ABSTRACT
BACKGROUND: Reactions between nitric oxide (NO), nitrite (NO2-), and unsaturated fatty acids give rise to electrophilic nitro-fatty acids (NO2 -FAs), such as nitro oleic acid (OA-NO2 ) and nitro linoleic acid (LNO2 ). Endogenous electrophilic fatty acids (EFAs) mediate anti-inflammatory responses by modulating metabolic and inflammatory signal transduction reactions. Hence, there is considerable interest in employing NO2 -FAs and other EFAs for the prevention and treatment of inflammatory disorders. Thus, we sought to determine whether OA-NO2 , an exemplary nitro-fatty acid, has the capacity to inhibit cutaneous inflammation. METHODS: We evaluated the effect of OA-NO2 on allergic contact dermatitis (ACD) using an established model of contact hypersensitivity in C57Bl/6 mice utilizing 2,4-dinitrofluorobenzene as the hapten. RESULTS: We found that subcutaneous (SC) OA-NO2 injections administered 18 h prior to sensitization and elicitation suppresses ACD in both preventative and therapeutic models. In vivo SC OA-NO2 significantly inhibits pathways that lead to inflammatory cell infiltration and the production of inflammatory cytokines in the skin. Moreover, OA-NO2 is capable of enhancing regulatory T-cell activity. Thus, OA-NO2 treatment results in anti-inflammatory effects capable of inhibiting ACD by inducing immunosuppressive responses. CONCLUSION: Overall, these results support the development of OA-NO2 as a promising therapeutic for ACD and provides new insights into the role of electrophilic fatty acids in the control of cutaneous immune responses potentially relevant to a broad range of allergic and inflammatory skin diseases.
Subject(s)
Dermatitis, Allergic Contact/immunology , Dermatitis, Allergic Contact/metabolism , Fatty Acids/metabolism , Nitric Oxide/metabolism , Nitrites/metabolism , Animals , Biomarkers , Dermatitis, Allergic Contact/genetics , Dermatitis, Allergic Contact/pathology , Disease Models, Animal , Female , Gene Expression Profiling , Mice , Neovascularization, Pathologic/immunology , Neovascularization, Pathologic/metabolism , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolismABSTRACT
The ability to deliver but hide immunogenic payloads and then reveal them at predetermined times could lead to autonomously boosting vaccine formulations or improved antigen-adjuvant vaccine designs. We used in silico modeling to determine the appropriate formulation and material properties for poly(lactic-co-glycolic) acid (PLGA) microparticles such that they would delay the in vitro"unmasking" of an ovalbumin-alum payload for precise and predetermined intervals. A preferred formulation was then tested in vivo. In vivo T cell proliferation data confirmed the presentation of antigen released through the programmed delayed burst while antibody subclass data demonstrated immunogenicity comparable to that observed with established multiple injection prime-boost regimens.
ABSTRACT
BACKGROUND: Microarray hybridization studies in Sézary syndrome (SS) have compared T lymphocytes from patients with cutaneous T-cell lymphoma with those of normal controls; a major limitation of this design is that significant inherent genetic variability of lymphocyte populations between individuals may produce differences in gene expression unrelated to disease state. OBJECTIVE: The objective of this study was to minimize the heterogeneity of information derived from whole-genome expression analysis and to identify specific genetic differences between highly purified malignant and nonmalignant (control) T cells from the same patient with SS. METHODS: Peripheral blood mononuclear cells were obtained from a patient with SS, stained with anti-T-cell receptor Vb (TCR-Vb) antibodies, and sorted by multiparameter flow cytometry. Malignant cells expressed the dominant TCR-Vb; control T cells lacked the dominant TCR-Vb but were otherwise phenotypically identical (CD3+CD4+CD45RO+). These cell populations were compared using the Illumina Inc. Sentrix Human-6 expression BeadChip system. RESULTS: Transcriptome analysis using the J5 test, which was selected for data analysis based on an efficiency analysis of competing statistical methods, showed differential expression of 44 genes between the malignant and nonmalignant cell subsets. Promyelocytic leukaemia zinc finger protein (ZBTB16) was the most profoundly upregulated gene in the malignant cell population, while interferon regulatory factor 3 (IRF3) and interferon-induced protein 35 (IFI35), which are important elements of the cellular response to viral infection, were significantly downregulated. CONCLUSIONS: The results of this study suggest the feasibility of this novel comparative approach to genomic profiling in SS. Using this method, we identified several differentially expressed genes and pathways not previously described in SS. While these findings require validation in larger studies, they may be important in SS pathogenesis.
Subject(s)
CD4-Positive T-Lymphocytes/metabolism , Gene Expression Profiling/methods , Sezary Syndrome/genetics , Skin Neoplasms/genetics , Antigens, CD/blood , Biomarkers/blood , CD4-Positive T-Lymphocytes/immunology , Flow Cytometry , Gene Frequency , Humans , Sezary Syndrome/immunology , Skin Neoplasms/immunologyABSTRACT
Dendritic cells (DC) engineered in vitro by DNA encoding OVAhsp70 and IL-15 up-regulated their expressions of CD80, CD86, CCR7 and IL-15Ralpha and promoted their productions of IL-6, IL-12 and TNF-alpha. Transcriptional IL-15-directed in vivo DC targeting DNA vaccine encoding OVAhsp70 elicited long-lasting Th1 and CTL responses and anti-B16OVA activity. CD8T cell-mediated primary tumor protection was abrogated by DC or CD4T cell depletion during the induction phase of immune responses. However, CD4T cell depletion during immunization did not impair CD8T cell-dependent long-lasting tumor protection. Furthermore, in vivo DC-derived IL-15 exerted the enhancements of cellular and humoral immune responses and antitumor immunity elicited by OVAhsp70 DNA vaccine. Importantly, the potency of this novel DNA vaccine strategy was proven using a self/tumor Ag (TRP2) in a clinically relevant B16 melanoma model. These findings have implications for developing next generation DNA vaccines against cancers and infectious diseases in both healthy and CD4 deficient individuals.
Subject(s)
Cancer Vaccines/immunology , Dendritic Cells/immunology , Interleukin-15/genetics , Melanoma, Experimental/prevention & control , Vaccines, DNA/immunology , Animals , CD4-Positive T-Lymphocytes/immunology , Cytotoxicity, Immunologic/immunology , Female , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/immunology , Interleukin-15/immunology , Melanoma, Experimental/immunology , Mice , Mice, Inbred C57BL , Ovalbumin/genetics , Ovalbumin/immunology , T-Lymphocytes, Cytotoxic/immunology , Th1 Cells/immunology , Transcription, GeneticABSTRACT
T-cell priming is strongly affected by the longevity of antigen-bearing dendritic cells (DCs), which are typically short-lived in lymphoid tissues. 'Survival gene' Bcl-xl is critical for the lifespan of DCs in vivo. Here, we showed that in vivo coadministration of Bcl-xl under control of the DC-specific promoter (CD11c-Bcl-xl) and TRP2hsp70 DNA prolonged T-cell stimulation by DCs and augmented TRP2-specific-IFN-gamma-producing CD8+ T-cell responses. Consistent with these findings, enhanced protection and significant therapeutic immunity to B16 melanoma was generated by this coimmunization strategy, which also augmented therapeutic immunity to GL-26 tumor. In this B16 melanoma model, results from animal experiments with depletion of immune cells indicate that CD8+ T cells and NK cells are important in the antitumor immunity induced by this coimmunization strategy. These observations suggest that 'survival gene' Bcl-xl potentiates the magnitude of antigen-specific-CD8+ T-cell responses and the efficacy of antitumor immunity induced by DNA vaccine, and is relevant for the design of in vivo targeted DC-based vaccine strategies to improve immunity against cancer.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Cancer Vaccines/administration & dosage , Genetic Therapy/methods , Melanoma, Experimental/therapy , Vaccines, DNA/administration & dosage , bcl-X Protein/genetics , Animals , Antigens, Neoplasm/immunology , Autoantigens , CD11c Antigen/genetics , Dendritic Cells/immunology , Female , Glioma/immunology , Glioma/therapy , HSP70 Heat-Shock Proteins/genetics , Intramolecular Oxidoreductases/genetics , Killer Cells, Natural/immunology , Lymphocyte Activation , Melanoma, Experimental/immunology , Mice , Mice, Inbred C57BL , Time FactorsABSTRACT
Epidermal Langerhans cells (LCs) show extraordinary immunostimulatory capacity and play a key role in the initiation and regulation of immune responses. Studies of LC biology are currently the focus of efforts to engineer immune responses and to better understand the immunopathology of cutaneous diseases. Here we identified and characterized a population of LC precursors that were resident in human skin. These immediate precursors expressed CD14, langerin and functional CCR6. When cultured with transforming growth factor-beta1 alone, they had the potential to differentiate into epidermal LCs; when cultured in the presence of granulocyte macrophage-colony-stimulating factor and interleukin 4 they differentiated into functionally mature dendritic cells. Identification and characterization of these LC precursors provided insight into LC biology and the mechanism(s) through which LCs repopulate the epidermis.
Subject(s)
Dermis/cytology , Langerhans Cells/immunology , Langerhans Cells/ultrastructure , Lipopolysaccharide Receptors/analysis , Skin/immunology , Antigens/metabolism , Cell Differentiation , Cell Division , Cells, Cultured , Chemokine CCL20 , Chemokines, CC/pharmacology , Chemotaxis , Cytokines/pharmacology , Endocytosis , Humans , Immunohistochemistry , Immunophenotyping , Langerhans Cells/classification , Lipopolysaccharide Receptors/immunology , Lymphocyte Activation , Macrophage Inflammatory Proteins/pharmacology , Receptors, CCR6 , Receptors, Chemokine/biosynthesis , Stem Cells/drug effects , Stem Cells/immunology , Stem Cells/ultrastructureABSTRACT
BACKGROUND: IL-12 is a potent antitumor cytokine for cancer gene therapy. Previously, we demonstrated that single systemic administration of naked DNA (encoding IL-12) could serve as a good model for in vivo evaluation of the antitumor effect of a candidate gene (unpublished data). In the present study, we propose that this gene delivery method could be a very useful model for in vivo evaluation of the toxicity of a given therapeutic gene (using IL-12 as an example). By comparing the toxicities and the effects of initial IL-12 administration on subsequent transgene expression, both IL-12 gene delivery and recombinant murine IL-12 protein (rmIL-12) administration showed similar toxicity profiles. METHODS: Naked DNA encoding murine IL-12 (mIL-12) was delivered into mice by systemic administration. Toxicity profiles of mice treated with DNA or rmIL-12 were compared. RESULTS: Systemic administration of naked DNA encoding mIL-12 resulted in very similar toxicity as rmIL-12 with respect to liver enzyme, hematological and immunological profiles. Repeated injection of mIL-12 gene did not recover a high level of mIL-12 production as the first injection. Moreover, initial mIL-12 administration resulted in inhibition of subsequent reporter gene expression with both viral and non-viral promoters (CMV, human alpha-antitrypsin or chicken beta-actin promoter). This transgene inhibition effect was entirely mediated by IFN-gamma as the transgene expression was fully recovered in IFN-gamma knockout mice. CONCLUSIONS: Systemic IL-12 therapy, with either a protein or gene therapy approach, resulted in comparable liver and systemic toxicities. Refractoriness of mIL-12 production by subsequent administration of mIL-12 gene was observed. The transgene attenuation effect of IL-12 pre-dosing (either by IL-12 or rmIL-12), mediated by IFN-gamma, provided important insights for the design of IL-12 combination gene therapy and the improvement of gene vectors for IL-12 therapy. The present results show that simple injection of naked DNA could serve as a good model for in vivo evaluation of the toxicity of a candidate therapeutic gene.
Subject(s)
Interleukin-12/genetics , Liver/physiopathology , Animals , Cytomegalovirus/genetics , Hematocrit , Interleukin-12/toxicity , Leukocytes/drug effects , Liver/drug effects , Liver/pathology , Liver Function Tests , Mice , Mice, Transgenic , Plasmids , Promoter Regions, Genetic , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/toxicity , alpha 1-Antitrypsin/geneticsABSTRACT
Although it is known that dendritic cells (DCs) produce cytokines, there is little information about how cytokine synthesis is regulated during DC development. A range of cytokine mRNA/proteins was analyzed in immature (CD86-) or mature (CD86+) murine bone marrow (BM)- derived DCs. Highly purified, flow-sorted, immature DCs exhibited higher amounts of interleukin-1alpha (IL-1alpha), IL-1beta, tumor necrosis factor-alpha (TNF-alpha), transforming growth factor beta1 (TGF-beta1), and macrophage migration inhibitory factor (MIF) mRNA/protein than mature DCs. After differentiation, DC up-regulated the levels of IL-6 and IL-15 mRNA/protein and synthesized de novo mRNA/protein for IL-12p35, IL-12p40, and IL-18. Although immature BM-derived DCs did not stimulate naive allogeneic T cells, mature DCs elicited a mixed population of T helper (Th) 1 (mainly) and Th2 cells in 3d-mixed leukocyte reactions. CD86+ BM DCs switched to different cytokine patterns according to whether they were terminally differentiated by lipopolysaccharide (LPS) or CD40 ligation. Although both stimuli increased IL-6, IL-12p40, IL-15, and TNF-alpha mRNA/protein levels, only LPS up-regulated transcription of IL-1alpha, IL-1beta, IL-12p35, and MIF genes. Although LPS and CD40 cross-linking increased the T-cell allostimulatory function of BM DCs, only LPS stimulation shifted the balance of naive Th differentiation to Th1 cells, a mechanism dependent on the up-regulation of IL-12p35 and not of IL-23. These results demonstrate that, depending on the stimuli used to terminally mature BM DCs, DCs synthesize a different pattern of cytokines and exhibit distinct Th cell-driving potential.
Subject(s)
CD40 Antigens/physiology , Cytokines/biosynthesis , Dendritic Cells/metabolism , Gene Expression Regulation/drug effects , Lipopolysaccharides/pharmacology , Animals , Antibodies, Monoclonal/pharmacology , Antigens, CD/biosynthesis , B7-2 Antigen , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , CD40 Antigens/immunology , Cell Differentiation/drug effects , Cells, Cultured/metabolism , Dendritic Cells/drug effects , Endocytosis , Gene Expression Profiling , Immunophenotyping , Interleukin-12/physiology , Interleukin-23 , Interleukin-23 Subunit p19 , Interleukins/biosynthesis , Interleukins/genetics , Interleukins/physiology , Lymphocyte Culture Test, Mixed , Macrophage Migration-Inhibitory Factors/biosynthesis , Macrophage Migration-Inhibitory Factors/genetics , Membrane Glycoproteins/biosynthesis , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , RNA, Messenger/biosynthesis , Specific Pathogen-Free Organisms , Th1 Cells/immunology , Th2 Cells/immunology , Transforming Growth Factor beta/biosynthesis , Transforming Growth Factor beta/genetics , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/geneticsABSTRACT
Aspirin is the most commonly used analgesic and antiinflammatory agent. In this study, at physiological concentrations, it profoundly inhibited CD40, CD80, CD86, and MHC class II expression on murine, GM-CSF + IL-4 stimulated, bone marrow-derived myeloid dendritic cells (DC). CD11c and MHC class I expression were unaffected. The inhibitory action was dose dependent and was evident at concentrations higher than those necessary to inhibit PG synthesis. Experiments with indomethacin revealed that the effects of aspirin on DC maturation were cyclooxygenase independent. Nuclear extracts of purified, aspirin-treated DC revealed a decreased NF-kappaB DNA-binding activity, whereas Ab supershift analysis indicated that aspirin targeted primarily NF-kappaB p50. Unexpectedly, aspirin promoted the generation of CD11c+ DC, due to apparent suppression of granulocyte development. The morphological and ultrastructural appearance of aspirin-treated cells was consistent with immaturity. Aspirin-treated DC were highly efficient at Ag capture, via both mannose receptor-mediated endocytosis and macropinocytosis. By contrast, they were poor stimulators of naive allogeneic T cell proliferation and induced lower levels of IL-2 in responding T cells. They also exhibited impaired IL-12 expression and did not produce IL-10 after LPS stimulation. Assessment of the in vivo function of aspirin-treated DC, pulsed with the hapten trinitrobenzenesulfonic acid, revealed an inability to induce normal cell-mediated contact hypersensitivity, despite the ability of the cells to migrate to T cell areas of draining lymphoid tissue. These data provide new insight into the immunopharmacology of aspirin and suggest a novel approach to the manipulation of DC for therapeutic application.
Subject(s)
Aspirin/pharmacology , Dendritic Cells/immunology , Growth Inhibitors/pharmacology , Immunosuppressive Agents/pharmacology , Lymphocyte Activation/drug effects , Myeloid Cells/immunology , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/enzymology , Bone Marrow Cells/immunology , Bone Marrow Transplantation , Cell Differentiation/drug effects , Cell Differentiation/immunology , Cell Movement/drug effects , Cell Movement/immunology , Cell Survival/drug effects , Cell Survival/immunology , Cells, Cultured , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/metabolism , Dendritic Cells/drug effects , Dendritic Cells/enzymology , Dendritic Cells/transplantation , Dermatitis, Contact/immunology , Dose-Response Relationship, Drug , Endocytosis/drug effects , Endocytosis/immunology , Immunity, Cellular/drug effects , Immunophenotyping , Injections, Subcutaneous , Integrin alphaXbeta2/biosynthesis , Interleukin-10/antagonists & inhibitors , Interleukin-10/metabolism , Interleukin-12/antagonists & inhibitors , Interleukin-12/biosynthesis , Interleukin-2/antagonists & inhibitors , Interleukin-2/biosynthesis , Lymphocyte Culture Test, Mixed , Lymphoid Tissue/immunology , Lymphoid Tissue/pathology , Macrophages/cytology , Macrophages/drug effects , Macrophages/immunology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Myeloid Cells/drug effects , Myeloid Cells/enzymology , Myeloid Cells/transplantation , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , NF-kappa B p50 Subunit , Prostaglandin-Endoperoxide Synthases/physiology , Signal Transduction/drug effects , Signal Transduction/immunologyABSTRACT
Gene therapy techniques can be important tools for the induction and control of immune responses. Antigen delivery is a critical challenge in vaccine design, and DNA-based immunization offers an attractive method to deliver encoded transgenic protein antigens. In the present study, we used a gene gun to transfect human skin organ cultures with a particular goal of expressing transgenic antigens in resident cutaneous dendritic cells. Our studies demonstrate that when delivered to human skin, gold particles are observed primarily in the epidermis, even when high helium delivery pressures are used. We demonstrate that Langerhans cells resident in the basal epidermis can be transfected, and that biolistic gene delivery is sufficient to stimulate the activation and migration of skin dendritic cells. RT-PCR analysis of dendritic cells, which have migrated from transfected skin, demonstrates the presence of transgenic mRNA, indicating direct transfection of cutaneous dendritic cells. Importantly, transfected epidermal Langerhans cells can efficiently present a peptide derived from the transgenic melanoma antigen MART-1 to a MART-1-specific CTL. Taken together, our results demonstrate direct transfection, activation, and antigen-specific stimulatory function of in situ transduced human Langerhans cells.
Subject(s)
Biolistics/methods , Dendritic Cells/immunology , Genetic Therapy/methods , Skin/immunology , Transfection , Cell Culture Techniques , Cell Movement/immunology , Epidermis/immunology , Epitopes/genetics , Epitopes/metabolism , Gene Transfer Techniques , Gold/pharmacokinetics , Humans , Langerhans Cells/immunology , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes, Cytotoxic/immunology , TransgenesABSTRACT
The tumor necrosis factor (TNF)-related apoptosis-inducing ligand, TRAIL, is a new member of the TNF family. It can specifically induce apoptosis in a variety of human tumors. To investigate the possibility of employing the TRAIL gene for systemic cancer therapy, we constructed a recombinant gene encoding the soluble form of the human Flt3L gene (hFlex) at the 5' end and the human TRAIL gene at the 3' end. Such design allows the TRAIL gene product to be secreted into the body circulation. We have also demonstrated that the addition of an isoleucine zipper to the N-terminal of TRAIL greatly enhanced the trimerization of the fusion protein and dramatically increased its anti-tumor activity. The fusion protein reached the level of 16-38 microg/ml in the serum after a single administration of the recombinant gene by hydrodynamic-based gene delivery in mice. A high level of the fusion protein correlated with the regression of a human breast tumor established in SCID mice. No apparent toxicity was observed in the SCID mouse model. In addition, the fusion protein caused an expansion of the dendritic cell population in the C57BL/6 recipient mice, indicating that the hFlex component of the fusion protein was functional. Thus, the hFlex-TRAIL fusion protein may provide a novel approach, with the possible involvement of dendritic cell-mediated anti-cancer immunity, for the treatment of TRAIL-sensitive tumors.
Subject(s)
Adenocarcinoma/therapy , Genetic Therapy , Mammary Neoplasms, Experimental/therapy , Membrane Glycoproteins/genetics , Membrane Proteins/genetics , Tumor Necrosis Factor-alpha/genetics , Adenocarcinoma/genetics , Adenocarcinoma/immunology , Animals , Apoptosis/drug effects , Apoptosis Regulatory Proteins , Cytotoxicity, Immunologic/genetics , Cytotoxicity, Immunologic/immunology , Dose-Response Relationship, Drug , Gene Expression Regulation, Neoplastic , Gene Transfer Techniques , Humans , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/immunology , Membrane Glycoproteins/metabolism , Membrane Glycoproteins/therapeutic use , Membrane Proteins/metabolism , Membrane Proteins/therapeutic use , Mice , Mice, SCID , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/blood , Recombinant Fusion Proteins/therapeutic use , TNF-Related Apoptosis-Inducing Ligand , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/therapeutic useABSTRACT
Intravenous (i.v.) administration of a lipopolyplex consisting of a ternary complex of DOTAP:cholesterol cationic liposomes, protamine sulfate, and noncoding plasmid DNA (LPD-pDNA) is capable of stimulating a potent Th-1 cytokine response and inhibiting the growth of established tumors in mice. Both activities are mainly elicited by unmethylated CpG motifs in the plasmid DNA (pDNA) component, which are bacterial in origin. Since oligodeoxynucleotides (ODN) that possess a consensus immunostimulatory CpG motif of RRCpGYY (R is purine and Y is pyrimidine) can mimic the immunostimulatory actions of bacterial DNA, we hypothesized that i.v. administration of LPD prepared with GpG-ODN would mimic the ability of LPD-pDNA to stimulate Th-1 cytokines and antitumor activity and provide an improved vector for probing the immune mechanisms underlying the observed antitumor effects. These hypotheses were tested for the treatment of established 24JK experimental pulmonary metastases that are syngeneic in C57BL/6 mice. Mice treated with LPD containing 25 microg of the prototypical phosphodiester (PO) CpG-ODN 1668 (tccatGACGTTcctgatgct, motif capitalized) demonstrated a dramatic reduction in lung tumor burden (>80% inhibition, P<0.01) compared to dextrose-treated controls. The antitumor effect was dependent on the CpG dinulceotide and correlated with the ability to stimulate serum Th-1 cytokines (TNF-alpha, IL-12, and IFN-gamma). Both activities required assembly of CpG-ODN in a cationic liposome/DNA complex (lipoplex) or the LPD lipopolyplex. LPD delivery of both PO-1668 and phosphorothioated (PS)-1668 stimulated a greater cytokine response compared to delivery of free ODN. Furthermore, within the LPD complex, both PO- and PS-1668 had similar ability to stimulate Th-1 cytokines with respect to potency and duration of response, thus eliminating the need for the PS modification. In tumor cell lysis assays, LPD-CpG DNA stimulated development of an acquired, tumor-specific CD8+ cytotoxic T-lymphocyte (CTL) activity that was dependent on CpG DNA. LPD was also capable of stimulating NK activity; however, this was not dependent on CpG DNA. Only formulations that concomitantly stimulated NK activity and CpG-specific, Th-1 cytokine were capable of stimulating the development of tumor-specific CTL activity and significant inhibition of tumor growth. Thus, we propose a model where CpG DNA in complex with cationic liposome-based lipoplexes or lipopolyplexes stimulates antitumor NK activity and CpG-stimulated Th-1 cytokine production. The combination of these two activities of the innate immune system subsequently direct the development of an acquired, tumor-specific CTL response that in total are effective for inhibiting the growth of established tumors in mice.
Subject(s)
CpG Islands/immunology , Cytotoxicity, Immunologic , Lung Neoplasms/immunology , Lung Neoplasms/prevention & control , Animals , Cholesterol , Fatty Acids, Monounsaturated , Liposomes , Lung Neoplasms/secondary , Mice , Mice, Inbred C57BL , Neoplasm Transplantation , Plasmids/immunology , Quaternary Ammonium Compounds , Th1 Cells/immunologyABSTRACT
The combination of immunization strategies with gene therapy methods constitutes a powerful tool for the purpose of genetic immunization. The cutaneous microenvironment, rich in professional antigen-presenting cells and accessory cells capable of initiating and controlling the intensity of specific immune responses, makes the skin a unique target for the expression of transgenic antigens. The fact that epidermal and dermal dendritic cells can be directly transfected using genetically engineered vectors allows in vivo manipulation of immune responses by modifying the function of these distinctive antigen-presenting cell populations. Importantly, coexpression of antigenic proteins together with immunostimulatory molecules, and/or adjuvant or leader sequences, makes possible the engineering of antigen-specific immune responses. Even though most of the mechanisms related to DNA immunization remain to be explored, the skin has emerged as an ideal target for evolving genetic vaccination techniques.
Subject(s)
Epidermis/immunology , Epidermis/metabolism , Gene Transfer Techniques , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Immune System , Immunization/methods , Skin/metabolism , Transgenes , Humans , Lymphocytes/immunology , TransfectionABSTRACT
Recombinant adenovirus (rAd) infection is one of the most effective and frequently employed methods to transduce dendritic cells (DC). Contradictory results have been reported recently concerning the influence of rAd on the differentiation and activation of DC. In this report, we show that, as a result of rAd infection, mouse bone marrow-derived immature DC upregulate expression of major histocompatibility complex class I and II antigens, costimulatory molecules (CD40, CD80, and CD86), and the adhesion molecule CD54 (ICAM-1). rAd-transduced DC exhibited increased allostimulatory capacity and levels of interleukin-6 (IL-6), IL-12p40, IL-15, gamma interferon, and tumor necrosis factor alpha mRNAs, without effects on other immunoregulatory cytokine transcripts such as IL-10 or IL-12p35. These effects were not related to specific transgenic sequences or to rAd genome transcription. The rAd effect correlated with a rapid increase (1 h) in the NF-kappaB-DNA binding activity detected by electrophoretic mobility shift assays. rAd-induced DC maturation was blocked by the proteasome inhibitor Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) or by infection with rAd-IkappaB, an rAd-encoding the dominant-negative form of IkappaB. In vivo studies showed that after intravenous administration, rAds were rapidly entrapped in the spleen by marginal zone DC that mobilized to T-cell areas, a phenomenon suggesting that rAd also induced DC differentiation in vivo. These findings may explain the immunogenicity of rAd and the difficulties in inducing long-term antigen-specific T-cell hyporesponsiveness with rAd-transduced DC.
Subject(s)
Adenoviridae/physiology , Dendritic Cells/physiology , NF-kappa B/physiology , Animals , Bone Marrow Cells/physiology , Cytokines/genetics , DNA/metabolism , Genetic Vectors , Intercellular Adhesion Molecule-1/genetics , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , RNA, Messenger/analysis , TransgenesABSTRACT
The trace number of dendritic cells (DCs) present in tissues has limited the study of DC biology and development of clinical applications utilizing DCs. Here we show that hydrodynamics-based gene delivery of naked DNA encoding secreted human flt3 ligand (hFLex) can dramatically increase the number of functional DCs and natural killer (NK) cells. After a single injection of the hFLex gene, hFLex levels in mouse serum reached approximately 40 microg/ml and remained above 1 microg/ml for 5-6 days. Sustained levels of serum hFLex correlated with significant increases in the size of the lymphoid organs and in the proportion of dendritic cells and NK cells in both lymph nodes and spleen. The increase in DC and NK cell numbers started from day 5, and reached peak levels between day 8 and day 12. The levels then returned to normal on day 20. These DCs and NK cells were functional as evidenced by mixed leukocyte reactions and lysis of YAC-1 cells, respectively. These results suggest that delivery of the hFLex gene provides a simple, efficient, and inexpensive way of increasing DC and NK cell populations in vivo, and may have broad applications in the further study of DC and NK cell biology and in the development of immunotherapy strategies.
Subject(s)
Cell Division/genetics , DNA/administration & dosage , Dendritic Cells/cytology , Killer Cells, Natural/cytology , Membrane Proteins/genetics , Animals , Female , Humans , Injections, Intravenous , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , TransfectionABSTRACT
Human dendritic cells (DC) have polarized responses to chemokines as a function of maturation state, but the effect of maturation on DC trafficking in vivo is not known. We have addressed this question in a highly relevant rhesus macaque model. We demonstrate that immature and CD40 ligand-matured monocyte-derived DC have characteristic phenotypic and functional differences in vitro. In particular, immature DC express CC chemokine receptor 5 (CCR5) and migrate in response to macrophage inflammatory protein-1alpha (MIP-1alpha), whereas mature DC switch expression to CCR7 and respond exclusively to MIP-3beta and 6Ckine. Mature DC transduced to express a marker gene localized to lymph nodes after intradermal injection, constituting 1.5% of lymph node DC. In contrast, cutaneous DC transfected in situ via gene gun were detected in the draining lymph node at a 20-fold lower frequency. Unexpectedly, the state of maturation at the time of injection had no influence on the proportion of DC that localized to draining lymph nodes, as labeled immature and mature DC were detected in equal numbers. Immature DC that trafficked to lymph nodes underwent a significant up-regulation of CD86 expression indicative of spontaneous maturation. Moreover, immature DC exited completely from the dermis within 36 h of injection, whereas mature DC persisted in large numbers associated with a marked inflammatory infiltrate. We conclude that in vitro maturation is not a requirement for effective migration of DC in vivo and suggest that administration of Ag-loaded immature DC that undergo natural maturation following injection may be preferred for DC-based immunotherapy.
Subject(s)
Cell Movement/immunology , Dendritic Cells/immunology , Monocytes/immunology , Vaccines/immunology , Adoptive Transfer/methods , Animals , CD40 Antigens/metabolism , CD40 Ligand , Cell Count , Cell Differentiation/immunology , Cells, Cultured , Dendritic Cells/cytology , Dendritic Cells/metabolism , Dendritic Cells/transplantation , Genetic Vectors/immunology , Green Fluorescent Proteins , Humans , Immunophenotyping , Injections, Intradermal , Ligands , Luminescent Proteins/administration & dosage , Luminescent Proteins/genetics , Luminescent Proteins/immunology , Lymph Nodes/cytology , Lymph Nodes/immunology , Lymph Nodes/metabolism , Macaca mulatta , Membrane Glycoproteins/pharmacology , Monocytes/cytology , Pinocytosis/immunology , Receptors, Chemokine/biosynthesis , Skin/cytology , Skin/immunology , TransfectionABSTRACT
The surprising observation that direct inoculation of an expression plasmid encoding a foreign protein into the skin of mice resulted in the induction of antibody responses, demonstrated that injection of "naked" DNA could result in antigen expression in an immunogenic form (1). This observation and the subsequent demonstration that intramuscular injections of plasmid DNA encoding influenza nucleoprotein could protect mice against a challenge with live influenza virus have opened up new avenues for vaccine development (2-3). Immunization with plasmid DNA has been shown to activate both humoral and cellular immune responses, including the generation of antigen-specific CD8(+) cytotoxic T cells as well as CD4(+) T helper cells (4). An increasing number of studies using experimental animal models have demonstrated that plasmid DNA immunization can promote effective immune responses against numerous viruses, including influenza, rabies, HIV, HBV, HCV, and HSV; several bacteria, including: Mycobacterium tuberculosis, Mycoplasma pulmonis, and Borrelia burgdorferi; as well as parasites, such as malaria and leishmania (4). Phase I clinical vaccine trials are currently being performed for HIV, HBV, and influenza virus. With the molecular identification of tumor antigens (5), there has also been increasing interest in the development of DNA-based immunization for cancer. Preclinical studies demonstrate that DNA-based immunizations targeting model tumor antigens such as chicken ovalbumin (6), ß-galactosidase (7), or CEA (8) induce protective immune responses leading to rejection of a subsequent, normally lethal challenge with antigen-expressing tumor cells.
ABSTRACT
One of the most promising applications of recent advances in gene therapy is the development of immunization strategies based on the delivery of antigen-encoding DNA. DNA-based vaccination, also referred to as genetic vaccination or polynucleotide vaccination, offers considerable promise for improvement over existing immunization strategies, and the skin offers unique potential as a target tissue for genetic vaccines. The expression of genetically introduced antigens in a cutaneous microenvironment rich in both professional antigen-presenting cells and accessory cells, which are capable of producing immunostimulatory cytokines, has the potential to overcome the historical limitations of vaccinology and immunotherapy. Though the precise molecular mechanisms of genetic immunization remain unclear, a general working model of the events through which antigen-encoding plasmids introduced into the skin initiate an immune response can be constructed. The finding that Langerhans cells can be transfected in vivo raises the exciting possibility that these migrating professional antigen-presenting cells can be genetically engineered in vivo. By designing strategies to codeliver genes encoding antigens with genes encoding immunoregulatory molecules to the same antigen-presenting cell, it may be possible to either induce or suppress antigen-specific immune responses in the host. Though many aspects of the biology of cutaneous DNA immunization remain unknown, the skin appears to offer unique potential for the application of advances in gene therapy to vaccination and genetic engineering of the immune response.
Subject(s)
Genetic Therapy/methods , Skin/immunology , Vaccines, DNA/pharmacology , Animals , Antigen Presentation , Genetic Engineering , Humans , Models, Biological , Skin/cytologyABSTRACT
Immunization regimens that induce a broadly reactive cytolytic T lymphocyte (CTL) response specific for lentiviral antigens have emerged as the leading candidates in efficacy trials conducted in both animal modelshumans. To date, lentivirus vaccination strategies have overlooked one such immunization strategy, namely the use of particulate antigens. To evaluate the efficacy of targeting antigen into the phagocytic pathway to elicit a cell-mediated immune response to lentiviral antigens, we initiated the first study of a particulate-based vaccination protocol using a large animal model system. Gradient-purified equine infectious anemia virus (EIAV) was covalently coupled to glutaraldehyde-activated iron oxide beads. In vitro studies demonstrated the effectiveness of the inactivated whole virus particulate to prime antigen presenting cells for the activationexpansion of virus-specific CD8(+) CTL. The in vivo effectiveness of the particulate antigen was evaluated by experimental immunization of ponies. Ponies receiving the viral particulate vaccinechallenged with infectious EIAV had a delayed progression to diseasea reduced viral load compared with infected ponies that had not been vaccinated. Interestingly, in vitro virus-specific CTL activity was detected in only one of four immunized animals at the day of challenge. The beneficial effects of the particulate vaccine regimen were not clearly associated with any in vitro measurable parameters of the virus-specific cellular or humoral immune responses elicited by the vaccine at the day of challenge. However, within 3 weeks after virus challenge, anamnestic humoral responses characterized by a rapid emergence of neutralizing activity in the seruma predominance of conformationally dependent epitopes recognized by virus-specific antibodies were observed in the vaccinates. Taken together, further studies are clearly warranted in large animal model systems using a particulate-based vaccine regimen considering the beneficial effects of this regimen in our studythe protective effects of particulate antigen delivery in the murine model.