ABSTRACT
Cancer is one of the main causes of death among humans, second only to cardiovascular diseases. In recent years, numerous studies have been conducted on the pathophysiology of cancer, and it has been established that this disease is developed by a group of stem cells known as cancer stem cells (CSCs). Thus, cancer is considered a stem cell disease; however, there is no comprehensive consensus about the characteristics of these cells. Several different signaling pathways including Notch, Hedgehog, transforming growth factor-ß (TGF-ß), and WNT/ß-catenin pathways cause the self-renewal of CSCs. CSCs change their metabolic pathways in order to access easy energy. Therefore, one of the key objectives of researchers in cancer treatment is to destroy CSCs. Nuclear factor erythroid 2-related factor 2 (Nrf2) plays an essential role in the protection of CSCs from reactive oxygen species (ROS) and chemotherapeutic agents by regulating antioxidants and detoxification enzymes. Human epidermal growth factor receptor 2 (HER2) is a member of the tyrosine kinase receptor family, which contributes to the protection of cancer cells against treatment and implicated in the invasion, epithelial-mesenchymal transition (EMT), and tumorigenesis. Aldehyde dehydrogenases (ALDHs) are highly active in CSCs and protect the cells against damage caused by active aldehydes through the regulation of aldehyde metabolism. On the other hand, ALDHs promote the formation and maintenance of tumor cells and lead to drug resistance in tumors through the activation of various signaling pathways, such as the ALDH1A1/HIF-1α/VEGF axis and Wnt/ß-catenin, as well as changing the intracellular pH value. Given the growing body of information in this field, in the present narrative review, we attempted to shed light on the function of Nrf2, HER2, and ALDH in CSCs.
Subject(s)
Aldehyde Dehydrogenase , NF-E2-Related Factor 2 , Neoplastic Stem Cells , Receptor, ErbB-2 , beta Catenin , Humans , Aldehyde Dehydrogenase/metabolism , Aldehydes/metabolism , beta Catenin/metabolism , Neoplasms/metabolism , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Receptor, ErbB-2/metabolismABSTRACT
Immunotherapies using immune checkpoint inhibitors (ICIs) and chimeric antigen receptor (CAR) T-cell therapy have achieved successful results against several types of human tumors, particularly hematological malignancies. However, their clinical results for the treatment of solid tumors remain poor and unsatisfactory. The immunosuppressive tumor microenvironment (TME) plays an important role by interfering with intratumoral T-cell infiltration, promoting effector T-cell exhaustion, upregulating inhibitory molecules, inducing hypoxia, and so on. Oncolytic viruses are an encouraging biocarrier that could be used in both natural and genetically engineered platforms to induce oncolysis in a targeted manner. Oncolytic virotherapy (OV) contributes to the reprogramming of the TME, thus synergizing the functional effects of current ICIs and CAR T-cell therapy to overcome resistant barriers in solid tumors. Here, we summarize the TME-related inhibitory factors affecting the therapeutic outcomes of ICIs and CAR T cells and discuss the potential of OV-based approaches to alleviate these barriers and improve future therapies for advanced solid tumors.
Subject(s)
Neoplasms , Oncolytic Virotherapy , Oncolytic Viruses , Receptors, Chimeric Antigen , Humans , Tumor Microenvironment , Immunotherapy, Adoptive/methods , Immunotherapy/methods , Neoplasms/pathology , Oncolytic Virotherapy/methodsABSTRACT
We have demonstrated previously that BM-derived DCs can prevent diabetes development and halt progression of insulitis in NOD mice, the mouse model of type 1 diabetes. The DC population that was most effective in this therapy had a mature phenotype, expressed high levels of costimulatory molecules, and secreted low levels of IL-12p70. The protective DC therapy induced Treg and Th2 cells in vitro and in vivo. Microarray analysis of therapeutic and nontherapeutic DC populations revealed differences in the expression of OX40L, CD200, Ym-1, CCL2, and CCL5, which could play important roles in the observed DC-mediated therapy. The unique pattern of costimulatory molecules and chemokines expressed by the therapeutic DCs was confirmed by flow cytometry and ELISA. Using a novel cell-labeling and (19)F NMR, we observed that the chemokines secreted by the therapeutic DCs altered the migration of diabetogenic Th1 cells in vivo and attracted Th2 cells. These results suggest that the therapeutic function of DCs is mediated by a combination of costimulatory and chemokine properties that results in the attraction of diabetogenic Th1 and the induction of Th2 and/or Treg differentiation.
Subject(s)
Biomarkers/metabolism , Chemokines/metabolism , Dendritic Cells/immunology , Diabetes Mellitus/immunology , Diabetes Mellitus/prevention & control , Th1 Cells/immunology , Th2 Cells/immunology , Animals , Blotting, Western , Cell Differentiation , Cell Movement , Cell Proliferation , Cells, Cultured , Dendritic Cells/cytology , Dendritic Cells/metabolism , Diabetes Mellitus/genetics , Female , Gene Expression Profiling , Mice , Mice, Inbred NOD , Mice, SCID , Mice, Transgenic , Oligonucleotide Array Sequence Analysis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes/immunologyABSTRACT
In this paper, a sensitive, easy, efficient, and suitable method for the calculation of K(f) values of complexation between one derivative of Dansyl chloride [5-(dimethylamino) naphthalene-1-sulfonyl 4-phenylsemicarbazide] (DMNP) and Lanthanide(III) (Ln) ions is proposed, using both spectrofluorometric and spectrophotometric methods. Determination of K(f) showed that DMNP was mostly selective towards the erbium (III) ion. The validity of the method was also confirmed calculating the Stern-Volmer fluorescence quenching constants (K(sv)) that resulted in the same consequence, obtained by calculating the K(f) of complexation values. In addition, the UV-vis spectroscopy was applied for the determination of K(f) only for the Ln ions that had interactions with DMNP. Finally, the DFT studies were done on Er(3+) and the DMNP complex for distinguishing the active sites and estimating the pair wise interaction energy. It can be concluded that this derivative of Dansyl chloride with inherent high fluorescence intensity is a suitable reagent for the selective determination of the Er(3+) ion which can be used in constructing selective Er(3+) sensors.
Subject(s)
Dansyl Compounds/chemical synthesis , Ions/chemistry , Lanthanoid Series Elements/chemistry , Macromolecular Substances/chemical synthesis , Dansyl Compounds/chemistry , Macromolecular Substances/chemistry , Models, Biological , Models, Molecular , Models, Theoretical , Semicarbazides/chemistry , Spectrometry, Fluorescence , Spectrophotometry, Ultraviolet , Spectroscopy, Near-InfraredABSTRACT
Allogeneic islet transplantation is an option to treat diabetes however there are obstacles that are limiting its clinical use. We have examined whether mesenchymal stem cells (MSC) improve islet graft survival and whether such therapy allows for better graft acceptance with reduced requirement for immunosuppression. In vitro-expanded syngeneic bone marrow-derived MSC were co-transplanted with islets into omental pouch in a rat model of streptozotocin-induced diabetes. Marginal mass syngeneic islet transplantation into the omentum with MSC promoted sustained normoglycemia. Interestingly, allogeneic islets +MSC, but not islets alone, with short-term use of immunosuppression enhanced long-term islet graft survival, insulin expression in the grafts and induced normal serum insulin levels and normoglycemia. T cells from recipients transplanted with allogeneic islets +MSC produced low levels of IFN-gamma and TNF-alpha upon ex-vivo activation, and this transplantation protocol promoted the generation of IL-10-secreting CD4(+) T cells. These data encourage further preclinical and eventually, clinical MSC-based islet transplantation to improve the outcome of allogeneic islet transplantation in the treatment of diabetes.
Subject(s)
Glucose Metabolism Disorders/immunology , Graft Survival/immunology , Islets of Langerhans Transplantation/immunology , Mesenchymal Stem Cell Transplantation , Animals , CD4-Positive T-Lymphocytes/immunology , Cells, Cultured , Glucose Metabolism Disorders/blood , Insulin/blood , Interleukin-10/metabolism , Male , Rats , Time Factors , Transplantation, Homologous/immunologyABSTRACT
Cell-based therapies offer considerable promise for prevention or cure of diabetes. We explored the potential of autologous, self-renewing, mesenchymal stem cells (MSC) as a clinically-applicable approach to promote glucose homeostasis. In vitro-expanded syngeneic bone marrow-derived MSC were administered following or prior to diabetes induction into a rat model of streptozotocin-induced beta cell injury. MSC were CD45(-)/CD44(+)/CD54(+)/CD90(+)/CD106(+). MSC spontaneously secreted IL-6, HGF, TGF-beta1 and expressed high levels of SDF-1 and low levels of VEGF, IL-1beta and PGE(2), but no EGF, insulin or glucagon. MSC homed to the pancreas and this therapy allowed for enhanced insulin secretion and sustained normoglycemia. Interestingly, immunohistochemistry demonstrated that, the islets from MSC-treated rats expressed high levels of PDX-1 and that these cells were also positive for insulin staining. In addition, peripheral T cells from MSC-treated rats exhibited a shift toward IL-10/IL-13 production and higher frequencies of CD4(+)/CD8(+) Foxp3(+) T cells compared to the PBS-treated rats. These data suggest that the bioactive factors secreted by MSC establish a tissue microenvironment that supports beta cell activation/survival in the pancreas. In addition, because of anti-inflammatory and immunoregulatory effects of MSC on T cells, this work can lead to clinical trial of autologous MSC to prevent/cure type-1 diabetes.
Subject(s)
Cytokines/metabolism , Diabetes Mellitus, Experimental/therapy , Homeodomain Proteins/metabolism , Insulin/metabolism , Islets of Langerhans/metabolism , Mesenchymal Stem Cell Transplantation , T-Lymphocytes/immunology , Trans-Activators/metabolism , Animals , Antigens, CD/analysis , Blood Glucose/metabolism , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , Cell Movement , Diabetes Mellitus, Experimental/prevention & control , Diabetes Mellitus, Type 1/prevention & control , Diabetes Mellitus, Type 1/therapy , Female , Forkhead Transcription Factors/analysis , Insulin/blood , Intercellular Signaling Peptides and Proteins/metabolism , Male , Mesenchymal Stem Cells/metabolism , Pancreas/cytology , Pancreas/metabolism , Rats , Rats, Inbred Lew , T-Lymphocytes/cytology , T-Lymphocytes/metabolism , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/immunology , Transplantation, AutologousABSTRACT
We investigated the effect of autologous mesenchymal stem cells (MSC) on multiple unmodified donor bone marrow (BM) infusions and vascularized skin graft outcome. BM-derived rat MSC were examined for phenotype and function. MSC/MSC-conditioned-medium suppressed IFN-gamma production by T cells and modified DC function. Infusions of MSC with one-time BM improved vascularized skin graft survival, while with one-two-times BM reversed graft versus host disease (GVHD). Mixed chimerism was enhanced in recipients given two-four-times BM with MSC infusions. Interestingly, four-times BM infusions with MSC delayed GVHD onset, reduced host tissue damage and enhanced vascularized skin allograft survival compared to four-times BM alone. These data demonstrate that, the co-infusion of MSC with unmodified BM limit the toxicity of allogeneic BM transplantation, enhance mixed chimerism and improve vascularized skin graft survival. These findings provide insights for the development of autologous MSC-based BM transplantation and prevention of graft rejection or treatment of autoimmunity.
Subject(s)
Bone Marrow Transplantation/immunology , Cytokines/metabolism , Graft Survival/immunology , Graft vs Host Disease/therapy , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/immunology , Skin Transplantation , Animals , Bone Marrow/immunology , Bone Marrow/metabolism , Culture Media, Conditioned , Cytokines/immunology , Graft vs Host Disease/immunology , Interferon-gamma/biosynthesis , Interferon-gamma/immunology , Mesenchymal Stem Cells/metabolism , Rats , Rats, Inbred Lew , Rats, Wistar , Transplantation ChimeraABSTRACT
BACKGROUND: Composite tissue allograft transplantation is limited by risks of long-term immunosuppression. The authors investigated whether short-term immunosuppression combined with recipient immature dendritic cells pulsed with donor antigens promotes composite tissue allograft survival. METHODS: Orthotopic hind-limb transplants were performed (day 0) from Wistar-Furth (RT1) to Lewis (RT1(u)) rats. Recipient dendritic cells were propagated from bone marrow with granulocyte-macrophage colony-stimulating factor (bone marrow-derived dendritic cells) and pulsed with or without donor splenic cell lysate. Recipients were as follows: group I, control; group II, cyclosporine (10 mg/kg/day, days 0 through 6, intraperitoneally); group III, antilymphocyte serum plus cyclosporine (days -4 and +1, intraperitoneally); and groups IV and V, cyclosporine plus antilymphocyte serum, combined with 7 x 10(6) untreated or donor cell lysate-pulsed bone marrow-derived dendritic cells (days +7 and +14, intravenously), respectively. Epidermolysis/desquamation of donor skin defined rejection. Mixed leukocyte reaction determined recipient T-cell reactivity to donor. Tissue samples were obtained at 3 weeks and on the day of rejection. Groups comprised six or seven rats. RESULTS: Donor alloantigen-pulsed bone marrow-derived dendritic cells (group V) significantly prolonged median composite tissue allograft survival time (32.0 days) compared with groups II (18.0 days, p = 0.0012), III (22.5 days, p = 0.0043), and IV (26.5 days, p = 0.0043). Splenic T cells in group V exhibited hyporesponsiveness to donor alloantigen in mixed leukocyte reaction. Interestingly, the graft muscle component in the bone marrow-derived dendritic cell-treated group (group V) showed significant reduction in mononuclear cell infiltration relative to group II (p = 0.0317). CONCLUSIONS: Donor alloantigen-pulsed recipient bone marrow-derived dendritic cells combined with transient T-cell-directed immunosuppression significantly prolonged composite tissue allograft survival across a full major histocompatibility complex barrier. This may represent the basis for a novel, clinically applicable strategy to promote composite tissue allograft survival with reduced systemic immunosuppression.
Subject(s)
Dendritic Cells/immunology , Graft Survival/drug effects , Graft Survival/immunology , Immunosuppressive Agents/pharmacology , Animals , Antigens/immunology , Antilymphocyte Serum/pharmacology , Cyclosporine/pharmacology , Disease Models, Animal , Male , Rats , Rats, Inbred Lew , Rats, Inbred WF , Spleen/cytology , Tissue Transplantation/physiology , Transplantation, Homologous/immunologyABSTRACT
BACKGROUND: We showed recently that limb allograft survival could be enhanced by administration of alloantigen (Ag)-pulsed immature dendritic cells (DC) after transplantation. Since indefinite graft survival was not achieved, we have further modified the DC by pharmacologic (rapamycin; Rapa) conditioning and ascertained their influence on graft survival, without continued immunosuppressive therapy. METHODS: We compared the ability of donor Ag-pulsed, Rapa-conditioned rat myeloid DC (Rapa DC) and control DC (CTR DC) to inhibit alloreactive T-cell responses after limb transplantation in antilymphocyte serum (ALS)-treated recipients given a short postoperative course of cyclosporine (CsA). RESULTS: Both DC populations expressed similar levels of major histocompatibility complex (MHC) II, CD40 and CD54, but Rapa DC expressed lower CD86. After toll-like receptor activation, both populations produced minimal interleukin (IL)-12p70, but Rapa DC secreted lower levels of IL-6 and IL-10. The capacity of DCs to stimulate T-cell proliferation in mixed leukocyte reactions was very low. Pulsing of the DC with donor Ag did not alter their phenotype or function. Interestingly, posttransplant administration of donor Ag-pulsed Rapa DC to rats given perioperative ALS and 21 days CsA significantly delayed graft rejection and promoted long-term (>125 days) graft survival. AlloAg-pulsed Rapa DC induced T-cell hyporesponsiveness and promoted the generation of IL-10-secreting CD4 T cells upon ex vivo challenge. CONCLUSIONS: Infusion of donor Ag-pulsed, Rapa-conditioned DC after composite tissue transplantation can prevent rejection of the grafts, including skin, across a full MHC mismatch and in the absence of continued immunosuppressive therapy.
Subject(s)
Dendritic Cells/immunology , Dendritic Cells/transplantation , Graft Survival , Hindlimb/transplantation , Isoantigens/pharmacology , Transplantation, Homologous/physiology , Animals , Bone Marrow Cells/immunology , Immunosuppression Therapy , Immunosuppressive Agents/pharmacology , Interferon-gamma/metabolism , Male , Rats , Rats, Inbred Lew , Rats, Inbred WF , Rats, Sprague-Dawley , Sirolimus/pharmacology , Transplantation ChimeraABSTRACT
Clinically-applicable protocols that promote tolerance to vascularized skin grafts may contribute to more widespread use of composite tissue transplantation. We compared the properties of alloantigen (Ag)-pulsed, rapamycin (Rapa)-conditioned and control bone marrow-derived host myeloid dendritic cells (DCs) and their potential, together with transient immunosuppression (anti-lymphocyte serum+cyclosporine), to promote long-term, vascularized skin graft survival in Lewis rats across a full MHC barrier. Both types of DCs expressed low levels of CD86, but Rapa DC expressed lower levels of MHC II and CD40 and were less stimulatory in MLR. While both Rapa and control DCs produced low levels of IL-12p70 and moderate levels of IL-6 and IL-10 following TLR ligation, Rapa DC secreted significantly lower levels of IL-6 and IL-10 in response to LPS. Donor Ag-pulsed Rapa DC, but not control DC, induced long-term skin graft survival (median survival time >133 days) when administered 7 and 14 days post-transplant. Circulating T cells in hosts with long-surviving grafts were hyporesponsive to donor alloAg stimulation, but proliferated in response to third-party stimulation and produced IFN-gamma and IL-10. When recipients of long-surviving grafts were challenged with skin grafts, donor but not third-party grafts were prolonged, suggesting underlying regulatory mechanisms. Both flow cytometry and immunohistochemical analysis revealed that donor Ag-pulsed Rapa DC infusion expanded CD4+ Foxp3+ Treg in recipients' spleens, graft-associated lymph nodes and the graft. These data demonstrate for the first time that pharmacologically-modified, donor Ag-pulsed host DC administered post-transplant can promote indefinite vascularized skin graft survival, associated with Treg expansion.
Subject(s)
Cell Proliferation , Dendritic Cells/immunology , Graft Survival/immunology , Immunosuppressive Agents/pharmacology , Isoantigens/immunology , Sirolimus/pharmacology , Skin Transplantation/immunology , T-Lymphocytes, Regulatory/immunology , Animals , CD40 Antigens/metabolism , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Histocompatibility Antigens Class II/metabolism , Male , Rats , Rats, Inbred Lew , Rats, Inbred WF , Rats, Sprague-Dawley , Skin/blood supply , Skin/immunology , T-Lymphocytes, Regulatory/cytology , Transplantation ConditioningABSTRACT
BACKGROUND: Dendritic cells (DC) have the unique ability to induce immunity against tumors and various pathogens or to promote tolerance in autoimmunity and transplantation. Hence, they are central to the regulation of immune responses. OBJECTIVE/METHODS: Due to the unique tolerogenic ability of DC, understanding some of the key molecules that regulate DC function may help with targeting the relevant signals in DC as therapeutic options for many disease conditions. DC are also targets of drugs, and many of the anti-inflammatory and pharmaceutical agents used to prevent autoimmunity or inhibit graft rejection interfere with DC function. RESULTS/CONCLUSION: The drug-induced changes in DC may provide information for the selection of drugs and further drug discovery along with the use of DC as adjuvant in the treatment of autoimmunity and prevention of graft rejection in transplantation.
ABSTRACT
Described by Strauch and Murray in 1967, the rodent epigastric free flap remains a versatile tool for microsurgery research and training. We report herein three sequential phases of our quest to improve efficiency and effectiveness of the original technique, making it more accessible to more microsurgeons. Ninety-six allotransplants were performed. Surgical technique, complication rates, clinical findings, and histopathologic correlation of each phase are reported. In phase I, two experienced microsurgeons employed the original technique and succeeded in 77% of the procedures. In phase II, two junior microsurgeons achieved a patency rate of 16.6% using the same technique, as opposed to 100% in phase III, utilizing the not-yet-described simplified flow-thru technique. Although patency rate using the original method varies from 9 to 78% (according to other reports), this technical modification can increase even the less experienced microsurgeons' success rates, perpetuating the use of Strauch's epigastric flap in experimental microsurgery.
Subject(s)
Skin Transplantation/methods , Surgical Flaps , Anastomosis, Surgical/methods , Animals , Femoral Artery/surgery , Femoral Vein/surgery , Graft Rejection/etiology , Graft Survival , Hematoma/etiology , Male , Microsurgery/adverse effects , Microsurgery/education , Microsurgery/methods , Necrosis , Postoperative Complications , Rats , Rats, Inbred Lew , Rats, Inbred WF , Seroma/etiology , Skin Transplantation/adverse effects , Surgical Flaps/adverse effects , Surgical Flaps/blood supply , Suture Techniques , Thrombosis/etiology , Tissue and Organ Harvesting/methods , Treatment Outcome , Vascular Patency/physiologyABSTRACT
Composite tissue allografts (CTAs) contain their own reservoir of vascularized bone marrow, offering novel aspects for the induction of donor-specific tolerance. Additionally, the manipulation of recipient dendritic cells, pulsed with donor allopeptide, has been shown to engender solid organ allograft survival. To exploit these modalities, we have developed a protocol utilizing injection of recipient bone marrow-derived dendritic cells (BMDCs) pulsed with a donor-derived peptide for use in CTA transplantation. Six days prior to orthotopic hind-limb transplantation, Lewis rats received IV injection of donor allopeptide-pulsed, recipient BMDCs, in conjunction with a single dose of anti-lymphocyte serum. Control groups displayed signs of allograft rejection within 5 days postoperatively. Animals within the primary experimental cohort demonstrated prolongation of graft survival to an average of 8 days, and exhibited low numbers of donor T cells. The use of BMDCs in conjunction with transient immunosuppression has potential therapeutic application for induction of donor-antigen-specific tolerance to hind limb allografts.
Subject(s)
Dendritic Cells/transplantation , Femur/transplantation , Graft Survival , Transplantation Conditioning/methods , Transplantation Tolerance , Animals , Bone Marrow Cells , Dendritic Cells/immunology , Graft Rejection/physiopathology , Male , Peptides/immunology , Rats , Rats, Inbred Lew , Rats, Inbred WF , Transplantation Tolerance/immunology , Transplantation, HomologousABSTRACT
The influence of GM+IL-4 and Flt3 ligand (FL) on phenotype and function of BM-derived DC from Lewis rats was investigated. GM+IL-4-induced DC, despite expression of CD80/CD86, were less stimulatory than FL-induced DC that expressed low CD80/CD86 and were efficient stimulators of allogeneic T cells. GM+IL-4 DC were CD11b+ OX62lo, whereas FL DC were CD11blo OX62+. Following activation, GM+IL-4 DC produced IL-10 and IL-6, but no IL-12p70, and were resistant to further maturation. FL DC produced IL-12p70, IFN-alpha/beta, IL-10 and IL-6 and underwent maturation. Repeated stimulation of T cells with GM+IL-4 DC inhibited proliferation, cytokine production and induced early T cell apoptosis. FL DC-activated T cells produced large amounts of IFN-gamma/IL-10 and exhibited late T cell apoptosis/necrosis. In vivo, GM+IL-4 DC induced alloAg-specific hyporesponsiveness following T cell restimulation. These results demonstrate that GM+IL-4 DC display intrinsic regulatory properties, inducing passive-cell-death in T cells with potential for inactivation/regulation of alloreactive T cells in transplantation.
Subject(s)
Dendritic Cells/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Interleukin-4/pharmacology , Membrane Proteins/pharmacology , T-Lymphocytes/immunology , Animals , Antibodies, Monoclonal/pharmacology , Antigen-Presenting Cells/drug effects , Antigen-Presenting Cells/immunology , Antigens, CD/analysis , Antigens, CD/metabolism , Apoptosis/drug effects , Apoptosis/immunology , Cell Differentiation/drug effects , Cell Differentiation/immunology , Cell Transplantation , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Dinucleoside Phosphates/pharmacology , Histocompatibility Antigens/analysis , Immune Tolerance/drug effects , Immune Tolerance/immunology , Interferons/metabolism , Isoantigens/immunology , Lipopolysaccharides/pharmacology , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Male , Rats , Rats, Inbred ACI , Rats, Inbred Lew , Rats, Inbred WF , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes/drug effects , T-Lymphocytes/metabolismABSTRACT
Dendritic cells (DC) play important roles in the initiation of immune responses and maintenance of self-tolerance. We have been studying the role of DC in the pathogenesis of type 1 diabetes and exploring the ability of specific DC subsets to prevent diabetes in non-obese diabetic (NOD) mice. DC subsets that prevent diabetes in this model have a mature phenotype and induce the production of regulatory Th2 cells. We review here recent advances in this area and highlight the importance of optimizing culture conditions and purification methods in the isolation of therapeutic DC.
Subject(s)
Cell Culture Techniques/methods , Dendritic Cells/immunology , Diabetes Mellitus, Type 1/immunology , Diabetes Mellitus, Type 1/prevention & control , Immunotherapy/methods , Animals , Humans , Mice , Mice, Inbred NODABSTRACT
Dendritic cells (DCs) activate T cells and regulate their differentiation into T helper cell type 1 (Th1) and/or Th2 cells. To identify DCs with differing abilities to direct Th1/Th2 cell differentiation, we cultured mouse bone marrow progenitors in granulocyte macrophage-colony stimulating factor (GM), GM + interleukin (IL)-4, or GM + IL-15 and generated three distinct DC populations. The GM + IL-4 DCs expressed high levels of CD80/CD86 and major histocompatibility complex (MHC) class II and produced low levels of IL-12p70. GM and GM + IL-15 DCs expressed low levels of CD80/CD86 and MHC class II. The GM + IL-15 DCs produced high levels of IL-12p70 and interferon (IFN)-gamma, whereas GM DCs produced only high levels of IL-12p70. Naive T cells stimulated with GM + IL-4 DCs secreted high levels of IL-4 and IL-5 in addition to IFN-gamma. In contrast, the GM + IL-15 DCs induced higher IFN-gamma production by T cells with little or no Th2 cytokines. GM DCs did not induce T cell polarization, despite producing large amounts of IL-12p70 following activation. A similar pattern of T cell activation was observed after in vivo administration of DCs. These data suggest that IL-12p70 production alone, although necessary for Th1 differentiation, is not sufficient to induce Th1 responses. These studies have implications for the use of DC-based vaccines in immunotherapy of cancer and other clinical conditions.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Dendritic Cells/cytology , Dendritic Cells/immunology , Immunotherapy , Th1 Cells/immunology , Th2 Cells/immunology , Animals , B7-1 Antigen/biosynthesis , B7-1 Antigen/drug effects , B7-2 Antigen/biosynthesis , B7-2 Antigen/drug effects , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/metabolism , Cell Differentiation/drug effects , Cell Differentiation/immunology , Cell Polarity/drug effects , Cell Polarity/immunology , Cell Polarity/physiology , Cells, Cultured , Dendritic Cells/drug effects , Female , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Interferon-gamma/biosynthesis , Interferon-gamma/drug effects , Interleukin-12/biosynthesis , Interleukin-15/biosynthesis , Interleukin-15/immunology , Interleukin-15/pharmacology , Interleukin-4/biosynthesis , Interleukin-4/immunology , Interleukin-4/pharmacology , Mice , Mice, Inbred NOD , Mice, Inbred Strains , Mice, Transgenic , Phenotype , Protein Subunits/biosynthesis , Protein Subunits/drug effects , Th1 Cells/drug effects , Th2 Cells/drug effectsABSTRACT
Our previous studies demonstrated that adoptive transfer of dendritic cells (DC) prevents diabetes in young nonobese diabetic (NOD) mice by inducing regulatory T(H)2 cells. In this report, as a means of treating NOD mice with more advanced insulitis, we infected DC with adenoviral vectors expressing interleukin (IL)-4 (Ad.IL-4), eGFP (Ad.eGFP), or empty vector (Ad psi 5). DC infected with any of the Ad vectors expressed higher levels of CD40, CD80, and CD86 molecules than uninfected DC and Ad.IL-4 DC produced IL-4 after lipopolysaccharide (LPS) and interferon (IFN)-gamma stimulation. Ad-infected DC efficiently stimulated allogeneic T cells, and cultures of T cells with Ad.IL-4 DC produced lower levels of IFN-gamma and marginally higher levels of IL-4. In vivo studies demonstrated that the Ad.eGFP DC trafficked to the pancreatic lymph nodes within 24 hr of intravenous administration, and could be visualized in the T cell areas of the spleen. The intrapancreatic IFN-gamma:IL-4 or IFN-gamma:IL-10 cytokine ratios were lower in 10-week-old mice treated with Ad.IL-4 DC, and these mice were significantly protected from disease. These results demonstrate, for the first time, that genetically modified DC can prevent diabetes in the context of advanced insulitis.
Subject(s)
Dendritic Cells/physiology , Diabetes Mellitus, Experimental/prevention & control , Genetic Therapy/methods , Interleukin-4/genetics , Pancreatitis/therapy , Adenoviridae/genetics , Animals , Antigens, CD/metabolism , B7-1 Antigen/metabolism , B7-2 Antigen , CD40 Antigens/metabolism , Cells, Cultured , Cytokines/metabolism , Dendritic Cells/drug effects , Dendritic Cells/virology , Female , Interferon-gamma/pharmacology , Interleukin-4/metabolism , Lymph Nodes/cytology , Lymph Nodes/metabolism , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred NOD , Pancreas/metabolism , Phantoms, Imaging , Spleen/immunology , Spleen/physiology , T-Lymphocytes/physiology , Th2 Cells/metabolism , Th2 Cells/physiology , Transduction, GeneticABSTRACT
We previously demonstrated that immunotherapy with dendritic cells (DC) prevented diabetes development in prediabetic NOD mice and that this effect was optimal when using a stimulatory DC population generated from bone marrow cells cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4. In this study, we have investigated the mechanism by which GM-CSF- and IL-4-cultured DC prevent diabetes in prediabetic NOD mice. Histological analysis of pancreatic tissue from DC-treated mice revealed a reduction in the severity of insulitis compared to controls. Analysisof the T cell response in DC-treated mice suggested a general shift towards a Th2-dominated response, as determined by cytokine production following either concanavalin A or anti-TCR stimulation. Furthermore, sorted CD45RB(lo) CD25+ CD4+ T cells from the spleen of DC-treated mice produced high amounts of Th2 cytokines following anti-TCR stimulation, suggesting that these cells are responsible for the apparent Th2 shift. We conclude that DC therapy may have corrected the immunoregulatory defect in the NOD mouse, thus restoring a balance between pathogenic Th1 cells and protective Th2 cells.