Estradiol acts directly on bone marrow myeloid progenitors to differentially regulate GM-CSF or Flt3 ligand-mediated dendritic cell differentiation

Estrogen receptor (ER) ligands modulate hemopoiesis and immunity in the normal state, during autoimmunity, and after infection or trauma. Dendritic cells (DC) are critical for initiation of innate and adaptive immune responses. We demonstrate, using cytokine-driven culture models of DC differentiation, that 17-beta-estradiol exerts opposing effects on differentiation mediated by GM-CSF and Flt3 ligand, the two cytokines that regulate DC differentiation in vivo. We also show that estradiol acts on the same highly purified Flt3+ myeloid progenitors (MP) to differentially regulate the DC differentiation in each model. In GM-CSF-supplemented cultures initiated from MP, physiological amounts of estradiol promoted differentiation of Langerhans-like DC. Conversely, in Flt3 ligand-supplemented cultures initiated from the same MP, estradiol inhibited cell survival in a dose-dependent manner, thereby decreasing the yield of plasmacytoid and conventional myeloid and lymphoid DC. Experiments with bone marrow cells from ER-deficient mice and the ER antagonist ICI182,780 showed that estradiol acted primarily via ERalpha to regulate DC differentiation. Thus, depending on the cytokine environment, pathways of ER signaling and cytokine receptor signaling can differentially interact in the same Flt3+ MP to regulate DC development. Because the Flt3 ligand-mediated differentiation pathway is important during homeostasis, and GM-CSF-mediated pathways are increased by inflammation, our data suggest that endogenous or pharmacological ER ligands may differentially affect DC development during homeostasis and disease, with consequent effects on DC-mediated immunity.     

Flt3 ligand preferentially increases the number of functionally active myeloid dendritic cells in the lungs of mice

In the present study, we investigated the effects of in vivo Flt3L administration on the generation, phenotype, and function of lung dendritic cells (DCs) to evaluate whether Flt3L favors the expansion and maturation of a particular DC subset. Injection of Flt3L into mice resulted in an increased number of CD11c-expressing lung DCs, preferentially in the alveolar septa. FACS analysis allowed us to quantify a 19-fold increase in the absolute numbers of CD11c-positive, CD45R/B220 negative DCs in the lungs of Flt3L-treated mice over vehicle-treated mice. Further analysis revealed a 90-fold increase in the absolute number of myeloid DCs (CD11c positive, CD45R/B220 negative, and CD11b positive) and only a 3-fold increase of lymphoid DCs (CD11c positive, CD45R/B220 negative, and CD11b negative) from the lungs of Flt3L-treated mice over vehicle-treated mice. Flt3L-treated lung DCs were more mature than vehicle-treated lung DCs as demonstrated by a significantly higher percentage of cells expressing MHC class II, CD86, and CD40. Freshly isolated Flt3L lung DCs were not fully mature, because after an overnight culture they continued to increase accessory molecule expression. Functionally, Flt3L-treated lung DCs were more efficient than vehicle-treated DCs at stimulating naive T cell proliferation. Our data show that administration of Flt3L favors the expansion of myeloid lung DCs over lymphoid DCs and enhanced their ability to stimulate naive lymphocytes.     

Increased dendritic cell numbers impair protective immunity to intracellular bacteria despite augmenting antigen-specific CD8+ T lymphocyte responses

Dendritic cells (DCs) reside in tissues, where they function as sentinels, providing an essential link between innate and adaptive immunity. Increasing the numbers of DCs in vivo augments T cell responses, and can cause dramatic CTL-dependent tumor regression. To determine whether greater DC numbers promoted T cell-mediated protection in the context of host defense against intracellular bacteria, we treated mice with Flt3 ligand (Flt3-L) to increase DCs in vivo and challenged them with Listeria monocytogenes. Unexpectedly, after primary challenge with Listeria, the overall control of Listeria infection was impaired in Flt3-L-treated mice, which had greater bacterial burden and mortality than controls. Similar results were obtained when DC numbers were increased by treatment with polyethylene glycol-conjugated GM-CSF rather than Flt3-L and in mice infected with Mycobacterium tuberculosis. Impaired protection was not due to dysfunctional T cell responses, as Flt3-L-treated mice had a greater frequency and absolute number of Ag-specific CD8+ T cells, which produced IFN-gamma, exhibited cytolytic activity, and transferred protection. The increased Listeria burden in Flt3-L-treated mice was preferentially associated with DCs, which were unable to kill Listeria and more resistant to CTL lysis compared with macrophages in vitro. Although we cannot exclude the possibility that other potential effects, in addition to increased numbers of DCs, are shared by Flt3-L and polyethylene glycol-conjugated GM-CSF and contributed to the increase in susceptibility observed in treated mice, these results support the notion that DC numbers must be properly controlled within physiological limits to optimize host defense to intracellular bacterial pathogens.     

The adjuvanticity of a mannosylated antigen reveals TLR4 functionality essential for subset specialization and functional maturation of mouse dendritic cells

The evidence that dendritic cell (DC) subsets produce differential cytokines in response to specific TLR stimulation is robust. However, the role of TLR stimulation in Ag presentation and phenotypic maturation among DC subsets is not clear. Through the adjuvanticity of a novel mannosylated Ag, mannosylated dendrimer OVA (MDO), as a pathogen-associated molecular pattern Ag, we characterized the functionality of GM-CSF/IL-4-cultured bone marrow DC and Flt3 ligand (Flt3-L) DC subsets by Ag presentation and maturation assays. It was demonstrated that both bone marrow DCs and Flt3-L DCs bound, processed, and presented MDO effectively. However, while Flt3-L CD24(high) (conventional CD8(+) equivalent) and CD11b(high) (CD8(-) equivalent) DCs were adept at MDO processing by MHC class I and II pathways, respectively, CD45RA(+) plasmacytoid DCs presented MDO poorly to T cells. Successful MDO presentation was largely dependent on competent TLR4 for Ag localization and morphological/phenotypic maturation of DC subsets, despite the indirect interaction of MDO with TLR4. Furthermore, Toll/IL-1 receptor-domain-containing adaptor-inducing IFN-beta, but not MyD88, as a TLR4 signaling modulator was indispensable for MDO-induced DC maturation and Ag presentation. Taken together, our findings suggest that DC subsets differentially respond to a pathogen-associated molecular pattern-associated Ag depending on the intrinsic programming and TLRs expressed. Optimal functionality of DC subsets in Ag presentation necessitates concomitant TLR signaling critical for efficient Ag localization and processing.     

Fms-Like Tyrosine Kinase 3 Ligand Controls Formation of Regulatory T Cells in Autoimmune Arthritis

Fms-like tyrosine kinase 3 ligand (Flt3L) is known as the primary differentiation and survival factor for dendritic cells (DCs). Furthermore, Flt3L is involved in the homeostatic feedback loop between DCs and regulatory T cell (Treg). We have previously shown that Flt3L accumulates in the synovial fluid in rheumatoid arthritis (RA) and that local exposure to Flt3L aggravates arthritis in mice, suggesting a possible involvement in RA pathogenesis. In the present study we investigated the role of Flt3L on DC populations, Tregs as well as inflammatory responses in experimental antigen-induced arthritis. Arthritis was induced in mBSA-immunized mice by local knee injection of mBSA and Flt3L was provided by daily intraperitoneal injections. Flow cytometry analysis of spleen and lymph nodes revealed an increased formation of DCs and subsequently Tregs in mice treated with Flt3L. Flt3L-treatment was also associated with a reduced production of mBSA specific antibodies and reduced levels of the pro-inflammatory cytokines IL-6 and TNF-α. Morphological evaluation of mBSA injected joints revealed reduced joint destruction in Flt3L treated mice. The role of DCs in mBSA arthritis was further challenged in an adoptive transfer experiment. Transfer of DCs in combination with T-cells from mBSA immunized mice, predisposed naïve recipients for arthritis and production of mBSA specific antibodies. We provide experimental evidence that Flt3L has potent immunoregulatory properties. Flt3L facilitates formation of Treg cells and by this mechanism reduces severity of antigen-induced arthritis in mice. We suggest that high systemic levels of Flt3L have potential to modulate autoreactivity and autoimmunity.     

Increased and long-term generation of dendritic cells with reduced function from IL-6-deficient bone marrow

The importance of IL-6 in dendritic cell (DC) development and function has not been well defined. To establish the role of IL-6, we studied bone marrow-derived DC (BMDC) and freshly isolated splenic DC from IL-6(-/-)-transgenic mice. We found that although IL-6(-/-) bone marrow had a similar composition to that of wild-type (WT) mice, it generated up to 10 times more DC when cultured in GM-CSF. The difference persisted even when IL-6(-/-) and WT bone marrow were cultured together, excluding the possibility that the effects were simply due to different cytokine microenvironments. In comparison to WT BMDC, IL-6(-/-) BMDC captured at least as much Ag, had an equivalent surface phenotype, and matured similarly in response to LPS or CpG. However, IL-6(-/-) BMDC induced less T cell allostimulation and Ag-specific T cell activation, but only the former was related to their inability to generate IL-6. Although WT bone marrow cultures died within 4 wk, IL-6(-/-) cultures continued to generate BMDC for >120 days, although the BMDC became immature and less functional. In vivo, we found that IL-6(-/-) mice had similar numbers and types of splenic DC as WT mice, both normally and after treatment with either Flt-3 ligand or GM-CSF. These findings demonstrate that IL-6 has profound effects on DC development in vitro, although the number and subtype composition of DC are unaffected by the absence of IL-6 in vivo. Furthermore, secretion of IL-6 is critical to certain DC functions.     

Organ-specific cellular requirements for in vivo dendritic cell generation

Bone marrow-derived dendritic cell (DC) precursors seed peripheral organs, where they encounter diverse cellular environments during their final differentiation into DCs. Flt3 ligand (Flt3-L) is critical for instructing DC generation throughout different organs. However, it remains unknown which cells produce Flt3-L and, importantly, which cellular source drives DC development in such a variety of organs. Using a novel BAC transgenic Flt3-L reporter mouse strain coexpressing enhanced GFP and luciferase, we show ubiquitous Flt3-L expression in organs and cell types. These results were further confirmed at the protein level. Although Flt3-L was produced by immune and nonimmune cells, the source required for development of the DC compartment clearly differed among organs. In lymphoid organs such as the spleen and bone marrow, Flt3-L production by hemopoietic cells was critical for generation of normal DC numbers. This was unexpected for the spleen because both immune and nonimmune cells equally contributed to the Flt3-L content in that organ. Thus, localized production rather than the total tissue content of Flt3-L in spleen dictated normal splenic DC development. No differences were observed in the number of DC precursors, suggesting that the immune source of Flt3-L promoted pre-cDC differentiation in spleen. In contrast, DC generation in the lung, kidney, and pancreas was mostly driven by nonhematopoietic cells producing Flt3-L, with little contribution by immune cells. These findings demonstrate a high degree of flexibility in Flt3-L-dependent DC generation to adapt this process to organ-specific cellular environments encountered by DC precursors during their final differentiation.     

Generation of mucosal dendritic cells from bone marrow reveals a critical role of retinoic acid

It is unknown how dendritic cells (DCs) become specialized as mucosal DCs and maintain intestinal homeostasis. We report that a subset of bone marrow cells freshly isolated from C57BL/6 mice express the retinoic acid (RA)-synthesizing enzyme aldehyde dehydrogenase family 1, subfamily A2 (ALDH1a2) and are capable of providing RA to DC precursors in the bone marrow microenvironment. RA induced bone marrow-derived DCs to express CCR9 and ALDH1a2 and conferred upon them mucosal DC functions, including induction of Foxp3(+) regulatory T cells, IgA-secreting B cells, and gut-homing molecules. This response of DCs to RA was dependent on a narrow time window and stringent dose effect. RA promoted bone marrow-derived DC production of bioactive TGF-β by inhibiting suppressor of cytokine signaling 3 expression and thereby enhancing STAT3 activation. These RA effects were evident in vivo, in that mucosal DCs from vitamin A-deficient mice had reduced mucosal DC function, namely failure to induce Foxp3(+) regulatory T cells. Furthermore, MyD88 signaling enhanced RA-educated DC ALDH1a2 expression and was required for optimal TGF-β production. These data indicate that RA plays a critical role in the generation of mucosal DCs from bone marrow and in their functional activity.     

Flt3 ligand-generated murine plasmacytoid and conventional dendritic cells differ in their capacity to prime naive CD8 T cells and to generate memory cells in vivo

Mature dendritic cells (DCs) have the capacity to induce efficient primary T cell response and effector cell differentiation. Thus, these cells are a major tool in the design of various immunotherapeutic protocols. We have tested the capacity of different subsets of matured DCs pulsed with a peptide to induce the differentiation of naive CD8 T cells into memory cells in vivo. Flt3 ligand (FL) induces the differentiation of conventional DCs (cDCs) and plasmacytoid DCs (PDCs) from murine bone marrow precursors in vitro. After maturation, both subsets become strong stimulators of Ag-specific T cell responses in vitro. However, the in vivo T cell stimulatory capacity of these DC subsets has not been studied in detail. In the present study, we demonstrate that mature FL-generated DCs induce efficient peptide-specific CD8 T cell response and memory cell differentiation in vivo. This is mainly due to the cDC subset because the PDC subset induced only a negligible primary CD8 response without detectable levels of memory CD8 T cell differentiation. Thus, in vitro FL-generated mature cDCs, but not PDCs, are potent stimulators of peptide-specific CD8 T cell responses and memory generation in vivo.     

Eradication of hepatoma and colon cancer in mice with Flt3L gene therapy in combination with 5-FU

We developed a recombinant defective adenovirus with an insert of gene encoding extracellular domain of mouse Flt3L (Ad-mFlt3L) under control of cytomegalovirus promoter to investigate the biological efficacy of Flt3L in combination with chemotherapeutical drug, 5-FU, in eliciting an effective anti-cancer immunity in mouse hepatoma and colon cancer model systems. The constructed Ad-mFlt3L efficiently infected hepatoma and colon cancer cells both in vitro and in vivo, leading to a high production of mFlt3L proteins in association with accumulation of DCs, NK cells and lymphocytes in local tumor tissues. Administration of Ad-mFlt3L can protect bone marrow injury caused by 5-Fu and stimulates proliferation and maturation of lymphocytes, APCs and NKs. Intratumoral injection of Ad-mFlt3L followed by an intraperitoneal administration of 5-Fu significantly inhibited tumor growth and cured established tumors. Adenovirus mediated Flt3L gene therapy synergies with chemotherapeutic drug, 5-Fu, in elicitation of long-lasting antitumor immunity. The tumor specific immunity can be adoptively transferred into naïve animals successfully by transfusion of CD3⁺CD8⁺ T cells from the treated mice. The data suggests that adenovirus mediated Flt3L gene therapy in combination with 5-Fu chemotherapy may open a new avenue for development of anti-cancer chemogenetherapy.     

Suppression of experimental autoimmune myasthenia gravis by granulocyte-macrophage colony-stimulating factor is associated with an expansion of FoxP3+ regulatory T cells

Dendritic cells (DCs) have the potential to activate or tolerize T cells in an Ag-specific manner. Although the precise mechanism that determines whether DCs exhibit tolerogenic or immunogenic functions has not been precisely elucidated, growing evidence suggests that DC function is largely dependent on differentiation status, which can be manipulated using various growth factors. In this study, we investigated the effects of mobilization of specific DC subsets-using GM-CSF and fms-like tyrosine kinase receptor 3-ligand (Flt3-L)-on the susceptibility to induction of experimental autoimmune myasthenia gravis (EAMG). We administered GM-CSF or Flt3-L to C57BL/6 mice before immunization with acetylcholine receptor (AChR) and observed the effect on the frequency and severity of EAMG development. Compared with AChR-immunized controls, mice treated with Flt3-L before immunization developed EAMG at an accelerated pace initially, but disease frequency and severity was comparable at the end of the observation period. In contrast, GM-CSF administered before immunization exerted a sustained suppressive effect against the induction of EAMG. This suppression was associated with lowered serum autoantibody levels, reduced T cell proliferative responses to AChR, and an expansion in the population of FoxP3+ regulatory T cells. These results highlight the potential of manipulating DCs to expand regulatory T cells for the control of autoimmune diseases such as MG.     

Dendritic Cell-Specific Delivery of Flt3L by Coronavirus Vectors Secures Induction of Therapeutic Antitumor Immunity

Efficacy of antitumor vaccination depends to a large extent on antigen targeting to dendritic cells (DCs). Here, we assessed antitumor immunity induced by attenuated coronavirus vectors which exclusively target DCs in vivo and express either lymphocyte- or DC-activating cytokines in combination with a GFP-tagged model antigen. Tracking of in vivo transduced DCs revealed that vectors encoding for Fms-like tyrosine kinase 3 ligand (Flt3L) exhibited a higher capacity to induce DC maturation compared to vectors delivering IL-2 or IL-15. Moreover, Flt3L vectors more efficiently induced tumor-specific CD8⁺ T cells, expanded the epitope repertoire, and provided both prophylactic and therapeutic tumor immunity. In contrast, IL-2- or IL-15-encoding vectors showed a substantially lower efficacy in CD8⁺ T cell priming and failed to protect the host once tumors had been established. Thus, specific in vivo targeting of DCs with coronavirus vectors in conjunction with appropriate conditioning of the microenvironment through Flt3L represents an efficient strategy for the generation of therapeutic antitumor immunity.     

The Lyn tyrosine kinase differentially regulates dendritic cell generation and maturation

The Src family kinase Lyn plays both stimulatory and inhibitory roles in hemopoietic cells. In this report we provide evidence that Lyn is involved in dendritic cell (DC) generation and maturation. Loss of Lyn promoted DC expansion in vitro from bone marrow precursors due to enhanced generation and accelerated differentiation of Lyn-deficient DC progenitors. Differentiated Lyn-deficient DCs also had a higher survival rate. Similarly, the CD11c-positive cell number was increased in aged Lyn-deficient mice in vivo. In contrast to their enhanced generation, lyn-/- DCs failed to mature appropriately in response to innate stimuli, resulting in DCs with lower levels of MHC class II and costimulatory molecules. In addition, IL-12 production and Ag-specific T cell activation were reduced in lyn-/- DCs after maturation, resulting in impaired Th1 responses. This is the first study to characterize Lyn-deficient DCs. Our results suggest that Lyn kinase plays uniquely negative and positive regulatory roles in DC generation and maturation, respectively.     

Lymphoid-related CD11c+ CD8alpha+ dendritic cells are involved in enhancing herpes simplex virus type 1 latency

The mechanism(s) by which herpes simplex virus type 1 (HSV-1) latency is established in neurons is not known. In this study, we examined the effect of dendritic cells (DCs) on the level of HSV-1 latency in trigeminal ganglia (TGs) of ocularly infected BALB/c and C57BL/6 mice. We found that immunization of wild-type mice with FMS-like tyrosine kinase 3 ligand (Flt3L) DNA, which increases the number of DCs, increased the amount of latency in infected mice. Conversely, depletion of DCs was associated with reduced latency. Latency was also significantly reduced in Flt3L^−/− and CD8^−/− mice. Interestingly, immunization of Flt3L^−/− but not CD8^−/− mice with Flt3L DNA increased latency. Transfer experiments using DCs expanded ex vivo with Flt3L or granulocyte-macrophage colony-stimulating factor suggested that increased latency was associated with the presence of lymphoid-related (CD11c⁺ CD8α⁺) DCs, while reduced latency was associated with myeloid-related (CD11c⁺ CD8α⁻) DCs. Modulation of DC numbers by Flt3L DNA immunization or depletion did not alter acute virus replication in the eye or TG or eye disease in ocularly infected mice. Our results suggest that CD11c⁺ CD8α⁺ DCs directly or indirectly increase the amount of HSV-1 latency in mouse TGs.     

Murine Flt3 ligand expands distinct dendritic cells with both tolerogenic and immunogenic properties

Human Flt3 ligand can expand dendritic cells (DC) and enhance immunogenicity in mice. However, little is known about the effects of murine Flt3 ligand (mFlt3L) on mouse DC development and function. We constructed a vector to transiently overexpress mFlt3L in mice. After a single treatment, up to 44% of splenocytes became CD11c(+) and the total number of DC increased 100-fold. DC expansion effects lasted for >35 days. mFlt3L DC were both phenotypically and functionally distinct. They had increased expression of MHC and costimulatory molecules and expressed elevated levels of B220 and DEC205 but had minimal CD4 staining. mFlt3L DC also had a markedly altered cytokine profile, including lowered secretion of IL-6, IL-10, IFN-gamma, and TNF-alpha, but had a slightly increased capacity to stimulate T cells in vitro. However, in a variety of in vivo models, DC expanded by mFlt3L induced tolerogenic effects on T cells. Adoptive transfer of Ag-pulsed mFlt3L splenic DC to naive mice actually caused faster rates of tumor growth and induced minimal CTL compared with control DC. mFlt3L also failed to protect against tumors in which human Flt3 ligand was protective, but depletion of CD4(+) T cells restored tumor protection. Our findings 1) demonstrate that mFlt3L has distinct effects on DC development, 2) suggest an important role for mFlt3L in generating DC that have tolerogenic effects on T cells, and 3) may have application in immunotherapy in generating massive numbers of DC for an extended duration.