cell lineage-specific markers has complicated the study of minor NK cell subpopulations.Using CD7 as an additional NK cell marker, we found that CD3(neg)CD56(neg)CD16? cells are a heterogeneous population comprised of CD7? NK cells and CD7(neg) non-classical myeloid cells. CD7?CD56(neg)CD16? NK cells are significantly expanded in HIV-1 infection. CD7?CD56(neg)CD16? NK cells are mature and express KIRs, the C-type lectin-like receptors NKG2A and NKG2C, and natural cytotoxicity receptors similar to CD7?CD56?CD16? NK cells. CD7?CD56(neg) NK cells in healthy donors produced minimal IFN? following K562 target cell or IL-12 plus IL-18 stimulation; however, they degranulated in response to K562 stimulation similar to CD7?CD56? NK cells. HIV-1 infection resulted in reduced IFN? secretion following K562 or cytokine stimulation by both NK cell subsets compared to healthy donors. Decreased granzyme B and perforin expression and increased expression of CD107a in the absence of stimulation, particularly in HIV-1-infected subjects, suggest that CD7?CD56(neg)CD16? NK cells may have recently engaged target cells. Furthermore, CD7?CD56(neg)CD16? NK cells have significantly increased expression of CD95, a marker of NK cell activation.Taken together, CD7?CD56(neg)CD16? NK cells are activated, mature NK cells that may have recently engaged target cells.
Project description:Natural killer (NK) cells are effector lymphocytes that are under clinical investigation for the adoptive immunotherapy of hematologic malignancies, especially acute myeloid leukemia. Recent work in mice has identified innate memory-like properties of NK cells. Human NK cells also exhibit memory-like properties, and cytokine-induced memory-like (CIML) NK cells are generated via brief preactivation with IL-12, IL-15, and IL-18, which later exhibit enhanced functionality upon restimulation. However, the optimal cytokine receptors and signals for maintenance of enhanced function and homeostasis after preactivation remain unclear. Here, we show that IL-12, IL-15, and IL-18 preactivation induces a rapid and prolonged expression of CD25, resulting in a functional high-affinity IL-2 receptor (IL-2R???) that confers responsiveness to picomolar concentrations of IL-2. The expression of CD25 correlated with STAT5 phosphorylation in response to picomolar concentrations of IL-2, indicating the presence of a signal-competent IL-2R???. Furthermore, picomolar concentrations of IL-2 acted synergistically with IL-12 to costimulate IFN-? production by preactivated NK cells, an effect that was CD25 dependent. Picomolar concentrations of IL-2 also enhanced NK cell proliferation and cytotoxicity via the IL-2R???. Further, after adoptive transfer into immunodeficient NOD-SCID-?c(-/-) mice, human cytokine-preactivated NK cells expand preferentially in response to exogenous IL-2. Collectively, these data demonstrate that human CIML NK cells respond to IL-2 via IL-2R??? with enhanced survival and functionality, and they provide additional rationale for immunotherapeutic strategies that include brief cytokine preactivation before adoptive NK cell transfer, followed by low-dose IL-2 therapy.