Thymic Stromal Lymphopoietin and Thymic Stromal Lymphopoietin–Conditioned Dendritic Cells Induce Regulatory T-Cell Differentiation and Protection of NOD Mice Against Diabetes

OBJECTIVE—Autoimmune diabetes in the nonobese diabetic (NOD) mouse model results from a breakdown of T-cell tolerance caused by impaired tolerogenic dendritic cell development and regulatory T-cell (Treg) differentiation. Re-establishment of the Treg pool has been shown to confer T-cell tolerance and protection against diabetes. Here, we have investigated whether murine thymic stromal lymphopoietin (TSLP) re-established tolerogenic function of dendritic cells and induced differentiation and/or expansion of Tregs in NOD mice and protection against diabetes

Dendritic Cells Transduced to Express Interleukin 4 Reduce Diabetes Onset in Both Normoglycemic and Prediabetic Nonobese Diabetic Mice

Background: We and others have previously demonstrated that treatment with bone marrow derived DC genetically modified to express IL-4 reduce disease pathology in mouse models of collagen-induced arthritis and delayed-type hypersensitivity. Moreover, treatment of normoglycemic NOD mice with bone marrow derived DC, genetically modified to express interleukin 4 (IL-4), reduces the onset of hyperglycemia in a significant number of animals. However, the mechanism(s) through which DC expressing IL-4 function to prevent autoimmune diabetes and whether this treatment can reverse disease in pre-diabetic NOD mice are unknown. Methodology/Principal Findings: DC were generated from the bone marrow of NOD mice and transduced with adenoviral vectors encoding soluble murine IL-4 (DC/sIL-4), a membrane-bound IL-4 construct, or empty vector control. Female NOD mice were segregated into normoglycemic (<150mg/dL) and prediabetic groups (between 150 and 250 mg/dL) on the basis of blood glucose measurements, and randomized for adoptive transfer of 106 DC via a single i.v. injection. A single injection of DC/sIL-4, when administered to normoglycemic 12-week old NOD mice, significantly reduced the number of mice that developed diabetes. Furthermore, DC/sIL-4, but not control DC, decreased the number of mice progressing to diabetes when given to prediabetic NOD mice 12-16 weeks of age. DC/sIL-4 treatment also significantly reduced islet mononuclear infiltration and increased the expression of FoxP3 in the pancreatic lymph nodes of a subset of treated animals. Furthermore, DC/sIL-4 treatment altered the antigen-specific Th2:Th1 cytokine profiles as determined by ELISPOT of splenocytes in treated animals. Conclusions: Adoptive transfer of DC transduced to express IL-4 into both normoglycemic and prediabetic NOD mice is an effective treatment for T1D. © 2010 Ruffner, Robbins

Gene expression analysis of dendritic cells that prevent diabetes in NOD mice: analysis of chemokines and costimulatory molecules.

Contains fulltext : 96948.pdf (publisher's version ) (Closed access)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.1 september 201

Influence of interleukin-4 on the phenotype and function of bone marrow-derived murine dendritic cells generated under serum-free conditions

Murine bone marrow-derived dendritic cells (DC) can be generated by culture in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF) alone or GM-CSF in conjunction with interleukin-4 (IL-4). However, these two culture methods result in the production of heterogeneous DC populations with distinct phenotypic and stimulatory properties. In this study, we investigated the properties of DC generated under serum-free conditions in the presence or absence of IL-4 and compared their yield and phenotype to that of DC generated in the presence of fetal calf serum (FCS) (±IL-4). We did not observe a significant difference in the total cell yield between these four culture conditions, although the proportion of CD11c DC in cultures that received FCS was higher than that of their counterparts generated under serum-free conditions. Also, the four culture conditions generated CD11c DC with comparable levels of major histocompatibility complex (MHC) class II, CD40, CD80 and CD86 expression, with the exception of cells cultured under serum-free conditions in the absence of IL-4, which displayed suboptimal levels of these markers. Moreover, we compared the functional and stimulatory properties of DC generated under serum-free conditions in the presence or absence of IL-4. DC cultured in the presence of IL-4 were stronger stimulators of allogeneic splenocytes in a primary mixed lymphocyte reaction (MLR) and of naïve antigen-specific OT-II transgenic T cells when pulsed with the class II ovalbumin (OVA) peptide or whole OVA protein than DC cultured in the absence of IL-4. However, both DC populations displayed a similar capacity to take up fluorescein isothiocyanate (FITC)-albumin by macropinocytosis and FITC-Dextran by the mannose receptor and to secrete IL-12 in response to stimulation with lipopolysaccharide (LPS) or an agonistic anti-CD40 monoclonal antibody. Therefore, we conclude that although both DC culture methods result in the production of DC with similar functional abilities, under serum-free conditions, DC cultured in GM-CSF and IL-4 show an increased stimulatory potential over DC cultured in GM-CSF alone. This is an important consideration in the design of experiments where DC are being exploited as immunotherapeutic vaccines

Dendritic cells, T cell tolerance and therapy of adverse immune reactions

Dendritic cells (DC) are uniquely able to either induce immune responses or to maintain the state of self tolerance. Recent evidence has shown that the ability of DC to induce tolerance in the steady state is critical to the prevention of the autoimmune response. Likewise, DC have been shown to induce several type of regulatory T cells including Th2, Tr1, Ts and NKT cells, depending on the maturation state of the DC and the local microenvironment. DC have been shown to have therapeutic value in models of allograft rejection and autoimmunity, although no success has been reported in allergy. Several strategies, including the use of specific DC subsets, genetic modification of DC and the use of DC at various maturation stages for the treatment of allograft rejection and autoimmune disease are discussed. The challenge for the future use of DC therapy in human disease is to identify the appropriate DC for the proposed therapy; a task made more daunting by the extreme plasticity of DC that has recently been demonstrated. However, the progress achieved to date suggests that these are not insurmountable obstacles and that DC may become a useful therapeutic tool in transplantation and autoimmune disease

Qualitative and quantitative abnormalities in splenic dendritic cell populations in NOD mice

The phenotype and function of splenic DC populations from diabetes-prone NOD mice were chara-cterized and compared to DC from diabetes-resistant strains in the presence or absence of Flt3 ligand (FL) treatment. NOD mice were found to have significantly fewer CD8α(+) DC than both B10.BR and C57BL/6 mice, and this defect was reversed by FL treatment. Freshly isolated CD8α(+) and CD8α(−) DC from all three strains were found to express similar levels of costimulatory molecules and this was similar in both FL-treated and untreated animals. IL-12 p40 production was significantly lower in purified CD11c(+) DC from NOD mice compared to DC from C57BL/6 or B10.BR mice. CD8α(+) DC isolated from NOD mice produced lower levels of IL-12p40 than CD8α(+) DC from C57CBL/6 and this was dependent on the nature of the stimulus given. In contrast both CD8α(+) and CD8α(−) DC from FL-treated mice produced high levels of IL-12p40 following activation, but only the CD8α(−) DC produced IL-12p70. Functionally, freshly isolated CD8α(−) DC were more stimulatory than CD8α(+) DC in a primary allogeneic mixed lymphocyte reaction. However, DC maturation resulted in increased T cell stimulatory capacity for both DC subsets, and this pattern was seen in all strains. These results demonstrate significant differences in phenotype and function of splenic NOD CD8α(+) DC, and further suggest that FL treatment may reverse some of these abnormalities