CD11c Promoter Research Articles

Cooperative but Distinct Role of Medullary Thymic Epithelial Cells and Dendritic Cells in the Production of Regulatory T Cells in the Thymus.

Regulatory T cells (Tregs) are produced in the thymus to establish self-tolerance, and agonistic stimuli by self-Ags play a pivotal role in this process. Although two types of APCs, medullary thymic epithelial cells (mTECs) and dendritic cells (DCs), are responsible for presenting self-Ags together with costimulatory/cytokine signals, the distinct role of each APC in producing Tregs remains enigmatic. We have approached this issue by depleting the mTECs and DCs using mice expressing diphtheria toxin receptors driven by Aire and CD11c promoters, respectively. Depletion of mTECs showed an effect on Treg production quantitatively and qualitatively more profound than that of DCs followed by the development of distinct organ-specific autoimmune lesions in the hosts. Because self-Ags produced by mTECs are transferable to DCs through a process known as Ag transfer, we monitored the process of Ag transfer using mice expressing GFP from TECs. Although GFP expressed from total TECs was effectively transferred to DCs, GFP expressed from cortical TECs was not, suggesting that mTECs are the predominant source of self-Ags. We also found that GFP expressed not only from mature mTECs but also from immature mTECs was transferred to DCs, suggesting that a broad spectrum of molecules were subjected to Ag transfer during mTEC development. Interestingly, the numbers of recirculating non-Tregs producing IL-2, an important source for Treg expansion in the thymus, were reduced only in the mTEC-depleted mice. These results suggested the cooperative but distinct role of mTECs and DCs in the production of Tregs to avoid autoimmunity.

Targeting of SIRPα as a potential therapy for Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is a rare neoplastic disorder characterized by inflammatory lesions arising from the anomalous accumulation of pathogenic CD1a+ CD207+ dendritic cells (DCs). SIRPα is a transmembrane protein highly expressed in myeloid cells such as DCs and macrophages. Here we show that SIRPα is a potential therapeutic target for LCH. We found that SIRPα is expressed in CD1a+ cells of human LCH lesions as well as in CD11c+ DCs in the spleen, liver, and lung of a mouse model of LCH (BRAFV600ECD11c mouse), in which an LCH-associated active form of human BRAF is expressed in a manner dependent on the mouse Cd11c promoter. BRAFV600ECD11c mice manifested markedly increased numbers of CD4+ T cells, regulatory T cells, and macrophages as well as of CD11c+ MHCII+ DCs in the spleen. Monotherapy with a mAb to SIRPα greatly reduced the percentage of CD11c+ MHCII+ DCs in peripheral blood, LCH-like lesion size in the liver, and the number of CD11c+ MHCII+ DCs in the spleen of the mutant mice. Moreover, this mAb promoted macrophage-mediated phagocytosis of CD11c+ DCs from BRAFV600ECD11c mice, whereas it had no effects on the viability or CCL19-dependent migration of such CD11c+ DCs or on their expression of the chemokine genes Ccl5, Ccl20, Cxcl11, and Cxcl12. Our results thus suggest that anti-SIRPα monotherapy is a promising approach to the treatment of LCH that is dependent in part on the promotion of the macrophage-mediated killing of LCH cells.

Validation of the CD11c‐DTR transgenic mouse as a model to study neuroimmune interactions in the cornea

AbstractPurpose: CD11c‐DTR+ mice have a transgene encoding for a simian diphtheria toxin (DT) receptor under the CD11c promoter that makes dendritic cells (DCs) sensitive to DT, which causes DC depletion. Preliminary results using this mouse suggest that there is a functional interaction between DCs and corneal sensory nerves, as DC depletion modifies nerve activity and nociceptive behaviour. The aim of this work was to validate the CD11c‐DTR+ mouse model proving that the observed effects are not due to the DT itself, using littermates negative for the transgene and therefore resistant to DC depletion by DT.Methods: CD11c‐DTR+ and CD11c‐DTR‐ mice (5 months, both sexes) were used. Both groups received single or repetitive (every 2 days for 8 days) subconjunctival injections of DT or its vehicle (PBS). Basal tearing (in mm) was measured using phenol red threads placed in the temporally side of the eye for 30 s in isoflurane‐anaesthetised mice. To quantify spontaneous eye pain, the eye closure ratio was calculated by dividing the height by the width of the lid opening measured in photographs of freely moving animals. Nerve terminal impulse (NTI) activity of corneal cold thermoreceptor endings was recorded ex vivo in superfused eyes; spontaneous and stimulus‐evoked activity was explored.Results: Basal tearing was higher in DT‐injected CD11c‐DTR‐ animals, especially after repetitive injections (Day 0: 2 ± 0.16 mm vs. day 2: 2.8 ± 0.37 mm vs. day 8: 4.7 ± 0.36 mm, n = 5, p ≤ 0.001, RM ANOVA). Repetitive PBS injections caused similar effects in CD11c‐DTR+ and CD11c‐DTR‐ mice tearing (DTR−: 4.7 ± 0.36 mm, n = 5 vs. DTR+: 3.7 ± 1.48 mm, n = 3, p = 0.399, t‐test). No signs of spontaneous pain were observed in DT‐injected CD11c‐DTR‐ mice (Day 0: 0.91 ± 0.01 vs. 48 h: 0.93 ± 0.01 vs. day 8: 0.95 ± 0.01, n = 5, p = 0.065, RM ANOVA). No significant differences were found in spontaneous and stimulus‐evoked NTI activity recorded after repetitive injections of DT in CD11c‐DTR− mice or PBS in CD11c‐DTR+ mice.Conclusions: CD11c‐DTR+ mice are a valid model to study neuroimmune interactions. DT has no effects on pain behaviour or NTI activity of animals lacking the DT receptor. The changes in tearing appear to be consequence of the subconjunctival injection process per se.Reference PID2020‐115934RB‐I00 from DOI: MCIN/AEI/10.13039/50110001103

JAK1 signaling in dendritic cells promotes peripheral tolerance in autoimmunity through PD-L1-mediated regulatory Tcell induction.

Dendritic cells (DCs) induce peripheral Tcell tolerance, but cell-intrinsic signaling cascades governing their stable tolerogenesis remain poorly defined. Janus Kinase 1 (JAK1) transduces cytokine-receptor signaling, and JAK inhibitors (Jakinibs), including JAK1-specific filgotinib, break inflammatory cycles in autoimmunity. Here, we report in heterogeneous DC populations of multiple secondary lymphoid organs that JAK1 promotes peripheral Tcell tolerance during experimental autoimmune encephalomyelitis (EAE). Mice harboring DC-specific JAK1 deletion exhibit elevated peripheral CD4+ Tcell expansion, less regulatory Tcells (Tregs), and worse EAE outcomes, whereas adoptive DC transfer ameliorates EAE pathogenesis by inducing peripheral Tregs, programmed cell death ligand 1 (PD-L1) dependently. This tolerogenic program is substantially reduced upon the transfer of JAK1-deficient DCs. DC-intrinsic IFN-γ-JAK1-STAT1 signaling induces PD-L1, which is required for DCs to convert CD4+ Tcells into Tregs invitro and attenuated upon JAK1 deficiency and filgotinib treatment. Thus, DC-intrinsic JAK1 promotes peripheral tolerance, suggesting potential unwarranted DC-mediated effects of Jakinibs in autoimmune diseases.

Helper T Cell Response to Factor VIII In Vivo Requires Several Anatomically Distinct Types of Antigen Presenting Cells

Inhibitor formation is the most serious complication of factor (F)VIII replacement therapy for hemophilia A. It has long been clear that FVIII inhibitors arise in a CD4 + T cell-dependent manner, but early events in the immune response leading to MHC-II presentation and CD4 + T cell activation remain obscure. Several types of antigen presenting cells (APCs) have been implicated in the uptake of FVIII, but their relevance in T cell activation has been unclear. This study aimed to pinpoint the roles of APCs in priming FVIII-specific CD4 + T cells in vivo.Several transgenic strains of mice on the C57BL/6J background were employed to perform in vivo antigen presentation assays involving one intravenous (IV) injection of 5 µg FVIII-OVA fusion protein (FOVA), which contained an MHC-II I-A b peptide epitope of chicken ovalbumin in place of the B domain of FVIII, and an adoptive transfer (via IV injection on the following day) of 5E6 transgenic OT-II CD4 + T cells, which express chicken ovalbumin-specific T cell receptor (TCR). Before adoptive transfers, OT-II T cells were stained with CellTrace Violet (CTV). Spleens were collected for flow cytometry analyses four days after the adoptive transfer. FOVA shows identical specific activity to that of B domain deleted FVIII.CD11c-DTR/GFP mice (which express diphtheria toxin receptor and green fluorescent protein under the CD11c promoter) (n=3) received two intraperitoneal (IP) injections of 100 ng diphtheria toxin (DT) or PBS only (n=3) one day before and on the day of FVIII-OVA injection to deplete CD11c high cells (i.e. dendritic cells, DCs, marginal zone, sinusoidal and metallophilic macrophages, MFs). DT treatment completely abrogated T cell proliferation in the animals (p=0.03), while on average 11.1% of CD4 +CTV + cells from the control animals were progenies of the transferred cells.XCR1-DTRvenus mice (which express DT receptor and the Venus variant of yellow fluorescent protein under the XCR1 promoter) were similarly treated with DT or PBS (n=4/group) one day before FOVA injection and one day after adoptive transfer to deplete type I conventional DCs (which make up ~80% of XCR1 + cells). XCR1 + cell depletion reduced T cell proliferation by ~50% (p=0.02). CD4 +CTV + cells from the DT-treated and control mice included 19.4% and 38.6% progenies, respectively.Hemophilia A (F8e16-/-) mice received 100 µg of marginal zone (MZ) B cell-depleting antibodies anti-CD11a and anti-CD49d (n=4) or isotype control antibodies (n=3) four and two days before FOVA injection. MZ B cell depletion completely abrogated T cell proliferation (p=0.02), while on average 31% of CD4 +CTV + cells from the control animals were progenies of the transferred cells. In another experiment, hemophilia A mice were injected with GdCl 3 (n=4), which inactivates MFs, or PBS (n=3) one day before and on the day of FOVA administration. MF inactivation completely abrogated T cell proliferation in all but 1 animal (p=0.03), while on average 15.9% of CD4 +CTV + cells from the control animals were progenies of the transferred cells.To visualize APC-T cell interactions in vivo, we performed multiphoton (MP) intravital microscopy (IVM) of inguinal lymph nodes (LNs) in CD11c-DTR/GFP mice. The animals received adoptive transfers of 1E7 CTV-stained OT-II CD4 + T cells IV ~24 hours and 5 µg FOVA 20, 5 or 1 hour before IVM delivered intradermally (ID) to target the skin-draining LNs. Control animals received adoptive cell transfer only. For IVM, the LNs were surgically exposed in live, anesthetized animals. Five hours after FOVA injection, multiple CTV + OT-II T cells formed clusters around GFP + cells throughout the T cell zone with several motile T cells surveying the B cell follicle. Twenty hours after FOVA injection, CTV + T cells densely populated and demarcated the T-B border in the LNs. CTV + T cells were nearly absent from the inguinal LNs in the control animals.We propose that activation of CD4 + T cells in response to FVIII requires a complex interaction of multiple types of APCs, which occupy distinct compartments in the secondary lymphoid organs that FVIII antigen traverses en route to cognate CD4 + T cells. In the spleen, where response to FVIII primarily takes place, antigens larger than 60 kDa (such as FVIII at 280 kDa) do not freely flow into the white pulp and instead need to be ferried by APCs. While DCs ultimately activate CD4 + T helper cells, MFs shuttle the antigen to DCs and may also provide innate activation immune signals. DisclosuresNo relevant conflicts of interest to declare.

In Vivo Expansion of Recipient DC Leads to Deletion of Donor Alloreactive CD8+ T Cells and Is an Alternative to Post-Transplant Cyclophosphamide for the Prevention of Graft-Versus-Host Disease after BMT

Allogeneic bone marrow transplantation (BMT) provides curative therapy for leukemia via immunological graft-versus-leukemia (GVL) effects. In practice, this must be balanced against graft-versus-host disease (GVHD) and opportunistic infection. We have investigated the role of recipient dendritic cells (DC) in the control of MHC class I restricted T cell responses after BMT. We first used the C3H.Sw → B6 miHA mismatched model of mouse BMT, with lethally irradiated B6.CD11c.DOG recipients (diphtheria toxin receptor, ovalbumin and GFP driven off the CD11c promoter) such that recipient DC can be deleted by diphtheria toxin, in combination with B6-derived MLL-AF9 induced primary acute myeloid leukemia to model clinical relapse. Depletion of recipient DC resulted in improved leukemic control (median survival 43 vs 31 days, P <0.001). The use of IRF8-/- BMT recipients (CD8α+ DC subset absent) confirmed that recipient CD8α+ DC were critical for regulating these GVL effects (median survival 43 vs 34 days, P = 0.0005). We confirmed that this accelerated relapse occurred as a consequence of recipient DC-induced donor T cell apoptosis resulting in a profound contraction of the donor T cell compartment. Using the same depletion strategies in an antigen-specific model (donor OT-I T cells, B6.CD11c.DOG x DBA/2 F1 recipients) we confirmed that recipient DC invoked effector donor CD8+ T cell hyperactivation, differentiation and apoptosis (Annexin V+ OT-I; 52.4% vs. 23.9% in DC replete vs. depleted recipients, P = 0.01).In light of the effects of recipient DC on antigen-specific donor T cell deletion, we next compared pre-transplant Flt-3L treatment (Pre-T Flt-3L; d -10 to d 0) to expand recipient CD8α+ DC (mean CD8α+ DC/spleen saline 0.14×106 vs Pre-T Flt-3L 23.6×106; P= 0.008) with post-transplant cyclophosphamide (PT-Cy at d 3,4), in a model of haploidentical BMT (B6 → B6D2F1). Consistent with our DC depletion models, Pre-T Flt-3L resulted in augmented antigen-specific donor T cell expansion 12 hours post-BMT, followed by deletion within 72 hrs (d 3 mean splenic OT-I: saline 15.94×106 vs Pre-T Flt-3L 0.01×106; P= 0.02). PT-Cy resulted in similar profound but alloantigen-independent polyclonal T cell deletion (d 7 mean splenic CD3+: saline 10.8×106 vs PT-Cy 0.8×106; P= 0.02). Pre-T Flt-3L offered better protection from GVHD than PT-Cy (median survival 36 vs 45 days, P= 0.02). In contrast, cyclosporine (CsA, administered to deliver clinically relevant serum levels of 200 - 300 ng/mL) did not result in major improvements in GVHD outcome (median survival 11 vs 13 days, P= 0.07). Both Pre-T Flt-3L and PT-Cy ablated GVL effects with relapse rates similar to corresponding TCD controls (Pre-T Flt3-L median time to relapse 11 days TCD vs 12 days BM+T; PT-Cy 22 days TCD vs 26 days BM+T). In contrast, GVL was maintained after CsA (with long-term survival seen in CsA and saline treated BM+T mice, median survival not reached; P >0.99). We confirmed that the effects with Pre-T Flt-3L were DC specific, and not related to the other populations by simultaneously deleting recipient DC (using diphtheria toxin) in Flt-3L treated recipients. This completely restored GVL effects and long-term survival to that observed in non-Flt-3L treated recipients (P >0.99). Importantly, despite effects on antigen-specific T cells, Pre-T Flt-3L facilitated the maintenance of third party CMV-specific donor T cells whereas PT-Cy did not (m38 tetramer+ cells 3 days post treatment; saline vs Pre-T Flt-3L P= 0.27; saline vs PT-Cy P= 0.028). These data highlight pre-transplant Flt-3L therapy as a potent alternative therapeutic strategy to PT-Cy to prevent GVHD, and moreover, that the effects of these strategies on attenuating GVL may have important clinical ramifications. DisclosuresLane:novartis: Consultancy; janssen: Consultancy, Research Funding.

Elucidating the Role of Ezh2 in Tolerogenic Function of NOD Bone Marrow-Derived Dendritic Cells Expressing Constitutively Active Stat5b.

Tolerogenic dendritic cells (toDCs) are crucial to controlling the development of autoreactive T cell responses and the prevention of autoimmunity. We have reported that NOD.CD11cStat5b-CA transgenic mice expressing a constitutively active (CA) form of Stat5b under the control of a CD11c promoter are protected from diabetes and that Stat5b-CA-expressing DCs are tolerogenic and halt ongoing diabetes in NOD mice. However, the molecular mechanisms by which Stat5b-CA modulates DC tolerogenic function are not fully understood. Here, we used bone marrow-derived DCs (BMDCs) from NOD.CD11cStat5b-CA transgenic mice (Stat5b-CA.BMDCs) and found that Stat5b-CA.BMDCs displayed high levels of MHC class II, CD80, CD86, PD-L1, and PD-L2 and produced elevated amounts of TGFβ but low amounts of TNFα and IL-23. Stat5b-CA.BMDCs upregulated Irf4 and downregulated Irf8 genes and protein expression and promoted CD11c+CD11b+ DC2 subset differentiation. Interestingly, we found that the histone methyltransferase Ezh2 and Stat5b-CA bound gamma-interferon activated site (GAS) sequences in the Irf8 enhancer IRF8 transcription, whereas Stat5b but not Ezh2 bound GAS sequences in the Irf4 promoter to enhance IRF4 transcription. Injection of Stat5b-CA.BMDCs into prediabetic NOD mice halted progression of islet inflammation and protected against diabetes. Importantly, inhibition of Ezh2 in tolerogenic Stat5b-CA.BMDCs reduced their ability to prevent diabetes development in NOD recipient mice. Taken together, our data suggest that the active form of Stat5b induces tolerogenic DC function by modulating IRF4 and IRF8 expression through recruitment of Ezh2 and highlight the fundamental role of Ezh2 in Stat5b-mediated induction of tolerogenic DC function.

Abstract 3322: SIRPα is a potential therapeutic target for Langerhans cell histiocytosis

Abstract Langerhans cell histiocytosis (LCH) is a rare disease, in which immature dendritic cells (DCs) with the same traits as Langerhans cells proliferate and develop various symptoms at various sites such as the skin, bone, lungs, and pituitary. Most patients with LCH are thought to have abnormalities in the mitogen-activated protein (MAP) kinase pathway, with more than 50% of BRAF mutations. It is rarely associated with morbidity and mortality in patients with single organ disease, but sometimes fatal in patients with multiple organ disease, and there is still no effective treatment for these patients. SIRPα is a transmembrane protein with three immunoglobulin-like domains in the extracellular region. This protein is especially abundant in myeloid cells such as DCs and macrophages. SIRPα interacts with its ligand CD47, another transmembrane protein, and we and others previously showed that the CD47-SIRPα interaction negatively regulates the phagocytosis by macrophages of target cells. We also showed that anti-SIRPα antibodies could promote the killing by macrophages of SIRPα-expressing tumor cells through dual mechanisms: direct induction of the antibody-dependent cellular phagocytosis of tumor cells by macrophages and blockade of CD47-SIRPα signaling that negatively regulates such phagocytosis. By the use of tissue sections from patients with LCH, here we showed that DCs positive for CD1a, a definitive diagnostic marker in LCH, expressed high levels of SIRPα, and inflammatory cells significantly infiltrated around such CD1a+ DCs in LCH regions. As a mouse model for LCH, BRAFV600ECD11c mice, in which BRAFV600E were expressed under the control of the CD11c promoter, displayed a severe LCH-like disease phenotype with a short lifespan and the increased infiltration of CD11c+ DCs into multiple organs. Among CD11c+ DCs, the number of SIRPα+ DCs (SIRPα+/CD11c+) increased in the spleen, liver, and lung. Moreover, an anti-SIRPα antibody that blocks the CD47-SIRPα interaction stimulated the phagocytosis by bone marrow-derived macrophages of CD11c+ DCs sorted from the spleen of BRAFV600ECD11c in vitro. Given the prominent expression of SIRPα in CD11c+ DCs from BRAFV600ECD11c mice, anti-SIRPα antibodies likely promote the phagocytosis of these cells by macrophages through above-mentioned dual mechanisms. In conclusion, we have first demonstrated that CD1a+ DCs in LCH regions expressed high levels of SIRPα. Moreover, it is also suggested that the anti-SIRPα antibody is a potential tool for the new therapy of LCH. Citation Format: Takeshi Okamoto, Yoji Murata, Daisuke Hazama, Mariko Sakamoto, Yuka Kakuchi, Yasuyuki Saito, Takenori Kotani, Yoshimasa Maniwa, Takashi Matozaki. SIRPα is a potential therapeutic target for Langerhans cell histiocytosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3322.

Importance of methodology in the evaluation of renal mononuclear phagocytes and analysis of a model of experimental nephritis with Shp1 conditional knockout mice

Tissue resident mononuclear phagocytes (Mophs), comprising monocytes, macrophages, and dendritic cells (DCs), play important roles under physiological and pathological conditions. The presence of these cells in the kidney has been known for decades, and studies of renal Mophs (rMophs) are currently underway. Since no unified procedure has been identified to isolate rMophs, results of flow cytometric analysis of rMophs have been inconsistent among studies. We therefore first evaluated a preparative method for rMophs using collagenous digestion. The yield of rMophs greatly increased after the collagenase digestion. In particular, F4/80high rMophs, which were positive for CD11c, a specific marker of DCs, dramatically increased. In addition, since neutrophils are sometimes mixed among rMophs in the analysis of flow cytometry, we established a gating strategy for eliminating neutrophils. To determine the contribution of rMophs to the development of autoimmune nephritis, we analyzed an experimental model of autoimmune nephritis that was applied to Shp1 conditional knockout mice (Shp1 CKO). This knockout strain is generated by crossing a mouse line carrying floxed Shp1 allele to mice expressing Cre recombinase under the control of the CD11c promoter. Shp1 CKO therefore specifically lack Shp1 in cells expressing CD11c. As a result, Shp1 CKO were susceptible to that experimental glomerulonephritis and F4/80high rMophs of Shp1 CKO increased dramatically. In conclusion, our preparative methods for collagenase digestion and gating strategy for neutrophils are necessary for the analysis of rMophs, and Shp1 suppresses the development of autoimmune nephritis through the control of rMophs.

Abstract P281: Artificial Dendritic Cells Identify a Major Histocompatibility Complex Class 1 Subset that Activates T Cells in Hypertension

We and others have shown an important role CD8+ cells in both experimental and human hypertension. CD8+ T cells are activated by antigens presented by major histocompatibility complex 1 (MHC1). C57BL/6 mice express two MHC1, referred to as H2-Kb and H2-Db. To identify antigenic peptides responsible for hypertension, we made two transgenic mice lacking the transmembrane domains of MHC1 and an added His-tag to the modified MHC1. These transgenes are driven by CD11c promoter, allowing expression in antigen presenting cells. The soluble Kb and Db (sKb and sDb) mice received 2 week infusions of sham or angiotensin II and their splenocytes placed in culture for two days. Ni-NTA beads were then used to bind the shed MHC-1 and these beads mixed with 10 6 T cells from other ang II infused mice (Figure panel A). CFSE dilution was used to monitor T cell proliferation. We found that sDb from ang II infused male mice, but not sham infused mice, potently stimulated proliferation of CD8+ T cells from ang II infused male mice. This degree of stimulation was significantly greater than that observed by sKb (Figure panel B). The shed MHC1 from female ang II-treated mice caused significantly less stimulation of female CD8+ T cells compared to male MHC1, in keeping with prior observations that female mice have less immune activation in hypertension. In summary, these data strongly suggest that unique antigens, presented in the context of H2-Db, are generated in hypertension. These data also suggest that there may be unique human lymphocyte antigens (HLAs), the analog of mouse MHC, that predispose to hypertension and related end-organ damage.

Optic nerve as a source of activated retinal microglia post-injury

Using mice expressing green fluorescent protein (GFP) from a transgenic CD11c promoter we found that a controlled optic nerve crush (ONC) injury attracted GFPhi retinal myeloid cells to the dying retinal ganglion cells and their axons. However, the origin of these retinal myeloid cells was uncertain. In this study we use transgenic mice in conjunction with ONC, partial and full optic nerve transection (ONT), and parabiosis to determine the origin of injury induced retinal myeloid cells. Analysis of parabiotic mice and fate mapping showed that responding retinal myeloid cells were not derived from circulating macrophages and that GFPhi myeloid cells could be derived from GFPlo microglia. Comparison of optic nerve to retina following an ONC showed a much greater concentration of GFPhi cells and GFPlo microglia in the optic nerve. Optic nerve injury also induced Ki67+ cells in the optic nerve but not in the retina. Comparison of the retinal myeloid cell response after full versus partial ONT revealed fewer GFPhi cells and GFPlo microglia in the retina following a full ONT despite it being a more severe injury, suggesting that full transection of the optic nerve can block the migration of responding myeloid cells to the retina. Our results suggest that the optic nerve can be a reservoir for activated microglia and other retinal myeloid cells in the retina following optic nerve injury.

SMAD4-deficient Dendritic Cells in the Murine Lung shows an Activated Phenotype

Abstract SMAD4 is a signaling intermediate that acts downstream of the TGFb receptor. This signaling pathway is known to regulate expression of the Itgae gene (CD103) in activated CD8 T cells. Whether SMAD4 plays a role in induction or maintenance of CD103+ dendritic cells (DCs) has not been investigated. To address this question, we crossed SMAD4 floxed mice with Cre under the control of the CD11c promoter. DCs in the lung, spleen and bone marrow of naïve CD11c-SMAD4 mice were analyzed by multi-parameter flow cytometry. The lungs of Smad4-deficient mice contained smaller numbers of CD11c+ cells than control animals, and the remaining cells expressed costimulatory molecules and CD11b at high levels. Lung suspensions were metal barcoded for time of flight cytometry (cyTOF) and viSNE maps were contracted using 25 parameters. Under these homeostatic conditions, Smad4-abalation caused a dramatic decrease in CD11c+ DCs with activation of pSTAT5, when compared to DCs from littermate controls. Activation of pSTAT5 in KO was specific to CD11c+ DCs as no difference was seen in other cell types including T cells. Our data indicate that the lungs of SMAD4-CD11c KO mice were depleted of migratory DCs, which migrated to the draining lymph nodes after spontaneous maturation. We currently are exploring whether DC-depletion undermines cell mediated immunity to respiratory virus infection.

A novel mechanism of NO synthase uncoupling involving isolevuglandin adduction

We have previously shown that hypertension leads to modification of self‐proteins in antigen presenting cells by isolevuglandin (isoLG) lipid oxidation products. These modified proteins seem to be immunogenic. IsoLG‐modified peptides are presented in major histocompatibility complex (MHC) class 1 and drive CD8+ T cell proliferation. To understand alterations in the peptidome presented by MHC1 in hypertension, we created transgenic mice expressing a truncated form of MHC1 Kb lacking the transmembrane domain, driven by the CD11c promoter. The lack of a transmembrane domain causes MHC1 to be shed into the media when DCs are cultured. We treated these mice with either 2 weeks sham or angiotensin (ang) II infusion, isolated splenic DCs and placed these in culture for 72 hours. The shed soluble MHC1 Kb was harvested and associated peptides were analyzed by mass spectroscopy. One peptide identified in DCs from hypertensive mice was an identical match for a portion of the neuronal nitric oxide synthase (nNOS) containing an isoLG adduct at lysine 225. Because this lysine is positioned between the heme and arginine binding sites, we hypothesized that its modification would disrupt normal NO production and instead lead to superoxide production, i.e. NOS uncoupling. In subsequent studies, we found that nNOS expression is increased 5‐fold in DCs of ang II‐treated compared to sham‐treated mice (n = 6). Importantly, ang II infusion increased superoxide production of DCs from 37±3 to 111±5 pmol/mg protein (mean ± SE, p = 0.0001), and this was completely blocked by treatment of the cells with the selective nNOS inhibitor L‐vinyl NIO, suggesting that nNOS is a source of superoxide in DCs in hypertension. Next we expressed wild type nNOS and a mutant nNOS in which lysine 225 was mutated to an alanine (nNOS K225A) in human epithelial kidney (HEK) cells. Untransfected HEK cells failed to produce NO, as measured by electron spin resonance and the spin trap Fe[DETC]2. HEK cells transfected with either the WT or K225A nNOS produced 152 ± 5 vs 156 ± 6 arbitrary units of NO/mg protein (p = NS). However, exposure of the transfected HEK cells to tertbutyl hydroperoxide, which promotes isoLG formation, reduced NO production by cells expressing the wild‐type enzyme to 86 ± 6 units/mg protein, while having no effect on cells transfected with the mutant K225A (p = 0.003, n = 5 – 7), further implicating lysine 225 as a site of oxidative modification in nNOS. In summary, hypertension increases nNOS expression in DCs and this enzyme is altered by isoLG adduction at lysine 225. This leads to an increase in superoxide and reduced NO production in these immune cells. We conclude that isoLG adduction represents a novel mechanism of NOS uncoupling and a source of reactive oxygen species in dendritic cells.Support or Funding InformationAmerican Heart Association grants POST290900 and SFRN204200, and National Institutes of Health grants K01HL130497, R01HL125865, R01HL039006 and P01HL129941This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Targeting Mycobacterium tuberculosis Antigens to Dendritic Cells via the DC-Specific-ICAM3-Grabbing-Nonintegrin Receptor Induces Strong T-Helper 1 Immune Responses.

Tuberculosis remains a major global health problem and efforts to develop a more effective vaccine have been unsuccessful so far. Targeting antigens (Ags) to dendritic cells (DCs) in vivo has emerged as a new promising vaccine strategy. In this approach, Ags are delivered directly to DCs via antibodies that bind to endocytic cell-surface receptors. Here, we explored DC-specific-ICAM3-grabbing-nonintegrin (DC-SIGN) targeting as a potential vaccine against tuberculosis. For this, we made use of the hSIGN mouse model that expresses human DC-SIGN under the control of the murine CD11c promoter. We show that in vitro and in vivo delivery of anti-DC-SIGN antibodies conjugated to Ag85B and peptide 25 of Ag85B in combination with anti-CD40, the fungal cell wall component zymosan, and the cholera toxin-derived fusion protein CTA1-DD induces strong Ag-specific CD4+ T-cell responses. Improved anti-mycobacterial immunity was accompanied by increased frequencies of Ag-specific IFN-γ+ IL-2+ TNF-α+ polyfunctional CD4+ T cells in vaccinated mice compared with controls. Taken together, in this study we provide the proof of concept that the human DC-SIGN receptor can be efficiently exploited for vaccine purposes to promote immunity against mycobacterial infections.

Constitutive CD40 Signaling in Dendritic Cells Limits Atherosclerosis by Provoking Inflammatory Bowel Disease and Ensuing Cholesterol Malabsorption

The costimulatory molecule CD40 is a major driver of atherosclerosis. It is expressed on a wide variety of cell types, including mature dendritic cells (DCs), and is required for optimal T-cell activation and expansion. It remains undetermined whether and how CD40 on DCs impacts the pathogenesis of atherosclerosis. Here, the effects of constitutively active CD40 in DCs on atherosclerosis were examined using low-density lipoprotein-deficient (Ldlr-/-) bone marrow chimeras that express a transgene containing an engineered latent membrane protein 1 (LMP)/CD40 fusion protein conferring constitutive CD40 signaling under control of the DC-specific CD11c promoter (DC-LMP1/CD40). As expected, DC-LMP1/CD40/Ldlr-/- chimeras (DC-LMP1/CD40) showed increased antigen-presenting capacity of DCs and increased T-cell numbers. However, the mice developed extensive neutrophilia compared to CD40wt/Ldlr-/- (CD40wt) chimeras. Despite overt T-cell expansion and neutrophilia, a reduction in conventional DC frequency and a dramatic (approximately 80%) reduction in atherosclerosis was observed. Further analyses revealed that cholesterol and triglyceride levels had decreased by 37% and 60%, respectively, in DC-LMP1/CD40 chimeras. Moreover, DC-LMP1/CD40 chimeras developed inflammatory bowel disease characterized by massive transmural influx of leukocytes and lymphocytes, resulting in villous degeneration and lipid malabsorption. Constitutive activation of CD40 in DCs results in inflammation of the gastrointestinal tract, thereby impairing lipid uptake, which consequently results in attenuated atherosclerosis.

Peripheral Autoimmune Regulator Induces Exhaustion of CD4+ and CD8+ Effector T Cells to Attenuate Autoimmune Diabetes in Non-Obese Diabetic Mice.

Autoimmune regulator (Aire) is one of the most crucial genes expressed in the thymus, where it orchestrates the promiscuous expression and presentation of tissue-specific antigens during thymocyte selection. The presence of Aire-expressing cells outside the thymus points toward its plausible extrathymic functions; however, the relative contribution of Aire-expressing cells of hematopoietic origin and their role in the modulation of autoimmune diseases are still obscure. Here, we report that non-obese diabetic mice with transgenic Aire expression under the control of the CD11c (integrin alpha X) promoter were significantly protected from autoimmune diabetes compared with their non-transgenic littermates. The protective effect of Aire transgene was mediated primarily by an increase in the “exhausted” populations of CD4+ and CD8+ T cells, both demonstrating poor expressions of interferon-γ and tumor necrosis factor-α. Both CD4+ and CD8+ effector T cells in transgenic mice displayed distinctive and differential expression of T-bet and Eomesodermin, respectively, in conjunction with high expression of programmed cell death protein-1 and other exhaustion-associated markers. Importantly, transgenic Aire expression did not result in any detectable changes in the population of Foxp3+ regulatory T (Treg) cells. Co-transfer experiments also demonstrated that Aire transgenic dendritic cells, as a “stand-alone” cell population, had the potential to suppress effector T cells in vivo without the support of Treg cells, but eventually failed to prevent the diabetogenesis in recipient mice. In conclusion, our study suggests that apart from its role in clonal deletion of autoreactive T cells or clonal diversion to Treg lineage, Aire can also contribute to tolerance by forcing effector T cells into a state of exhaustion with poor effector functions, thereby effectively containing autoimmune diseases.

A subpopulation of activated retinal macrophages selectively migrated to regions of cone photoreceptor stress, but had limited effect on cone death in a mouse model for type 2 Leber congenital amaurosis.

Studies of antigen presentation in retina using mice that expressed green fluorescent protein (GFP) from a transgenic CD11c promoter found that retinal GFPhi cells possessed antigen presentation function. Subsequent studies found that these high GFPhi cells preferentially localized to sites of retinal injury, consistent with their APC function. Interest in the roles of macrophages in degenerative CNS diseases led us to study the GFPhi cells in a retinal model of neurodegeneration. We asked if apoptotic cone photoreceptor cell death in Rpe65-/- knockout mice induced the GFPhi cells, explored their relationship to resident microglia (MG), and tested their role in cone survival. Rpe65-/- mice were bred to CD11cGFP mice on the B6/J background. CD11cGFPRpe65-/- mice were also backcrossed to CX3CR1YFP-creERROSADTA mice so that CX3CR1+ mononuclear cells could be depleted by Tamoxifen. Retinas were analyzed by immunohistochemistry, confocal microscopy, fluorescence fundoscopy and flow cytometry. Elevated numbers of GFPhi cells were concentrated in photoreceptor cell layers of CD11cGFPRpe65-/- mice coinciding with the peak of cone death at 2 to 4weeks of age, and persisted for at least 14months. After the initial wave of cone loss, a slow progressive loss of cones was found that continued to retain GFPhi cells in the outer retina. Sustained, four-week Tamoxifen depletions of the GFPhi cells and MG in Rpe65-/- mice from day 13 to day 41, and from day 390 to day 420 promoted a small increase in cone survival. We found no evidence that the GFPhi cells were recruited from the circulation; all data pointed to a MG origin. MG and GFPhi cells were well segregated in the dystrophic retina; GFPhi cells were foremost in the photoreceptor cell layer, while MG were concentrated in the inner retina. The expression of GFP on a subset of retinal mononuclear cells in CD11cGFP mice identified a distinct population of cells performing functions previously attributed to MG. Although GFPhi cells dominated the macrophage response to cone death in the photoreceptor cell layer, their ablation led to only an incremental increase in cone survival. The ability to identify, ablate, and isolate these cells will facilitate analysis of this activated, antigen-presenting subset of MG.

Abstract 592: Constitutive CD40-Signaling in Dendritic Cells Limits Atherosclerosis by Provoking Inflammatory Bowel Disease and Ensuing Cholesterol Malabsorption

The co-stimulatory molecule CD40 is a major driver of atherosclerosis. It is expressed on a wide variety of cell types including mature dendritic cells (DCs) and required for optimal T cell activation and expansion. It remains undetermined if and how CD40 on DCs impacts the pathogenesis of atherosclerosis. Here we examined the effects of constitutively active CD40 in DCs on atherosclerosis, using low-density lipoprotein-deficient (Ldlr -/- ) bone-marrow chimeras that express an engineered latent membrane protein 1 (LMP)/CD40 fusion protein conferring constitutive CD40 signaling under control of the CD11c promoter ( DC-CD40ca ). As expected, DC-CD40ca/Ldlr -/- chimeras showed increased antigen presenting capacity on DCs and increased T cell numbers. However, they developed extensive neutrophilia compared to wt / ldlr -/- chimeras. Despite overt T cell expansion and neutrophilia we observed a reduction in cDC frequency and a dramatic reduction in atherosclerosis ( CD40wt/ldlr -/- 22076±3763 μm 2 vs. DC-CD40ca/ldlr -/- 2511±1256 μm 2 ). Further analyses revealed that cholesterol and triglyceride levels decreased by 37% and 60%, respectively, in DC-CD40ca/Ldlr -/- chimeras. Moreover, DC-CD40ca/Ldlr -/- chimeras developed inflammatory bowel disease characterized by massive transmural influx of leukocytes and lymphocytes, resulting in villous degeneration and lipid malabsorption. Constitutive activation of CD40 in DCs results in inflammation of the gastrointestinal tract, thereby impairing lipid uptake, which consequently results in attenuated atherosclerosis.

Constitutively active Stat5b signaling confers tolerogenic functions to dendritic cells of NOD mice and halts diabetes progression

Defects in dendritic cells (DCs) development and function lead to autoimmune disorders. Autoimmune diabetes in humans and NOD mice results from a breakdown of self-tolerance, ending in T cell-mediated β-cell destruction. DCs dysfunction in NOD mice results in part from a defect in the JAK-STAT5 signaling pathway associated with the idd4 susceptibility locus. The involvement of Stat5b in DCs tolerogenic functions remains unknown. We have generated transgenic mice (NOD.CD11cStat5b−CA) expressing a constitutively active form of the Stat5b gene (Stat5b-CA) under control of CD11c promoter. All NOD.CD11cStat5b−CA mice were protected against diabetes. Protection was associated with an increased in the pool and suppressive function of Tregs, a promotion of Th2 and Tc2 immune response and a decreased percentage of CD8+ T cells. Splenic DCs of NOD.CD11cStat5b−CA mice acquired a mature phenotype, promoted and induced better conversion of CD4+CD25-Foxp3- T cells into Tregs (CD4+CD25+Foxp3+ T cells) than DCs of NOD mice. Stat5b-CA.DC-educated CD4+CD25− T cells delayed diabetes onset whereas Stat5b-CA.DC-educated Tregs blocked ongoing diabetes in 8–10 weeks old NOD recipient mice. Importantly, injection of Stat5b.CA.DC to 8–10-week old NOD mice halted diabetes progression and educated their splenocytes to loose their diabetogenic potential when transferred to NOD.SCID mice. Our work is the first to report that an active form of Stat5b restored DCs tolerogenic functions that re-educated Tregs to re-establish and to sustain long-term protective immune response against diabetes in NOD mice.

Dendritic cell-specific Mgat2 knockout mice show antigen presentation defects but reveal an unexpected CD11c expression pattern.

Zwitterionic polysaccharide antigens such as polysaccharide A (PSA) from Bacteroides fragilis have been shown to activate CD4+ T cells upon presentation by class II major histocompatibility complex (MHCII) on professional antigen presenting cells. For T cell recognition and activation, high affinity binding between MHCII and PSA is required, and complex N-glycans on conserved MHCII asparagine residues play a central role in controlling this interaction. By truncating these glycans in a myeloid-specific knockout of Mgat2, created using the LyzM-CRE mouse (M-cKO), we previously reported defects in PSA responses in vivo. Unfortunately, the M-cKO also showed a propensity to develop common variable immunodeficiencywith autoimmune hemolytic anemia features. Here, we describe a novel murine model in which Mgat2 was targeted for ablation using the dendritic cell (DC)-specific CD11c-CRE-GFP strain in order to develop a more specific and robust in vivo model of PSA presentation defects (DC-cKO). This study shows that Mgat2 deficient DCs from DC-cKO mice show ablation of PSA presentation and downstream T cell activation in vitro. However, the CD11c promoter was unexpectedly active and triggered Mgat2 deletion within multiple hematopoietic lineages, showed remarkably poor penetrance within native DC populations, and produced almost undetectable levels of green fluorescent proteinsignal. These findings show that the CD11c promoter is not DC-specific, and extreme care should be taken in the interpretation of data using any mouse created using the CD11c-CRE model.