Population Of CD11c Research Articles

Abstract 499: A Novel En Face Ex Vivo Live Imaging Approach For The Visualization Of Intimal Myeloid Cell Dynamics

Populations of CD11c + intimal myeloid cells (MCs) are known to reside in regions of the vasculature predisposed to atherosclerosis, such as the lesser curvature of the ascending arch. In Ldlr-/- mice, these cells have been shown to take up lipid and contribute to the formation of a nascent lesion under conditions of hypercholesterolemia. The role of resident CD11c+ cells in normal, healthy aortas has only recently been investigated. One function for these MCs may be the clearance of pathogens from the subendothelial space via reverse transendothelial migration (RTM). RTM can be triggered by systemic stimulation of cytokine and toll-like receptors (such as toll-like receptor 4 by lipopolysaccharide [LPS]), and induces a reduction in the number of resident intimal MCs in the lesser curvature. Our investigation of intimal RTM suggests that this cell loss is due to egress rather than cell death and is dependent on CCL19-CCR7 signaling, however we have yet to directly visualize this process. We endeavour to visualize resident intimal MC dynamics, including RTM, using ex vivo live imaging of the aortic intima. Ascending aortic arches were harvested from mice with fluorescent intimal myeloid cells (CD11c-eYFP mice or LyzM Cre -TdTomato reporter mice), stained with an endothelial marker (VE-Cadherin), and mounted en face in an imaging system that allows for constant perfusion of buffer across the tissue. Using a confocal microscope, images of the lesser curvature were continually acquired over a period of several hours following different stimuli to visualize cell behaviour. Intimal MCs were observed extending and retracting their dendrites under conditions of fluid flow, as well as undergoing cell division. Following the addition of the inflammatory stimulus LPS, cells can be seen migrating across and disappearing from the field of view. Thus, this method allows for direct visualization of intimal myeloid cell behaviour following perturbation by either physical or pharmacological stimuli. This approach, in tandem with pulsed perfusion of CD45-targeted antibodies, could provide a valuable toolkit to study immune cell egress from the intima of the lesser curvature of the aortic arch, under homeostasis and during the progression of atherosclerosis.

Investigating the role of BHLHE40 in host responses to Mycobacterium tuberculosisinfection and myeloid cell development.

Abstract We currently lack a complete understanding of the immune responses required to control Mycobacterium tuberculosis (Mtb) infection. Our lab has shown that the transcription factor BHLHE40 is essential during Mtb infection. Deletion of Bhlhe40 in CD11c +lung macrophages and dendritic cells (DCs) is sufficient to increase mouse susceptibility to Mtb infection, though the role of BHLHE40 in these cells during infection is unknown. Murine bone marrow cultured in the presence of GM-CSF, an abundant cytokine in the lung, develop into populations of CD11c +macrophages, DCs, and granulocytes, all of which express Bhlhe40. Using this system, we examined cytokine and transcriptional responses before and after exposure to heat-killed Mtb to understand how BHLHE40 impacts cell development and inflammatory responses. We found that in WT mice, GM-CSF-cultured cells develop into 9 distinct populations and expression of Bhlhe40 was required for GM-CSF-mediated pro-inflammatory cytokine and transcriptional programs. Additionally, loss of Bhlhe40 led to decreased DCs accompanied by upregulation of the transcription factor C/EBPβ in DC progenitor populations, which negatively impacts DC development. ChIP-seq analysis revealed BHLHE40 binds directly to C/EBPβ’s gene locus, indicating that BHLHE40 may directly repress Cebpb. In contrast, loss of Bhlhe40 did not affect macrophage development, but led to a transcriptional shift towards an anti-inflammatory state. These findings demonstrate that in the presence of GM-CSF, BHLHE40 is required for DC development and inflammatory macrophage polarization. Defects in either of these aspects could contribute to the essential role for BHLHE40 in lung macrophages and DCs during Mtb infection in vivo.

Bronchus-associated macrophages efficiently capture and present soluble inhaled antigens and are capable of local Th2 cell activation.

In allergic asthma, allergen inhalation leads to local Th2 cell activation and peribronchial inflammation. However, the mechanisms for local antigen capture and presentation remain unclear. By two-photon microscopy of the mouse lung, we established that soluble antigens in the bronchial airway lumen were efficiently captured and presented by a population of CD11c+ interstitial macrophages with high CX3CR1-GFP and MHC class II expression. We refer to these cells as Bronchus-Associated Macrophages (BAMs) based on their localization underneath the bronchial epithelium. BAMs were enriched in collagen-rich regions near some airway branchpoints, where inhaled antigens are likely to deposit. BAMs engaged in extended interactions with effector Th2 cells and promoted Th2 cytokine production. BAMs were also often in contact with dendritic cells (DCs). After exposure to inflammatory stimuli, DCs migrated to draining lymph nodes, whereas BAMs remained lung resident. We propose that BAMs act as local antigen presenting cells in the lung and also transfer antigen to DCs.

Clemastine Induces an Impairment in Developmental Myelination.

Abnormalities in myelination are associated to behavioral and cognitive dysfunction in neurodevelopmental psychiatric disorders. Thus, therapies to promote or accelerate myelination could potentially ameliorate symptoms in autism. Clemastine, a histamine H1 antagonist with anticholinergic properties against muscarinic M1 receptor, is the most promising drug with promyelinating properties. Clemastine penetrates the blood brain barrier efficiently and promotes remyelination in different animal models of neurodegeneration including multiple sclerosis, ischemia and Alzheimer’s disease. However, its role in myelination during development is unknown. We showed that clemastine treatment during development increased oligodendrocyte differentiation in both white and gray matter. However, despite the increase in the number of oligodendrocytes, conduction velocity of myelinated fibers of corpus callosum decreased in clemastine treated mice. Confocal and electron microscopy showed a reduction in the number of myelinated axons and nodes of Ranvier and a reduction of myelin thickness in corpus callosum. To understand the mechanisms leading to myelin formation impairment in the presence of an excess of myelinating oligodendrocytes, we focused on microglial cells that also express muscarinic M1 receptors. Importantly, the population of CD11c+ microglia cells, necessary for myelination, as well as the levels of insulin growth factor-1 decrease in clemastine-treated mice. Altogether, these data suggest that clemastine impact on myelin development is more complex than previously thought and could be dependent on microglia-oligodendrocyte crosstalk. Further studies are needed to clarify the role of microglia cells on developmental myelination.

Altered function and differentiation of age-associated B cells contribute to the female bias in lupus mice

Differences in immune responses to viruses and autoimmune diseases such as systemic lupus erythematosus (SLE) can show sexual dimorphism. Age-associated B cells (ABC) are a population of CD11c+T-bet+ B cells critical for antiviral responses and autoimmune disorders. Absence of DEF6 and SWAP-70, two homologous guanine exchange factors, in double-knock-out (DKO) mice leads to a lupus-like syndrome in females marked by accumulation of ABCs. Here we demonstrate that DKO ABCs show sex-specific differences in cell number, upregulation of an ISG signature, and further differentiation. DKO ABCs undergo oligoclonal expansion and differentiate into both CD11c+ and CD11c− effector B cell populations with pathogenic and pro-inflammatory function as demonstrated by BCR sequencing and fate-mapping experiments. Tlr7 duplication in DKO males overrides the sex-bias and further augments the dissemination and pathogenicity of ABCs, resulting in severe pulmonary inflammation and early mortality. Thus, sexual dimorphism shapes the expansion, function and differentiation of ABCs that accompanies TLR7-driven immunopathogenesis.

Novel biomarker panel based on cellular and soluble checkpoint proteins for PD-1/PD-L1 blockade treatment efficacy.

3045 Background: Although anti-PD-1/PD-L1 therapy has become one of the standard treatments for advanced cancers, its low treatment efficacy (10-30%) has remained a major issue. We sought to perform a detailed immune profiling of cells and soluble proteins in order to characterize key regulators and signaling molecules and identify therapeutic targets and biomarkers that may improve treatment efficacy and diagnosis. Methods: This observational study enrolled 49 advanced cancer patients treated with PD-1/PD-L1 blockade monotherapy. Treatment response was assessed by RECIST 1.1. PBMC and plasma samples were collected at baseline and every 6 weeks following initial treatment. Immune profiling of PBMC was done by multi-parametric flow cytometer, and t-SNE analysis was used to identify key immune subtypes. Soluble proteins were evaluated by LUMINEX assays. Cut-off values were determined by ROC curve analysis. Results: Three unique subtypes of immune cells were identified. The population of CD11c+HLA-DRlowCD80+CD86− CD274+ cells (regDC) at baseline was significantly higher in patients with progressive disease (PD, n=28) than in patients showing clinical benefit (non-PD, n=21; p=0.030). The higher regDC population also correlated with higher levels of IL-8, IL-10, CXCL1, CXCL5, and CXCL11 in plasma. The population of CD4+CD25+CD62L+ T cells (Treg) was also higher in PD patients (p<0.001). A unique subtype of CD4+CD28− T cell, however, was higher in non-PD patients (p<0.001). For the soluble proteins, the levels of sLAG-3 and sGITR in plasma correlated with better clinical outcome in low regDC patients (p=0.004 and 0.044, respectively). The combined biomarker panel (cellular and protein markers) yields high sensitivity (90.5 %) and specificity (82.1 %) for predicting treatment efficacy. Disease control rate (DCR) and median progression free survival (PFS) are shown in the Table. Conclusions: To our knowledge, this pilot study is the first to detect three immune cell subtypes, regDC, Treg and CD4+CD28− cells, associated with clinical outcome in the treatment of PD-1/PD-L1 blockade. Profiling of immune cell subtypes and soluble immune checkpoint proteins can serve to identify therapeutic targets and biomarkers for treatment efficacy. We will report the data with further enrollment. [Table: see text]

Identification of dendritic cell precursors in the culture of bone marrow with hematopoietic cytokines

Abstract Dendritic cells (DCs) play crucial parts in a variety of immune responses by capturing and presenting antigens to naïve T cells and by producing the subsequent T-cell activation or tolerance. In vitro culture systems of DCs have been widely used. The culture of bone marrow (BM) in the presence of a hematopoietic cytokine, Fms-like tyrosine kinase 3 ligand (FLT3L), produces plasmacytoid DCs (pDCs) and classical DCs (cDCs), whereas the culture of BM with granulocyte-macrophage colony-stimulating factor (GM-CSF) generates DCs and macrophages considered to possess inflammatory phenotypes. In the present study, we examined the culture of BM with both cytokines, i.e., FLT3L and GM-CSF in combination and identified 4 different subsets in CD11c⁺ MHCII+ cells. Meanwhile, in the culture of BM with FLT3L, we discovered a novel population of CD11c⁺ MHCIIhi cells that possess a limited capacity of antigen-presenting activity, suggesting precursors for DCs. This novel population of DC precursors from the BM culture with FLT3L was isolated and evaluated for their potential to differentiate to DCs in the BM culture containing both FLT3L and GM-CSF. Consequently, we demonstrated that the presence of a novel population of DC precursors in the in vitro culture system of DCs.

Cathepsin S Is Involved in Th17 Differentiation Through the Upregulation of IL-6 by Activating PAR-2 after Systemic Exposure to Lipopolysaccharide from Porphyromonas gingivalis.

Positive links have been found between periodontitis and numerous diseases in humans via persistent inflammation throughout the body. However, the main factors responsible for maintaining this pro-inflammatory condition are poorly understood. The spleen, the largest secondary immune organ, is a central hub regulating the immune response/inflammation due to the dendritic cell (DC) response to CD4+ T cell subtype differentiation, and lysosomal proteinase cathepsin S (CatS) is known to be involved in DC functions. In the present study, we found that CatS-induced IL-6 production by splenic DCs subsequently promotes Th17 differentiation, in response to systemic exposure to lipopolysaccharide derived from Porphyromonas gingivalis (PgLPS). The population of CD11c+ DCs was significantly increased in the splenic marginal zone (MZ) locally of wild-type (DBA/2) mice with splenomegaly but not in that of CatS deficient (CatS-/-) mice after systemic exposure to PgLPS for 7 consecutive days (5 mg/kg/day, intraperitoneal). Similarly, the population of Th17+CD4+ T cells was also significantly increased in the splenic MZ of wild-type mice but not in that of CatS-/- mice after PgLPS exposure. Furthermore, the increase in the Th17+ CD4+ T cell population paralleled increases in the levels of CatS and IL-6 in CD11c+ cells in the splenic MZ. In isolated primary splenic CD11c+ cells, the mRNA expression and the production of IL-6 was dramatically increased in wild-type mice but not in CatS-/- mice after direct stimulation with PgLPS (1 μg/ml), and this PgLPS-induced increase in the IL-6 expression was completely abolished by pre-treatment with Z-Phe-Leu-COCHO (Z-FL), the specific inhibitor of CatS. The PgLPS activated protease-activated receptor (PAR) 2 in the isolated splenic CD11c+ cells was also significantly inhibited by CatS deficiently. In addition, the PgLPS-induced increase in the IL-6 production by splenic CD11c+ cells was completely abolished by pre-treatment with FSLLRY-NH2, a PAR2 antagonist, as well as Akti, a specific inhibitor of Akt. These findings indicate that CatS plays a critical role in driving splenic DC-dependent Th17 differentiation through the upregulation of IL-6 by activating PAR2 after exposure to components of periodontal bacteria. Therefore, CatS-specific inhibitors may be effective in alleviating periodontitis-related immune/inflammation.

IgE-mediated enhancement of CD4(+) T cell responses requires antigen presentation by CD8α(-) conventional dendritic cells.

IgE, forming an immune complex with small proteins, can enhance the specific antibody and CD4+ T cell responses in vivo. The effects require the presence of CD23 (Fcε-receptor II)+ B cells, which capture IgE-complexed antigens (Ag) in the circulation and transport them to splenic B cell follicles. In addition, also CD11c+ cells, which do not express CD23, are required for IgE-mediated enhancement of T cell responses. This suggests that some type of dendritic cell obtains IgE-Ag complexes from B cells and presents antigenic peptides to T cells. To elucidate the nature of this dendritic cell, mice were immunized with ovalbumin (OVA)-specific IgE and OVA, and different populations of CD11c+ cells, obtained from the spleens four hours after immunization, were tested for their ability to present OVA. CD8α− conventional dendritic cells (cDCs) were much more efficient in inducing specific CD4+ T cell proliferation ex vivo than were CD8α+ cDCs or plasmacytoid dendritic cells. Thus, IgE-Ag complexes administered intravenously are rapidly transported to the spleen by recirculating B cells where they are delivered to CD8α− cDCs which induce proliferation of CD4+ T cells.

Quantitative and Qualitative Deficits in Neonatal Lung-Migratory Dendritic Cells Impact the Generation of the CD8+ T Cell Response

CD103+ and CD11b+ populations of CD11c+MHCIIhi murine dendritic cells (DCs) have been shown to carry antigens from the lung through the afferent lymphatics to mediastinal lymph nodes (MLN). We compared the responses of these two DC populations in neonatal and adult mice following intranasal infection with respiratory syncytial virus. The response in neonates was dominated by functionally-limited CD103+ DCs, while CD11b+ DCs were diminished in both number and function compared to adults. Infecting mice at intervals through the first three weeks of life revealed an evolution in DC phenotype and function during early life. Using TCR transgenic T cells with two different specificities to measure the ability of CD103+ DC to induce epitope-specific CD8+ T cell responses, we found that neonatal CD103+ DCs stimulate proliferation in a pattern distinct from adult CD103+ DCs. Blocking CD28-mediated costimulatory signals during adult infection demonstrated that signals from this costimulatory pathway influence the hierarchy of the CD8+ T cell response to RSV, suggesting that limited costimulation provided by neonatal CD103+ DCs is one mechanism whereby neonates generate a distinct CD8+ T cell response from that of adults.

CpG vaccination induces cytolytic T cells to the lung and heterosubtypic protection against lethal IAV infection (P4289)

Abstract With the emergence of pandemic influenza A virus (IAV) strains such as H1N1 and H5N1, the development of a universal vaccine has become more necessary than ever. In contrast to the highly mutable outer coat proteins, internal proteins of IAV are more conserved among different strains and are therefore a target for developing vaccines that induce strong T cell responses. Using a Toll-like receptor 9 agonist with unmethylated CpG motifs, we have shown that intranasal immunization with CpG and inactivated x31 (H3N2) IAV protects mice following a heterosubtypic challenge with a lethal dose of PR8 virus (H1N1). Vaccination with CpG and inactivated IAV cause a stronger inflammatory response than IAV infection alone, with significant levels of antiviral cytokines such as MIP-1β, CXCL 10, and IL-6 produced in the lungs. Furthermore, CpG immunization induces a substantial influx of CD11b+ cells into the draining lymph nodes as well as a population of CD11c+ cells that express high levels of the costimulatory molecule CD86, providing favorable conditions for T cell activation. At 6 days post immunization, effector CD4 and CD8 T cells expressing Granzyme B and IFN-γ are observed in the lungs of vaccinated mice. These experiments suggest that TLR agonists can act as potent immune adjuvants to generate robust T cell immunity capable of combating IAV and other viral infections where successful vaccines have not yet been developed.

Development of Two Distinct Dendritic-Like APCs in the Context of Splenic Stroma

Murine splenic stroma has been found to provide an in vitro niche for hematopoiesis of dendritic-like APC. Two distinct cell types have been characterized. The novel “L-DC” subset has cross-presenting capacity, leading to activation of CD8+ T cells, but not activating CD4+ T cells, which is consistent with their CD11cloCD11bhiMHC-II− phenotype. For L-DC, an equivalent tissue-specific APC has been found only in spleen. A second population of CD11chiCD11bloMHC-II+ cells resembling conventional dendritic cells (cDC) can activate both CD4 and CD8 T cells. Production of L-DC but not cDC-like cells is now shown to be dependent on contact between the L-DC progenitor and stroma such that the presence of a Transwell membrane can prevent L-DC development. Since L-DC can be produced continuously in vitro in stromal co-cultures overlaid with bone marrow (BM) progenitors, it was hypothesized that L-DC progenitors are self-renewing. The L-DC progenitor is shown here to be defined by the Flt3−c-kit+Lin−Sca-1+ (F−KLS) subset of adult BM which contains primitive HSC. Since the less primitive F+KLS HSC subset also contains L-DC progenitors, Flt3 does not appear to be a defining marker for this progenitor. Precursors of the cDC-like subset are found only within the F+KLS subset and seed production of a transient population of APC. All data identify differentiation of L-DC from HSC, and of cDC-like cells from DC precursors, which occurs independently of inflammatory signals and is dependent on a splenic stromal microenvironment.

Intranasal vaccination with messenger RNA as a new approach in gene therapy: Use against tuberculosis

BackgroundmRNAs are highly versatile, non-toxic molecules that are easy to produce and store, which can allow transient protein expression in all cell types. The safety aspects of mRNA-based treatments in gene therapy make this molecule one of the most promising active components of therapeutic or prophylactic methods. The use of mRNA as strategy for the stimulation of the immune system has been used mainly in current strategies for the cancer treatment but until now no one tested this molecule as vaccine for infectious disease.ResultsWe produce messenger RNA of Hsp65 protein from Mycobacterium leprae and show that vaccination of mice with a single dose of 10 μg of naked mRNA-Hsp65 through intranasal route was able to induce protection against subsequent challenge with virulent strain of Mycobacterium tuberculosis. Moreover it was shown that this immunization was associated with specific production of IL-10 and TNF-alpha in spleen. In order to determine if antigen presenting cells (APCs) present in the lung are capable of capture the mRNA, labeled mRNA-Hsp65 was administered by intranasal route and lung APCs were analyzed by flow cytometry. These experiments showed that after 30 minutes until 8 hours the populations of CD11c+, CD11b+ and CD19+ cells were able to capture the mRNA. We also demonstrated in vitro that mRNA-Hsp65 leads nitric oxide (NO) production through Toll-like receptor 7 (TLR7).ConclusionsTaken together, our results showed a novel and efficient strategy to control experimental tuberculosis, besides opening novel perspectives for the use of mRNA in vaccines against infectious diseases and clarifying the mechanisms involved in the disease protection we noticed as well.

The origin and development of nonlymphoid tissue CD103+ DCs

CD103+ dendritic cells (DCs) in nonlymphoid tissues are specialized in the cross-presentation of cell-associated antigens. However, little is known about the mechanisms that regulate the development of these cells. We show that two populations of CD11c+MHCII+ cells separated on the basis of CD103 and CD11b expression coexist in most nonlymphoid tissues with the exception of the lamina propria. CD103+ DCs are related to lymphoid organ CD8+ DCs in that they are derived exclusively from pre-DCs under the control of fms-like tyrosine kinase 3 (Flt3) ligand, inhibitor of DNA protein 2 (Id2), and IFN regulatory protein 8 (IRF8). In contrast, lamina propria CD103+ DCs express CD11b and develop independently of Id2 and IRF8. The other population of CD11c+MHCII+ cells in tissues, which is CD103−CD11b+, is heterogenous and depends on both Flt3 and MCSF-R. Our results reveal that nonlymphoid tissue CD103+ DCs and lymphoid organ CD8+ DCs derive from the same precursor and follow a related differentiation program.

Fms-like tyrosine kinase 3 ligand increases a lung DC subset with regulatory properties in allergic airway inflammation

Dendritic cell (DC) subsets display different functional roles in regulating immune responses and lead to various outcomes, including T(H)1 versus T(H)2 or regulatory versus immunologic responses. Administration of Fms-like tyrosine kinase 3 (Flt3) ligand prevents and reverses allergic airway inflammation and airway hyperresponsiveness in a mouse model. However, the underlying mechanisms are unclear. We characterized and examined the role of lung DC subsets in the therapeutic effect of Flt3 ligand. DCs were isolated from the lungs of ovalbumin (OVA)-sensitized and OVA-challenged mice treated with recombinant human Flt3 ligand. Two populations of CD11c+ cells labeled with fluorochrome-conjugated antibodies were sorted. The ability of the purified cells to stimulate T-cell proliferation and cytokine secretion patterns by different DC subsets was examined. Also, DCs were adoptively transferred in mice to examine their effect on pulmonary function. Two DC populations, CD11c(high)CD11b(low) and CD11c(low)CD11b(high), were identified in the lungs of naive and OVA-sensitized and OVA-challenged mice with and without treatment with Flt3 ligand. The expression levels of CD8alpha, B220, CD19, F4/80, MHC II, CCR7, CD40, programmed death ligand 1, programmed death ligand 2, CD80, and CD86 were distinctly different between the 2 DC populations, which supports the notion that CD11c(high)CD11b(low) and CD11c(low)CD11b(high) DCs potentially have regulatory and immunogenic properties, respectively. Administration of Flt3 ligand increased the DCs with regulatory potential in the lungs of antigen-sensitized mice, and CD11c(high)CD11b(low) DCs acquired a maximum degree of regulatory capacity after Flt3 ligand treatment. These data suggest that Flt3 ligand reverses airway hyperresponsiveness by regulating the function of lung DCs in a mouse model of allergic airway inflammation.

Effects of CpG- oligodeoxynucleotides on dendritic cell development

Dendritic cell (DC) development begins in the bone marrow and immature progenitors reach their sites of residence in lymphoid organs. The mechanism of DC development in the bone marrow and in peripheral lymphoid organs is poorly understood. Here, we examined the effects of synthetic oligodeoxynucleotides containing a CpG motif (CpG-ODNs) on the development of DC from the bone marrow cells. Approximately 15% of bone marrow cells expressed CD11c surface antigen in the in vitro culture for 8 days in the presence of IL-3. However, the addition of a phosphorothioate-modified CpG-ODN (PTO-CpG-ODN), but not a phosphodiester-modified ODN (PO-CpG-ODN), suppressed the expression of CD11c surface antigen. Also, we examined the effects of CpG-ODNs on the maintenance of DCs resident in the gastrointestinal lymphoid tissues, where immature progenitors may be challenged by a variety of ODN derived from microflora. The population of CD11c(+)B220(int)Gr-1(low) cells decreased by the addition of PTO-CpG-ODN when the lymphocytes from mesenteric lymph nodes as well as spleen are cultured for 2 days in the presence of GM-CSF. In contrast, this population increased in the case of the lymphocytes from Peyer's patches. It thus follows that PTO-CpG-ODN plays a regulatory role in the differentiation of bone marrow cells into DCs and in the functional maintenance of DCs at peripheral lymphoid organs.

Supplementation of CXCL12 (CXCL12) induces homing of CD11c+ dendritic cells to the spleen and enhances control of Plasmodium berghei malaria in BALB/c mice

In malaria, parasitaemia is controlled in the spleen, a multicomponent organ that undergoes changes in its cellular constituents to control the parasite. During this process, dendritic cells (DCs) orchestrate the positioning of effector cells in a timely manner for optimal parasite clearance. We have recently demonstrated that CXCL12 [stromal cell-derived factor-1 (CXCL12)] supplementation partially restores the ability to control parasitaemia in Plasmodium berghei-infected mice. In the present study, we investigated the nature of the DCs involved by flow cytometry and immunohistochemistry of CD11c(+) cells. Flow cytometry of bone marrow cells showed that infection with P. berghei did not alter the proportion of CD11c(+) cells present in this haematopoietic compartment, while CXCL12 supplementation of naïve uninfected mice induced only minor increases in the population of CD11c(+) cells. In the spleen, P. berghei infection alone resulted in an increase in CD11c(+) cells as compared with naïve animals. Exogenously administered CXCL12 in the absence of infection resulted in a significant expansion of the splenic CD11c(+) population, and this effect was even more pronounced in infected and supplemented mice. Immunohistochemistry revealed that CD11c(+) cells infiltrated the perivascular areas and marginal zone of the spleen in infected animals treated with CXCL12, suggesting that this chemokine induces homing of CD11c(+) dendritic cells to the splenic compartment. Our results show that small amounts of CXCL12 supplementation are effective in recruiting DCs to the spleens of both uninfected and infected mice, suggesting the participation of CXCL12 and CD11c(+) cells in the establishment of an adequate environment in the spleen for malaria control.

GM-CSF expands dendritic cells and their progenitors in mouse liver

Dendritic cells (DCs) are rare but ubiquitous antigen-presenting cells situated in lymphoid and nonlymphoid organs throughout the body. The study of DCs located in the liver has been restricted by their relative scarcity and the difficulty of their isolation. Because granulocyte-macrophage colony–stimulating factor (GM-CSF) is a critical growth factor for DCs in vitro, we postulated that it would expand hepatic DCs in vivo. We found that adenoviral-mediated GM-CSF overexpression in normal mice increased the number of liver DCs 400-fold to more than 100 million cells. GM-CSF–recruited DCs were CD11c +DEC205 − and had high expression of major histocompatibility complex (MHC) class II, CD54, and CD80 but low CD40 and CD86 staining. Further maturation occurred after overnight culture. In addition to CD11c +DEC205 − DCs, a population of CD11c −DEC205 low/− cells resembling DC progenitors described previously in normal mice was expanded as serum GM-CSF levels increased. GM-CSF–recruited CD11c +DEC205 − DCs and CD11c −DEC205 low/− cells had different functional capabilities. CD11c +DEC205 − DCs captured far more protein antigen in vivo, produced higher amounts of interleukin (IL)-6 and tumor necrosis factor (TNF)-α, and induced greater allogeneic and antigen-specific T-cell stimulation. A proportion of CD11c −DEC205 low/− cells differentiated into CD11c + cells and gained T-cell stimulatory ability when cultured in the presence of GM-CSF. In conclusion, our findings show that GM-CSF can profoundly influence recruitment and development of DCs in murine liver. (H EPATOLOGY 2003;37:641-652.)