Classical Dendritic Cells Research Articles

CD103+ CD11b- salivary gland dendritic cells have antigen cross-presenting capacity.

Dendritic cells (DCs) in lymphoid and non-lymphoid tissues are professional antigen-presenting cells that are essential for effective immunity and tolerance. However, the presence and characteristics of DCs in steady-state salivary glands (SGs) currently remain unknown. We herein identified CD64- CD11c+ classical DCs (cDCs) as well as CD64+ macrophages among CD45+ MHC class II+ antigen-presenting cells in steady-state murine SGs. SG cDCs were divided into CD103+ CD11b- and CD103- CD11b+ cDCs. CD103+ CD11b- cDCs expressed XCR1, CLEC9A, and interferon regulatory factor 8, whereas CD103- CD11b+ cDCs strongly expressed CD172a. Both cDC subsets in SGs markedly expanded in response to the Flt3 ligand (Flt3L), were replenished by bone marrow-derived precursors, and differentiated from common DC precursors, but not monocytes. Furthermore, ovalbumin-pulsed SG CD103+ CD11b- cDCs induced the proliferation of naïve ovalbumin-specific CD8+ T cells and production of interferon-γ from proliferating T cells. SG CD103+ CD11b- cDCs expanded by Flt3L in vivo exhibited the same properties. These results indicate that bona fide cDCs reside in steady-state murine SGs and cDCs with the CD103+ CD11b- phenotype possess antigen cross-presenting capacity. Moreover, Flt3L enhances protective immunity by expanding cDCs. Taken together, SG cDCs might play an important role in maintaining immune homeostasis in the tissues.

Transcription factor Zeb2 regulates commitment to plasmacytoid dendritic cell and monocyte fate

Dendritic cells (DCs) and monocytes develop from a series of bone-marrow-resident progenitors in which lineage potential is regulated by distinct transcription factors. Zeb2 is an E-box-binding protein associated with epithelial-mesenchymal transition and is widely expressed among hematopoietic lineages. Previously, we observed that Zeb2 expression is differentially regulated in progenitors committed to classical DC (cDC) subsets in vivo. Using systems for inducible gene deletion, we uncover a requirement for Zeb2 in the development of Ly-6Chi monocytes but not neutrophils, and we show a corresponding requirement for Zeb2 in expression of the M-CSF receptor in the bone marrow. In addition, we confirm a requirement for Zeb2 in development of plasmacytoid DCs but find that Zeb2 is not required for cDC2 development. Instead, Zeb2 may act to repress cDC1 progenitor specification in the context of inflammatory signals.

BRAFV600E Transduced Human CD34+ Cells Establish Aggressive Langerhans Cell Histiocytosis in Humanized Mice

IntroductionLangerhans cell histiocytosis (LCH) is the most common histiocytic disease in humans with a complex pathophysiology and unknown etiology. LCH has been recently reclassified from an inflammatory disease to a neoplastic disorder due to the high prevalence of mutations in the MAP kinase pathway. Recurrent mutation in BRAF kinase (BRAFV600E) was detected in more than 50% of patients. We recently showed that BRAFV600E mutation can be detected in the circulating CD34+ cells in LCH patients and that the expression of the mutation in distinct hematopoietic compartments defines the stage and the clinical risk in LCH disease (Berres et al. J Exp Med 2014). Knowledge on induction and progression of LCH is limited due to appropriate models resembling the human disease. We recently generated so called MISTRG mice where the mouse cytokines M-CSF, IL-3, GM-CSF, and thrombopoietin were replaced by their human variants and that in addition express a human transgene of SIRPa (Rongvaux et al. Nat Biotech 2014). These mice efficiently support human myelopoiesis from human hematopoietic stem and progenitor cells (HSPCs). We here tested if xeno-transplantation of BRAFV600E transduced human CD34+ cells in these mice would result in LCH.Materials and MethodsHuman CD34+ HSPCs obtained from healthy donor cord blood samples were lentivirally transduced using a bidirectional promoter vector harboring BRAFV600E and GFP (as a marker for transduction efficiency) in the presence of supportive growth factors. As control, BRAFV600E construct was substituted by luciferase gene. The In vivo experiments were performed by injection of transduced CD34+ into newborn (n=10, of which 5 control and BRAFV600E respectively) and adult (n=8, of which 4 control and BRAFV600E respectively) MISTRG mice, which were sublethally irradiated prior to transplantation. Mice were sacrificed once symptoms of sickness were observed and analyzed using flow cytometry and immunohistochemistry staining for investigating the development of LCH-like disease. The influence of BRAFV600E mutation on proliferation and lineage commitment of human HSPCs was investigated using colony forming unit (CFU) assay and short term liquid cultures. For CFU assays, the transduced human CD34+ cells were plated in M4230 medium in the presence of supportive growth factors. After 14-16 days the obtained colonies were scored microscopically and measured by flow cytometry for further characterization of the cells. For short term liquid cultures, transduced cells were kept in culture in the presence of supportive growth factors and measured by flow cytometry for expression of human myeloid and lymphoid markers.ResultsMice transplanted with human CD34+ HSPCs expressing BRAFV600E developed anemia and reduced WBC and RBC counts. Analysis of lymphoid and non-lymphoid organs demonstrated the accumulation of ''atypical'' BRAFV600E expressing dendritic cells in granuloma-like lesions. Using immunohistochemistry staining we observed the expression of LCH-associated markers, CD1a and CD207 to a varying extent in different organs (Figure 1). We did not observe development of hairy cell leukemia. The in vitro studies revealed that the expression of BRAFV600E blocks the commitment of erythroid progeny toward mature erythrocytes shown by lower number of BFU-E colony type and reduced CD235ahigh-expressing cells. BRAFV600E seemed to increase monocytosis and generation of classical dendritic cells in short term liquid cultures, without affecting the proliferation potential of human HSPCs.Conclusions and outlookWe demonstrate that expression of BRAFV600E in human HSPCs induces LCH-like disease in MISTRG mice. The involvement of wide range of organs resembles high-risk LCH in humans and confirms our already reported data with aggressive LCH being a HSPC disease. Using this model system will further support basic understanding of LCH patho-biology and testing of targeted therapies in LCH. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

RAB43 facilitates cross-presentation of cell-associated antigens by CD8α+ dendritic cells.

In this study, to examine cross-presentation by classical dendritic cells (DCs; cDCs), we evaluated the role of RAB43, a protein found to be selectively expressed by Batf3-dependent CD8α+ and CD103+ compared with other DC subsets and immune lineages. Using a specific monoclonal antibody, we localized RAB43 expression to the Golgi apparatus and LAMP1- cytoplasmic vesicles. Mice with germline or conditional deletion of Rab43 are viable and fertile and have normal development of cDCs but show a defect for in vivo and in vitro cross-presentation of cell-associated antigen. This defect is specific to cDCs, as Rab43-deficient monocyte-derived DCs showed no defect in cross-presentation of cell-associated antigen. These results suggest that RAB43 provides a specialized activity used in cross-presentation selectively by CD8α+ DCs but not other antigen-presenting cells.

Mafb lineage tracing to distinguish macrophages from other immune lineages reveals dual identity of Langerhans cells.

Current systems for conditional gene deletion within mouse macrophage lineages are limited by ectopic activity or low efficiency. In this study, we generated a Mafb-driven Cre strain to determine whether any dendritic cells (DCs) identified by Zbtb46-GFP expression originate from a Mafb-expressing population. Lineage tracing distinguished macrophages from classical DCs, neutrophils, and B cells in all organs examined. At steady state, Langerhans cells (LCs) were lineage traced but also expressed Zbtb46-GFP, a phenotype not observed in any other population. After exposure to house dust mite antigen, Zbtb46-negative CD64+ inflammatory cells infiltrating the lung were substantially lineage traced, but Zbtb46-positive CD64- cells were not. These results provide new evidence for the unique identity of LCs and challenge the notion that some inflammatory cells are a population of monocyte-derived DCs.

In Vivo Gene Delivery to Lymph Node Stromal Cells Leads to Transgene-specific CD8+ T Cell Anergy in Mice.

Lymph node stromal cells play a role in self-tolerance by presenting tissue antigens to T cells. Yet, immunomodulatory properties of lymphoid tissue stroma, particularly toward CD4+ T cells, remain insufficiently characterized by lack of tools to target antigens for presentation by stromal cells. A lentiviral vector was therefore designed for antigen delivery to MHC class II+ cells of nonhematopoietic origin. Following intravenous vector delivery, the transgene was detected in lymph node gp38+ stromal cells which were CD45- MHCII+ and partly positive for CD86 and CTLA4 or B7-H4. The transgene was not detected in classical dendritic cells of lymph nodes or spleen. Transgene-specific CD4+ and CD8+ T cell responses were primed and regulatory T cells were also induced but effector T cell response did not develop, even after a peptide boost. Antigen-specific CD8+ T cells were not cytolytic in vivo. Thus, expressing a neo-antigen in MHC-II+ lymph node stroma seems to trigger blunt CD4 T cell responses leading to antigen-specific CD8+ T cell anergy. These results open up new perspectives to further characterize lymph node stromal cell functional properties and to develop gene transfer protocols targeting lymph node stroma to induce peripheral tolerance.

Classical dendritic cells mediate fibrosis directly via the retinoic acid pathway in severe eye allergy.

Fibrosis is a shared end-stage pathway to lung, liver, and heart failure. In the ocular mucosa (conjunctiva), fibrosis leads to blindness in trachoma, pemphigoid, and allergy. The indirect fibrogenic role of DCs via T cell activation and inflammatory cell recruitment is well documented. However, here we demonstrate that DCs can directly induce fibrosis. In the mouse model of allergic eye disease (AED), classical CD11b+ DCs in the ocular mucosa showed increased activity of aldehyde dehydrogenase (ALDH), the enzyme required for retinoic acid synthesis. In vitro, CD11b+ DC-derived ALDH was associated with 9-cis-retinoic acid ligation to retinoid x receptor (RXR), which induced conjunctival fibroblast activation. In vivo, stimulating RXR led to rapid onset of ocular mucosal fibrosis, whereas inhibiting ALDH activity in DCs or selectively depleting DCs markedly reduced fibrosis. Collectively, these data reveal a profibrotic ALDH-dependent pathway by DCs and uncover a role for DC retinoid metabolism.

Abstract LB-089: Imprime PGG, a β-glucan PAMP (pathogen-associated molecular pattern), effectively elicits in vivo maturation of antigen presenting cells in mice and humans, suggesting potential synergy with checkpoint inhibitor therapy

Abstract Recognition of PAMPs via pattern recognition receptors is central to immune recognition of foreign threats and to the generation of a coordinated innate and adaptive immune response. Cancers lack PAMPs and are poorly immunogenic. Consequently, the immune system often fails to mount an effective, coordinated anti-cancer immune attack. Though immunotherapies (e.g. checkpoint inhibitors) are effective in some cancer patients, the majority of patients fail to achieve substantial therapeutic benefit. To fully realize the potential of immune checkpoint inhibitors, there is substantial interest in developing therapeutically-viable PAMPs capable of enabling the maturation and function of professional antigen presenting cells (e.g. dendritic cells, DCs). Bacterial and viral PAMPs (i.e. TLR and STING agonists) can elicit DC maturation but are poorly tolerated systemically and are thereby limited to intra-tumoral delivery. The soluble yeast β-1,3/1,6 glucan, Imprime PGG (Imprime), is a PAMP that has been successfully administered intravenously (IV), is well-tolerated and shows promising efficacy in a series of clinical trials in > 400 total subjects. We sought to determine whether Imprime could drive maturation and enhanced function of antigen presenting cells in vivo. We now show that Imprime binds in vivo to various dendritic cell (DC) subsets in both human and mouse. In mice dosed IV, Imprime also elicits DC maturation as indicated by CD86 upregulation and successfully induces a type I interferon transcriptional profile. In C57Bl/6 mice immunized with the OVA257-264 model antigen, Imprime treatment elicits the specific expansion of adoptively transferred OT-I CD8 T cells (transgenic T cells engineered to recognize the OVA antigen). These OT-I T cells are functional effector cells, showing enhanced degranulation and increased capacity to produce IFN-γ and IL-2 when compared to OT-I cells isolated from mice challenged with OVA peptide alone. In ex vivo human whole blood, Imprime also enables DC maturation (enhanced expression of CD86, CD83, MHC class II), T cell expansion and production of the potent anti-tumor cytokine IFN-γ. Importantly, we now show preliminary data that peripheral blood monocytes and classical DCs from Imprime-treated cancer patients show elevated CD86 expression. Collectively, these data show that Imprime, a novel, systemically-administered, well-tolerated PAMP, can effectively elicit the maturation and function of antigen presenting cells in vivo and may thereby enhance T cell priming and anti-tumor immune response elicited by checkpoint inhibitors. Citation Format: Ross B. Fulton, Steven M. Leonardo, Adria B. Jonas, Kathryn A. Fraser, Anissa S.H. Chan, Nadine R. Ottoson, Michael E. Danielson, Nandita Bose, Jeremy R. Graff, Keith Gorden. Imprime PGG, a β-glucan PAMP (pathogen-associated molecular pattern), effectively elicits in vivo maturation of antigen presenting cells in mice and humans, suggesting potential synergy with checkpoint inhibitor therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-089.

Allergen-induced Changes in Bone Marrow and Airway Dendritic Cells in Subjects with Asthma.

Dendritic cells (DCs) are antigen-presenting cells essential for the initiation of T-cell responses. Allergen inhalation increases the number of airway DCs and the release of epithelial-derived cytokines, such as IL-33 and thymic stromal lymphopoietin (TSLP), that activate DCs. To examine the effects of inhaled allergen on bone marrow production of DCs and their trafficking into the airways in subjects with allergic asthma, and to examine IL-33 and TSPL receptor expression on DCs. Bone marrow, peripheral blood, bronchoalveolar lavage (BAL), and bronchial biopsies were obtained before and after inhalation of diluent and allergen from subjects with asthma that develop allergen-induced dual responses. Classical DCs (cDCs) were cultured from bone marrow CD34(+) cells. cDC1s, cDC2s, and plasmacytoid DCs were measured in bone marrow aspirates, peripheral blood, and BAL by flow cytometry, and cDCs were quantified in bronchial biopsies by immunofluorescence staining. Inhaled allergen increased the number of cDCs grown from bone marrow progenitors, and cDCs and plasmacytoid DCs in bone marrow aspirates 24 hours after allergen. Allergen also increased the expression of the TSLP receptor, but not the IL-33 receptor, on bone marrow DCs. Finally, inhaled allergen increased the percentage of cDC1s and cDC2s in BAL but only cDC2s in bronchial tissues. Inhaled allergen increases DCs in bone marrow and trafficking of DCs into the airway, which is associated with the development airway inflammation in subjects with allergic asthma. Inhaled allergen challenge also increases expression of TSLP, but not IL-33, receptors on bone marrow DCs.

Abstract 4534: Taxol therapy-induced catalytic EZH2 limits therapeutic responses in PTEN null prostate cancer

Abstract Prostate cancer (PCa) is the most common cancer and a major cause of cancer related deaths in men in North America. The phosphatase and tensin homolog gene (PTEN) is frequently inactivated in PCa. Approximately 40% of primary and 70% of metastatic PCa's have genomic alterations in the PI3K-signaling, mostly through the loss of PTEN. Majority of metastatic PCa associated with PTEN loss are treated with docetaxel; an FDA approved therapy for recurrent PCa. However, in PTEN null PCa docetaxel treatment enhances senescence and does not significantly impact tumor volume. These findings were further confirmed in PCa patients with decreased levels of PTEN. In order to understand the global impact of docetaxel in PTEN null PCa we performed an unbiased RNA-seq of prostate tissues harvested from docetaxel treated prostate specific PTEN null mice. Data analysis showed strong enrichment of chromatin modifying enzymes reactome, mainly PRC2 as one of the top ranked categories. Among them EZH2, an oncogenic methyltransferase was found to be significantly upregulated. Quantitative PCR analysis confirmed statistically significant upregulation of EZH2 in docetaxel treated mice compared to vehicle control. EZH2 upregulation was independent of androgen levels as both androgen dependent and independent cells showed striking upregulation of EZH2 when treated with docetaxel. Next, we confirmed the nature of the upregulated EZH2 (catalytic or non-catalytic) by docetaxel. EZH2 methyltransferase activity assay data showed significant induction of methyltransferase activity by docetaxel in both androgen dependent and independent conditions and in PTEN null mouse tissues. Further, we measured other methyltransferase marks i.e. H3K27me1&2, H3K4me3, H3K9me3 in the presence of docetaxel and found only specific upregulation of H3K27me3, confirming upregulation of only catalytic EZH2 by docetaxel. These data suggested a need for co-targeting EZH2 during docetaxel treatment in PTEN null PCa conditions. In order to provide a proof-of-principle we performed a tumor xenograft study in nude mice implanted with PTEN null PC3 cells. Combination of DZNeP (an EZH2 inhibitor) and docetaxel significantly inhibited tumor growth compared to each agent alone. Results obtained from the xenograft studies prompted us to perform a preclinical study in prostate specific PTEN null mice. Data obtained from this study clearly showed a significant reduction in number of high grade lesions in PTEN null tumors in combination group when compared to either docetaxel or DZNeP treated mice. We also observed clearance of senescent cells and accumulation of CD8+ T cells and classical dendritic (CD8+ Clec9a+ positive) cells. Our data suggests the necessity of designing a combination therapy of targeting EZH2 during docetaxel treatment specifically, which will likely increase the efficacy of docetaxel in PTEN null PCa. Citation Format: Mohammad Imran Khan, Abid Hamid, Vaqar Mustafa Adhami, Chloe Lang, Hasan Mukhtar. Taxol therapy-induced catalytic EZH2 limits therapeutic responses in PTEN null prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4534.

Targeting CD1c-expressing classical dendritic cells to prevent thymus and activation-regulated chemokine (TARC)-mediated T-cell chemotaxis in rheumatoid arthritis

Objectives: Thymus and activation-regulated chemokine (TARC) attracts cells that express the C-C chemokine receptor type 4 (CCR4), including CD4 T cells. As expression of CCR4 is increased on peripheral T cells and intra-articular interleukin (IL)-17-producing cells in patients with rheumatoid arthritis (RA), we investigated whether TARC plays a role in the attraction of T cells to the synovial compartment. In addition, we assessed the role of classical dendritic cells (cDCs) in the production of TARC in RA.Method: TARC was measured in synovial fluid (SF) samples from RA and osteoarthritis (OA) patients. Spontaneous and thymic stromal lymphopoietin (TSLP)-induced TARC production by mononuclear cells (MCs) and CD1c cDCs from peripheral blood (PB) and SF was assessed. The role of TARC in CD4 T-cell migration towards cDCs was assessed and the contribution of CD1c-expressing cells to TARC production was studied.Results: TARC concentrations were higher in SF of RA patients compared to OA patients. MCs from SF produced TARC spontaneously and produced more TARC upon stimulation than paired PBMCs. Blocking TARC strongly inhibited CD4 T-cell chemotaxis by TSLP-stimulated cDCs, associated with decreased production of tumour necrosis factor (TNF)-α. Depletion of cDCs from SFMCs strongly reduced TARC production.Conclusions: TARC levels are increased in RA SF and our data indicate that this results from production by SFMCs and in particular CD1c cDCs. TARC attracts T cells and TARC secretion by MCs is crucially dependent on the presence of CD1c cDCs. Considering the potential of SF cDCs to activate T cells and induce pro-inflammatory cytokine secretion, targeting intra-articular cDCs constitutes a novel therapeutic approach in RA.

Distinct Transcriptional Programs Control Cross-Priming in Classical and Monocyte-Derived Dendritic Cells.

Both classical DCs (cDCs) and monocyte-derived DCs (Mo-DCs) are capable of cross-priming CD8(+) Tcells in response to cell-associated antigens. Wefound that Ly-6C(hi)TREML4(-) monocytes can differentiate into Zbtb46(+) Mo-DCs in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) but that Ly-6C(hi)TREML4(+) monocytes were committed to differentiate into Ly-6C(lo)TREML4(+) monocytes. Differentiation of Zbtb46(+) Mo-DCs capable of efficient cross-priming required both GM-CSF and IL-4 and was accompanied by the induction of Batf3 and Irf4. However, monocytes require IRF4, but not BATF3, to differentiate into Zbtb46(+) Mo-DCs capable of cross-priming CD8(+) Tcells. Instead, Irf4(-/-) monocytes differentiate into macrophages in response to GM-CSF and IL-4. Thus, cDCs and Mo-DCs require distinct transcriptional programs of differentiation in acquiring the capacity to prime CD8(+) Tcells. These differences may be of consideration in the use of therapeutic DC vaccines based on Mo-DCs.

Oral CD103-CD11b+ classical dendritic cells present sublingual antigen and induce Foxp3+ regulatory T cells in draining lymph nodes.

Sublingual immunotherapy (SLIT) is a safe and efficient treatment for type 1 allergies; however, the underlying immunological mechanisms, particularly the phenotype of oral antigen-presenting cells (APCs) responsible for the induction of regulatory T (Treg) cells, remain unclear. We show here that the sublingual application of ovalbumin (OVA) induced antigen-specific Foxp3+ Treg cells in draining submandibular lymph nodes (ManLNs). Oral APCs were classified into macrophages, classical dendritic cells (cDCs), and Langerhans cells by flow cytometry. A major subset of oral cDCs with the CD103-CD11b+ phenotype showed retinoic acid (RA)-producing activity and converted naive CD4+ T cells to Foxp3+ Treg cells in a transforming growth factor-β- and RA-dependent manner in vitro. In the ManLNs, migratory CD103-CD11b+ cDCs also showed RA-producing activity. After the sublingual application of fluorescent OVA, fluorescence was detected in oral macrophages in tissues, followed by migratory CD103-CD11b+ cDCs in ManLNs and migratory CD103-CD11b+ cDCs were the main APCs responsible for the induction of sublingual antigen-specific Treg cells. The transfer of OVA-SLIT-induced Treg cells suppressed the OVA-induced hypersensitivity response. These results suggest that oral CD103-CD11b+ cDCs transport sublingual antigens to draining ManLNs and induce antigen-specific Foxp3+ Treg cells, and, thus, provide a rationale for developing cDC-based therapeutic approaches in SLIT.

Ly6C+ monocyte efferocytosis and cross-presentation of cell-associated antigen

Abstract Recently it was shown that circulating Ly6C+ monocytes traffic from tissue to the draining lymph nodes (LNs) with minimal alteration in their overall phenotype. Furthermore, in the steady state, Ly6C+ monocytes are as abundant as classical dendritic cells (DCs) within the draining LNs, and even more abundant during inflammation. However, little is known about the functional roles of constitutively trafficking Ly6C+ monocytes. In this study we investigated whether Ly6C+ monocytes can efferocytose (acquire dying cells) and cross-present cell-associated antigen, a functional property specially attributed to Batf3+ DCs. We demonstrated that Ly6C+ monocytes intrinsically efferocytose and cross-present cell-associated antigen to CD8+ T cells. In addition, efferocytosis was enhanced upon direct activation of the Ly6C+ monocytes through its corresponding TLRs, TLR4 and TLR7. However, only ligation of TLR7, and not TLR4, enhanced cross-presentation by Ly6C+ monocytes. Overall, this study outlines two functional roles, among others, that Ly6C+ monocytes have during an adaptive immune response.

Crystal structure of human dendritic cell inhibitory receptor C-type lectin domain reveals the binding mode with N-glycan.

Human dendritic cell inhibitory receptor (DCIR) is a C-type lectin receptor expressed in classical dendritic cells and accepts several oligosaccharide ligands including N-glycans. Here, we report the crystal structures of human DCIR C-type lectin domains in the absence and presence of a branched N-glycan unit. The domain has a typical C-type lectin fold and two bound calcium ions. In the ligand-bound form, the disaccharide unit (GlcNAcβ1-2Man) acceptably fits the electron density map, indicating that it forms the main epitope. The recognition of the nonterminal N-glycan unit explains the relatively broad specificity of this lectin.

Zeb2 is required for development of plasmacytoid dendritic cells

Abstract Recent work has shown that the transcription factor Zeb2 is regulated by and cooperates with T-bet to promote terminal differentiation of NK cells and cytotoxic T cells. In the mouse, Zeb2 is expressed throughout dendritic cell (DC) development and in mature plasmacytoid DCs (pDCs) and CD11b+ classical DCs (cDCs), but not in CD8+/CD103+ cDCs. We hypothesized that downregulation of Zeb2 expression would be essential for the development of CD8+/CD103+ cDCs. Upon conditional deletion of Zeb2, we observed a substantial decrease in pDC frequency and an increased ratio of CD8+:CD11b+ cDCs; meanwhile, Zeb2 haploinsufficiency yielded a partial defect in pDC development. Additional experiments demonstrated that this defect was cell-intrinsic and durable, and that it could not be rescued by administration of type I interferon, which increased pDC frequency in cultures of wild-type DC progenitors but directed Zeb2-deficient DC progenitors almost entirely to the CD8+/CD103+ cDC lineage. Deletion of Zeb2 induced by poly(I:C) treatment in an Mx1-Cre-driven model also resulted in perturbations in other myeloid lineages. Although poly(I:C) treatment led to an acute loss of Ly-6Chi monocytes both in Zeb2fl/fl;Mx1-Cre(tg) mice and in control mice, those monocytes were replenished to baseline frequencies in control mice but not in Zeb2-deficient mice. Similar decreases in monocyte frequency were observed in other organs, and further experiments suggested that this defect was also cell-intrinsic, gene dosage-dependent, durable, and observable in model systems that do not require use of poly(I:C). Thus, we have identified an essential regulator of development in multiple myeloid lineages.

Rab43 facilitates cross-presentation of cell-associated antigens by CD8a+ dendritic cells

Abstract Batf3-dependent classical dendritic cells (cDCs) are essential for cross-presentation and clearance of intracellular pathogens through priming of CD8 T cells. By analyzing differences in gene expression between cDC subsets we identified vesicular transport molecules unique to cross-presenting dendritic cell subsets. RAB43, a GTPase that is localized to the golgi, is selectively expressed in Batf3-dependent CD8a+ and CD103+ cDCs and was highly conserved between mouse and human. Mice with germline and conditional Rab43 deletions are viable and fertile with normal hematopoiesis. However, mice lacking Rab43 showed reduced cross-presentation of cell-associated, but not soluble, antigens both in vitro and in vivo. Bone-marrow derived DCs (BMDCs) from Rab43 deficient mice show normal cross-presentation efficiency, suggesting that Rab43 is involved in optimizing cross-presentation in vivo through a pathway unique to CD8a+ DCs.

Crystal structure of human dendritic cell inhibitory receptor C‐type lectin domain reveals the binding mode with N‐glycan

Human dendritic cell inhibitory receptor (DCIR) is a C-type lectin receptor expressed in classical dendritic cells and accepts several oligosaccharide ligands including N-glycans. Here, we report the crystal structures of human DCIR C-type lectin domains in the absence and presence of a branched N-glycan unit. The domain has a typical C-type lectin fold and two bound calcium ions. In the ligand-bound form, the disaccharide unit (GlcNAcβ1-2Man) acceptably fits the electron density map, indicating that it forms the main epitope. The recognition of the nonterminal N-glycan unit explains the relatively broad specificity of this lectin.

Classical dendritic cells are required for dietary antigen-mediated induction of peripheral T(reg) cells and tolerance.

Oral tolerance prevents pathological inflammatory responses towards innocuous foreign antigens via peripheral regulatory T cells (pTreg cells). However, whether a particular subset of antigen-presenting cells (APCs) is required during dietary antigen exposure to instruct naïve CD4+ T cells to differentiate into pTreg cells has not been defined. Using myeloid lineage-specific APC depletion in mice, we found that monocyte-derived APCs are dispensable, while classical dendritic cells (cDCs) are critical for pTreg cell induction and oral tolerance. CD11b− cDCs from the gut-draining lymph nodes efficiently induced pTreg cells, and conversely, loss of IRF8-dependent CD11b− cDCs impaired their polarization, although oral tolerance remained intact. These data reveal the hierarchy of cDC subsets in pTreg cell induction and their redundancy during oral tolerance development.

Ly6C(+) monocyte efferocytosis and cross-presentation of cell-associated antigens.

Recently it was shown that circulating Ly6C+ monocytes traffic from tissue to the draining lymph nodes (LNs) with minimal alteration in their overall phenotype. Furthermore, in the steady state, Ly6C+ monocytes are as abundant as classical dendritic cells (DCs) within the draining LNs, and even more abundant during inflammation. However, little is known about the functional roles of constitutively trafficking Ly6C+ monocytes. In this study we investigated whether Ly6C+ monocytes can efferocytose (acquire dying cells) and cross-present cell-associated antigen, a functional property particularly attributed to Batf3+ DCs. We demonstrated that Ly6C+ monocytes intrinsically efferocytose and cross-present cell-associated antigen to CD8+ T cells. In addition, efferocytosis was enhanced upon direct activation of the Ly6C+ monocytes through its corresponding TLRs, TLR4 and TLR7. However, only ligation of TLR7, and not TLR4, enhanced cross-presentation by Ly6C+ monocytes. Overall, this study outlines two functional roles, among others, that Ly6C+ monocytes have during an adaptive immune response.