Interleukin-15-Induced CD56+ Myeloid Dendritic Cells Combine Potent Tumor Antigen Presentation with Direct Tumoricidal Potential

Dendritic cells (DCs) are the quintessential antigen-presenting cells of the human immune system and play a prime role in coordinating innate and adaptive immune responses, explaining the strong and still growing interest in their application for cancer immunotherapy. Much current research in the field of DC-based immunotherapy focuses on optimizing the culture conditions for in vitro DC generation in order to assure that DCs with the best possible immunogenic qualities are being used for immunotherapy. In this context, monocyte-derived DCs that are alternatively induced by interleukin-15 (IL-15 DCs) have attracted recent attention due to their superior immunostimulatory characteristics. In this study, we show that IL-15 DCs, in addition to potent tumor antigen-presenting function, possess tumoricidal potential and thus qualify for the designation of killer DCs. Notwithstanding marked expression of the natural killer (NK) cell marker CD56 on a subset of IL-15 DCs, we found no evidence of a further phenotypic overlap between IL-15 DCs and NK cells. Allostimulation and antigen presentation assays confirmed that IL-15 DCs should be regarded as bona fide myeloid DCs not only from the phenotypic but also from the functional point of view. Concerning their cytotoxic activity, we demonstrate that IL-15 DCs are able to induce apoptotic cell death of the human K562 tumor cell line, while sparing tumor antigen-specific T cells. The cytotoxicity of IL-15 DCs is predominantly mediated by granzyme B and, to a small extent, by tumor necrosis factor-α (TNF-α)-related apoptosis-inducing ligand (TRAIL) but is independent of perforin, Fas ligand and TNF-α. In conclusion, our data provide evidence of a previously unappreciated role for IL-15 in the differentiation of human monocytes towards killer DCs. The observation that IL-15 DCs have killer DC capacity lends further support to their implementation in DC-based immunotherapy protocols.     

IL-15 activated human peripheral blood dendritic cell kill allogeneic and xenogeneic endothelial cells via apoptosis

IL-15 is a pleotropic cytokine, which plays an important role in natural killer (NK) cell activity, T cell proliferation, and T cell cytotoxic activity. Dendritic cells (DCs) are the major antigen presenting cells in the immune system and presumed to play an important role in immune recognition of allo and xenotransplantation. We showed that IL-15 activated human peripheral blood DC is cytotoxic to human and porcine aortic endothelial cells. Unlike DCs, CD14+ monocytes show no cytotoxicity against the endothelial cells. This cytotoxic potential of IL-15 activated DC against endothelial cells is dose dependent and increases significantly upon treatment of endothelial cells with inflammatory cytokines like TNF-α or IFN-γ. The cytotoxic potential of IL-15 activated DC is associated with apoptosis of endothelial cells, as indicated by the increased Annexin V staining, caspase activation and loss of mitochondrial membrane potential. Further it was observed that DC mediated cytotoxicity against endothelial cell is mediated via granzyme B possibly secreted by the activated DCs.     

Dendritic cell-induced activation of adaptive and innate antitumor immunity

While studying Ag-pulsed syngeneic dendritic cell (DC) immunization, we discovered that surprisingly, unpulsed DCs induced protection against tumor lung metastases resulting from i.v. injection of a syngeneic BALB/c colon carcinoma CT26 or a syngeneic C57BL/6 lung carcinoma LL/2. Splenocytes or immature splenic DCs did not protect. The protection was mediated by NK cells, in that it was abrogated by treatment with anti-asialo-GM1 but not anti-CD8, and was induced by CD1(-/-) DCs unable to stimulate NKT cells, but did not occur in beige mice lacking NK cells. Protection correlated with increased NK activity, and increased infiltration of NK but not CD8(+) cells in lungs of tumor-bearing mice. Protection depended on the presence of costimulatory molecules CD80, CD86, and CD40 on the DCs, but surprisingly did not require DCs that could make IL-12 or IL-15. Unexpectedly, protection sensitive to anti-asialo-GM1 and increased NK activity were still present 14 mo after DC injection. As NK cells lack memory, we found by depletion that CD4(+) not CD8(+) T cells were required for induction of the NK antitumor response. The role of DCs and CD4(+) T cells provides a novel mechanism for NK cell induction and innate immunity against cancer that may have potential in preventing clinical metastases.     

MHC-I Molecules Selectively Inhibit Cell-Mediated Cytotoxicity Triggered by ITAM-Coupled Activating Receptors and 2B4

NK cell effector functions are controlled by a combination of inhibitory receptors, which modulate NK cell activation initiated by stimulatory receptors. Most of the canonical NK cell inhibitory receptors recognize allelic forms of classical and non-classical MHC class I molecules. Furthermore, high expression of MHC-I molecules on effector immune cells is also associated with reverse signaling, giving rise to several immune-regulatory functions. Consequently, the inhibitory function of MHC class I expressed on a human NKL cell line and activated primary NK and T cells on different activating receptors are analyzed in this paper. Our results reveal that MHC-I molecules display specific patterns of “selective” inhibition over cytotoxicity and cytokine production induced by ITAM-dependent receptors and 2B4, but not on NKG2D. This contrasts with the best known “canonical” inhibitory receptors, which constitutively inhibit both functions, regardless of the activating receptor involved. Our results support the existence of a new fine-tuner inhibitory function for MHC-I molecules expressed on cytotoxic effector cells that could be involved in establishing self-tolerance in mature activated NK cells, and could also be important in tumor and infected cell recognition.     

Toll-Like Receptor 4 Engagement Drives Differentiation of Human and Murine Dendritic Cells from a Pro- into an Anti-Inflammatory Mode

The dendritic cell (DC) coordinates innate and adaptive immunity to fight infections and cancer. Our observations reveal that DCs exposed to the microbial danger signal lipopolysaccharide (LPS) in the presence of interferon-γ (IFN-γ) acquire a continuously changing activation/maturation phenotype. The DCs’ initial mode of action is pro-inflammatory via up-regulation among others of the signaling molecule interleukin (IL) 12, which polarizes IFN-γ secreting type 1 helper T-cells (Th1). Within 24 hours the same DC switches from the pro- into an anti-inflammatory phenotype. This is mediated by autocrine IL-10 release and secretion of soluble IL-2 receptor alpha (sIL-2RA) molecules. T-cells, when contacted with DCs during their anti-inflammatory phase loose their proliferative capacity and develop regulatory T-cell (Treg) -like anti-inflammatory functions indicated by IL-10 secretion and elevated FoxP3 levels. Studying the kinetics of IL-12 and IL-10 expression from LPS/IFN-γ activated myeloid DCs on a single cell level confirmed these observations. When T-cells are separated from DCs within 24 hours, they are spared from the anti-inflammatory DC activity. We conclude that, in addition to differentiation of DCs into distinct subsets, the observed sequential functional phases of DC differentiation permit the fine-tuning of an immune response. A better understanding of time-kinetic DC features is required for optimally exploiting the therapeutic capacity of DCs in cancer immune therapy.     

Novel IL-15 dendritic cells have a potent immunomodulatory effect in immunotherapy of multiple myeloma

Dendritic cells (DCs) are the most potent antigen-presenting cells, and have thus been used in clinical cancer vaccines. However, the effects of DC vaccines are still limited, leading researchers to explore novel ways to make them effective. In this study, we investigated whether human monocyte-derived DCs generated via the addition of interleukin 15 (IL-15) had a higher capacity to induce antigen-specific T cells compared to conventional DCs. We isolated CD14⁺ monocytes from peripheral blood from multiple myeloma (MM) patients, and induced immature DCs with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 in the presence or absence of IL-15 for 4–6 days. Then we generated mature DCs (mDCs) with lipopolysaccharide for another 2 days [IL-15 mDCs (6 days), IL-15 mDCs (8 days), and conventional mDCs (8 days)]. IL-15 mDCs (6 days) showed higher expression of MHC I and II, CD40, CD86, and CCR7, and the secretion of IFN-γ was significantly higher compared to conventional mDCs. IL-15 mDCs (6 days) showed superior polarization of naïve T cells toward Th1 cells and a higher proportion of activated T cells, cytokine-induced killer (CIK) cells, and natural killer (NK) cells for inducing strong cytotoxicity against myeloma cells, and lower proportion of regulatory T cells compared to conventional mDCs. These data imply that novel multipotent mDCs generated by the addition of IL-15, which can be cultivated in 6 days, resulted in outstanding activation of T cells, CIK cells and NK cells, and may facilitate cellular immunotherapy for cancer patients.     

Cutting edge: murine dendritic cells require IL-15R alpha to prime NK cells

NK cells protect hosts against viral pathogens and transformed cells, and dendritic cells (DCs) play important roles in activating NK cells. We now find that murine IL-15Ralpha-deficient DCs fail to support NK cell cytolytic activity and elaboration of IFN-gamma, despite the fact that these DCs express normal levels of costimulatory molecules and IL-12. By contrast, IL-15Ralpha expression on NK cells is entirely dispensable for their activation by DCs. In addition, blockade with anti-IL-15Ralpha and anti-IL-2Rbeta but not anti-IL-2Ralpha-specific Abs prevents NK cell activation by wild-type DCs. Finally, presentation of IL-15 by purified IL-15Ralpha/Fc in trans synergizes with IL-12 to support NK cell priming. These findings suggest that murine DCs require IL-15Ralpha to present IL-15 in trans to NK cells during NK cell priming.     

An Optimized Method for Manufacturing a Clinical Scale Dendritic Cell-Based Vaccine for the Treatment of Glioblastoma

Immune-based treatments represent a promising new class of therapy designed to boost the immune system to specifically eradicate malignant cells. Immunotherapy may generate specific anti-tumor immune responses, and dendritic cells (DC), professional antigen-presenting cells, are widely used in experimental cancer immunotherapy. Several reports describe methods for the generation of mature, antigen-pulsed DC for clinical use. Improved quality and standardization are desirable to obtain GMP-compliant protocols. In this study we describe the generation of DC from 31 Glioblastoma (GB) patients starting from their monocytes isolated by immunomagnetic CD14 selection using the CliniMACS® device. Upon differentiation of CD14+ with IL-4 and GM-CSF, DC were induced to maturation with TNF-α, PGE₂, IL-1β, and IL-6. Whole tumor lysate was obtained, for the first time, in a closed system using the semi-automated dissociator GentleMACS®. The yield of proteins improved by 130% compared to the manual dissociation method. Interestingly the Mean Fluorescence Intensity for CD83 increased significantly in DC pulsed with “new method” lysate compared to DC pulsed with “classical method” lysate. Our results indicate that immunomagnetic isolation of CD14⁺ monocytes using the CliniMACS® device and their pulsing with whole tumor lysate proteins is a suitable method for clinical-scale generation of high quality, functional DC under GMP-grade conditions.     

Photodynamic-therapy Activates Immune Response by disrupting Immunity Homeostasis of Tumor Cells,which Generates Vaccine for Cancer Therapy

Photodynamic therapy (PDT), a regulatory approved cancer treatment, is reported to be capable of causing immunogenic apoptosis. The current data reveal PDT can cause the dysregulation of “eat me” and “don''t eat me” signal by generating reactive oxygen species (ROS) -mediated endoplasmic reticulum (ER) stress. This dysregulation probably contribute to the increased uptake of PDT-killed Lewis lung carcinoma (LLC) cells by homologous dendritic cells (DCs), accompanied by phenotypic maturation (CD80^high, CD86^high, and CD40^high) and functional stimulation (NO^high, IL-10^absent) of dendritic cells as well as subsequent T-cell responses. Morevover, C57BL/6 mice vaccinated with dendritic cells (DCs) pulsed with PDT-treated LLCs (PDT-DCs) or PDT-treated LLCs alone (PDT-LLCs) exhibited potent immunity against LLC tumors. In the current study, the PDT-induced immune response was characterized as a process related with the dysregulation of “eat me” signal and “don''t eat me” signal, revealing the possibility for developing PDT into an antitumor vaccination strategy for personalized cancer immunotherapy.     

Distinct Licensing of IL-18 and IL-1β Secretion in Response to NLRP3 Inflammasome Activation

Inflammasome activation permits processing of interleukins (IL)-1β and 18 and elicits cell death (pyroptosis). Whether these responses are independently licensed or are “hard-wired” consequences of caspase-1 (casp1) activity has not been clear. Here, we show that that each of these responses is independently regulated following activation of NLRP3 inflammasomes by a “non-canonical” stimulus, the secreted Listeria monocytogenes (Lm) p60 protein. Primed murine dendritic cells (DCs) responded to p60 stimulation with reactive oxygen species (ROS) production and secretion of IL-1β and IL-18 but not pyroptosis. Inhibitors of ROS production inhibited secretion of IL-1β, but did not impair IL-18 secretion. Furthermore, DCs from caspase-11 (casp11)-deficient 129S6 mice failed to secrete IL-1β in response to p60 but were fully responsive for IL-18 secretion. These findings reveal that there are distinct licensing requirements for processing of IL-18 versus IL-1β by NLRP3 inflammasomes.     

Natural Killer Cell Signal Integration Balances Synapse Symmetry and Migration

Natural killer (NK) cells discern the health of other cells by recognising the balance of activating and inhibitory ligands expressed by each target cell. However, how the integration of activating and inhibitory signals relates to formation of the NK cell immune synapse remains a central question in our understanding of NK cell recognition. Here we report that ligation of LFA-1 on NK cells induced asymmetrical cell spreading and migration. In contrast, ligation of the activating receptor NKG2D induced symmetrical spreading of ruffled lamellipodia encompassing a dynamic ring of f-actin, concurrent with polarization towards a target cell and a “stop” signal. Ligation of both LFA-1 and NKG2D together resulted in symmetrical spreading but co-ligation of inhibitory receptors reverted NK cells to an asymmetrical migratory configuration leading to inhibitory synapses being smaller and more rapidly disassembled. Using micropatterned activating and inhibitory ligands, signals were found to be continuously and locally integrated during spreading. Together, these data demonstrate that NK cells spread to form large, stable, symmetrical synapses if activating signals dominate, whereas asymmetrical migratory “kinapses” are favoured if inhibitory signals dominate. This clarifies how the integration of activating and inhibitory receptor signals is translated to an appropriate NK cell response.     

Growth Differentiation Factor-15 Suppresses Maturation and Function of Dendritic Cells and Inhibits Tumor-Specific Immune Response

Dendritic cells (DCs) play a key role in the initiation stage of an antigen-specific immune response. A variety of tumor-derived factors (TDFs) can suppress DC maturation and function, resulting in defects in the tumor-specific immune response. To identify unknown TDFs that may suppress DCs maturation and function, we established a high-throughput screening technology based on a human liver tumor T7 phage cDNA library and screened all of the proteins derived from hepatoma cells that potentially interact with immature DCs. Growth/differentiation factor-15 (GDF-15) was detected and chosen for further study. By incubation of DCs cultures with GDF-15, we demonstrate that GDF-15 can inhibit surface protrusion formation during DC maturation; suppress the membrane expression of CD83, CD86 and HLA-DR on DCs; enhance phagocytosis by DCs; reduce IL-12 and elevate TGF-β1 secretion by DCs; inhibit T cell stimulation and cytotoxic T lymphocyte (CTL) activation by DCs. By building tumor-bearing mouse models, we demonstrate that GDF-15 can inhibit the ability of DCs to stimulate a tumor-specific immune response in vivo. These results indicate that GDF-15 may be one of the critical molecules that inhibit DC maturation and function and are involved in tumor immune escape. Thus, GDF-15 may be a novel target in tumor immunotherapy.     

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.     

Identification of a Human Cyclin D1-Derived Peptide that Induces Human Cytotoxic CD4 T Cells

Cyclin D1 is over-expressed in various human tumors and therefore can be a potential oncogenic target antigen. However, only a limited number of T cell epitopes has been characterized. We aimed at identifying human cyclin D1-derived peptides that include both CD4 and CD8 T cell epitopes and to test if such multi-epitope peptides could yield improved cytotoxic CD8 T cell responses as well as cytotoxic CD4 T cells. Five HLA-DR.B1-binding peptides containing multiple overlapping CD4 epitopes and HLA-A0201-restricted CD8 T cell epitopes were predicted by computer algorithms. Immunogenicity of the synthetic peptides was assessed by stimulating T cells from healthy donors in vitro and the epitope recognition was measured by IFN-γ ELISPOT and ⁵¹Chromium release assays. A HLA-DR.B1 peptide, designed “DR-1”, in which a HLA-A0201-binding epitopes (D1-1) was imbedded, induced CD3 T cell responses against both DR-1 and D1-1 peptides in IFN-γ ELISPOT assay. This suggested processing of the shorter D1-1 epitope from the DR-1 sequence. However, only DR-1-stimulated CD4 or CD3 T cells possessed cytotoxicity against peptide-pulsed autologous DCs and a cancer cell line, that expresses a high level of cyclin D1. Monoclonal antibody to HLA-DR abrogated the epitope-specific responses of both CD3 and CD4 T cells, demonstrating class II-mediated killing. Our studies suggest a possible role of CD4 T cells in anti-tumor immunity as cytotoxic effectors against HLA-DR expressing cancers and provide a rationale for designing peptide vaccines that include CD4 epitopes.     

Dendritic cells in innate immune responses against HIV

Dendritic cells (DCs) were recently found to be innate immunity effectors against tumoral cells and viruses. (i) In response to most viruses, including HIV, plasmacytoid DCs are responsible for most of the type I IFN secretion, which is strongly anti-viral and induces TH1 type responses. Myeloid DCs secrete IL-12, which is also important for TH1-type and cytotoxic responses. In HIV patient blood, both DC population numbers decrease as early as the primary stage. Plasmacytoid DC numbers correlate with type I IFN secretion, which is a prognosis predictor, particularly under treatment. IL-12 secretion is also defective. Immunotherapies to replace the defective cytokines or to restore a potentially defective DC-T lymphocyte feed-back might help patients restore their immune responses under antiviral therapy. (ii) After measles and other viral infections, or incubation with dsRNA, DCs become cytotoxic and consequently exhibit natural killer function, through upregulation of type I IFN secretion which enhances TRAIL expression. In HIV infection, this mechanism was not demonstrated yet, but it might a) be responsible for the massive apoptosis of uninfected lymphocytes, and b) increase specific immunity through cross-presentation of antigens from infected cells killed by DCs. (iii) DCs direct expansion and effector functions of NK cells in the absence of adaptive-type cytokines and modulate NKT cell IFN-gamma production. Reciprocally, NK activation triggers DC maturation. HIV-1 Tat inhibits NK cell cytotoxicity directly and probably through inhibition of IL-12 secretion by DC. Therefore, understanding the functions of DCs in innate immune responses and in pathogenesis will help obtain better HIV replication control.     

Generation of a novel regulatory NK cell subset from peripheral blood CD34+ progenitors promoted by membrane-bound IL-15

Background

NK cells have been long time considered as cytotoxic lymphocytes competent in killing virus-infected cells and tumors. However, NK cells may also play essential immuno-regulatory functions. In this context, the real existence of a defined NK subset with negative regulatory properties has been hypothesized but never clearly demonstrated.

Methodology/Principal Findings

Herein, we show the in vitro generation from human peripheral blood haematopoietic progenitors (PB-HP), of a novel subset of non-cytolytic NK cells displaying a mature phenotype and remarkable immuno-regulatory functions (NK-ireg). The main functional hallmark of these NK-ireg cells is represented by the surface expression/release of HLA-G, a major immunosuppressive molecule. In addition, NK-ireg cells secrete two powerful immuno-regulatory factors: IL-10 and IL-21. Through these factors, NK-ireg cells act as effectors of the down-regulation of the immune response: reconverting mature myeloid DC (mDC) into immature/tolerogenic DC, blocking cytolytic functions on conventional NK cells and inducing HLA-G membrane expression on PB-derived monocytes. The generation of “NK-ireg” cells is obtained, by default, in culture conditions favouring cell-to-cell contacts, and it is strictly dependent on reciprocal trans-presentation of membrane-bound IL-15 forms constitutively and selectively expressed by human CD34⁺ PB-HP. Finally, a small subset of NKp46⁺ HLA-G⁺ IL-10⁺ is detected within freshly isolated decidual NK cells, suggesting that these cells could represent an in vivo counterpart of the NK-ireg cells.

Conclusions/Significance

In conclusion, NK-ireg cells represent a novel truly differentiated non-cytolytic NK subset with a self-sustainable phenotype (CD56⁺ CD16⁺ NKp30⁺ NKp44⁺ NKp46⁺ CD94⁺ CD69⁺ CCR7⁺) generated from specific pSTAT6⁺ GATA3⁺ precursors. NK-ireg cells could be employed to develop new immuno-suppressive strategies in autoimmune diseases, transplant rejection or graft versus host diseases. In addition, NK-ireg cells can be easily derived from peripheral blood of the patients and could constitute an autologous biotherapic tool to be used combined or in alternative to other immuno-regulatory cells.     

Innate direct anticancer effector function of human immature dendritic cells. I. Involvement of an apoptosis-inducing pathway

Dendritic cells (DCs) mediate cross-priming of tumor-specific T cells by acquiring tumor Ags from dead cancer cells. The process of cross-priming would be most economical and efficient if DCs also induce death of cancer cells. In this study, we demonstrate that normal human in vitro generated immature DCs consistently and efficiently induce apoptosis in cancer cell lines, freshly isolated noncultured cancer cells, and normal proliferating endothelial cells, but not in most normal cells. In addition, in vivo generated noncultured peripheral blood immature DCs mediate similar tumoricidal activity as their in vitro counterpart, indicating that this DC activity might be biologically relevant. In contrast to immature DCs, freshly isolated monocytes (myeloid DC precursors) and in vitro generated mature DCs are not cytotoxic or are less cytotoxic, respectively, suggesting that DC-mediated killing of cancer cells is developmentally regulated. Comparable cytotoxic activity is mediated by untreated DCs, paraformaldehyde-fixed DCs, and soluble products of DCs, and is destructible by proteases, indicating that both cell membrane-bound and secreted proteins mediate this DC function. Overall, our data demonstrate that human immature DCs are capable of inducing apoptosis in cancer cells and thus to both directly mediate anticancer activity and initiate processing of cellular tumor Ags.     

Isoflavones,Genistein and Daidzein,Regulate Mucosal Immune Response by Suppressing Dendritic Cell Function

Lipopolysaccharide (LPS), a component of gram-negative bacterial cell walls, has been shown to have a strong adjuvant effect towards inhaled antigens contributing to airway inflammation. Isoflavones are anti-inflammatory molecules present in abundant quantities in soybeans. We investigated the effect of isoflavones on human dendritic cell (DC) activation via LPS stimulation and subsequent DC-mediated effector cell function both in vitro and in a mouse model of upper airway inflammation. Human monocyte-derived DCs (MDDC) were matured with LPS (or TNF-α) +/− isoflavones (genistein or daidzein). The surface expression levels of DC activation markers were analyzed by flow cytometry. Mature DCs +/− isoflavones were washed and cultured with freshly-isolated allogenic naïve CD4⁺ T cells for 5 days or with autologous natural killer (NK) cells for 2 hours. The percentages of proliferating IFN-γ⁺ CD4⁺ T cells and cytokine levels in culture supernatants were assessed. NK cell degranulation and DC cytotoxicity were measured by flow cytometry. Isoflavones significantly suppressed the activation-induced expression of DC maturation markers (CD83, CD80, CD86) and MHC class I but not MHC class II molecules in vitro. Isoflavone treatment inhibited the ability of LPS-DCs to induce IFN-γ in CD4⁺ T cells. NK cell degranulation and the percentage of dead DCs were significantly increased in isoflavone-treated DC-NK co-culture experiments. Dietary isoflavones suppressed the mucosal immune response to intra-nasal sensitization of mice to ovalbumin. Similar results were obtained when isoflavones were co-administered during sensitization. These results demonstrate that soybean isoflavones suppress immune sensitization by suppressing DC-maturation and its subsequent DC-mediated effector cell functions.     

Artificial feeder cells expressing ligands for killer cell immunoglobulin-like receptors and CD94/NKG2A for expansion of functional primary natural killer cells with tolerance to self

Background aimsNatural killer (NK) cells are promising cells for immunotherapy of cancer, and there are ongoing efforts to improve their ex vivo expansion to clinically relevant numbers. This study focused on the development of a C1-, C2-, Bw4 killer cell immunoglobulin-like receptor (KIR) ligand and NKG2A ligand-containing feeder cell line for autonomous expansion of functional NK cells.MethodsPC3^PSCA-derived feeder cells expressing IL-2, 4-1BBL and membrane-bound IL-15-mutDAP12 (mIL-15d) fusion protein in combinations or alone were generated and used for expansion. Expanded NK cells were analyzed with respect to subpopulations, expression of NK cell receptors and immune checkpoint molecules as well as their cytotoxicity against K562 cells, cetuximab-marked tumor cells and autologous B cells.ResultsOnly combinatorial expression of IL-2 plus 4-1BBL or IL-2, 4-1BBL plus mIL-15d in feeder cells efficiently expanded NK cells and supported selective outgrowth of NK cells from peripheral blood mononuclear cell samples. Best expansion of NK cells was achieved using PC3^PSCA-IL-2-4-1BBL-mIL-15d feeder cells. Such expanded NK cells exhibited upregulation of natural cytotoxicity receptors, DNAM-1 and NKG2C and induced expression of high affinity IL-2 receptor, which were paralleled by attenuated KIR and increased expression of NKG2A and ILT2. In addition, elevated TIM-3 levels were noted and PD-1 and T cell immunoreceptor with Ig and ITIM domain (TIGIT) levels remained low. Expanded NK cells were highly cytolytic when encountering K562 cells and cetuximab-marked target cells but remained unresponsive to autologous B cells and target cells with protective levels of human leukocyte antigen.ConclusionsCollectively, the results demonstrate the feasibility of PC3^PSCA-IL-2-4-1BBL-mIL-15d feeder cells for robust expansion of NK cells, which remain tolerant to self and could be used in the future for adoptive cell therapy of cancer.     

Effect of interleukin-15 on depressed splenic dendritic cell functions following trauma-hemorrhage

Although trauma-hemorrhage (T-H) induces suppressed splenic dendritic cell (DC) maturation and antigen presentation capacity, it remains unclear whether IL-15 modulates splenic DC functions. The aim of this study therefore was to investigate the effect of IL-15 on splenic DC functions after T-H. Male C3H/HeN mice (6-8 wk old) were randomly assigned to T-H or sham operation. T-H was induced by midline laparotomy and approximately 90 min of hemorrhagic shock (blood pressure 35 mmHg), followed by fluid resuscitation (4x the shed blood volume in the form of Ringer lactate). Two hours later, mice were killed, splenic DCs were isolated, and the effects of exogenous IL-15 on their costimulatory factors, major histocompatibility class II expression, ability to produce cytokines, and antigen presentation were measured. The results indicate that IL-15 production capacity of splenic DCs was reduced following T-H. Ex vivo exposure to IL-15 attenuated the suppressed production of TNF-alpha, IL-6, and IFN-gamma from splenic DCs following T-H. In addition, expression of surface antigen studies demonstrate that exogenous IL-15 attenuated T-H-induced downregulation of the activation of DC. The suppressed splenic DC antigen presentation function following T-H was also attenuated by IL-15 treatment. Moreover, IL-15 enhanced IL-12-induced IFN-gamma production and antigen presentation by splenic DCs. These data suggest that ex vivo treatment with IL-15 following T-H provides beneficial effects on splenic DCs. The depression in IL-15 production by splenic DCs could contribute to the host's enhanced susceptibility to infections following T-H.