Increased Cytokine Secretion Research Articles

Dental implant surface treatments may modulate cytokine secretion in Porphyromonas gingivalis‐stimulated human gingival fibroblasts: A comparative study

Peri-implantitis is an inflammation that affects dental implants and can lead to implant loss. The aim of this study was to analyze the in vitro effect of different implant surface treatments on cytokine production by human gingival fibroblasts (HGFs) stimulated or not with Porphyromonas gingivalis lipopolysaccharide (PgLPS). Six different titanium implants were tested: turned, sandblasted, anodized, acid-etched, TiO2-blasted/acid-etched, and grit-blasted/acid-etched. HGFs were seeded with each implant in a 6-well plate and assayed before LPS treatment (-LPS) or after 36 h of LPS (+LPS) treatment. Protein concentrations were measured using a Pierce bicinchoninic acid (BCA) assay and cytokine secretions were analyzed using a multiplex cytokine array. Scanning electron microscopy was performed for sterile implants and after cell attachment. Protein levels were consistent across all implants indicating that cell growth was uniform (p > 0.05). Sandblasted and turned surfaces significantly increased the secretion of interleukin (IL)-6, -8, -10, MCP-1 and VEGF (p < 0.05) when compared with the other surfaces. PgLPS stimulus increased cytokine secretion in all tested surfaces. In conclusion, different implant surfaces had various effects on HGFs' cytokine secretion. The findings may provide insights into the progression of peri-implantitis.

The Innate Immune Response in the Airways and in Blood is Altered by Training in Horses

IntroductionLower airway diseases are common problems in sports and racing horses. In humans, exercise causes increased susceptibility to respiratory infections associated with down‐regulated expression of Toll‐like receptors (TLRs), co‐stimulatory and antigen‐presenting molecules. Because numerous cell types are implicated in important defense mechanisms against airborne infections, we aimed to assess the effect of training on innate immune responses of pulmonary alveolar macrophages (PAM), equine bronchial epithelial cells (EBEC) and blood monocytes.MethodsPAM, EBEC and monocytes were collected at rest and after a standardized exercise test (SET) from eight untrained, trained and deconditioned horses. We compared their respective expression of TLR1‐9 by qPCR and their cytokinic responses following exposure to various TLR ligands by ELISA. Data were reported as least square means and analyzed by a global linear mixed model.ResultsThe mRNA expression of TLR3 was down‐regulated by training in PAM (P&lt;0.0001) and monocytes (P&lt;0.015), whereas the mRNA expression of TLR4 was up‐regulated in monocytes (P = 0.0004) only. Monocytes stimulated with TLR2‐ and TLR4‐ligands showed increased cytokine secretion after training and deconditioning (P&lt;0.025). EBEC from trained horses produced less TNF‐alpha (P&lt;0.05) after treatment with ligands of TLR2 and TLR3 compared with EBEC from untrained horses. The production of TNF‐alpha (P&lt;0.001) and IFN‐beta (P&lt;0.001) in non‐stimulated and stimulated PAM was decreased in trained and deconditioned horses.ConclusionsThe impaired cytokine production in PAM and EBEC after a training period might explain the increased susceptibility to respiratory viral infections and is associated with the activation of cells at the systemic level.Ethical Animal ResearchThis work was approved by the Animal Ethics Committee of the National Veterinary School of Alfort (agreement number: 13/12/11‐9). Sources of funding: Hippolia Foundation, the Conseil Regional de Basse‐Normandie, the European Regional Development Fund and the Fonds unique interministeriel. Competing interests: none.

Proinflammatory effect of trivalent arsenical species in a co-culture of Caco-2 cells and peripheral blood mononuclear cells.

Chronic exposure to inorganic arsenic (As) is associated with type 2 diabetes, cardiovascular diseases and cancer. Ingested inorganic As is transformed within the gastrointestinal tract and can give rise to more toxic species such as monomethylarsonous acid [MMA(III)] and dimethylarsinous acid [DMA(III)]. Thus, the intestinal epithelium comes into contact with toxic arsenical species, and the effects of such exposure upon epithelial function are not clear. The present study has evaluated the effect of 1 µM arsenite [As(III)], 0.1 µM MMA(III) and 1 µM DMA(III) upon the release of cytokines [interleukin-6 (IL6), IL8, tumor necrosis factor alpha (TNFα)], using a compartmentalized co-culture model with differentiated Caco-2 cells in the apical compartment and peripheral blood mononuclear cells in the basolateral compartment. In addition, the combined effect of arsenical species and lipopolysaccharide (LPS), both added into the apical compartment, has been analyzed. The results indicate that exposure to the arsenical forms induces a proinflammatory response. An increase in cytokine secretion into the basolateral compartment was observed, particularly as regards TNFα (up to 1,600 %). The cytokine levels on the apical side also increased, though to a lesser extent. As/LPS co-exposure significantly affected the proinflammatory response as compared to treatment with As alone. Treatment with DMA(III) and As/LPS co-exposure increased the permeability of the intestinal monolayer. In addition, As/LPS treatments enhanced As(III) and MMA(III) transport through the intestinal monolayer.

Immune responses to vaccines involving a combined antigen–nanoparticle mixture and nanoparticle-encapsulated antigen formulation

Many physicochemical characteristics significantly influence the adjuvant effect of micro/nanoparticles; one critical factor is the kinetics of antigen exposure to the immune system by particle-adjuvanted vaccines. Here, we investigated how various antigen–nanoparticle formulations impacted antigen exposure to the immune system and the resultant antigen-specific immune responses. We formulated antigen with poly(lactic-co-glycolic acid) (PLGA) nanoparticles by encapsulating antigen within nanoparticles or by simply mixing soluble antigen with the nanoparticles. Our results indicated that the combined formulation (composed of antigen encapsulated in nanoparticles and antigen mixed with nanoparticles) induced more powerful antigen-specific immune responses than each single-component formulation. Mice immunized with the combined vaccine formulation displayed enhanced induction of antigen-specific IgG antibodies with high avidity, increased cytokine secretion by splenocytes, and improved generation of memory T cell. Enhanced immune responses elicited by the combined vaccine formulation might be attributed to the antigen-depot effect at the injection site, effective provision of both adequate initial antigen exposure and long-term antigen persistence, and efficient induction of dendritic cell (DC) activation and follicular helper T cell differentiation in draining lymph nodes. Understanding the effect of antigen–nanoparticle formulations on the resultant immune responses might have significant implications for rational vaccine design.

ASET study: Long-term follow-up data of patients with metastatic renal cell carcinoma treated with MGN1601.

LBA399 Background: The cell-based therapeutic cancer vaccine MGN1601 is a fixed combination of allogeneic tumor cells modified via MIDGE vectors to express IL-7, GM-CSF, CD80, and CD154 plus the immunomodulator dSLIM. In the ASET trial heavily pretreated patients with metastatic renal cell carcinoma (mRCC) were enrolled to assess safety, immunological effects, and clinical efficacy of MGN1601. Methods: The ASET study was a multicenter, single-arm phase 1/2 study. The treatment phase consisted of 8 intradermal vaccinations over 12 weeks. Following evaluation, patients with at least disease stabilization entered an extension phase (EP, 5 vaccinations over 120 weeks). Overall, 19 patients were included and received at least one MGN1601 injection (ITT); the majority (N=17) had ≥3 lines of previous therapy. Results: Of 19 patients, 10 (55%) completed the treatment phase per-protocol (PP); 9 patients discontinued the study due to disease progression (PD) or rapid deterioration. Overall, 110 adverse events were documented (grade 1-2: 80.9%) of which only 10 (9.1%) were drug-related. No drug-related serious adverse event was reported. PBMCs from patients increased cytokine secretion after re-stimulation with standard antigens, confirming an improved cellular immune function. Two patients achieved disease control (PR, SD) after 12 treatment weeks and continued in the extension phase: One patient had PD after 60 weeks, the other remains in sustained partial remission after completion of the EP. Median overall survival (OS) was 24.8 weeks in the ITT population and 115.3 weeks in the PP population. Univariate analysis of pre-treatment characteristics revealed absolute lymphocyte counts, neutrophil to lymphocyte ratios, platelets, MSKCC score, and liver metastasis as putative predictive factors of longer overall survival. Conclusions: MGN1601 administration was safe and showed promising OS in a population of heavily pretreated RCC patients who could receive therapy over 12 weeks (PP population). These results warrant further evaluation in a larger, controlled clinical trial. Baseline biomarkers and factors may allow identifying patients more likely to benefit from this innovative vaccination approach. Clinical trial information: NCT01265368.

Interference with PD-L1/PD-1 co-stimulation during antigen presentation enhances the multifunctionality of antigen-specific T cells

The release of cytokines by T cells strongly defines their functional activity in vivo. The ability to produce multiple cytokines has been associated with beneficial immune responses in cancer and infectious diseases, while their progressive loss is associated with T-cell exhaustion, senescence and anergy. Consequently, strategies that enhance the multifunctional status of T cells are a key for immunotherapy. Dendritic cells (DCs) are professional antigen presenting cells that regulate T-cell functions by providing positive and negative co-stimulatory signals. A key negative regulator of T-cell activity is provided by binding of programmed death-1 (PD-1) receptor on activated T cells, to its ligand PD-L1, expressed on DCs. We investigated the impact of interfering with PD-L1/PD-1 co-stimulation on the multifunctionality of T cells, by expression of the soluble extracellular part of PD-1 (sPD-1) or PD-L1 (sPD-L1) in human monocyte-derived DCs during antigen presentation. Expression, secretion and binding of these soluble molecules after mRNA electroporation were demonstrated. Modification of DCs with sPD-1 or sPD-L1 mRNA resulted in increased levels of the co-stimulatory molecule CD80 and a distinct cytokine profile, characterized by the secretion of IL-10 and TNF-α, respectively. Co-expression in DCs of sPD-1 and sPD-L1 with influenza virus nuclear protein 1 (Flu NP1) stimulated Flu NP1 memory T cells, with a significantly higher number of multifunctional T cells and increased cytokine secretion, while it did not induce regulatory T cells. These data provide a rationale for the inclusion of interfering sPD-1 or sPD-L1 in DC-based immunotherapeutic strategies.

Alcoholism: a systemic proinflammatory condition.

Excessive ethanol consumption affects virtually any organ, both by indirect and direct mechanisms. Considerable research in the last two decades has widened the knowledge about the paramount importance of proinflammatory cytokines and oxidative damage in the pathogenesis of many of the systemic manifestations of alcoholism. These cytokines derive primarily from activated Kupffer cells exposed to Gram-negative intestinal bacteria, which reach the liver in supra-physiological amounts due to ethanol-mediated increased gut permeability. Reactive oxygen species (ROS) that enhance the inflammatory response are generated both by activation of Kupffer cells and by the direct metabolic effects of ethanol. The effects of this increased cytokine secretion and ROS generation lie far beyond liver damage. In addition to the classic consequences of endotoxemia associated with liver cirrhosis that were described several decades ago, important research in the last ten years has shown that cytokines may also induce damage in remote organs such as brain, bone, muscle, heart, lung, gonads, peripheral nerve, and pancreas. These effects are even seen in alcoholics without significant liver disease. Therefore, alcoholism can be viewed as an inflammatory condition, a concept which opens the possibility of using new therapeutic weapons to treat some of the complications of this devastating and frequent disease. In this review we examine some of the most outstanding consequences of the altered cytokine regulation that occurs in alcoholics in organs other than the liver.

Soluble complement complex C5b-9 promotes microglia activation

Soluble C5b-9 has been described as a pro-inflammatory mediator that triggers cell activation rather than inducing cell death. Microglia is the most important immune cell involved in inflammatory response in the CNS. Although microglia activation induced by various stimuli has been well characterized, the role of C5b-9 in microglia has not been well studied. In the current experiment, we utilized assembled functional C5b-9 to treat microglia and analyzed the function. We found that soluble C5b-9 could promote microglia activation by up-regulation of costimulatory molecules and increase cytokine secretion. Our results suggested that soluble C5b-9 possessed immunoregulatory potential on microglia.

Subinhibitory concentrations of tetracyclines induce lipopolysaccharide shedding by Porphyromonas gingivalis and modulate the host inflammatory response

Antibiotics at below minimal inhibitory concentrations (MICs) may induce various biological responses in bacteria. In this study, we hypothesized that subinhibitory concentrations (subICs) of tetracycline and doxycycline induce the shedding of lipopolysaccharide (LPS) by Porphyromonas gingivalis and, as a consequence, may contribute to enhancing the host inflammatory response associated with periodontitis. A polymyxin-based enzyme-linked immunosorbent assay was used to quantify LPS shedding by P.gingivalis grown in the presence of subICs of tetracycline and doxycycline. A macrophage model was used to show that tetracycline- and doxycycline-mediated LPS shedding by P.gingivalis can induce cytokine secretion. The secretion of interleukin (IL)-1β, IL-8, and tumor necrosis factor-α was quantified by enzyme-linked immunosorbent assay. LPS was shed spontaneously in a time-dependent way by P.gingivalis during growth. LPS shedding was significantly increased by growth in the presence of subICs of tetracycline and doxycycline corresponding to 1/20 of their MICs (0.025μg/mL for tetracycline and 0.0125μg/mL for doxycycline). This shedding was not associated with an increased rate of bacterial cell lysis. Stimulating macrophages with a P.gingivalis culture supernatant induced the secretion of IL-1β, IL-8 and tumor necrosis factor-α when the bacteria were grown in the presence of 1/20 MIC of the antibiotics. Our study showed that growing P.gingivalis in the presence of subICs of either tetracycline or doxycycline induces LPS shedding. Shed LPS may in turn increase cytokine secretion in a macrophage model.

The composition of cigarette smoke determines inflammatory cell recruitment to the lung in COPD mouse models

COPD (chronic obstructive pulmonary disease) is caused by exposure to toxic gases and particles, most often CS (cigarette smoke), leading to emphysema, chronic bronchitis, mucus production and a subsequent decline in lung function. The disease pathogenesis is related to an abnormal CS-induced inflammatory response of the lungs. Similar to active (mainstream) smoking, second hand (sidestream) smoke exposure severely affects respiratory health. These processes can be studied in vivo in models of CS exposure of mice. We compared the acute inflammatory response of female C57BL/6 mice exposed to two concentrations [250 and 500 mg/m3 TPM (total particulate matter)] of sidestream and mainstream CS for 3 days and interpreted the biological effects based on physico-chemical differences in the gas and particulate phase composition of CS. BAL (bronchoalveolar lavage fluid) was obtained to perform differential cell counts and to measure cytokine release. Lung tissue was used to determine mRNA and protein expression of proinflammatory genes and to assess tissue inflammation. A strong acute inflammatory response characterized by neutrophilic influx, increased cytokine secretion [KC (keratinocyte chemoattractant), TNF-α (tumour necrosis factor α), MIP-2 (macrophage inflammatory protein 2), MIP-1α and MCP-1 (monocyte chemoattractant protein-1)], pro-inflammatory gene expression [KC, MIP-2 and MMP12 (matrix metalloproteinase 12)] and up-regulated GM-CSF (granulocyte macrophage colony-stimulating factor) production was observed in the mainstream model. After sidestream exposure there was a dampened inflammatory reaction consisting only of macrophages and diminished GM-CSF levels, most likely caused by elevated CO concentrations. These results demonstrate that the composition of CS determines the dynamics of inflammatory cell recruitment in COPD mouse models. Different initial inflammatory processes might contribute to COPD pathogenesis in significantly varying ways, thereby determining the outcome of the studies.

DNA Damage in Mammalian Neural Stem Cells Leads to Astrocytic Differentiation Mediated by BMP2 Signaling through JAK-STAT

SummaryThe consequences of DNA damage generation in mammalian somatic stem cells, including neural stem cells (NSCs), are poorly understood despite their potential relevance for tissue homeostasis. Here, we show that, following ionizing radiation-induced DNA damage, NSCs enter irreversible proliferative arrest with features of cellular senescence. This is characterized by increased cytokine secretion, loss of stem cell markers, and astrocytic differentiation. We demonstrate that BMP2 is necessary to induce expression of the astrocyte marker GFAP in irradiated NSCs via a noncanonical signaling pathway engaging JAK-STAT. This is promoted by ATM and antagonized by p53. Using a SOX2-Cre reporter mouse model for cell-lineage tracing, we demonstrate irradiation-induced NSC differentiation in vivo. Furthermore, glioblastoma assays reveal that irradiation therapy affects the tumorigenic potential of cancer stem cells by ablating self-renewal and inducing astroglial differentiation.

Slack Length Reduces the Contractile Phenotype of the Swine Carotid Artery

Contraction is the primary function of adult arterial smooth muscle. However, in response to vessel injury or inflammation, arterial smooth muscle is able to phenotypically modulate from the contractile state to several ‘synthetic' states characterized by proliferation, migration and/or increased cytokine secretion. We examined the effect of tissue length (L) on the phenotype of intact, isometrically held, initially contractile swine carotid artery tissues. Tissues were studied (1) without prolonged incubation at the optimal length for force generation (1.0 Lo, control), (2) with prolonged incubation for 17 h at 1.0 Lo, or (3) with prolonged incubation at slack length (0.6 Lo) for 16 h and then restoration to 1.0 Lo for 1 h. Prolonged incubation at 1.0 Lo minimally reduced the contractile force without substantially altering the mediators of contraction (crossbridge phosphorylation, shortening velocity or stimulated actin polymerization). Prolonged incubation of tissues at slack length (0.6 Lo), despite return of length to 1.0 Lo, substantially reduced contractile force, reduced crossbridge phosphorylation, nearly abolished crossbridge cycling (shortening velocity) and abolished stimulated actin polymerization. These data suggest that (1) slack length treatment significantly alters the contractile phenotype of arterial tissue, and (2) slack length treatment is a model to study acute phenotypic modulation of intact arterial smooth muscle.

The Lack of Maturation of Ebola Virus-Infected Dendritic Cells Results from the Cooperative Effect of at Least Two Viral Domains

Ebola virus (EBOV) infections are characterized by deficient T lymphocyte responses, T lymphocyte apoptosis, and lymphopenia in the absence of direct infection of T lymphocytes. In contrast, dendritic cells (DC) are infected but fail to mature appropriately, thereby impairing the T cell response. We investigated the contributions of EBOV proteins in modulating DC maturation by generating recombinant viruses expressing enhanced green fluorescent protein and carrying mutations affecting several potentially immunomodulating domains. They included envelope glycoprotein (GP) domains, as well as innate response antagonist domains (IRADs) previously identified in the VP24 and VP35 proteins. GP expressed by an unrelated vector, but not the wild-type EBOV, was found to strongly induce DC maturation, and infections with recombinant EBOV carrying mutations disabling GP functional domains did not restore DC maturation. In contrast, each of the viruses carrying mutations disabling any IRAD in VP35 induced a dramatic upregulation of DC maturation markers. This was dependent on infection, but not interaction with GP. Disabling of IRADs also resulted in up to a several hundredfold increase in secretion of cytokines and chemokines. Furthermore, these mutations induced formation of homotypic DC clusters, which represent close correlates of their maturation and presumably facilitate transfer of antigen from migratory DC to lymph node DC. Thus, an individual IRAD is insufficient to suppress DC maturation; rather, the suppression of DC maturation and the "immune paralysis" observed during EBOV infections results from a cooperative effect of two or more individual IRADs.

Leishmania Dices Away Cholesterol for Survival

Host lipid alterations are centrally involved in Leishmania donovani infection, and infected patients exhibit hypocholesterolemia. In this issue of Cell Host & Microbe, Ghosh etal. (2013) show that the metalloprotease GP63 released by L.donovani in the liver cleaves DICER1, inhibiting miR-122 maturation, which regulates cholesterol metabolism. These events decrease serum cholesterol and promote parasite growth.

Varicella zoster-specific CD4+Foxp3+ T cells accumulate after cutaneous antigen challenge in humans.

We investigated the relationship between varicella zoster virus (VZV)-specific memory CD4(+) T cells and CD4(+)Foxp3(+) regulatory T cells (Tregs) that accumulate after intradermal challenge with a VZV skin test Ag. VZV-specific CD4(+) T cells were identified with a MHC class II tetramer or by intracellular staining for either IFN-γ or IL-2 after Ag rechallenge in vitro. VZV-specific T cells, mainly of a central memory (CD45RA(-)CD27(+)) phenotype, accumulate at the site of skin challenge compared with the blood of the same individuals. This resulted in part from local proliferation because >50% of tetramer defined Ag-specific CD4(+) T cells in the skin expressed the cell cycle marker Ki67. CD4(+)Foxp3(+) T cells had the characteristic phenotype of Tregs, namely CD25(hi)CD127(lo)CD39(hi) in both unchallenged and VZV challenged skin and did not secrete IFN-γ or IL-2 after antigenic restimulation. The CD4(+)Foxp3(+) T cells from unchallenged skin had suppressive activity, because their removal led to an increase in cytokine secretion after activation. After VZV Ag injection, Foxp3(+)CD25(hi)CD127(lo)CD39(hi) T cells were also found within the VZV tetramer population. Their suppressive activity could not be directly assessed by CD25 depletion because activated T cells in the skin were also CD25(+). Nevertheless, there was an inverse correlation between decreased VZV skin responses and proportion of CD4(+)Foxp3(+) T cells present, indicating indirectly their inhibitory activity in vivo. These results suggest a linkage between the expansion of Ag-specific CD4(+) T cells and CD4(+) Tregs that may provide controlled responsiveness during Ag-specific stimulation in tissues.

Cowpox virus but not Vaccinia virus induces secretion of CXCL1, IL-8 and IL-6 and chemotaxis of monocytes in vitro

Orthopoxviruses are large DNA viruses which can cause disease in numerous host species. Today, after eradication of Variola virus and the end of vaccination against smallpox, zoonotic Orthopoxvirus infections are emerging as potential threat to human health. The most common causes of zoonotic Orthopoxvirus infections are Cowpox virus in Europe, Monkeypox virus in Africa and Vaccinia virus in South America. Although all three viruses are genetically and antigenically closely related, the human diseases caused by each virus differ considerably. This observation may reflect different capabilities of these viruses to modulate the hosts’ immune response. Therefore, we aimed at characterizing the specific cytokine response induced by Orthopoxvirus infection in vitro. We analysed the gene expression of nine human pro-inflammatory cytokines and chemokines in response to infection of HeLa cells and could identify an upregulation of cytokine gene expression following Cowpox virus and Monkeypox virus infection but not following Vaccinia virus infection. This was verified by a strong induction of especially IL-6, IL-8 and CXCL1 secretion into the cell culture supernatant following Cowpox virus infection. We could further show that supernatants derived from Cowpox virus-infected cells exhibit an increased chemotactic activity towards monocytic and macrophage-like cells. On the one hand, increased cytokine secretion by Cowpox virus-infected cells and subsequent monocyte/macrophage recruitment may contribute to host defence and facilitate clearance of the infection. On the other hand, given the assumed important role of circulating macrophages in viral spread, this may also point towards a mechanism facilitating delivery of the virus to further tissues in vivo.

Abstract Title Submitted by Hollie Pegram to the 2012 ASH Annual Meeting: Expansion and Modification of Umbilical Cord Blood T Cells with a Chimeric Antigen Receptor and IL-12

AbstractAbstract 1907Transplantation of unrelated umbilical cord blood (CB) derived stem cells is often used to treat adult patients with B-cell acute lymphoblastic leukemia (B-ALL). However, patients can relapse post transplant and prognosis of those with advanced or refractory disease is poor. We hypothesize that additional therapy involving adoptive transfer of CB derived T cells modified to express a CD19-specific chimeric antigen receptor (CAR) could improve patient outcome following CB transplantation in this population. We have previously demonstrated that human T cells which express the anti-CD19 1928z CAR, containing the signaling domains of the co-stimulatory CD28 receptor and CD3z chain, effectively eradicate CD19+ tumors in SCID-Beige mice, and have shown promise in early clinical trials.In this study, we demonstrate the ability to effectively isolate and expand T cells from CB, necessary for application of adoptive CB T cell therapy. We compared T cell expansion in in vitro cultures with the addition of exogenous stimulatory cytokines, including IL-2, IL-7, IL-12, IL-15 or combinations thereof. We demonstrate that in vitro culture in the context of exogenous IL-12 combined with IL-15 resulted in optimal expansion of CB T cells (over 150-fold). In addition, expansion of T cells in the context of exogenous IL-12 and IL-15 resulted in a favorable phenotype, with maximal expression of memory T cell markers, CD62L and CCR7. Furthermore, these cells were shown to produce high levels of IFNγ and express high levels of CD107a following stimulation with PHA. CB T cells expanded in IL-12 and IL-15 were therefore shown to have a unique memory cell phenotype combined with effector T cell function.Previous studies in our laboratory have demonstrated that expression of IL-12 in tumor targeted T cells resulted in increased anti-tumor function. Given these studies and the favorable effect of IL-12 on CB T cell expansion we used retroviral modification to express both 1928z CAR and IL-12 in CB T cells. CB T cells modified with both IL-12 and 1928z were shown to have increased cytokine secretion when cultured with CD19+ tumor cells, compared to UCB T cells modified with a CAR alone. Additionally, these cells were demonstrated to have increased lytic function, resulting in increased specific lysis of CD19+ tumor cells in a 51Cr release assay.Our data suggest that CB T cells modified to express both CAR and IL-12 will have improved anti-tumor function, and could therefore decrease relapse and improve overall survival following CB transplant for the treatment of B-ALL. Disclosures:Giralt:Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Membership on an entity's Board of Directors or advisory committees, Research Funding.

The preparation of HL-60 cells vaccine expressing BCG heat shock protein 70 and detection of its immunogenicity in vitro

Gene-modified cell vaccines are the best way to achieve the immunotherapy for all types of acute leukemia. In this study, the recombinant eukaryotic expression vector (pDisplay-HSP70) of heat shock protein 70 (HSP70) of Bacille Calmette-Guérin (BCG) was constructed by amplifying the whole BCG HSP70 gene using polymerase chain reaction (PCR) and sub-cloning into the polyclone endonuclease sites in pDisplay. Then the HL-60 cell vaccine expressing the protein onto the cell surface was prepared by lipofectamine transfection and its anti-tumor effect and mechanism were further studied. Results showed that the fragment of BCG HSP70 was consistent with Mycobacterium tuberculosis HSP70 gene published in GeneBank. DNA sequencing showed that the recombinant vector was correctly constructed and named pDisplay-HSP70. After BCG HSP70 gene transfection, the yellow-green fluorescence on the HL-60 cells surface was observed under a fluorescence microscope. The immunogenicity of HSP70-transfected HL-60 cells exhibited upregulated proliferation of lymphocytes, increased cytokine secretion (IFN-γ) and enhanced killing activity. These results suggested that gene transfection of BCG HSP70 could significantly enhance the immunogenicity of HL-60 cells. It may be used as a suitable candidate gene-modified cell vaccine for cancer immunotherapy.

Intestinal Epithelial Cells Modulate Antigen-Presenting Cell Responses to Bacterial DNA

Intestinal epithelial cells and antigen-presenting cells orchestrate mucosal innate immunity. This study investigated the role of bacterial DNA in modulating epithelial and bone marrow-derived antigen-presenting cells (BM-APCs) and subsequent T-lymphocyte responses. Murine MODE-K epithelial cells and BM-APCs were treated with DNA from either Bifidobacterium breve or Salmonella enterica serovar Dublin directly and under coculture conditions with CD4(+) T cells. Apical stimulation of MODE-K cells with S. Dublin DNA enhanced secretion of cytokines from underlying BM-APCs and induced interleukin-17 (IL-17) and gamma interferon (IFN-γ) secretion from CD4(+) T cells. Bacterial DNA isolated from either strain induced maturation and increased cytokine secretion from BM-APCs. Conditioned medium from S. Dublin-treated MODE-K cells elicited an increase in cytokine secretion similar to that seen for S. Dublin DNA. Treatment of conditioned medium from MODE-K cells with RNase and protease prevented the S. Dublin-induced increased cytokine secretion. Oral feeding of mice with B. breve DNA resulted in enhanced levels of colonic IL-10 and transforming growth factor β (TGFβ) compared with what was seen for mice treated with S. Dublin DNA. In contrast, feeding mice with S. Dublin DNA increased levels of colonic IL-17 and IL-12p70. T cells from S. Dublin DNA-treated mice secreted high levels of IL-12 and IFN-γ compared to controls and B. breve DNA-treated mice. These results demonstrate that intestinal epithelial cells are able to modulate subsequent antigen-presenting and T-cell responses to bacterial DNA with pathogenic but not commensal bacterial DNA inducing effector CD4(+) T lymphocytes.

Autocrine secretion of hemokinin-1 promotes the innate immunostimulatory function of mast cells. (177.21)

Abstract Mast cells (MC), with pro- and anti-inflammatory functions, control innate and adaptive immune responses. Mechanisms causing such disparate functions are only starting to be elucidated. Recent advances in neuroimmune regulation suggest that MC function may be regulated, in part, by neuropeptides. MC express the neurokinin-1 receptor (NK1R), the preferred receptor for the inflammatory neuropeptides, Substance P (SP) and Hemokinin-1 (HK1). We hypothesized that agonistic signaling through the NK1R regulates MC function. We compared the ability of C57BL/6 wild type (WT) or NK1RKO bone marrow-derived MC stimulated via FcϵRI or TLR4 to secrete pro-inflammatory IL-1β, IL-6, IL-33, and TNFα, or anti-inflammatory, IL-10. NK1RKO MC secreted significantly less TNFα and IL-33 but equal levels of IL-10, IL-6 and IL-1β compared to WT-MC, results that were further confirmed with WT-MC incubated with the NK1R antagonist L760,330. As our experimental conditions lack nerve fibers and exogenous SP or HK1 did not increase cytokine secretion we postulated that the observed effects were caused by autocrine secretion of NK1R agonists by MC. Using quantitative RT-PCR, we found high expression of the tac4 gene encoding for HK1 which was further increased following MC stimulation while the levels of SP mRNA remained significantly lower in spite of MC simulation. Together our results demonstrate that autocrine HK1 and NK1R signaling are potent stimulators of the pro-inflammatory function of MC.