Granulocyte macrophage-colony stimulating factor: A key modulator of renal mononuclear phagocyte plasticity

Macrophage-colony stimulating factor (M-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF) play key roles in the differentiation of macrophages and dendritic cells (DCs). We examined the effect of treatment with M-CSF-containing macrophage medium or GM-CSF-containing DC medium upon the phenotype of murine bone marrow-derived macrophages and DCs. Culture of macrophages for 5 days in DC medium reduced F4/80 expression and increased CD11c expression with cells effectively stimulating T cell proliferation in a mixed lymphocyte reaction. DC medium treatment of macrophages significantly reduced phagocytosis of both apoptotic cells and latex beads and strongly induced the expression of the chemokine receptor CCR7 known to be involved in DC trafficking to lymph nodes. Lysates of obstructed murine kidneys expressed both M-CSF and GM-CSF though M-CSF expression was dominant (M-CSF:GM-CSF ratio ～30:1). However, combination treatment with both M-CSF and GM-CSF (ratio 30:1) indicated that small amounts of GM-CSF skewed macrophages towards a DC–like phenotype. To determine whether macrophage phenotype might be modulated in vivo we tracked CD45.1⁺ bone marrow-derived macrophages intravenously administered to CD45.2⁺ mice with unilateral ureteric obstruction. Flow cytometry of enzyme dissociated kidneys harvested 3 days later indicated CD11c and MHC Class II upregulation by adoptively transferred CD45.1⁺ cells with CD45.1⁺ cells evident in draining renal lymph nodes. Our data suggests that GM-CSF modulates mononuclear phagocyte plasticity, which likely promotes resolution of injury and healing in the injured kidney.     

4种细胞因子组合方式对小鼠树突状细胞分化发育的影响

目的观察在体外培养时4种细胞因子(CK)组合方式对小鼠骨髓源树突状细胞(DC)分化、增殖、发育的影响.方法用不同的CK定向诱导小鼠骨髓细胞分化为DC,通过流式细胞仪(荧光抗体双标记法)测定CD11c+细胞比例、MHC-Ⅱ类分子的表达及在脂多糖(LPS)刺激后CD86表达的变化.结果GM-CSF+IL-3+SCF促进DC分化、增殖的能力明显高于其他3组(P＜0.05).该组CK所诱导的DC在LPS刺激后,CD86表达增加的幅度明显低于GM-CSF+IL-4组(P＜0.01).结论GM-CSF、IL-3和SCF对于促进小鼠骨髓细胞向DC定向分化、增殖有协同作用,分化后的DC多数处于发育早期,DC前体所占的比例较大.     

Disease-modifying capability of murine Flt3-ligand DCs in experimental autoimmune encephalomyelitis

Dendritic cells (DCs) bridge the innate and adaptive immune response, are uniquely capable of priming na?ve T cells, and play a critical role in the initiation and regulation of autoimmune and immune-mediated disease. At present, in vivo expansion of DC populations is accomplished primarily through the administration of the recombinant human growth factor fms-like tyrosine kinase 3 ligand (hFL), and in vitro DCs are generated using cytokine cocktails containing GM-CSF +/- IL-4. Although hFL has traditionally been used in mice, differences in amino acid sequence and biological activity exist between murine FL (mFL) and hFL, and resultant DC populations differ in phenotype and immunoregulatory functional capabilities. This study developed and characterized mFL-generated DCs and determined the therapeutic capability of mFL DCs in the autoimmune disease experimental autoimmune encephalomyelitis (EAE). Our findings demonstrate that mFL and hFL expand splenic DCs equally in vivo but that mFL-expanded, splenic DCs more closely resemble normal, resting, splenic DCs. In addition, a novel method for generating mFL-derived bone marrow-derived DCs (BM-DCs) was developed, and comparison of mFL with hFL BM-DCs found mFL BM-DCs to be less mature (i.e., lower MHC Class II, CD80, and CD86) than hFL BM-DCs. These immature mFL DCs up-regulated costimulatory molecules in response to maturation stimuli LPS and TNF-alpha. Mature mFL BM-DCs were immunogenic and exacerbated the clinical disease course of EAE.     

Generation of blood-derived dendritic cells in dogs with oral malignant melanoma

Advances in treatment of human melanoma indicate that immunotherapy, particularly dendritic cell (DC) immunization, may prove useful. The aim of this study was to investigate whether blood-derived DCs could be generated from canine melanoma patients. Peripheral blood mononuclear cells were isolated from three such dogs and cultured with recombinant canine granulocyte-macrophage colony stimulating factor (GM-CSF), canine interleukin 4 and human Flt3-ligand for 7 days. The resulting cells demonstrated a typical dendritic morphology, and were enriched for cells expressing CD1a, CD11c and MHC II by flow cytometric analysis. Thus, canine blood-derived DCs can be generated in vitro and DC immunization should be feasible in dogs.     

TLR2- and 4-independent immunomodulatory effect of high molecular weight components from Ascaris suum

Components of high molecular-weight (PI) obtained from Ascaris suum extract down-regulate the Th1/Th2-related immune responses induced by ovalbumin (OVA)-immunization in mice. Furthermore, the PI down-modulates the ability of dendritic cells (DCs) to activate T lymphocytes by an IL-10-mediated mechanism. Here, we evaluated the role of toll like receptors 2 and 4 (TLR2 and 4) in the modulatory effect of PI on OVA-specific immune response and the PI interference on DC full activation. An inhibition of OVA-specific cellular and humoral responses were observed in wild type (WT) or in deficient in TLR2 (TLR2^−/−) or 4 (TLR4^−/−) mice immunized with OVA plus PI when compared with OVA-immunized mice. Low expression of class II MHC, CD40, CD80 and CD86 molecules was observed in lymph node (LN) cells from WT, TLR2^−/− or TLR4^−/− mice immunized with OVA plus PI compared with OVA-primed cells. We also verified that PI was able to modulate the activation of DCs derived from bone marrow of WT, TLR2^−/− or TLR4^−/− mice induced in vitro by agonists of TLRs, as observed by a decreased expression of class II MHC and costimulatory molecules and by low secretion of pro-inflammatory cytokines. Its effect was accompanied by IL-10 synthesis. In this sense, the modulatory effect of PI on specific-immune response and DC activation is independent of TLR2 or TLR4.     

A defect in bone marrow derived dendritic cell maturation in the nonobesediabetic mouse

The pathogenesis of diabetes in the nonobese diabetic (NOD) mouse is characterized by a selective destruction of the insulin-producing β-cells in the islets of Langerhans mediated by autoreactive T cells. The function of T cells is controlled by dendritic cells (DC), which are not only the most potent activators of naïve T cells, but also contribute significantly to the establishment of central and peripheral tolerance. In this study, we demonstrate that the NOD mouse (H2: K^d, A^g7, E°, D^b) shows selective phenotypic and functional abnormalities in DC derived from bone marrow progeny cells in response to GM-CSF (DC^NOD). NOD DC, in contrast to CBA DC, have very low levels of intracellular I-A molecules and cell surface expression of MHC class II, CD80, CD86 and CD40 but normal β2-microglobulin expression. Incubation with the strong inflammatory stimulus of LPS and IFN-γ does not increase class II MHC, CD80 or CD86, but upregulates the level of CD40. The genetic defect observed in the DC^NOD does not map to the MHC, because the DC from the MHC congenic NOD.H2^h4 mouse (H2: K^k, A^k, E^k, D^k) shares the cell surface phenotype of the DC^NOD. DC from these NOD.H2^h4 also fail to present HEL or the appropriate HEL-peptide to an antigen-specific T cell hybridoma. However all the DC irrespective of origin were able to produce TNF-α, IL-6, low levels of IL-12(p70) and NO in response to LPS plus IFN-γ. A gene or genes specific to the NOD strain, but outside the MHC region, therefore must regulate the differentiation of DC in response to GM-CSF. This defect may contribute to the complex genetic aetiology of the multifactorial autoimmune phenotype of the NOD strain.     

Generation or large numbers of immature and mature dendritic cells from rat bone marrow cultures

We have defined conditions for generating large numbers of dendritic cells (DC) in marrow cultures from 10 – 12-week-old ACI or WF rats. The combination of granulocyte-macrophage colony-stimulating factor (GM-CSF) and TNF-α, known to induce DC from human CD34⁺ progenitors, was not effective with rat. In contrast, GM-CSF plus IL-4 generated DC in high yield, corresponding to 30 – 40 % of the initial number of plated marrow cells. The DC proliferated in distinctive aggregates, in which most cells had an immature phenotype marked by undetectable surface B7 and high levels of MHC class II products within intracellular lysosomes. When dislodged and dispersed, the aggregates gave rise to mature stellate DC with abundant surface MHC class II and B7, sparse MHC class II^– lysosomes, and strong T cell-stimulating capacity. Therefore, rat marrow progenitors can generate large numbers of immature DC, with abundant intracellular MHC class II compartments, and potent, stimulatory, mature DC.     

Oxygen tension regulates the in vitro maturation of GM-CSF expanded murine bone marrow dendritic cells by modulating class II MHC expression

Conventional culture conditions for GM-CSF expanded murine bone marrow derived dendritic cells (BMDCs) uses ambient (hyperoxic) oxygen pressure (20% v/v, 152 Torr) and medium supplemented with the thiol 2-mercaptoethanol (2-Me). Given the redox activities of O2 and 2-Me, the effects of 2%, 5%, 10%, and 20% v/v O2 atmospheres and omitting 2-Me from the medium were tested upon the generation of GM-CSF expanded BMDCs. DC yield, phenotype and function were compared to BMDCs grown using conventional conditions. All cultures yielded DC subsets with CD11c+ MHC II(NEG), CD11c+ MHC II(INT), CD11c+ MHC II(HI) expression phenotypes, classed as precursor, immature, and mature DCs (IDC, MDC). Low O2 tensions generated significantly fewer precursor DCs, and more IDCs and MDCs. Cytometer sorted precursor DCs expressed surface class II MHC after transfer to low, but not high O2 atmospheres. Expression of myeloid markers was similar between BMDC cultures generated in 5% O2 or conventional conditions, and MDCs from low O2 cultures had the morphology typical of mature myeloid DCs. IDCs and MDCs from low O2 and conventional culture conditions were similarly potent allostimulatory APCs. The O2 tension (but not 2-Me addition) in vitro significantly influences overall DC subset frequencies and yield, and governs DC maturation by regulating the surface class II MHC expression of GM-CSF expanded BMDC cultures.     

小鼠骨髓树突状细胞制备方法的比较研究

为了比较GM CSF与IL 4 (IL 4DC )以及GM CSF与IL 3(IL 3DC )共刺激培养制备的两种树突状细胞 (DC )的差异 ,采用GM CSF (10 0 0U/ml)和IL 4 (10～ 2 0ng/ml)或IL 3(10～ 2 0ng/ml)从小鼠骨髓培养制备DC ,流式细胞仪分析表型 ,体外饲以颗粒化抗原 (与Latexbead交联的Ovalbumin ,微粒 OVA )或抗原多肽 (Ovalbumin的SL8表位 ,即SIIFEKL )后 ,测定两种DC体外对颗粒化抗原的摄取能力和对抗原多肽的递呈能力 ,以及体内对特异性CTL的诱导能力。结果显示 ,IL 3DC较IL 4DC胞体略大 ,但树突状分支略少 ,表达更高的F4 / 80和更低的NLDC14 5、CD4 0 ,而两者的共刺激分子CD80、CD86和MHCI类分子的表达率无显著差异。体外IL 3DC对颗粒化抗原具有更高的摄取能力和对SIIFEKL多肽有更强的递呈能力 ,但体内对特异性CTL的诱导能力二者无显著差异。研究表明 ,两种方法制备的DC在形态、表型、体外抗原多肽递呈和抗原摄取能力方面存在一定差别 ,但具有相似的体内细胞免疫应答诱导能力     

The influence of granulocyte/macrophage colony-stimulating factor on dendritic cell levels in mouse lymphoid organs

To ascertain whether the development of dendritic cells (DC) in mouse lymphoid organs is dependent on granulocyte/macrophage colony-stimulating factor (GM-CSF), we determined the number of DC in the thymus, spleen and lymph nodes of normal mice, of mice with the genes coding for GM-CSF or its receptor inactivated, and of transgenic mice with excessive levels of GM-CSF. DC were extracted from the tissues and enriched prior to flow cytometric analysis. The total DC level and the incidence of DC expressing lymphoid-related markers (CD8^hiCD11b^lo) and myeloid-related markers (CD8^loCD11b^hi) were monitored. Both in GM-CSF null mice, and GM-CSF receptor null mice, DC of all surface phenotypes were present in all lymphoid organs; only small decreases in DC levels were recorded, except for the lymph nodes of GM-CSF receptor null mice which showed a more pronounced (threefold) decrease in DC numbers. Since the GM-CSF receptor null mice lacked the β chain common to the GM-CSF, interleukin (IL)-3 and IL-5 receptors, the development of DC in the absence of GM-CSF was not due to common β chain mediated developmental signals elicited by IL-3 or IL-5. In GM-CSF transgenic mice, there was only a 50 % increase in DC numbers in thymus and spleen, paralleling an increase in overall cellularity, but a more pronounced (threefold) increase in DC numbers in lymph nodes. There was no evidence that GM-CSF had a selective effect on any particular DC subpopulation defined by CD8 or CD11b expression. We conclude that the development of most lymphoid tissue DC can proceed in the absence of GM-CSF, although this cytokine can produce some elevation of DC levels. It is not clear whether the enhancing effect of GM-CSF is direct or an indirect effect mediated by other cytokines.     

Immunophenotypical and functional heterogeneity of dendritic cells generated from murine bone marrow cultured with different cytokine combinations: implications for anti-tumoral cell therapy

Dendritic cells (DC) are professional antigen-presenting cells that can be used as immune adjuvant for anti-tumoural therapies. This approach requires the generation of large quantities of DC that are fully characterized on the immunophenotypical and functional levels. In a murine model, we analysed the in vitro effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) alone or combined with interleukin-4 (IL-4) or Flt3 ligand (Flt3-L) on the number, immunophenotype and functions of bone marrow-derived DC. In GM-CSF cultures, we have identified two populations based on their level of expression of major histocompatibility complex (MHC) class II molecules: MHC-IIhi cells, exhibiting the typical morphology and immunophenotype of myeloid DC (CD11c+ 33D1+ DEC-205+ F4/80+), and MHC-IIlo cells, heterogeneous for DC markers (30% CD11c+; 50% 33D1+; DEC-205-; F4/80+). The addition of Flt3-L to GM-CSF induced a twofold increase in MHC-IIhi DC number; besides, the MHC-IIlo cells lost all DC markers. In contrast, after addition of IL-4 to GM-CSF, the two populations displayed a very similar phenotype (CD11c+ 33D1- DEC-205+ F4/80-), differing only in their expression levels of MHC class II and costimulatory molecules, and showed similar stimulatory activity in mixed leucocyte reaction. We next analysed the migration of these cultured cells after fluorescent labelling. Twenty-four hours after injection into the footpads of mice, fluorescent cells were detected in the draining popliteal lymph nodes, with an enhanced migration when cells were cultured with GM-CSF+Flt3-L. Finally, we showed that MHC-IIhi were more efficient than MHC-IIlo cells in an anti-tumoral vaccination protocol. Altogether, our data highlight the importance of characterizing in vitro-generated DC before use in immunotherapy.     

Analysis of the CCR7 expression on murine bone marrow-derived and spleen dendritic cells

About 40% of bone marrow-derived dendritic cells (BM-DCs) generated from stem cells of C57BL/6 (B6.WT) mice differentiate in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF) without further stimuli to mature DCs. These cells are characterized by high levels of major histocompatibility complex class II, CD40, and CD86 on their surface. Recent studies have revealed that tumor necrosis factor (TNF) is crucial for maturation of BM-DCs. However, once matured, the phenotype of mature TNF-negative C57BL/6 (B6.TNF-/-) and B6.WT BM-DCs is comparable. Both expressed high levels of CD40 and CD86 and were positive for mRNA of the chemokine receptor (CCR)7. To extend our studies, we generated a monoclonal antibody (mAb) specific for mouse CCR7. This mAb allowed us to analyze the surface expression of CCR7 during maturation of B6.WT and B6.TNF-/- BM-DCs in the presence of GM-CSF and stimulated with TNF or lipopolysaccharide (LPS) and to compare it with the CCR7 expression on ex vivo-isolated splenic DCs with or without additional stimulation. Our results showed that CCR7 expression on murine BM-DCs is an indication of cell maturity. Incubation with LPS induced the maturation of all BM-DCs in culture but increased the number of mature CCR7+ splenic DCs only marginally.     

Flt-3 ligand (FL) drives differentiation of rat bone marrow-derived dendritic cells expressing OX62 and/or CD161 (NKR-P1)

Bone marrow-derived dendritic cells (DC) of the rat have not been as well characterized as those from the mouse. Here, large quantities of bone marrow-derived rat DC were generated when Flt-3 ligand (FL) was used as an adjunct to granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). These cells displayed a typical DC phenotype, expressing MHC class II, CD54, CD80, CD86, and CD11b/c. These DC also uniformly expressed low levels of CD161 and expressed OX62 in a bimodal distribution. Few cells were recovered from cultures grown without FL, and they failed to express OX62 or CD161. The DC generated with FL were more potent antigen-presenting cells in mixed lymphocyte cultures than cells grown without FL, and among FL-derived cells, the OX62+ cells were slightly more stimulatory than OX62- cells. Thus, FL is a useful cytokine for obtaining large quantities of functional rat DC subsets in vitro.     

MHC class I-mediated exogenous antigen presentation by exosomes secreted from immature and mature bone marrow derived dendritic cells

Exosomes are 50-90 nm vesicles with antigen presenting ability carrying major histocompatibility complex (MHC) class I, class II, abundant co-stimulatory molecules and some tetraspan proteins. Although dendritic cells (DCs) are one of the professional antigen presenting cells capable of presenting exogenous antigens in MHC class I-mediated antigen specific manner (cross-presentation), the cross-presentation ability by exosomes from immature or mature DCs are unknown. Here we show that exosomes released from ovalbumin (OVA) protein-pulsed bone marrow derived dendritic cells (BM-DCs) weakly present the peptide determinants to OVA specific MHC class I-restricted CD8(+) T cell hybridomas. The exosomes secreted by OVA(257-264) peptide- or OVA protein-pulsed mature BM-DCs activated OVA specific MHC class I-restricted T cell hybridomas more efficiently than those from immature BM-DCs. Transporters associated with antigen processing (TAP) deficient mice-derived BM-DCs were also used to examine whether functional TAP activity was required for cross-presentation by exosomes. The exosomes obtained from OVA(257-264) peptide-pulsed BM-DCs derived from TAP(-/-) mice showed a significant antigen presenting ability to OVA specific MHC class I-restricted T cell hybridomas. Altogether, our data indicate that BM-DCs secrete exosomes with weak cross-presentation ability.     

Interferon γ Stimulates Cellular Maturation of Dendritic Cell Line DC2.4 Leading to Induction of Efficient Cytotoxic T Cell Responses and Antitumor Immunity

Dendritic cells （DCs） are the most potent antigen-presenting cells （APCs） for the initiation of antigen （Ag）-specific immune responses. In most studies, mature DCs are generated from bone marrow cells or peripheral monocytes; in either case, the harvested cells are then cultured in medium containing recombinant GM-CSF, IL-4 and TNF-α for 7-10 days and stimulated with lipopolysaccharide （LPS）. However, this approach is time-consuming and expensive. There is another less cost approach of using immobilized DC cell lines, which can easily grow in the medium. A disadvantage with the immobilized DC cell lines, however, is that they are immature DCs and lack expression of MHC class Ⅱ and costimulatory CD40 and CD80 molecules. This, therefore, limits their capacity for inducing efficient antitumor immunity. In the current study, we investigated the possible efficacy of various stimuli （IL-1β, IFN-γ, TNF-α CpG and LPS） in converting the immature dendritic cell line DC2.4 to mature DCs. Our findings were quite interesting since we demonstrated for the first time that IFN-γ was able to stimulate the maturation of DC2.4 cells. The IFN-γ-activated ovalbumin （OVA）-pulsed DC2.4 cells have capacity to upregulate MHC class Ⅱ, CD40, CD80 and CCR7, and to more efficiently stimulate in vitro and in vivo OVA-specific CD8^＋ T cell responses and antitumor immunity. Therefore, IFN-γ-activated immortal DC2.4 ceils may prove to be useful in the study of DC biology and antitumor immunity.     

Phenotypic characterisation of intestinal dendritic cells in sheep

The present study was undertaken to identify dendritic cells (DCs) in the ileum and rectum of lambs and adult sheep. The distribution of these cells in four different intestinal compartments, i.e. lamina propria, lymphoid follicles, domes and interfollicular areas was assessed, and the presence of these cells in lambs and adult sheep was compared. Specimens were examined by using a number of potential DC markers (CD11c, CD205, MHC class II (MHCII), CD1b and CD209) in immunohistochemical and multicolour immunofluorescent procedures. The ovine ileal and rectal mucosa contain many CD11c+/CD205+ cells with a dendritic morphology, and the majority of these cells co-expressed MHCII. These double-positive cells were also labelled with the CD209 antibody in the lamina propria and interfollicular regions. Only very few cells expressed CD1b. In conclusion, a major DC population in ileum and rectum of sheep co-expressed the CD11c, CD205 and MHCII molecules. The CD209 antibody appeared to be a novel marker for a subpopulation of ovine intestinal DCs.     

Development of Dendritic Cells from GM-CSF-/- Mice in vitro : GM-CSF Enhances Production and Survival of Cells

The production of dendritic cells (DC) from haemopoietic progenitors maintained in long term stroma-dependent cultures (LTC) of spleen or bone marrow (BM) occurs independently of added granulocyte/macrophage colony stimulating factor (GM-CSF). The possibility that cultures depend on endogenous GM-CSF produced in low levels was tested by attempting to generate LTC from spleen and BM of GM-CSF^-/- mice. Multiple cultures from GM-CSF^-/- and wild type mice were established and compared for cell production. GM-CSF^-/- LTC developed more slowly, but by 16 weeks produced cells resembling DC in numbers comparable to wild type cultures. LTC maintained distinct populations of small and large cells, the latter resembling DC. Cells collected from GM-CSF^-/- LTC were capable antigen presenting cells (APC) for T cell stimulation and morphologically resembled DC. Large cells expressed the CD11b, CD11c, CD86, 33D1 and Dec-205 markers of DC. Addition of GM-CSF to GM-CSF^-/- LTC increased the proportion of large, mature DC present in culture. Stromal cells from GM-CSF^-/- LTC could support the differentiation of DC from early progenitors maintained in LTC without addition of GM-CSF. However, GM-CSF is not a critical factor in the in vitro generation of DC from progenitors. It can, however, substitute for stromal cells in increasing the survival of mature DC.     

Role of the cytokine environment and cytokine receptor expression on the generation of functionally distinct dendritic cells from human monocytes

Myeloid dendritic cells (DC) and macrophages evolve from a common precursor. However, factors controlling monocyte differentiation toward DC or macrophages are poorly defined. We report that the surface density of the GM-CSF receptor (GM-CSFR) alpha subunit in human peripheral blood monocytes varies among donors. Although no correlation was found between the extent of GM-CSFR and monocyte differentiation into DC driven by GM-CSF and IL-4, GM-CSFR expression strongly influenced the generation of CD1a(+) dendritic-like cells in the absence of IL-4. CD1a(+) cells generated in the presence of GM-CSF express CD40, CD80, MHC class I and II, DC-SIGN, MR, CCR5, and partially retain CD14 expression. Interestingly, they spontaneously induce the expansion of CD4(+) and CD8(+) allogeneic T lymphocytes producing IFN-gamma, and migrate toward CCL4 and CCL19. Upon stimulation with TLR ligands, they acquire the phenotypic features of mature DC. In contrast, the allostimulatory capacity is not further increased upon LPS activation. However, by blocking LPS-induced IL-10, a higher T cell proliferative response and IL-12 production were observed. Interestingly, IL-23 secretion was not affected by endogenous IL-10. These results highlight the importance of GM-CSFR expression in monocytes for cytokine-induced DC generation and point to GM-CSF as a direct player in the generation of functionally distinct DC.     

Phosphatidylinositol-3-kinase activity during in vitro dendritic cell generation determines suppressive or stimulatory capacity

Modulating PI3K at different stages of dendritic cells (DC) generation could be a novel means to balance the generation of immunosuppressive versus immunostimulatory DC. We show that PI3K inhibition during mouse DC generation in vitro results in cells that are potently immunosuppressive and characteristic of CD8alpha- CD11c+ CD11b+ DC. These DC exhibited low surface class I and class II MHC, CD40, and CD86 and did not produce TNF-alpha. In allogeneic MLR, these DC were suppressive. Although in these mixed cultures, there was no increase in the frequency of CD4+ CD25+ Foxp3+ cells, the Foxp3 content on a per cell basis was significantly increased. Sustained TLR9 signaling in the presence of PI3K inhibition during DC generation overrode the cells' suppressive phenotype.