Lactococcus lactis subsp. lactis JCM 5805 activates natural killer cells via dendritic cells

Lactococcus lactis subsp. lactis JCM 5805 (JCM5805) has been shown to stimulate plasmacytoid dendritic cells (pDC). Here, we investigated the effect of JCM5805 on NK cells. In vitro studies suggested that JCM5805 activated natural killer (NK) cells via dendritic cells including pDC. Furthermore, the oral administration of JCM5805 enhanced the cytotoxic activity of NK cells     

NK cells eliminate Cryptococcus neoformans by potentiating the fungicidal activity of macrophages rather than by directly killing them upon stimulation with IL-12 and IL-18

In the present study, we examined whether natural killer (NK) cells have direct fungicidal activity against Cryptococcus neoformans. Splenic NK cells were obtained from SCID mice and stimulated with a combination of interleukin (IL)-12 and IL-18 in flat culture plates or round tubes. They were then or at the same time cultured with the yeast cells and the number of viable yeast cells was examined. We could not detect direct fungicidal activity by NK cells under any culture condition, although they produced a large amount of IFN-gamma and exerted marked cytotoxic activity against YAC-1 cells. On the other hand, NK cells significantly potentiated the nitric oxide-mediated cryptococcocidal activity of thioglycolate-elicited peritoneal macrophages obtained from SCID mice upon stimulation with IL-12 and IL-18. The culture supernatants of NK cells stimulated with IL-12 and IL-18 provided similar results when used in place of NK cells. The induction of macrophage anticryptococcal activity by NK cells and NK cell culture supernatants were both mediated by IFN-gamma because the specific mAb almost completely abrogated such effect. Considered collectively, our results suggested that NK cells may play a regulatory role in potentiating macrophage-mediated fungicidal mechanisms in host resistance to infection with C. neoformans rather than exerting a direct killing activity against the fungal pathogen.     

IRBP-specific Th1 cells from peripheral blood were predominant in the experimental autoimmune uveitis

Experimental autoimmune uveitis (EAU) is a Th1-cell-mediated autoimmune disease. In this study, the correlation between IRBP-specific Th1 cells in PBLs and the histological grading in the eyes was evaluated kinetically during EAU induction. EAU was induced in B10.A mice with IRBP immunization and the eyes were enucleated for histological examination on days 0, 3, 7, 15, and 21 after immunization. To determine the Th1-cell-mediated immune response, Th1 cytokines (IL-12p40 and IFN-gamma) were measured by RT-PCR in inflamed eyes. At mean time, CD4(+) and IFN-gamma(+) double positive T cells (Th1 cells) from PBLs were analyzed by flow cytometry. The level of the IRBP-specific Th1 cells was significantly increased and kinetically changed during EAU induction, but the cells reached peak time early before the disease was onset. Those IRBP-specific Th1 cells in the PBLs were evidence for EAU disease, but its peak time was different from EAU disease in the eyes. Our data suggested that it is very important to collect blood from patients at a suitable time point and the Th1 cells measured by flow cytometry are good marker for disease diagnosis.     

HPV vaccine stimulates cytotoxic activity of killer dendritic cells and natural killer cells against HPV‐positive tumour cells

Cervarix™ is approved as a preventive vaccine against infection with the human papillomavirus (HPV) strains 16 and 18, which are causally related to the development of cervical cancer. We are the first to investigate in vitro the effects of this HPV vaccine on interleukin (IL)-15 dendritic cells (DC) as proxy of a naturally occurring subset of blood DC, and natural killer (NK) cells, two innate immune cell types that play an important role in antitumour immunity. Our results show that exposure of IL-15 DC to the HPV vaccine results in increased expression of phenotypic maturation markers, pro-inflammatory cytokine production and cytotoxic activity against HPV-positive tumour cells. These effects are mediated by the vaccine adjuvant, partly through Toll-like receptor 4 activation. Next, we demonstrate that vaccine-exposed IL-15 DC in turn induce phenotypic activation of NK cells, resulting in a synergistic cytotoxic action against HPV-infected tumour cells. Our study thus identifies a novel mode of action of the HPV vaccine in boosting innate immunity, including killing of HPV-infected cells by DC and NK cells.     

CD137 signaling interferes with activation and function of CD4+CD25+ regulatory T cells in induced tolerance to experimental autoimmune thyroiditis

Experimental autoimmune thyroiditis (EAT), a model for Hashimoto's thyroiditis, is a T cell-mediated disease inducible with mouse thyroglobulin (mTg). Pretreatment with mTg, however, can induce CD4+ T cell-mediated tolerance to EAT. We demonstrate that CD4+CD25+ regulatory cells are critical for the tolerance induction, as in vivo depletion of CD25+ cells abrogated established tolerance, and CD4+CD25+ cells from tolerized mice suppressed mTg-responsive cells in vitro. Importantly, administration of an agonistic CD137 monoclonal antibody (mAb) inhibited tolerance development, and the mediation of established tolerance. CD137 mAb also inhibited the suppression of mTg-responsive cells by CD4+CD25+ cells in vitro. Signaling through CD137 likely resulted in enhancement of the responding inflammatory T cells, as anti-CD137 did not enable CD4+CD25+ T cells to proliferate in response to mTg in vitro.     

CCR7 ligand-enhanced phagocytosis of various antigens in mature dendritic cells-time course and antigen distribution different from phagocytosis in immature dendritic cells

CC chemokine receptor 7 (CCR7) is selectively expressed on mature dendritic cells (DC). The CCR7 ligands, CC chemokine ligand (CCL) 19 and CCL21, facilitate migration of mature DC from the peripheral tissues to regional lymph nodes. We previously demonstrated that CCR7 ligands induced rapid receptor-mediated endocytosis of dextran in mature DC. In the present study, we further examined the effects of CCR7 ligands on endocytosis of other kinds of antigen, mannosilated bovine serum albumin (Mann-BSA), Escherichia coli(E. coli), or ovalbumin-containing immune complex (OVA-IC), by mature DC. We found that CCR7 ligands enhanced the endocytosis of Mann BSA, E. coli, and OVA-IC in mature DC but not in immature DC. The endocytosis of BSA was not enhanced by CCR7 ligands. Furthermore, the phagocytosis of OVA-IC was significantly inhibited by anti-Fcgamma receptor III/II antibody. These results demonstrate that CCR7 ligands enhance only receptor-mediated endocytosis by mature DC. When rapidly phagocytosed E. coli were traced in CCL19-treated mature DC, most of the phagocytosed E. coli did not colocalize with the lysosomal marker: lysosome-associated membrane protein-1 (Lamp-1), whereas most of E. coli taken up relatively slowly by immature DC colocalized with Lamp-1. These results suggest that phagocytosis of antigens by immature and mature DC plays different functional roles.     

Role of CD25+ dendritic cells in the generation of Th17 autoreactive T cells in autoimmune experimental uveitis

In the current study, we showed that in vivo administration of an anti-CD25 Ab (PC61) decreased the Th17 response in C57BL/6 mice immunized with the uveitogenic peptide interphotoreceptor retinoid-binding protein, while enhancing the autoreactive Th1 response. The depressed Th17 response was closely associated with decreased numbers of a splenic dendritic cell (DC) subset expressing CD11c(+)CD3(-)CD25(+) and decreased expansion of γδ T cells. We demonstrated that ablation of the CD25(+) DC subset accounted for the decreased activation and the expansion of γδ T cells, leading to decreased activation of IL-17(+) interphotoreceptor retinoid-binding protein-specific T cells. Our results show that an enhanced Th17 response in an autoimmune disease is associated with the appearance of a DC subset expressing CD25 and that treatment of mice with anti-CD25 Ab causes functional alterations in a number of immune cell types, namely DCs and γδ T cells, in addition to CD25(+)αβTCR(+) regulatory T cells.     

Aging impacts CD103+CD8+ T cell presence and induction by dendritic cells in the genital tract

As women age, susceptibility to systemic and genital infections increases. Tissue‐resident memory T cells (TRMs) are CD103⁺CD8⁺ long‐lived lymphocytes that provide critical mucosal immune protection. Mucosal dendritic cells (DCs) are known to induce CD103 expression on CD8⁺ T cells. While CD103⁺CD8⁺ T cells are found throughout the female reproductive tract (FRT), the extent to which aging impacts their presence and induction by DCs remains unknown. Using hysterectomy tissues, we found that endometrial CD103⁺CD8⁺ T cells were increased in postmenopausal compared to premenopausal women. Endometrial DCs from postmenopausal women were significantly more effective at inducing CD103 expression on allogeneic naïve CD8⁺ T cells than DCs from premenopausal women; CD103 upregulation was mediated through membrane‐bound TGFβ signaling. In contrast, cervical CD103⁺ T cells and DC numbers declined in postmenopausal women with age. Decreases in DCs correlated with decreased CD103⁺ T cells in endocervix, but not ectocervix. Our findings demonstrate a previously unrecognized compartmentalization of TRMs in the FRT of postmenopausal women, with loss of TRMs and DCs in the cervix with aging, and increased TRMs and DC induction capacity in the endometrium. These findings are relevant to understanding immune protection in the FRT and to the design of vaccines for women of all ages.     

Induction of peripheral tolerance to experimental autoimmune myasthenia gravis by acetylcholine receptor-pulsed dendritic cells

Dendritic cells (DC) are usually regarded as antigen-presenting cells involved in T cell activation, but DC also directly and indirectly affect B cell activation, antibody synthesis, and isotype switch. In the present study, bone marrow (BM)-derived DC from healthy rats were pulsed in vitro with acetylcholine receptor (AChR) and injected subcutaneously into healthy Lewis rats. No clinical signs of the first phase of experimental autoimmune myasthenia gravis (EAMG) were observed during 3 weeks of observation. Upon immunization with AChR and complete Freund's adjuvant, the rats that had received AChR-pulsed DC did not develop clinical EAMG. This tolerance of rats injected with AChR-pulsed DC was associated with reduced expression of B cell-activating factor (BAFF) and by reduced numbers of B cells among splenic mononuclear cells (MNC) compared to rats injected with medium or unpulsed DC. Anti-AChR IgG antibody-secreting cells were decreased, while the ratio of IgG1:IgG2b isotypes was enhanced in rats treated with AChR-pulsed DC compared to control EAMG rats. These results demonstrate that AChR-pulsed DC induce peripheral tolerance to EAMG by possibly inhibiting the expression of BAFF and production of anti-AChR antibodies, providing a possible potential for immunotherapy of antibody-mediated autoimmune diseases.     

Hidden talents of natural killers: NK cells in innate and adaptive immunity

Natural killer (NK) cells are innate immune lymphocytes capable of killing target cells and producing immunoregulatory cytokines. Herein, we discuss recent studies that indicate that NK cells span the conventional boundaries between innate and adaptive immunity. For example, it was recently discovered that NK cells have the capacity for memory‐like responses, a property that was previously thought to be limited to adaptive immunity. NK cells have also been identified in multiple tissues, and a subset of cells that specialize in the production of the T_H17 cytokine IL‐22, NK‐22s, was recently described in mucosal‐associated lymphoid tissue. Finally, we review work that shows that NK cells develop at sites that were traditionally thought to be occupied only by adaptive immune cells, including the thymus and lymph nodes.     

Tolerogenic semimature dendritic cells suppress experimental autoimmune thyroiditis by activation of thyroglobulin-specific CD4+CD25+ T cells

Ex vivo treatment of bone marrow-derived dendritic cells (DCs) with TNF-alpha has been previously shown to induce partial maturation of DCs that are able to suppress autoimmunity. In this study, we demonstrate that i.v. administration of TNF-alpha-treated, semimature DCs pulsed with thyrogloblin (Tg), but not with OVA Ag, inhibits the subsequent development of Tg-induced experimental autoimmune thyroiditis (EAT) in CBA/J mice. This protocol activates CD4(+)CD25(+) T cells in vivo, which secrete IL-10 upon specific recognition of Tg in vitro and express regulatory T cell (Treg)-associated markers such as glucocorticoid-induced TNFR, CTLA-4, and Foxp3. These CD4(+)CD25(+) Treg cells suppressed the proliferation and cytokine release of Tg-specific, CD4(+)CD25(-) effector cells in vitro, in an IL-10-independent, cell contact-dependent manner. Prior adoptive transfer of the same CD4(+)CD25(+) Treg cells into CBA/J hosts suppressed Tg-induced EAT. These results demonstrate that the tolerogenic potential of Tg-pulsed, semimature DCs in EAT is likely to be mediated through the selective activation of Tg-specific CD4(+)CD25(+) Treg cells and provide new insights for the study of Ag-specific immunoregulation of autoimmune diseases.     

Phosphodiesterase 4A confers resistance to PGE2‐mediated suppression in CD25+/CD54+ NK cells

Inadequate persistence of tumor‐infiltrating natural killer (NK) cells is associated with poor prognosis in cancer patients. The solid tumor microenvironment is characterized by the presence of immunosuppressive factors, including prostaglandin E2 (PGE2), that limit NK cell persistence. Here, we investigate if the modulation of the cytokine environment in lung cancer with IL‐2 or IL‐15 renders NK cells resistant to suppression by PGE2. Analyzing Cancer Genome Atlas (TCGA) data, we found that high NK cell gene signatures correlate with significantly improved overall survival in patients with high levels of the prostaglandin E synthase (PTGES). In vitro, IL‐15, in contrast to IL‐2, enriches for CD25⁺/CD54⁺ NK cells with superior mTOR activity and increased expression of the cAMP hydrolyzing enzyme phosphodiesterase 4A (PDE4A). Consequently, this distinct population of NK cells maintains their function in the presence of PGE2 and shows an increased ability to infiltrate lung adenocarcinoma tumors in vitro and in vivo. Thus, strategies to enrich CD25⁺/CD54⁺ NK cells for adoptive cell therapy should be considered.     

Adoptive transfer of CD3+ T cells and CD4+CD44high memory T cells induces autoimmune pancreatitis in MRL/MpJ mice

The immunopathogenesis of autoimmune pancreatitis (AIP) is poorly understood. Here, we have used MRL/MpJ mice, a model of spontaneous AIP, to address the role of cellular autoimmune processes in the initiation and progression of the disease. Therefore, different T cell subpopulations were adoptively transferred from sick to still healthy (but susceptible) MRL/MpJ mice. Unpurified splenocytes and CD3⁺ T cells both efficiently induced AIP, while CD4⁺ and CD8⁺ T cells alone, as well as splenocytes from healthy mice, were insufficient to trigger the disease. Strikingly, CD4⁺CD44^high memory T cells, although transferred at lower numbers than other T cells, also induced AIP in recipient mice. Employing a modified experimental design, we also evaluated the effects of regulatory T cells (T_regs) on the progression of AIP in already diseased mice. Under the given experimental conditions, there was no significant suppressive effect of adoptively transferred T_regs on pancreatic histopathology. The results of our studies suggest a key role of T cell‐mediated processes in murine AIP. The effects of CD4⁺CD44^high memory T cells are in accordance with genetic studies of our group, which had previously implicated this cell type into the pathogenesis of AIP. In follow‐up studies, we will focus on the interplay of cellular and humoral autoimmunity in the context of AIP.     

Tumor cells prevent mouse dendritic cell maturation induced by TLR ligands

Tumor cells can evade the immune system through several mechanisms, one of which is to block DC maturation. It has been suggested that signaling via Toll-like receptors (TLR) may be involved in the induction of prophylactic anti-cancer immunity and in the treatment of established tumors. In the present study we found that high numbers of tumor cells interfere with BMDC activation induced by the TLR ligands LPS and poly IC. Tumor cells blocked TLR3- and TLR4-mediated induction of MHCII and the co-stimulatory molecules CD40 and CD86, as well as the cytokines IL-12, TNF-α and IL-6. Importantly, tumor cells induced inhibitory molecules (B7-DC, B7-H1 and CD80) on spleen DC in vivo and on BMDC, even in the presence of TLR ligands. Moreover, after a long exposure with tumor cells, purified BMDC were unable to respond to a second challenge with TLR ligands. The failure of tumor exposed-BMDC to express co-stimulatory molecules and cytokines in the presence of TLR ligands has implications for the future development of DC-based cancer immune therapies using TLR ligands as adjuvants for the activation of DC.     

Forced IDO1 expression in dendritic cells restores immunoregulatory signalling in autoimmune diabetes

Indoleamine 2,3‐dioxygenase (IDO1), a tryptophan catabolizing enzyme, is recognized as an authentic regulator of immunity in several physiopathologic conditions. We have recently demonstrated that IDO1 does not merely degrade tryptophan and produce immunoregulatory kynurenines, but it also acts as a signal‐transducing molecule, independently of its enzymic function. IDO1 signalling activity is triggered in plasmacytoid dendritic cells (pDCs) by transforming growth factor‐β (TGF‐β), an event that requires the non‐canonical NF‐κB pathway and induces long‐lasting IDO1 expression and autocrine TGF‐β production in a positive feedback loop, thus sustaining a stably regulatory phenotype in pDCs. IDO1 expression and catalytic function are defective in pDCs from non‐obese diabetic (NOD) mice, a prototypic model of autoimmune diabetes. In the present study, we found that TGF‐β failed to activate IDO1 signalling function as well as up‐regulate IDO1 expression in NOD pDCs. Moreover, TGF‐β‐treated pDCs failed to exert immunosuppressive properties in vivo. Nevertheless, transfection of NOD pDCs with Ido1 prior to TGF‐β treatment resulted in activation of the Ido1 promoter and induction of non‐canonical NF‐κB and TGF‐β, as well as decreased production of the pro‐inflammatory cytokines, interleukin 6 (IL‐6) and tumour necrosis factor‐α (TNF‐α). Overexpression of IDO1 in TGF‐β‐treated NOD pDCs also resulted in pDC ability to suppress the in vivo presentation of a pancreatic β‐cell auto‐antigen. Thus, our data suggest that a correction of IDO1 expression may restore its dual function and thus represent a proper therapeutic manoeuvre in this autoimmune setting.     

IL‐35Ig–expressing dendritic cells induce tolerance via Arginase 1

The cytokine interleukin IL‐35 is known to exert strong immunosuppressive functions. Indoleamine 2,3‐dioxygenase 1 (IDO1) and Arginase 1 (Arg1) are metabolic enzymes that, expressed by dendritic cells (DCs), contribute to immunoregulation. Here, we explored any possible link between IL‐35 and the activity of those enzymes. We transfected a single chain IL‐35Ig gene construct in murine splenic DCs (DC₃₅) and assessed any IDO1 and Arg1 activities as resulting from ectopic IL‐35Ig expression, both in vitro and in vivo. Unlike Ido1, Arg1 expression was induced in vitro in DC₃₅, and it conferred an immunosuppressive phenotype on those cells, as revealed by a delayed‐type hypersensitivity assay. Moreover, the in vivo onset of a tolerogenic phenotype in DC₃₅ was associated with the detection of CD25⁺CD39⁺, rather than Foxp3⁺, regulatory T cells. Therefore, Arg1, but not Ido1, expression in DC₃₅ appears to be an early event in IL‐35Ig–mediated immunosuppression.     

The limitation of IL-10-exposed dendritic cells in the treatment of experimental autoimmune myasthenia gravis and myasthenia gravis

Dendritic cells (DC) are highly specialized antigen presenting cells that play critical roles as instigators and regulators of immune responses including B cell function, antibody synthesis and isotype switch. In this study, we compared immunotherapeutic effect of IL-10-treated DC (IL-10-DC) via both intraperitoneal (i.p.) and subcutaneous (s.c.) delivery in rats with incipient experimental autoimmune myasthenia gravis (EAMG). Spleen DC were isolated from onset of EAMG on day 39 post-immunization, exposed in vitro to IL-10, and then injected into incipient EAMG at dose of 1 x 10(6) cells/rat on day 5 after immunization. Intraperitoneal administration of IL-10-DC suppressed clinical scores, anti-acetylcholine receptors (AChR) antibody secreting cells, antigen-specific IL-10/IFN-gamma production and T cell proliferation compared to control EAMG rats. Importantly, IL-10-DC, if given by s.c. route, failed to ameliorate clinical sign of EAMG. Simultaneously, T cell proliferation, anti-AChR antibody secreting cells and IL-10/IFN-gamma production had no alteration, as compared to control EAMG rats. Both in vitro and in vivo experiments showed that treatment of IL-10 inhibited the migration of DC toward MIP-3beta and lymph node, indicating that in vitro manipulation of DC with IL-10 alters the migration of DC that influences the therapeutic effect in the treatment of autoimmune diseases. In MG patients, neither the improvement of clinical symptom nor the alteration of immunological parameter was observed through s.c. delivery of IL-10-DC, suggesting the limitation of IL-10-DC in the treatment of MG patients.