Role of IFN-alpha/beta and IL-12 in the activation of natural killer cells and interferon-gamma production during experimental infection with Trypanosoma cruzi

Control of Trypanosoma cruzi infection depends largely upon the production of interferon (IFN)-gamma. During experimental infection this cytokine is produced early, mainly by natural killer (NK) cells and later by T cells. As NK cells have been reported to participate in defence against T. cruzi, it is of importance to study the regulation of NK cell functions during infection with the parasite. Several innate cytokines regulate NK cell activity, among them being interferon (IFN)-alpha and IFN-beta (type 1 IFNs) and interleukin (IL)-12, which have all been reported to be involved in protection against T. cruzi. The role of these cytokines in regulation of NK cell functions and disease outcome were studied by infection of mutant mice lacking the IFN-alpha/beta receptor (IFNalpha/betaR-/-) or IL-12 (IL-12-/-) with T. cruzi. IFNalpha/betaR-/- mice were unable to activate the cytotoxic response but produced IFN-gamma, and were not more susceptible than controls. IL-12-/- mice were extremely susceptible and failed to produce T cell-derived IFN-gamma and nitric oxide (NO), although NK cytotoxicity was induced. The results indicate that IL-12 protects against T. cruzi by initiating T cell-mediated production of IFN-gamma, but that endogenous IFN-alpha/beta and NK cell cytotoxicity are not of major importance in defence.     

CD38 is expressed on human mature monocyte-derived dendritic cells and is functionally involved in CD83 expression and IL-12 induction

Dendritic cell (DC) maturation is characterized by the gain or loss of immunological functions and by expression of distinctive surface receptors. CD38 is an ectoenzyme that catalyzes the synthesis of cyclic ADP ribose (a potent second messenger for Ca(2+) release), as well as a receptor that initiates transmembrane signaling upon engagement with its counter-receptor CD31 or with agonistic monoclonal antibodies. Since CD38 is expressed by resting monocytes, we aimed to monitor CD38 expression during the differentiation of human monocyte-derived DC (MDDC) and to investigate the possibility that CD38 plays a functional role during DC maturation. CD38 is down-modulated during differentiation into immature MDDC and expressed again upon maturation. The extent of CD38 expression is dependent on the stimulus adopted (LPS > IFN-gamma > CD40 cross-linking). Although weak, IFN-gamma consistently induces DC maturation. De novo-synthesized CD38 is enzymatically active, and its expression in mature (m) MDDC is dependent on NF-kappa B activity. However, CD38 is not merely a maturation marker but also mediates signaling in mMDDC, where it maintains its functions as a receptor. Activation via agonistic anti-CD38 mAb induces up-regulation of CD83 expression and IL-12 secretion, whereas disruption of CD38/CD31 interaction inhibits CD83 expression, IL-12 secretion and MDDC-induced allogeneic T cell proliferation.     

gammadelta T cells condition dendritic cells in vivo for priming pulmonary CD8 T cell responses against Mycobacterium tuberculosis

gammadelta T cells and dendritic cells are quickly recruited to the lungs shortly after intranasal vaccination with BCG, but the functional in vivo interplay between these two cell populations and its role in the induction of adaptive immune responses is unclear. Using TCR-deficient mice and bone marrow chimeras, we show here that gammadelta T cells provide a non-redundant early source of IFN-gamma in vivo, which enhances IL-12 production by lung dendritic cells. The in vivo-conditioned dendritic cells, in turn, prime a more efficient lung CD8 T cell response against Mycobacterium tuberculosis. Thus, strategies exploiting gammadelta T cell function and IFN-gamma production could be valuable for the design and testing of mucosal vaccines.     

The natural killer cell-mediated killing of autologous dendritic cells is confined to a cell subset expressing CD94/NKG2A,but lacking inhibitory killer Ig-like receptors

The cognate NK-DC interaction in inflamed tissues results in NK cell activation and acquisition of cytotoxicity against immature DC (iDC). This may represent a mechanism of DC selection required for the control of downstream adaptive immune responses. Here we show that killing of monocyte-derived iDC is confined to the NK cell subset that expresses CD94/NKG2A, but not killer Ig-like receptors (KIR). Consistent with these data, the expression of HLA-E (i.e. the cellular ligand of CD94/NKG2A) was down-regulated in iDC. On the other hand, HLA-B and HLA-C down-regulation in iDC was not sufficient to induce cytotoxicity in NK cells expressing KIR3DL1 or KIR2DL. Remarkably, CD94/NKG2A(+)KIR(-) NK cells were heterogeneous in their ability to kill iDC and an inverse correlation existed between their CD94/NKG2A surface density and the magnitude of their cytolytic activity. It is conceivable that the reduced CD94/NKG2A surface density enables these cells to efficiently sense the decrease of HLA-E surface expression in iDC. Finally, most NK cells that lysed iDC did not kill mature DC that express higher amounts of HLA class I molecules (including HLA-E)as compared with iDC. However, a small NK cell subset was capable of killing not only iDC but also mature DC.     

Endotoxin hyperresponsiveness upon CD4+ T cell reconstitution in lymphopenic mice: control by natural regulatory T cells

Natural CD4(+)CD25(+) regulatory T cells (nTreg) have been shown to control graft-versus-host disease after hematopoietic stem cell transplantation (HSCT). Herein, we considered the possibility that the beneficial action of nTreg upon immune reconstitution in lymphopenic hosts involves dampening of the inflammatory response induced by bacterial products. We first observed that transfer of syngeneic CD4(+)CD25(-) T cells in RAG-deficient mice dramatically enhanced release of inflammatory cytokines and associated pathology upon endotoxin injection. Interferon (IFN)-gamma produced by T cells undergoing homeostatic proliferation was shown to be involved in the endotoxin hyperresponsiveness induced by CD4(+) T cell reconstitution. Co-transfer of CD4(+)CD25(+) nTreg with CD4(+)CD25(-) T cells inhibited the expansion of IFN-gamma-producing T cells and reduced endotoxin responses in RAG(-/-) mice. We conclude that (1) CD4(+) T cell reconstitution sensitizes lymphopenic hosts to endotoxin-induced pathology and (2) nTreg prevent this process by limiting the emergence of IFN-gamma-producing cells.     

Lack of anti-tumour reactivity despite enhanced numbers of circulating natural killer T cells in two patients with metastatic renal cell carcinoma

Natural killer T (NK T) cells play a central role as intermediates between innate and adaptive immune responses important to induce anti-tumour reactivity in cancer patients. In two of 14 renal cell carcinoma (RCC) patients, treated with interferon (IFN)-α, we detected significantly enhanced numbers of circulating NK T cells which were typed phenotypically and analysed for anti-tumour reactivity. These NK T cells were T cell receptor (TCR) Vα24/Vβ11(+), 6B11(+) and bound CD1d tetramers. No correlation was observed between NK T frequencies and regulatory T cells (T(regs)), which were also enhanced. NK T cells expressed CD56, CD161, CD45RO and CD69 and were predominantly CD8(+), in contrast to the circulating T cell pool that contained both CD4(+) and CD8(+) T cells, as is found in healthy individuals. It is unlikely that IFN-α triggered the high NK T frequency, as all other patients expressed low to normal NK T numbers. A parallel was observed in IFN-α-related increase in activation of NK T cells with that in conventional T and non-T cells. Normal interleukin (IL)-7, IL-12 and IL-15 plasma levels were found. In one of the patients sporadic NK T cells were detected at the tumour site. α-Galactosylceramide (αGalCer) stimulation of peripheral blood mononuclear cells or isolated NK T cell lines from both patients induced IFN-γ, but no IL-4 and no response towards autologous tumour cells or lysates. The clinical course of disease in both patients was not exceptional with regard to histological subtype and extent of metastatic disease. Therefore, despite a constitutive high peripheral frequency and in vitroαGalCer responsiveness, the NK T cells in the two RCC patients did not show anti-tumour responsiveness.     

Induction of interferon-gamma from natural killer cells by immunostimulatory CpG DNA is mediated through plasmacytoid-dendritic-cell-produced interferon-alpha and tumour necrosis factor-alpha

Immunostimulatory sequences (ISS) that contain CpG motifs have been demonstrated to exert antipathogen and antitumour immunity in animal models through several mechanisms, including the activation of natural killer (NK) cells to secrete interferon-gamma (IFN-gamma) and to exert lytic activity. Since NK cells lack the ISS receptor TLR9, the exact pathway by which NK cells are activated by ISS is unclear. We determined that ISS-induced IFN-gamma from NK cells is primarily dependent upon IFN-alpha release from plasmacytoid dendritic cells (PDCs), which directly activates the NK cell. However, further analysis indicated that other PDC-released soluble factor(s) may contribute to IFN-gamma induction. Indeed, tumour necrosis factor-alpha (TNF-alpha) was identified as a significant contributor to ISS-mediated activation of NK cells and was observed to act in an additive fashion with IFN-alpha in the induction of IFN-gamma from NK cells and to up-regulate CD69 expression on NK cells. This activity of TNF-alpha, however, was dependent upon the presence of PDC-derived factors such as type I interferon. These results illustrate an important function for type I interferon in innate immunity, which is not only to activate effectors like NK cells directly, but also to prime them for enhanced activation by other factors such as TNF-alpha.     

Loss of the CD56hiCD16- NK cell subset and NK cell interferon-gamma production during antiretroviral therapy for HIV-1: partial recovery by human growth hormone

Previous studies have shown that human natural killer (NK) cells are lost from the periphery and are functionally suppressed during HIV-1 infection, and that the administration of highly active antiretroviral therapy (HAART) results in a recovery of NK cell numbers in HIV-1-infected individuals. However, despite this recovery, interleukin (IL)-2 + IL-12-driven interferon (IFN)-gamma production by NK cells has been shown to remain suppressed after HAART. Here we show that the innate immune factor IL-15 in combination with IL-12 is also unable to recover NK cell IFN-gamma production in HAART-treated individuals. Furthermore, we also demonstrate an imbalance in the distribution of CD56loCD16hi and CD56hiCD16- NK subsets after successful HAART, CD56hiCD16- cells being reduced substantially in HIV-1 patients on HAART. Treatment of patients with combined human growth hormone and antiretroviral therapy resulted in further enhancement in the absolute numbers and the proportion of NK cells in some individuals in the absence of parallel effects on CD4+ T cells. Furthermore, in these individuals HAART with growth hormone resulted in an enhancement of cytokine-driven NK cell activation and IFN-gamma production compared to the HAART-only baseline.     

IFN-gamma production in human NK cells through the engagement of CD8 by soluble or surface HLA class I molecules

The engagement of CD8 on NK cell surface by either surface or soluble HLA class I (sHLA-I) molecules induces synthesis and secretion of IFN-gamma. HLA-I-mediated effects were inhibited by the covering of CD8 with specific anti-CD8 monoclonal antibodies, indicating a direct interaction of HLA-I and CD8. That CD8 ligation induces IFN-gamma production was further supported by the finding that cross-linking of CD8 led to release of IFN-gamma at similar levels to those obtained with HLA-I. The sHLA-I-induced IFN-gamma production via CD8 was strongly down-regulated by the engagement of the inhibitory isoforms of either CD94/NKG2 complex by sHLA-I-non-(A,B,C,G) (putative sHLA-E) or CD158b by sHLA-I-Cw3 allele. Ligation of CD8 did not elicit, different from other activating NK cell surface molecules such as CD16 or CD69, triggering of NK cell-mediated cytolysis. Cyclosporin A, but not concanamycin A, an H+-ATPase vacuolar inhibitor which affects perforin and granzyme release, strongly reduced the sHLA-I-mediated CD8-dependent IFN-gamma production but did not affect cytolytic activity of NK cells, suggesting that different biochemical pathways are involved. Altogether, these findings indicate that CD8 engagement by sHLA-I activates a cyclosporin A-dependent pathway leading to production and secretion of IFN-gamma which may play a role in the regulation of innate immune responses in humans.     

Preferential apoptosis of CD56dim natural killer cell subset in patients with cancer

Natural killer (NK) cells (CD56(+)/CD3(-)) in the circulation of cancer patients were reported to have low NK activity and undergo spontaneous apoptosis. A possible relationship between apoptosis and impaired NK activity was studied by Annexin V-binding and NK-cell assays performed with peripheral blood mononuclear cells of patients with head and neck cancer (HNC), breast cancer (BC) and normal controls (NC). Cells stained with Annexin V (Anx) and antibodies to CD56, CD3, CD95, CD25, CD122 or CD132 were examined by flow cytometry. NK activity was tested against K562 targets in 4-h (51)Cr-release assays. The ratio of CD56(dim)/CD56(bright) NK cells was significantly different in patients vs. controls (10 vs. 16; p<0.01). A significantly greater percentage of CD56(dim) NK cells bound Anx in HNC patients (27+/-17%, median +/- SD) or BC (46+/-18%) than in NC (15+/-18%, p<0.04 and p<0.0002, respectively). CD56(dim) NK cells were preferentially targeted for apoptosis. NK activity was significantly lower in patients with HNC and BC than in NC (p<0.009). An inverse correlation between NK activity and the percent of Anx(+)CD56(dim) NK cells was observed in cancer patients (p =0.002) but not in NC. In patients, circulating CD56(dim) NK cells were targeted for apoptosis, leading to low levels of NK activity.     

Vaccination with cell immunoglobulin mucin-1 antibodies and inactivated influenza enhances vaccine-specific lymphocyte proliferation, interferon-gamma production and cross-strain reactivity

Influenza virus causes a contagious and potentially serious infection of the upper respiratory tract. While neutralizing antibodies are protective against infection, the problem of antigenic drift remains, requiring the constant monitoring and development of new vaccines. The magnitude of this situation is underscored by the emergence of new potentially human pathogenic influenza strains, avian H5N1 being the most recent example. We present evidence that antibodies against T cell immunoglobulin mucin-1 (TIM-1), a recently identified immunomodulatory molecule, stimulate cellular immunity against influenza viruses and cross-strain immune reactivity. To determine potential immunostimulatory properties of anti-TIM-1, mice were vaccinated with inactivated influenza virus in the presence or absence of TIM-1-specific monoclonal antibodies. Development of cellular immunity against both the influenza strain used for immunization and serotypically distinct virus strains was monitored 3 weeks after vaccination by determining antigen-specific lymphocyte proliferation and cytokine production. Results show that TIM-1 antibodies enhance antigen-specific cellular proliferation (P < 0.05) and interferon (IFN)-gamma production (P < 0.01). Using blocking anti-CD4 and CD8 antibodies, it was observed that antigen-specific cellular proliferation is CD4-dependent and that the majority of proliferating cells are CD4+. Finally, vaccination with inactivated influenza virus with TIM-1 antibody results in the significant (P < 0.001) induction of proliferation and IFN-gamma production upon stimulation with one of three serologically distinct strains. TIM-1 antibodies demonstrate an adjuvant effect promoting antigen-specific cellular proliferation and IFN-gamma production, which are important for the promotion of cell-mediated immunity. These results are the first to suggest that TIM-1 antibody may serve as a potent adjuvant in the development of new influenza virus vaccines.     

树突状细胞与自然杀伤细胞在免疫应答中的相互作用

树突状细胞(dendritic cells,DC)是专职的抗原提呈细胞,它们在免疫应答中发挥的作用越来越受到重视。NK细胞是固有免疫中的一种重要的效应细胞。当两者共培养后,在免疫应答中具有互惠作用。现就两者的生物学活性以及在免疫应答中相互作用的机制等方面作一综述。     

IFN-alpha-conditioned dendritic cells are highly efficient in inducing cross-priming CD8(+) T cells against exogenous viral antigens

Dendritic cells (DC) generated after a short-term exposure of monocytes to IFN-alpha and GM-CSF (IFN-DC) are highly effective in inducing cross-priming of CD8(+ )T cells against viral antigens. We have investigated the mechanisms responsible for the special attitude of these DC and compared their activity with that of reference DC. Antigen uptake and endosomal processing capabilities were similar for IFN-DC and IL-4-derived DC. Both DC types efficiently cross-presented soluble HCV NS3 protein to the specific CD8(+) T cell clone, even though IFN-DC were superior in cross-presenting low amounts of viral antigens. Moreover, when DC were pulsed with inactivated HIV-1 and injected into hu-PBL-SCID mice, the generation of virus-specific CD8(+ )T cells was markedly higher in animals immunized with IFN-DC than in mice immunized with CD40L-matured IL-4-DC. Of interest, in experiments with purified CD8(+ )T cells, IFN-DC were superior with respect to CD40L-matured IL-4-DC in inducing in vitro cross-priming of HIV-specific CD8(+ )T cells. This property correlated with enhanced potential to express the specific subunits of the IL-23 and IL-27 cytokines. These results suggest that IFN-DC are directly licensed for an efficient CD8(+) T cell priming by mechanisms likely involving enhanced antigen presentation and special attitude to produce IL-12 family cytokines.     

Differential effects of ifosfamide on dendritic cell-mediated stimulation of T cell interleukin-2 production, natural killer cell cytotoxicity and interferon-gamma production

Ifosfamide is a DNA-alkylating agent used frequently in chemotherapy of human malignancies. Ifosfamide and its major decomposition products deplete intracellular glutathione (GSH). Glutathione is the major intracellular thiol reductant that protects cells against oxidative injury. Ifosfamide depletion of intracellular GSH in human dendritic cells (DC), T cells and natural killer (NK) cells impairs their functional activity which can be restored by reconstituting GSH. Here we assessed the effect of ifosfamide on DC-mediated stimulation of NK cell proliferation via T cells and on direct DC stimulation of NK cell cytotoxicity and interferon (IFN)-gamma production. Indirect DC stimulation of NK cell proliferation via T cells and T cell-derived interleukin (IL)-2 were reduced by ifosfamide treatment of DC and reconstitution of GSH in DC restored both responses. When DC and NK cells were treated with ifosfamide, DC could overcome the negative effect of ifosfamide on NK cytotoxic function whereas NK cell IFN-gamma production was less efficiently restored. The ability of IL-2 activated NK cells to kill autologous immature DC or to induce DC maturation was reduced moderately by treatment of both cell types with ifosfamide. Overall, our results suggest that DC may stimulate anti-tumour effector cells in patients even if they had received treatment with chemotherapeutic agents such as ifosfamide.     

Invariant natural killer T cells in lupus patients promote IgG and IgG autoantibody production

IgG autoantibodies, including antibodies to double‐stranded DNA (dsDNA), are pathogenic in systemic lupus erythematosus (SLE), but the mechanisms controlling their production are not understood. To assess the role of invariant natural killer T (iNKT) cells in this process, we studied 44 lupus patients. We took advantage of the propensity of PBMCs from patients with active disease to spontaneously secrete IgG in vitro. Despite the rarity of iNKT cells in lupus blood (0.002–0.05% of CD3‐positive T cells), antibody blockade of the conserved iNKT TCR or its ligand, CD1d, or selective depletion of iNKT cells, inhibited spontaneous secretion of total IgG and anti‐dsDNA IgG by lupus PBMCs. Addition of anti‐iNKT or anti‐CD1d antibody to PBMC cultures also reduced the frequency of plasma cells, suggesting that lupus iNKT cells induce B‐cell maturation. Like fresh iNKT cells, expanded iNKT‐cell lines from lupus patients, but not healthy subjects, induced autologous B cells to secrete antibodies, including IgG anti‐dsDNA. This activity was inhibited by anti‐CD40L antibody, as well as anti‐CD1d antibody, confirming a role for CD40L‐CD40 and TCR‐CD1d interactions in lupus iNKT‐cell‐mediated help. These results reveal a critical role for iNKT cells in B‐cell maturation and autoantibody production in patients with lupus.     

gammadelta-T cells expressing NK receptors predominate over NK cells and conventional T cells in the innate IFN-gamma response to Plasmodium falciparum malaria

Rapid production of interferon-gamma (IFN-gamma) in response to malaria by the innate immune system may determine resistance to infection, or inflammatory disease. However, conflicting reports exist regarding the identity of IFN-gamma-producing cells that rapidly respond to Plasmodium falciparum. To clarify this area, we undertook detailed phenotyping of IFN-gamma-producing cells across a panel of naive human donors following 24-h exposure to live schizont-infected red blood cells (iRBC). Here, we show that NK cells comprise only a small proportion of IFN-gamma-responding cells and that IFN-gamma production is unaffected by NK cell depletion. Instead, gammadelta-T cells represent the predominant source of innate IFN-gamma, with the majority of responding gammadelta-T cells expressing NK receptors. Malaria-responsive gammadelta-T cells more frequently expressed NKG2A compared to non-responding gammadelta-T cells, while non-responding gammadelta-T cells more frequently expressed CD158a/KIR2DL1. Unlike long-term gammadelta-T cell responses to iRBC, alphabeta-T cell help was not required for innate gammadelta-T cell responses. Diversity was observed among donors in total IFN-gamma output. This was positively associated with CD94 expression on IFN-gamma(+) NK-like gammadelta-T cells. Applied to longitudinal cohort studies in endemic regions, similar comparative phenotyping should allow assessment of the contribution of diverse cell populations and regulatory receptors to risk of infection and disease.     

In vivo activation of invariant V alpha 14 natural killer T cells by alpha-galactosylceramide sequentially induces Fas-dependent and -independent cytotoxicity

The present study was designed to clarify the cytotoxic capacities of invariant V alpha 14 natural killer T (iNKT) cells activated in vivo. We found that as early as 2 h after a single injection of alpha-galactosylceramide (alpha-GalCer), sorted iNKT splenocytes from treated mice kill Fas-transfected target cells. The implication of the Fas pathway in this lysis was strengthened by both the blockage of cytotoxicity in the presence of anti-Fas ligand (FasL) monoclonal antibody (mAb) and the up-regulation of FasL expression on iNKT cells. Sorted NK cells did not participate in the lytic activity at this time point. Yet, they became cytotoxic later on, 24 h post-treatment, when target cell lysis was mainly independent of the Fas pathway. This type of cell killing was predominant at this later time point, even though iNKT cells conserved a slight Fas-dependent cytotoxicity. NK cells failed to acquire the ability to kill target cells when IFN-gamma production in alpha-GalCer-injected mice was blocked by anti-IFN-gamma mAb, underscoring the major role of this cytokine. In conclusion, our findings provide the first direct evidence that iNKT cells can exert Fas-dependent cytotoxicity very shortly after in vivo alpha-GalCer activation and later, through IFN-gamma secretion, enable NK cells to kill target cells in a Fas-independent pathway.     

Differential recruitment and activation of natural killer cell sub‐populations by Mycobacterium bovis‐infected dendritic cells

Through complex interplay with APCs, subsets of NK cells play an important role in shaping adaptive immune responses. Bovine tuberculosis, caused by Mycobacterium bovis, is increasing in incidence and detailed knowledge of host–pathogen interactions in the natural host is essential to facilitate disease control. We investigated the interactions of NK‐cell sub‐populations and M. bovis‐infected DCs to determine early innate mechanisms in the response to infection. A sub‐population of NK cells (NKp46⁺CD2^?) selectively expressing lymphoid homing and inflammatory chemokine receptors were induced to migrate towards M. bovis‐infected DCs. This migration was associated with increased expression of chemokines CCL3, 4, 5, 20 and CXCL8 by M. bovis‐infected DCs. Activation of NKp46⁺CD2^? NK cells and secretion of IFN‐γ was observed, a response reliant on localised IL‐12 release and direct cellular interaction. In a reciprocal manner, NKp46⁺CD2^? cells induced an increase in the intensity of cell surface MHC class II expression on DCs. In contrast, NKp46⁺CD2⁺ NK cells were unable to secrete IFN‐γ and did not reciprocally affect DCs. This study provides novel evidence to demonstrate distinct effector responses between bovine NK‐cell subsets during mycobacterial infection.