The role of TNF-alpha in the pathogenesis of type 1 diabetes in the nonobese diabetic mouse: analysis of dendritic cell maturation

TNF-alpha has been linked to the development of type 1 diabetes (T1D). We previously reported that neonatal treatment of nonobese diabetic (NOD) mice with TNF-alpha accelerated the onset of T1D, whereas TNF-alpha blockade in the same time period resulted in a complete absence of diabetes. The mechanisms by which TNF-alpha modulates development of T1D in NOD mice remain unclear. Here we tested the effects of TNF-alpha on the maturation of dendritic cells (DCs) in the NOD mouse. We found that neonatal treatment with TNF-alpha caused an increase in expression of maturation markers on CD11c(+)CD11b(+) DC subpopulations, whereas treatment with anti-TNF-alpha resulted in a decrease in expression of maturation markers in the CD11c(+)CD11b(+) subset. Moreover, neonatal treatment with TNF-alpha resulted in skewed development of a CD8alpha(+)CD11b(-)CD11c(+) DC subset such that TNF-alpha decreases the CD8alpha(+)CD11c(+) DC subset, increases the CD11c(+)CD11b(+) subset, and causes an increase in the expression of CD40 and CD54 on mature DCs capable of inducing immunity. Anti-TNF-alpha-treated mice had an increase in the CD8alpha(+)CD11c(+) DCs. Notably, adoptively transferred na?ve CD4(+) T cells from BDC2.5 T cell receptor transgenic mice proliferated in the pancreatic lymph nodes in TNF-alpha-treated NOD mice but not in anti-TNF-alpha-treated mice. Finally, we show that anti-TNF-alpha-treated mice showed immunological tolerance to islet cell proteins. We conclude that TNF-alpha plays an important role in the initiation of T1D in the NOD mouse by regulating the maturation of DCs and, thus, the activation of islet-specific pancreatic lymph node T cells.     

Mouse CD11c(+) B220(+) Gr1(+) plasmacytoid dendritic cells develop independently of the T-cell lineage

The developmental origin of dendritic cells (DCs) is controversial. In the mouse CD8alpha(+) and CD8alpha(-) DC subsets are often considered to be of lymphoid and myeloid origin respectively, although evidence on this point is conflicting. Very recently a novel CD11c(+) B220(+) DC subset has been identified that appears to be the murine counterpart to interferon alpha (IFNalpha)-producing human plasmacytoid DCs (PDCs). We show here that CD11c(+) B220(+) mouse PDCs, like human PDCs, are present in the thymus and express T lineage markers such as CD8alpha and CD4. However, the intrathymic development of PDCs can be completely dissociated from immature T lineage cells in mixed chimeras established with bone marrow cells from mice deficient for either Notch-1 or T-cell factor 1, two independent mutations that severely block early T-cell development. Our data indicate that thymic PDCs do not arise from a bipotential T/DC precursor.     

Flt3 ligand and granulocyte-macrophage colony-stimulating factor preferentially expand and stimulate different dendritic and T-cell subsets

OBJECTIVE: Mechanisms of T-cell stimulation by Flt3 ligand (Flt3L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) remain unclear. Herein, we compared the effects of Flt3L and GM-CSF on the expansion of dendritic cells (DC) and T-cell subsets and cytokine expression. METHODS: Na?ve and effector/memory T cells were analyzed by flow cytometry (FC). CD4(+) and CD8(+) T cells and CD11c(+)CD11b(dull/-)(DC1) and CD11c(+)CD11b(+) (DC2) subsets were isolated and the frequency of IFN-gamma-, IL-12- (type 1) and IL-4-, IL-10 (type 2)-producing cells and cytokine mRNA expression evaluated. RESULTS: Flt3L expanded both DC1 and DC2 subsets with a significantly higher percentage and number of DC1 than DC2, while GM-CSF preferentially expanded the DC2 subset. Isolated DC1 from Flt3L-injected mice had significantly higher levels of IL-12 (p40) than IL-10, while the converse occurred with DC2. The numbers of na?ve and memory T cells were elevated in mice that received Flt3L or GM-CSF. However, the number of memory CD4(+) and CD8(+) T cells was significantly increased in Flt3L as compared to GM-CSF cohorts. While GM-CSF increased the frequency of both type 1 and type 2 cytokine-producing cells, Flt3L significantly augmented the frequency of type 1 T cells. CONCLUSIONS: In contrast to GM-CSF, Flt3L preferentially induces the expansion of type 1 T cells. The mechanism of Flt3L-induced T-cell stimulation is associated with the expansion of the IL-12 (p40)-producing DC1 and memory T cells.     

Generation of murine dendritic cells from flt3-ligand-supplemented bone marrow cultures

Murine dendritic cells (DCs) can be classified into at least 2 subsets, "myeloid-related" (CD11b(bright), CD8alpha(-)) and "lymphoid-related" (CD11b(dull), CD8alpha(+)), but the absolute relationship between the 2 remains unclear. Methods of generating DCs from bone marrow (BM) precursors in vitro typically employ granulocyte-macrophage colony-stimulating factor (GM-CSF) as the principal growth factor, and the resultant DCs exhibit a myeloidlike phenotype. Here we describe a flt3-ligand (FL)-dependent BM culture system that generated DCs with more diverse phenotypic characteristics. Murine BM cells cultured at high density in recombinant human FL for 9 days developed into small lymphoid-sized cells, most of which expressed CD11c, CD86, and major histocompatibility complex (MHC) class II. The CD11c(+) population could be divided into 2 populations on the basis of the level of expression of CD11b, which may represent the putative myeloid- and lymphoid-related subsets. The FL in vitro-derived DCs, when treated with interferon-alpha or lipopolysaccharide during the final 24 hours of culture, expressed an activated phenotype that included up-regulation of MHC class II, CD1d, CD8alpha, CD80, CD86, and CD40. The FL-derived DCs also exhibited potent antigen-processing and antigen-presenting capacity. Neutralizing anti-interleukin-6 (IL-6) antibody, but not anti-GM-CSF, significantly reduced the number of DCs generated in vitro with FL, suggesting that IL-6 has a role in the development of DCs from BM precursors. Stem cell factor, which exhibits some of the same bioactivities as FL, was unable to replace FL to promote DC development in vitro. This culture system will facilitate detailed analysis of murine DC development.     

CD8alpha-11b+ dendritic cells but not CD8alpha+ dendritic cells mediate cross-tolerance toward intestinal antigens

Cross-presentation is a critical process by which antigen is displayed to CD8 T cells to induce tolerance. It is believed that CD8alpha+ dendritic cells (DCs) are responsible for cross-presentation, suggesting that the CD8alpha+ DC population is capable of inducing both cross-priming and cross-tolerance to antigen. We found that cross-tolerance against intestinal soluble antigen was abrogated in C57BL/6 mice lacking mesenteric lymph nodes (MLNs) and Peyer patches (PPs), whereas mice lacking PPs alone were capable of developing CD8 T-cell tolerance. CD8alpha-CD11b+ DCs but not CD8alpha+ DCs in the MLNs present intestinal antigens to relevant CD8 T cells, while CD8alpha+ DCs but not CD8alpha-CD11b+ DCs in the spleen exclusively cross-present intravenous soluble antigen. Thus, CD8alpha-CD11b+ DCs in the MLNs play a critical role for induction of cross-tolerance to dietary proteins.     

Phenotypic analysis of circulating and intrahepatic dendritic cell subsets in patients with chronic liver diseases

BACKGROUND/AIMS: Dendritic cells (DCs) are the most potent professional antigen-presenting cells. Although two subsets of circulating DCs, lineage(-)CD11c(+)CD4(low) (CD11c(+)DCs) and lineage (-)CD11c(-)CD4(+)CD123(+) (CD123(+)DCs) are identified in humans, the role of each DC subset in the immunopathogenesis of liver diseases is unknown.METHODS: We examined the numbers and activation status of each DC subset in the circulation and in the inflamed livers in patients with chronic liver diseases by flow cytometry and immunohistochemistry.RESULTS: The numbers of circulating CD11c(+)DCs were inversely correlated with serum alanine aminotransferase (ALT) levels in patients with chronic viral hepatitis, and that the expression of costimulatory molecules on circulating CD11c(+)DCs in patients with chronic viral hepatitis was significantly up-regulated in patients with high serum levels of ALT. Both DCs are also identified in the livers by flow cytometry, and the expression of costimulatory molecule CD40 on those DCs was significantly higher in liver DCs than that in circulating DCs. Moreover, the ratios of CD11c(+)DCs/CD123(+)DCs were higher in liver DCs (mean+/-SD, 7.2+/-6.0) than those of circulating DCs (4.0+/-4.6). Immunohistochemically, CD11c(+) or CD123(+) cells and CD83(+) activated DCs were observed mostly in portal areas with mononuclear cell infiltration in various liver diseases. These overall data suggest that DCs, especially CD11c(+)DCs, could be associated with the necroinflammatory response in the liver of chronic viral liver diseases.CONCLUSIONS: DCs, especially CD11c(+)DCs, may be involved in the immunopathogenesis of chronic liver diseases.     

Two immunogenic passenger dendritic cell subsets in the rat liver have distinct trafficking patterns and radiosensitivities

The aim of this study was to investigate the trafficking patterns, radiation sensitivities, and functions of conventional dendritic cell (DC) subsets in the rat liver in an allotransplantation setting. We examined DCs in the liver, hepatic lymph, and graft tissues and recipient secondary lymphoid organs after liver transplantation from rats treated or untreated by sublethal irradiation. We identified two distinct immunogenic DC subsets. One was a previously reported population that underwent blood-borne migration to the recipient's secondary lymphoid organs, inducing systemic CD8(+) T-cell responses; these DCs are a radiosensitive class II major histocompatibility complex (MHCII)(+) CD103(+) CD172a(+) CD11b(-) CD86(+) subset. Another was a relatively radioresistant MHCII(+) CD103(+) CD172a(+) CD11b(+) CD86(+) subset that steadily appeared in the hepatic lymph. After transplantation, the second subset migrated to the parathymic lymph nodes (LNs), regional peritoneal cavity nodes, or persisted in the graft. Irradiation completely eliminated the migration and immunogenicity of the first subset, but only partly suppressed the migration of the second subset and the CD8(+) T-cell response in the parathymic LNs. The grafts were acutely rejected, and intragraft CD8(+) T-cell and FoxP3(+) regulatory T-cell responses were unchanged. The radioresistant second subset up-regulated CD25 and had high allostimulating activity in the mixed leukocyte reaction, suggesting that this subset induced CD8(+) T-cell responses in the parathymic LNs and in the graft by the direct allorecognition pathway, leading to the rejection. Conclusion: Conventional rat liver DCs contain at least two distinct immunogenic passenger subsets: a radiosensitive blood-borne migrant and a relatively radioresistant lymph-borne migrant. LNs draining the peritoneal cavity should be recognized as a major site of the intrahost T-cell response by the lymph-borne migrant. This study provides key insights into liver graft rejection and highlights the clinical implications of immunogenic DC subsets. (HEPATOLOGY 2012).     

Negative regulation of IFN-alpha/beta signaling by IFN regulatory factor 2 for homeostatic development of dendritic cells

The development and cooperation of distinct subsets of antigen-presenting cells, particularly dendritic cells (DCs), may be critical for maintaining homeostatic immune responses. Recently, much attention has been focused on IFN-alpha/beta, the cytokines induced en masse by virus infection or the activation of Toll-like receptors, in the context of DC activation. Here, we show that mice deficient in IFN regulatory factor 2 exhibit selective loss of CD8alpha- DCs, the so-called myeloid DCs, which is accompanied by a notable increase in CD11c-CD11bhigh other myeloid lineage cells. Such deficiency is intrinsic to the bone marrow precursors, in which the abnormal induction of IFN-alpha/beta genes causes excessive IFN signaling. The critical function of IFN regulatory factor 2 in the negative regulation of IFN-alpha/beta signaling is underscored by the observation that the deficiency is rescued by introducing an additional null mutation for the IFN receptor complex. In view of accumulating evidence of the critical role of IFN-alpha/beta signaling in DC activation, our present study offers a unique example in that the magnitude of a cytokine signal should be properly balanced in a stage-specific manner during the differentiation and activation of DCs.     

Effect of aging on bone marrow-derived murine CD11c+CD4-CD8alpha- dendritic cell function

Dendritic cells (DCs) are actively used as cellular adjuvant in cancer immunotherapy. However, although DC immunotherapies primarily target the elderly population, little is known about the effect of aging on DC functions. Here, we compared the T-cell stimulation, cytokine production, and tumor surveillance functions of bone marrow-derived CD11c(+)CD4(-)CD8alpha(-) DCs of old and young C57BL/6 mice. Old immature bone marrow-derived CD4(-)CD8alpha(-) DCs (imDCs) were 4 times less effective than were young DCs in stimulating syngeneic CD4(+) T-cell proliferation. Old imDCs also have decreased DC-specific/intracellular adhesion molecule type 3-grabbing, nonintegrin (DC-SIGN) expression compared to young DCs. Interestingly, mice treated with the ovalbumin peptide-pulsed young DCs exhibited significantly greater tumor regression than with ovalbumin peptide-pulsed old DCs. Old terminally differentiated bone marrow-derived DCs (tDC) also have increased interleukin-10, but decreased interleukin-6 and tumor necrosis factor-alpha production. Taken together, these results have important implications in the clinical application of DC-based tumor immunotherapy in elderly persons.     

Concept of lymphoid versus myeloid dendritic cell lineages revisited: both CD8alpha(-) and CD8alpha(+) dendritic cells are generated from CD4(low) lymphoid-committed precursors

Two dendritic cell (DC) subsets have been identified in the murine system on the basis of their differential CD8alpha expression. CD8alpha(+) DCs and CD8alpha(-) DCs are considered as lymphoid- and myeloid-derived, respectively, because CD8alpha(+) but not CD8alpha(-) splenic DCs were generated from lymphoid CD4(low) precursors, devoid of myeloid reconstitution potential. Although CD8alpha(-) DCs were first described as negative for CD4, our results demonstrate that approximately 70% of them are CD4(+). Besides CD4(-) CD8alpha(-) and CD4(+) CD8alpha(-) DCs displayed a similar phenotype and T-cell stimulatory potential in mixed lymphocyte reaction (MLR), although among CD8alpha(-) DCs, the CD4(+) subset appears to have a higher endocytic capacity. Finally, experiments of DC reconstitution after irradiation in which, in contrast to previous studies, donor-type DCs were analyzed without depleting CD4(+) cells, revealed that both CD8alpha(+) DCs and CD8alpha(-) DCs were generated after transfer of CD4(low) precursors. These data suggest that both CD8alpha(+) and CD8alpha(-) DCs derive from a common precursor and, hence, do not support the concept of the CD8alpha(+) lymphoid-derived and CD8alpha(-) myeloid-derived DC lineages. However, because this hypothesis has to be confirmed at the clonal level, it remains possible that CD8alpha(-) DCs arise from a myeloid precursor within the CD4(low) precursor population or, alternatively, that both CD8alpha(+) and CD8alpha(-) DCs derive from an independent nonlymphoid, nonmyeloid DC precursor. In conclusion, although we favor the hypothesis that both CD8alpha(+) and CD8alpha(-) DCs derive from a lymphoid-committed precursor, a precise study of the differentiation process of CD8alpha(+) and CD8alpha(-) DCs is required to define conclusively their origin.     

Etoposide (VP-16) plus G-CSF mobilizes different dendritic cell subsets than does G-CSF alone

Dendritic cells (DCs) are antigen-presenting cells that are critical to the generation of immunologic tumor responses. Myeloid DCs (DC1) express myeloid antigen CD11c; lymphoid DCs (DC2) express CD123(+) and are CD11c(-). Analysis of DC subsets from peripheral blood progenitor cells (PBPC) collected from normal donors mobilized with G-CSF shows a predominance of DC2 cells. Whether PBPCs mobilization by chemotherapy yields different subsets of DCs has not been studied. We analyzed DC subsets in apheresis products from 44 patients undergoing autologous stem cell transplantation from 6/00 to 5/01. Patients received either G-CSF alone (10 microg/kg per day, n=11) or etoposide (2 g/m(2)) plus G-CSF (n=33) for progenitor cell mobilization. The patients were apheresed for 2-10 days (median 3) to reach a minimum of 2.0 x 10(6) CD34(+) cells/kg. Patients receiving G-CSF alone mobilized significantly more total DC2s than did those receiving etoposide plus G-CSF (median 6.2 x 10(6)/kg vs 2.9 x 10(6)/kg, P=0.001). The DC2/DC1 ratio was also significantly different in the two groups, with the G-CSF group having a higher ratio (median 1.2 vs 0.4, P<0.001). We conclude that the combination of chemotherapy plus G-CSF yields different mobilized dendritic cell subsets than does G-CSF alone.     

Galectin-3 deficiency prevents concanavalin A-induced hepatitis in mice

We used concanavalin A (Con A)-induced liver injury to study the role of galectin-3 (Gal-3) in the induction of inflammatory pathology and hepatocellular damage. We tested susceptibility to Con A-induced hepatitis in galectin-3-deficient (Gal-3(-/-)) mice and analyzed the effects of pretreatment with a selective inhibitor of Gal-3 (TD139) in wild-type (WT) C57BL/6 mice, as evaluated by a liver enzyme test, quantitative histology, mononuclear cell (MNC) infiltration, cytokine production, intracellular staining of immune cells, and percentage of apoptotic MNCs in the liver. Gal-3(-/-) mice were less sensitive to Con A-induced hepatitis and had a significantly lower number of activated lymphoid and dendritic cells (DCs) in the liver. The level of tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), and interleukin (IL)-17 and -4 in the sera and the number of TNFα-, IFNγ-, and IL-17- and -4-producing cluster of differentiation (CD)4(+) cells as well as IL-12-producing CD11c(+) DCs were lower, whereas the number of IL-10-producing CD4(+) T cells and F4/80(+) macrophages were significantly higher in livers of Gal-3(-/-) mice. Significantly higher percentages of late apoptotic Annexin V(+) propidium-idodide(+) liver-infiltrating MNCs and splenocytes were observed in Gal-3(-/-) mice, compared to WT mice. Pretreatment of WT C57BL/6 mice with TD139 led to the attenuation of liver injury and milder infiltration of IFNγ- and IL-17- and -4-producing CD4(+) T cells, as well as an increase in the total number of IL-10-producing CD4(+) T cells and F4/80(+) CD206(+) alternatively activated macrophages and prevented the apoptosis of liver-infiltrating MNCs. CONCLUSIONS: Gal-3 plays an important proinflammatory role in Con A-induced hepatitis by promoting the activation of T lymphocytes and natural killer T cells, maturation of DCs, secretion of proinflammatory cytokines, down-regulation of M2 macrophage polarization, and apoptosis of MNCs in the liver.