Opsonization of Apoptotic Cells by Autologous iC3b Facilitates Clearance by Immature Dendritic Cells, Down-regulates DR and CD86, and Up-regulates CC Chemokine Receptor 7

Immature dendritic cells (iDCs) do not mature after uptake of apoptotic cells and may play a role in the induction of peripheral tolerance to self antigens derived from apoptotic material. The integrins, αvβ3, αvβ5, and the scavenger receptor, CD36, have been shown to mediate uptake of apoptotic cells by iDCs. However, it is not known whether the complement system, also takes part in this process. In this study we investigated the ability of iDCs to bind to apoptotic cells opsonized by iC3b. Monocyte-derived dendritic cells were offered apoptotic Jurkat cells opsonized by autologous iC3b and labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanineperchlorate. A significant increase (P < 0.001) in the amount of cleared apoptotic cells was seen at low ratios. Despite increased efficiency of uptake, interaction between iC3b-opsonized apoptotic cells and iDCs down-regulated the expression of major histocompatibility complex class II, CD86, CC chemokine receptor (CCR)2, CCR5, and β2-integrins (P < 0.001), and up-regulated expression of CCR7 (P < 0.001). In addition, iDC maturation responses to CD40L and lipopolysaccharide were significantly inhibited. We conclude that opsonization of apoptotic cells by iC3b induces tolerant iDCs that are able to migrate to lymph nodes

Thrombospondin-1-N-Terminal Domain Induces a Phagocytic State and Thrombospondin-1-C-Terminal Domain Induces a Tolerizing Phenotype in Dendritic Cells

In our previous study, we have found that thrombospondin-1 (TSP-1) is synthesized de novo upon monocyte and neutrophil apoptosis, leading to a phagocytic and tolerizing phenotype of dendritic cells (DC), even prior to DC-apoptotic cell interaction. Interestingly, we were able to show that heparin binding domain (HBD), the N-terminal portion of TSP-1, was cleaved and secreted simultaneously in a caspase- and serine protease- dependent manner. In the current study we were interested to examine the role of HBD in the clearance of apoptotic cells, and whether the phagocytic and tolerizing state of DCs is mediated by the HBD itself, or whether the entire TSP-1 is needed. Therefore, we have cloned the human HBD, and compared its interactions with DC to those with TSP-1. Here we show that rHBD by itself is not directly responsible for immune paralysis and tolerizing phenotype of DCs, at least in the monomeric form, but has a significant role in rendering DCs phagocytic. Binding of TSP-1-C-terminal domain on the other hand induces a tolerizing phenotype in dendritic cells

CR3 is the dominant phagocytotic complement receptor on human dendritic cells.

Dendritic cells (DCs) play a decisive role in immunity; they interact with various pathogens via several pattern recognition and different opsonophagocytotic receptors, including Fc- and complement-receptors. beta2-integrins, including complement receptors CR3 (CD11b/CD18) and CR4 (CD11c/CD18) participate in many immunological processes, especially those involving cell migration, adherence, and phagocytosis. Human monocyte derived dendritic cells (MDCs) are known to express CR3 as well as CR4, however possible differences regarding the role of these receptors has not been addressed so far. Our aim was to explore whether there is a difference between the binding and uptake of various complement-opsonized microorganisms, mediated by CR3 and CR4. Studying the expression of receptors during differentiation of MDCs we found that the appearance of CD11b decreased, whereas that of CD11c increased. Interestingly, both receptors were present in the cell membrane in an active conformation. Here we demonstrate that ligation of CD11b directs MDCs to enhanced phagocytosis, while the maturation of the cells and their inflammatory cytokine production are not affected. Blocking CD11c alone did not change the uptake of opsonized yeast or bacteria by MDCs. We confirmed these results using siRNA; namely downregulation of CD11b blocked the phagocytosis of microbes while silencing CD11c had no effect on their uptake. Our data clearly demonstrate that complement C3-dependent phagocytosis of MDCs is mediated mainly by CR3

The Induction of APC with a Distinct Tolerogenic Phenotype via Contact-Dependent STAT3 Activation

BACKGROUND: Activation of the signal transducer and activator of transcription 3 (STAT3) within antigen presenting cells (APCs) is linked to abnormal APCs differentiation and function. We have previously shown that STAT3 is activated within APC by a novel contact-dependent mechanism, which plays a key role in mediating the immunomodulatory effects of hMSC. In order to better understand the underlying mechanisms that control APC maturation in a contact dependent manner, we extended our observation to tumor cells. Tumors were shown to secrete a variety of tumor-derived factors that activate STAT3 within infiltrating APCs. We now tested whether tumor cells can activate STAT3 within APC using the contact-dependent mechanism, in addition to soluble factors, and compared these two STAT3 activating pathways. PRINCIPAL FINDINGS: We demonstrate that in addition to tumor-derived secreted factors tumor cells activate STAT3 by a mechanism that is based on cell-cell interaction. We further demonstrate that these two STAT3 activating mechanisms differ in their JAK usage and their susceptibility to JSI-124 inhibition thereby representing two distinct pathways. Significantly, although both pathways activate STAT3, they modulate DCs maturation in a different manner that results in disparate phenotypic outcomes. Whereas the soluble-dependent pathway results in an immature phenotype, the contact-dependent pathway results in an apparently mature phenotype. Albeit their mature-like phenotype these latter cells express the tolerogenic markers ILT3 and ILT4 and possess T cell inhibitory activity. SIGNIFICANCE: This data suggests that, in at least certain cellular microenvironments, cell:cell interactions represent a novel way to activate STAT3 signaling, uncouple APC activation events and consequently regulate immunity and tolerance. Significantly, we have now demonstrated that this contact-dependent signaling pathway differs from that mediated by soluble factors and cytokines, inducing disparate phenotypic outcome, suggesting these two mechanisms have different and possibly complementary biological functions

L14. Immunomodulatory properties of apoptotic cells.

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Acquisition and presentation of follicular dendritic cell–bound antigen by lymph node–resident dendritic cells

Lymph node–resident dendritic cells sample antigen from follicular dendritic cells for presentation to CD8+ T cells

Dissociation between Mature Phenotype and Impaired Transmigration in Dendritic Cells from Heparanase-Deficient Mice

To reach the lymphatics, migrating dendritic cells (DCs) need to interact with the extracellular matrix (ECM). Heparanase, a mammalian endo-β-D-glucuronidase, specifically degrades heparan sulfate proteoglycans ubiquitously associated with the cell surface and ECM. The role of heparanase in the physiology of bone marrow-derived DCs was studied in mutant heparanase knock-out (Hpse-KO) mice. Immature DCs from Hpse-KO mice exhibited a more mature phenotype; however their transmigration was significantly delayed, but not completely abolished, most probably due to the observed upregulation of MMP-14 and CCR7. Despite their mature phenotype, uptake of beads was comparable and uptake of apoptotic cells was more efficient in DCs from Hpse-KO mice. Heparanase is an important enzyme for DC transmigration. Together with CCR7 and its ligands, and probably MMP-14, heparanase controls DC trafficking

The versatile functions of complement C3-derived ligands

The complement system is a major component of immune defense. Activation of the complement cascade by foreign substances and altered self-structures may lead to the elimination of the activating agent, and during the enzymatic cascade, several biologically active fragments are generated. Most immune regulatory effects of complement are mediated by the activation products of C3, the central component. The indispensable role of C3 in opsonic phagocytosis as well as in the regulation of humoral immune response is known for long, while the involvement of complement in T-cell biology have been revealed in the past few years. In this review, we discuss the immune modulatory functions of C3-derived fragments focusing on their role in processes which have not been summarized so far. The importance of locally synthesized complement will receive special emphasis, as several immunological processes take place in tissues, where hepatocyte-derived complement components might not be available at high concentrations. We also aim to call the attention to important differences between human and mouse systems regarding C3-mediated processes. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Lt

In Situ-Targeting of Dendritic Cells with Donor-Derived Apoptotic Cells Restrains Indirect Allorecognition and Ameliorates Allograft Vasculopathy

Chronic allograft vasculopathy (CAV) is an atheromatous-like lesion that affects vessels of transplanted organs. It is a component of chronic rejection that conventional immuno-suppression fails to prevent, and is a major cause of graft loss. Indirect allo-recognition through T cells and allo-Abs are critical during CAV pathogenesis. We tested whether the indirect allo-response and its impact on CAV is down-regulated by in situ-delivery of donor Ags to recipient's dendritic cells (DCs) in lymphoid organs in a pro-tolerogenic fashion, through administration of donor splenocytes undergoing early apoptosis. Following systemic injection, donor apoptotic cells were internalized by splenic CD11chi CD8α+ and CD8− DCs, but not by CD11cint plasmacytoid DCs. Those DCs that phagocytosed apoptotic cells in vivo remained quiescent, resisted ex vivo-maturation, and presented allo-Ag for up to 3 days. Administration of donor apoptotic splenocytes, unlike cells alive, (i) promoted deletion, FoxP3 expression and IL-10 secretion, and decreased IFN-γ-release in indirect pathway CD4 T cells; and (ii) reduced cross-priming of anti-donor CD8 T cells in vivo. Targeting recipient's DCs with donor apoptotic cells reduced significantly CAV in a fully-mismatched aortic allograft model. The effect was donor specific, dependent on the physical characteristics of the apoptotic cells, and was associated to down-regulation of the indirect type-1 T cell allo-response and secretion of allo-Abs, when compared to recipients treated with donor cells alive or necrotic. Down-regulation of indirect allo-recognition through in situ-delivery of donor-Ag to recipient's quiescent DCs constitutes a promising strategy to prevent/ameliorate indirect allorecognition and CAV

Apoptotic cell-based therapies against transplant rejection: role of recipient’s dendritic cells

One of the ultimate goals in transplantation is to develop novel therapeutic methods for induction of donor-specific tolerance to reduce the side effects caused by the generalized immunosuppression associated to the currently used pharmacologic regimens. Interaction or phagocytosis of cells in early apoptosis exerts potent anti-inflammatory and immunosuppressive effects on antigen (Ag)-presenting cells (APC) like dendritic cells (DC) and macrophages. This observation led to the idea that apoptotic cell-based therapies could be employed to deliver donor-Ag in combination with regulatory signals to recipient’s APC as therapeutic approach to restrain the anti-donor response. This review describes the multiple mechanisms by which apoptotic cells down-modulate the immuno-stimulatory and pro-inflammatory functions of DC and macrophages, and the role of the interaction between apoptotic cells and APC in self-tolerance and in apoptotic cell-based therapies to prevent/treat allograft rejection and graft-versus-host disease in murine experimental systems and in humans. It also explores the role that in vivo-generated apoptotic cells could have in the beneficial effects of extracorporeal photopheresis, donor-specific transfusion, and tolerogenic DC-based therapies in transplantation