Reciprocal activating interaction between dendritic cells and pamidronate-stimulated gammadelta T cells: role of CD86 and inflammatory cytokines

We investigated the interactions between human monocyte-derived dendritic cells (DCs) and Ag-activated circulating TCR-gammadelta-expressing lymphocytes (Vdelta2). Coculture of immature DCs (iDCs) with peripheral blood Vdelta2 T cells activated with either pyrophosphomonoesters (isopentenyl pyrophosphate; IPP) or aminobiphosphonates (pamidronate; PAM) led to a significant up-modulation of CD86 and MHC class I molecules and to the acquisition of functional features typical of activated DCs. DC activation induced by both IPP- and PAM-stimulated gammadelta T cells was mostly mediated by TNF-alpha and IFN-gamma secreted by activated lymphocytes. However, the effect of PAM-activated gammadelta T cells, but not that of IPP-activated cells, required cell-to-cell contact. Reciprocally, activation of Vdelta2 T cells by PAM, but not by IPP, was dependent on cell contact with iDCs. In fact, when PAM-stimulated DC-gammadelta T cell cocultures were separated by a semipermeable membrane or treated with blocking anti-CD86 Abs, induction of CD25 and CD69 as well as IFN-gamma and TNF-alpha secretion by Vdelta2 cells were strongly reduced. These results demonstrate for the first time a bidirectional activating interaction between iDCs and PAM-stimulated gammadelta T lymphocytes, thus suggesting a potential adjuvant role of this early cross-talk in the therapeutic activity of aminobiphosphonate drugs.     

Human dendritic cells very efficiently present a heterologous antigen expressed on the surface of recombinant gram-positive bacteria to CD4+ T lymphocytes.

Recombinant Streptococcus gordonii expressing on the surface the C-fragment of tetanus toxin was tested as an Ag delivery system for human monocyte-derived dendritic cells (DCs). DCs incubated with recombinant S. gordonii were much more efficient than DCs pulsed with soluble C-fragment of tetanus toxin at stimulating specific CD4+ T cells as determined by cell proliferation and IFN-gamma release. Compared with DCs treated with soluble Ag, DCs fed with recombinant bacteria required 102- to 103-fold less Ag and were at least 102 times more effective on a per-cell basis for activating specific T cells. S. gordonii was internalized in DCs by conventional phagocytosis, and cytochalasin D inhibited presentation of bacteria-associated Ag, but not of soluble Ag, suggesting that phagocytosis was required for proper delivery of recombinant Ag. Bacteria were also very potent inducers of DC maturation, although they enhanced the capacity of DCs to activate specific CD4+ T cells at concentrations that did not stimulate DC maturation. In particular, S. gordonii dose-dependently up-regulated expression of membrane molecules (MHC I and II, CD80, CD86, CD54, CD40, CD83) and reduced both phagocytic and endocytic activities. Furthermore, bacteria promoted in a dose-dependent manner DC release of cytokines (IL-6, TNF-alpha, IL-1beta, IL-12, TGF-beta, and IL-10) and of the chemokines IL-8, RANTES, IFN-gamma-inducible protein-10, and monokine induced by IFN-gamma. Thus, recombinant Gram-positive bacteria appear a powerful tool for vaccine design due to their extremely high capacity to deliver Ags into DCs, as well as induce DC maturation and secretion of T cell chemoattractans.     

Interactions of Haemophilus ducreyi and purified cytolethal distending toxin with human monocyte-derived dendritic cells, macrophages and CD4+ T cells

To evaluate the early stages of the host response to chancroid bacterium Haemophilus ducreyi, we investigated the in vitro responses of monocyte-derived dendritic cells (DCs) and macrophages (MQs) to this pathogen and Haemophilus influenzae. The phagocytic activities and pro-inflammatory cytokine secretion profiles of the antigen-presenting cells (APCs) were analyzed after exposure to gentamycin-killed bacteria, H. ducreyi lipooligosaccharide (LOS), and purified cytolethal distending toxin (HdCDT). T-cell proliferation and cytokine release were examined after co-culturing isolated autologous CD4+ T cells with antigen-pulsed APCs. Both the DCs and MQs phagocytosed H. ducreyi and H. influenzae, as estimated by flow cytometry. All of the strains induced APC secretion of TNF-alpha, IL-6, IL-8, and IL-12, as measured by ELISA. Other human cells, particularly endothelial cells and fibroblasts, also produced cytokines when stimulated with these bacteria. Purified LOS at concentration 1 microg/ml induced two to threefold lower levels of cytokines than the whole bacteria, which indicates that other components are involved in immune activation. HdCDT inhibited partially the production of the aforementioned cytokines. High levels of IFN-gamma, but not of IL-4 and IL-13, were secreted by T cells after activation by either DCs or MQs that were pre-exposed to bacteria, indicating the Th1 nature of the immune response. The levels of T-cell proliferation induced by H. ducreyi were lower than those induced by H. influenzae. HdCDT-treated APCs did not display cytokine responses or T-cell proliferation. These results indicate that HdCDT intoxication, which results in progressive apoptosis of APCs, may hamper early stage immune responses.     

Dendritic cells and NK cells stimulate bystander T cell activation in response to TLR agonists through secretion of IFN-alpha beta and IFN-gamma

Recognition of conserved features of infectious agents by innate pathogen receptors plays an important role in initiating the adaptive immune response. We have investigated early changes occurring among T cells after injection of TLR agonists into mice. Widespread, transient phenotypic activation of both naive and memory T cells was observed rapidly after injection of molecules acting through TLR3, -4, -7, and -9, but not TLR2. T cell activation was shown to be mediated by a combination of IFN-alphabeta, secreted by dendritic cells (DCs), and IFN-gamma, secreted by NK cells; notably, IFN-gamma-secreting NK cells expressed CD11c and copurified with DCs. Production of IFN-gamma by NK cells could be stimulated by DCs from TLR agonist-injected mice, and although soluble factors secreted by LPS-stimulated DCs were sufficient to induce IFN-gamma, maximal IFN-gamma production required both direct contact of NK cells with DCs and DC-secreted cytokines. In vitro, IFN-alphabeta, IL-18, and IL-12 all contributed to DC stimulation of NK cell IFN-gamma, whereas IFN-alphabeta was shown to be important for induction of T cell bystander activation and NK cell IFN-gamma production in vivo. The results delineate a pathway involving innate immune mediators through which TLR agonists trigger bystander activation of T cells.     

Extracellular ATP induces a distorted maturation of dendritic cells and inhibits their capacity to initiate Th1 responses

Dendritic cells (DCs) express functional purinergic receptors, but the effects of purine nucleotides on DC functions have been marginally investigated. In this study, we report on the ability of micromolar concentrations of ATP to affect the maturation and Ag-presenting function of monocyte-derived DCs in vitro. Chronic stimulation (24 h) of DCs with low, noncytotoxic ATP doses increased membrane expression of CD54, CD80, CD86, and CD83, slightly reduced the endocytic activity of DCs, and augmented their capacity to promote proliferation of allogeneic naive T lymphocytes. Moreover, ATP enhanced LPS- and soluble CD40 ligand-induced CD54, CD86, and CD83 expression. On the other hand, ATP markedly and dose-dependently inhibited LPS- and soluble CD40 ligand-dependent production of IL-1alpha, IL-1beta, TNF-alpha, IL-6, and IL-12, whereas IL-1 receptor antagonist and IL-10 production was not affected. As a result, T cell lines generated from allogeneic naive CD45RA(+) T cells primed with DCs matured in the presence of ATP produced lower amounts of IFN-gamma and higher levels of IL-4, IL-5, and IL-10 compared with T cell lines obtained with LPS-stimulated DCs. ATP inhibition of TNF-alpha and IL-12 production by mature DCs was not mediated by PGs or elevation of intracellular cAMP and did not require ATP degradation. The inability of UTP and the similar potency of ADP to reproduce ATP effects indicated that ATP could function through the P2X receptor family. These results suggest that extracellular ATP may serve as an important regulatory signal to dampen IL-12 production by DCs and thus prevent exaggerated and harmful immune responses.     

The schistosome oligosaccharide lacto-N-neotetraose expands Gr1(+) cells that secrete anti-inflammatory cytokines and inhibit proliferation of naive CD4(+) cells: a potential mechanism for immune polarization in helminth infections.

Immunomodulatory oligosaccharides found on helminths also are found in human milk, and both helminths and milk have been shown to be immunosuppressive. We have been examining the immunomodulatory capabilities of two oligosaccharides expressed in milk and on helminth parasites, lacto-N-fucopentaose III and lacto-N-neotetraose (LNnT). In an attempt to dissect mechanisms that lead to Th2 polarization and immune suppression, we examined the early response in mice to the glycoconjugate LNnT-Dextran (LNnT-Dex). We found that injection of LNnT-Dex expanded a cell population, phenotypically defined as Gr1(+)/CD11b(+)/F4/80(+), as early as 2 h after injection. Examination of spontaneous cytokine production showed that this Gr1(+)/F4/80(+) population of cells spontaneously produced low levels of proinflammatory cytokines, but higher levels of IL-10 and TGF-beta ex vivo, compared to peritoneal cells from mice injected with Dex. Gr1(+) cells adoptively suppressed naive CD4(+) T cell proliferation in vitro in response to anti-CD3/CD28 Ab stimulation. Suppression of naive CD4(+) cells involved cell contact and was dependent on IFN-gamma and NO, with a discrete role played by IL-10. Coculture of naive CD4(+)T cells with Gr1(+) suppressor cells did not lead to CD4(+) T cell apoptosis, although it did imprint on naive CD4(+) T cells a response characterized by lower levels of IFN-gamma, coincident with increased IL-13 production. Our results suggest that both human milk and helminth parasites may share a ligand-specific mechanism involved in the generation of anti-inflammatory mediators that suppress Th1-type and inflammatory responses.     

Expression of CD161 (NKR-P1A) defines subsets of human CD4 and CD8 T cells with different functional activities

A subset of T cells in human peripheral blood expresses CD161 (NKR-P1A) receptors that are primarily associated with NK cells. In the current study we isolated blood T cell subsets according to the expression of CD161 and examined their contents of naive, central memory, and effector memory cells and their capacities for proliferation, cytokine secretion, and natural cytolysis. We found that CD4+CD161- and CD8+CD161- subsets contained predominantly naive T cells that secreted high levels of IL-2 after in vitro stimulation, and CD4+CD161int and CD8+CD161int subsets contained predominantly effector and central memory T cells that secreted high levels of IFN-gamma and TNF-alpha. All of these subsets showed vigorous proliferation after stimulation in vitro, but none had NK lytic activity. Unexpectedly, the CD8+CD161+ cells contained an anergic CD8alpha+CD8betalow/-CD161high T cell subset that failed to proliferate, secrete cytokines, or mediate NK lytic activity.     

Mature dendritic cells derived from human monocytes within 48 hours: a novel strategy for dendritic cell differentiation from blood precursors

It is widely believed that generation of mature dendritic cells (DCs) with full T cell stimulatory capacity from human monocytes in vitro requires 5-7 days of differentiation with GM-CSF and IL-4, followed by 2-3 days of activation. Here, we report a new strategy for differentiation and maturation of monocyte-derived DCs within only 48 h of in vitro culture. Monocytes acquire immature DC characteristics by day 2 of culture with GM-CSF and IL-4; they down-regulate CD14, increase dextran uptake, and respond to the inflammatory chemokine macrophage inflammatory protein-1alpha. To accelerate DC development and maturation, monocytes were incubated for 24 h with GM-CSF and IL-4, followed by activation with proinflammatory mediators for another 24 h (FastDC). FastDC expressed mature DC surface markers as well as chemokine receptor 7 and secreted IL-12 (p70) upon CD40 ligation in the presence of IFN-gamma. The increase in intracellular calcium in response to 6Ckine showed that chemokine receptor 7 expression was functional. When FastDC were compared with mature monocyte-derived DCs generated by a standard 7-day protocol, they were equally potent in inducing Ag-specific T cell proliferation and IFN-gamma production as well as in priming autologous naive T cells using tetanus toxoid as a model Ag. These findings indicate that FastDC are as effective as monocyte-derived DCs in stimulating primary, Ag-specific, Th 1-type immune responses. Generation of FastDC not only reduces labor, cost, and time required for in vitro DC development, but may also represent a model more closely resembling DC differentiation from monocytes in vivo.     

Flt3-ligand and granulocyte colony-stimulating factor mobilize distinct human dendritic cell subsets in vivo

Dendritic cells (DCs) have a unique ability to stimulate naive T cells. Recent evidence suggests that distinct DC subsets direct different classes of immune responses in vitro and in vivo. In humans, the monocyte-derived CD11c+ DCs induce T cells to produce Th1 cytokines in vitro, whereas the CD11c- plasmacytoid T cell-derived DCs elicit the production of Th2 cytokines. In this paper we report that administration of either Flt3-ligand (FL) or G-CSF to healthy human volunteers dramatically increases distinct DC subsets, or DC precursors, in the blood. FL increases both the CD11c+ DC subset (48-fold) and the CD11c- IL-3R+ DC precursors (13-fold). In contrast, G-CSF only increases the CD11c- precursors (>7-fold). Freshly sorted CD11c+ but not CD11c- cells stimulate CD4+ T cells in an allogeneic MLR, whereas only the CD11c- cells can be induced to secrete high levels of IFN-alpha, in response to influenza virus. CD11c+ and CD11c- cells can mature in vitro with GM-CSF + TNF-alpha or with IL-3 + CD40 ligand, respectively. These two subsets up-regulate MHC class II costimulatory molecules as well as the DC maturation marker DC-lysosome-associated membrane protein, and they stimulate naive, allogeneic CD4+ T cells efficiently. These two DC subsets elicit distinct cytokine profiles in CD4+ T cells, with the CD11c- subset inducing higher levels of the Th2 cytokine IL-10. The differential mobilization of distinct DC subsets or DC precursors by in vivo administration of FL and G-CSF offers a novel strategy to manipulate immune responses in humans.     

CD47 engagement inhibits cytokine production and maturation of human dendritic cells

Upon encounter with bacterial products, immature dendritic cells (iDCs) release proinflammatory cytokines and develop into highly stimulatory mature DCs. In the present study, we show that human monocyte-derived DCs functionally express the CD47 Ag, a thrombospondin receptor. Intact or F(ab')2 of CD47 mAb suppress bacteria-induced production of IL-12, TNF-alpha, GM-CSF, and IL-6 by iDCs. 4N1K, a peptide derived from the CD47-binding site of thrombospondin, also inhibits cytokine release. The inhibition of IL-12 and TNF-alpha is IL-10-independent inasmuch as IL-10 production is down-modulated by CD47 mAb and blocking IL-10 mAb fails to restore cytokine levels. CD47 ligation counteracts the phenotypic and functional maturation of iDCs in that it prevents the up-regulation of costimulatory molecules, the loss of endocytic activity, and the acquisition of an increased capacity to stimulate T cell proliferation and IFN-gamma production. Interestingly, regardless of CD47 mAb treatment during DC maturation, mature DC restimulated by soluble CD40 ligand and IFN-gamma, to mimic DC/T interaction, produce less IL-12 and more IL-18 than iDCs. Finally, CD47 ligation on iDCs does not impair their capacity to phagocytose apoptotic cells. We conclude that following exposure to microorganisms, CD47 ligation may limit the intensity and duration of the inflammatory response by preventing inflammatory cytokine production by iDCs and favoring their maintenance in an immature state.     

The intracellular granzyme B inhibitor,proteinase inhibitor 9, is up-regulated during accessory cell maturation and effector cell degranulation,and its overexpression enhances CTL potency

Granzyme B (grB) is a serine proteinase released by cytotoxic lymphocytes (CLs) to kill abnormal cells. GrB-mediated apoptotic pathways are conserved in nucleated cells; hence, CLs require mechanisms to protect against ectopic or misdirected grB. The nucleocytoplasmic serpin, proteinase inhibitor 9 (PI-9), is a potent inhibitor of grB that protects cells from grB-mediated apoptosis in model systems. Here we show that PI-9 is present in CD4(+) cells, CD8(+) T cells, NK cells, and at lower levels in B cells and myeloid cells. PI-9 is up-regulated in response to grB production and degranulation, and associates with grB-containing granules in activated CTLs and NK cells. Intracellular complexes of PI-9 and grB are evident in NK cells, and overexpression of PI-9 enhances CTL potency, suggesting that cytoplasmic grB, which may threaten CL viability, is rapidly inactivated by PI-9. Because dendritic cells (DCs) acquire characteristics similar to those of target cells to activate naive CD8(+) T cells and therefore may also require protection against grB, we investigated the expression of PI-9 in DCs. PI-9 is evident in thymic DCs (CD3(-), CD4(+), CD8(-), CD45(+)), tonsillar DCs, and DC subsets purified from peripheral blood (CD16(+) monocytes and CD123(+) plasmacytoid DCs). Furthermore, PI-9 is expressed in monocyte-derived DCs and is up-regulated upon TNF-alpha-induced maturation of monocyte-derived DCs. In conclusion, the presence and subcellular localization of PI-9 in leukocytes and DCs are consistent with a protective role against ectopic or misdirected grB during an immune response.     

Lutzomyia longipalpis salivary peptide maxadilan alters murine dendritic cell expression of CD80/86, CCR7, and cytokine secretion and reprograms dendritic cell-mediated cytokine release from cultures containing allogeneic T cells

Leishmania protozoan parasites, the etiologic agent of leishmaniasis, are transmitted exclusively by phlebotomine sand flies of the genera Phlebotomus and Lutzomyia. In addition to parasites, the infectious bite inoculum contains arthropod salivary components. One well-characterized salivary component from Lutzomyia longipalpis is maxadilan (MAX), a vasodilator acting via the type I receptor for the pituitary cyclic AMP activating peptide. MAX has been shown to elicit immunomodulatory effects potentially dictating immune responses to Leishmania parasites. When exposed to MAX, both resting and LPS-stimulated dendritic cells (DCs) show reduced CD80 and CD86 expression on most DCs in vitro. However, CD86 expression is increased significantly on a subpopulation of DCs. Furthermore, MAX treatment promoted secretion of type 2 cytokines (IL-6 and IL-10) while reducing production of type 1 cytokines (IL-12p40, TNF-alpha, and IFN-gamma) by LPS-stimulated DCs. A similar trend was observed in cultures of MAX-treated DCs containing naive allogeneic CD4(+) T cells: type 2 cytokines (IL-6 and IL-13) increased while type 1 cytokines (TNF-alpha and IFN-gamma) decreased. Additionally, the proinflammatory cytokine IL-1beta was increased in cultures containing MAX-treated mature DCs. MAX treatment of LPS-stimulated DCs also prevented optimal surface expression of CCR7 in vitro. These MAX-dependent effects were evident in DCs from both Leishmania major-susceptible (BALB/c) and -resistant (C3H/HeN) murine strains. These data suggest that modification of DC phenotype and function by MAX likely affects crucial cellular components that determine the pathological response to infection with Leishmania.     

A soluble form of lymphocyte activation gene-3 (IMP321) induces activation of a large range of human effector cytotoxic cells

The principal antitumor immune response is mediated through the activation of type 1 cytotoxic (Tc1) CD8 T cells, NK cells, and monocytes/macrophages. In this study, we investigated the potency of a clinical-grade soluble form of lymphocyte activation gene-3 protein (IMP321), a physiological high-affinity MHC class II binder, at inducing in PBMCs an appropriate cytotoxic-type response in short-term ex vivo assays. We found that IMP321 binds to a minority (<10%) of MHC class II + cells in PBMCs, including all myeloid dendritic cells, and a small fraction of monocytes. Four hours after addition of IMP321 to PBMCs, these myeloid cells produce TNF-alpha and CCL4 as determined by intracellular staining. At 18 h, 1% of CD8+ T cells and 3.7% NK cells produce Tc1 cytokines such as IFN-gamma and/or TNF-alpha (mean values from 60 blood donors). Similar induction was observed in metastatic cancer patient PBMCs, but the values were lower for the NK cell subset. Early APC activation by IMP321 is needed for this Tc1-type activation because pure sorted CD8+ T cells could not be activated by IMP321. Only Ag-experienced, fully differentiated granzyme+ CD8 T cells (effector and effector memory but not naive or central memory T cells) are induced by IMP321 to full Tc1 activation. In contrast to IMP321, TLR1-9 agonists induce IL-10 and are therefore unable to induce this Tc1 IFN-gamma+ response. Thus, IMP321 has many properties that confirm its potential to be a new class of immunopotentiator in cancer patients.     

Granulysin, a cytolytic molecule, is also a chemoattractant and proinflammatory activator

Granulysin, a cationic protein produced by activated human CTL and NK cells, is cytolytic against microbial and tumor targets. In this study we show that granulysin also functions as a chemoattractant and activates monocytes to produce cytokines/chemokines. Although granulysin-mediated cytotoxicity occurs at micromolar concentrations, chemoattraction occurs in the nanomolar range, and immune activation occurs over a wide range of concentrations (nanomolar to micromolar). Granulysin causes a 2- to 7-fold increase in chemotaxis of monocytes, CD4(+), and CD8(+) memory (CD45RO) but not naive (CD45RA) T cells, NK cells, and mature, but not immature, monocyte-derived dendritic cells. Pertussis toxin treatment abrogates chemoattraction by granulysin, indicating involvement of G-protein-coupled receptor(s). At low concentrations (10 nM), granulysin promotes a 3- to 10-fold increase in MCP-1 and RANTES produced by monocytes and U937 cells, while a 2-fold increase in TNF-alpha production by LPS-stimulated monocytes requires higher concentrations of granulysin (micromolar). Taken together, these data indicate that the local concentration of granulysin is critical for the biologic activity, with high concentrations resulting in cytotoxicity while lower concentrations, presumably further from the site of granulysin release, actively recruit immune cells to sites of inflammation.     

Sustained impairment of IFN-gamma secretion in suppressed HIV-infected patients despite mature NK cell recovery: evidence for a defective reconstitution of innate immunity

The impairment of NK cell functions in the course of HIV infection contributes to a decreased resistance against HIV and other pathogens. We analyzed the proportion of mature and immature NK cell subsets, and measured subsets of IFN-gamma and TNF-alpha-producing NK and T cells in viremic or therapy-suppressed HIV-infected subjects, and noninfected control donors. Viremic HIV(+) individuals had significantly lower proportions of mature CD3(-)/CD161(+)/CD56(+) NK cells and of IFN-gamma-producing NK cells compared with noninfected donors, independent of CD4(+) T cell counts. HIV-infected subjects with undetectable viral load recovered mature CD3(-)/CD161(+)/CD56(+) NK cells and cytotoxicity against tumor (K562) and HSV-infected target cells to percentages comparable with those of uninfected individuals, but their NK cells remained impaired in their ability to produce IFN-gamma. In parallel to these ex vivo findings, in vitro NK cell differentiation of CD34-positive cord blood precursors in the presence of R5 or X4 HIV-1 resulted in the production of NK cells with a normal mature phenotype, but lacking the ability to produce IFN-gamma, whereas coculture of uninfected PBMC with HIV failed to affect mature NK cell properties or IFN-gamma secretion. Altogether, our findings support the hypothesis that mature NK cell phenotype may be uncoupled from some mature functions following highly active antiretroviral therapy-mediated suppression of HIV-1, and indicate that relevant innate immune functions of NK cell subsets may remain altered despite effective viral suppression following antiretroviral treatment.     

Tumoricidal potential of native blood dendritic cells: direct tumor cell killing and activation of NK cell-mediated cytotoxicity

Dendritic cells (DCs) are characterized by their unique capacity for primary T cell activation, providing the opportunity for DC-based cancer vaccination protocols. Novel findings reveal that besides their role as potent inducers of tumor-specific T cells, human DCs display additional antitumor effects. Most of these data were obtained with monocyte-derived DCs, whereas studies investigating native blood DCs are limited. In the present study, we analyze the tumoricidal capacity of M-DC8(+) DCs, which represent a major subpopulation of human blood DCs. We demonstrate that IFN-gamma-stimulated M-DC8(+) DCs lyse different tumor cell lines but not normal cells. In addition, we show that tumor cells markedly enhance the production of TNF-alpha by M-DC8(+) DCs via cell-to-cell contact and that this molecule essentially contributes to the killing activity of M-DC8(+) DCs. Furthermore, we illustrate the ability of M-DC8(+) DCs to promote proliferation, IFN-gamma production, and tumor-directed cytotoxicity of NK cells. The M-DC8(+) DC-mediated enhancement of the tumoricidal potential of NK cells is mainly dependent on cell-to-cell contact. These results reveal that, in addition to their crucial role in activating tumor-specific T cells, blood DCs exhibit direct tumor cell killing and enhance the tumoricidal activity of NK cells. These findings point to the pivotal role of DCs in triggering innate and adaptive immune responses against tumors.     

Enhancement of human cord blood CD34+ cell-derived NK cell cytotoxicity by dendritic cells

NK cells and dendritic cells (DCs) are both important in the innate host defense. However, the role of DCs in NK cell-mediated cytotoxicity is unclear. In this study, we designed two culture systems in which human cord blood CD34(+) cells from the same donor were induced to generate NK cells and DCs, respectively. Coculture of the NK cells with DCs resulted in significant enhancement of NK cell cytotoxicity and IFN-gamma production. However, NK cell cytotoxicity and IFN-gamma production were not increased when NK cells and DCs were grown together separated by a transwell membrane. Functional studies demonstrated that 1) concanamycin A, a selective inhibitor of perforin/granzyme B-based cytolysis, blocked DC-stimulated NK cytotoxicity against K562 cells; and 2) neutralizing mAb against Fas ligand (FasL) significantly reduced DC-stimulated NK cytotoxicity against Fas-positive Jurkat cells. In addition, a marked increase of FasL mRNA and FasL protein expression was observed in DC-stimulated NK cells. The addition of neutralizing mAb against IL-18 and IL-12 significantly suppressed DC-stimulated NK cell cytotoxicity. Neutralizing IFN-gamma Ab almost completely inhibited NK cell cytotoxicity against Jurkat cells. These observations suggest that DCs enhance NK cell cytotoxicity by up-regulating both perforin/granzyme B- and FasL/Fas-based pathways. Direct interaction between DCs and NK cells is necessary for DC-mediated enhancement of NK cell cytotoxicity. Furthermore, DC-derived IL-18 and IL-12 were involved in the up-regulation of NK cell cytotoxicity, and endogenous IFN-gamma production plays an important role in Fas-mediated cytotoxicity.     

IFN-gamma and R-848 dependent activation of human monocyte-derived dendritic cells by Neisseria meningitidis adhesin A

A soluble recombinant form of Neisseria meningitidis adhesin A (NadADelta351-405), proposed as a constituent of anti-meningococcal B vaccines, is here shown to specifically interact with and immune-modulate human monocyte-derived dendritic cells (mo-DCs). After priming with IFN-gamma and stimulation with NadADelta351-405, mo-DCs strongly up-regulated maturation markers CD83, CD86, CD80, and HLA-DR, secreted moderate quantities of TNF-alpha, IL-6, and IL-8, and produced a slight, although significant, amount of IL-12p70. Costimulation of mo-DCs with NadADelta351-405 and the imidoazoquinoline drug R-848, believed to mimic bacterial RNA, increased CD86 in an additive way, but strongly synergized the secretion of IL-12p70, IL-1, IL-6, TNF-alpha, and MIP-1alpha, especially after IFN-gamma priming. CD86/CD80 overexpression correlated with the occupation of high-(kd approximately 80 nM) and low-(kd approximately 4 muM) affinity binding sites for NadADelta351-405. Alternatively, secretion of IL-12p70 and TNF-alpha, IL-6, and IL-8 corresponded to the occupation of high- or low-affinity receptors, respectively. Mo-DCs matured by IFN-gamma and NadADelta351-405 supported the proliferation of naive CD4+ T lymphocytes, inducing the differentiation of both IFN-gamma and IL-4 producing phenotypes. Our data show that NadA not only is a good immunogen but is as well endowed with a proimmune, self-adjuvating, activity.