Cutting edge: Autocrine TGF-beta sustains default tolerogenesis by IDO-competent dendritic cells

CD8(-) and CD8(+) dendritic cells (DCs) are distinct subsets of mouse splenic accessory cells with opposite but flexible programs of Ag presentation, leading to immunogenic and tolerogenic responses, respectively. In this study, we show that the default tolerogenic function of CD8(+) DCs relies on autocrine TGF-beta, which sustains the activation of IDO in response to environmental stimuli. CD8(-) DCs do not produce TGF-beta, yet externally added TGF-beta induces IDO and turns those cells from immunogenic into tolerogenic cells. The acquisition of a suppressive phenotype by CD8(-) DCs correlates with activation of the PI3K/Akt and noncanonical NF-kappaB pathways. These data are the first to link TGF-beta signaling with IDO in controlling spontaneous tolerogenesis by DCs.     

Transfer of regulatory properties from tolerogenic to proinflammatory dendritic cells via induced autoreactive regulatory T cells

Infectious tolerance is a term generally assigned to the process through which regulatory T cells (Tregs) transfer immunoregulatory properties to other T cells. In this study, we demonstrated that a similar process applies to human dendritic cells (DCs), albeit through a different mechanism. We induced and cloned proinsulin-specific Tregs using tolerogenic DCs and investigated mechanisms by which induced Ag-specific regulatory T cells (iaTregs) endorse the suppressive effects. iaTregs expressed FOXP3, programmed death-1, and membrane-bound TGF-β and upregulated IL-10 and CTLA-4 after stimulation with the cognate Ag. The iaTregs suppressed effector T cells only when both encountered the cognate Ags on the same APCs (linked suppression). This occurred independently of IL-10, TGF-β, programmed death-1, or CTLA-4. Instead, iaTregs used a granzyme B-mediated mechanism to kill B cells and monocytes, whereas proinflammatory DCs that resisted being killed were induced to upregulate the inhibitory receptors B7 (family) homolog 3 and ICOS ligand. These re-educated mature monocyte-derived dendritic cells (mDCs) suppressed effector T cells and induced IL-10-producing cells from the naive T cell pool. Our data indicated that human tolerogenic DCs confer infectious tolerance by inducing Ag-specific Tregs, which, in turn, re-educate proinflammatory mature DCs into DCs with regulatory properties.     

The neuropeptide vasoactive intestinal peptide generates tolerogenic dendritic cells

Tolerogenic dendritic cells (DCs) play an important role in maintaining peripheral tolerance through the induction/activation of regulatory T cells (Treg). Endogenous factors contribute to the functional development of tolerogenic DCs. In this report, we present evidence that two known immunosuppressive neuropeptides, the vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP), contribute to the development of bone marrow-derived tolerogenic DCs in vitro and in vivo. The VIP/PACAP-generated DCs are CD11c(low)CD45RB(high), do not up-regulate CD80, CD86, and CD40 following LPS stimulation, and secrete high amounts of IL-10. The induction of tolerogenic DCs is mediated through the VPAC1 receptor and protein kinase A, and correlates with the inhibition of IkappaB phosphorylation and of NF-kappaBp65 nuclear translocation. The VIP/PACAP-generated DCs induce functional Treg in vitro and in vivo. The VIP/DC-induced Treg resemble the previously described Tr1 in terms of phenotype and cytokine profile, suppress primarily Th1 responses including delayed-type hypersensitivity, and transfer suppression to naive hosts. The effect of VIP/PACAP on the DC-Treg axis represents an additional mechanism for their general anti-inflammatory role, particularly in anatomical sites which exhibit immune deviation or privilege.     

Short-term lipopolysaccharide stimulation induces differentiation of murine bone marrow-derived dendritic cells into a tolerogenic phenotype

Dendritic cells (DCs) are professional, antigen-presenting cells, which induce and regulate T cell reactivity. DCs are crucial in innate and adaptive immune responses, and are also involved in central and peripheral tolerance induction. Tolerance can be mediated by immature and semi-mature DCs expressing low levels of co-stimulator and major histocompatibility complex (MHC) molecules. The aim of this study was to investigate the ability of short-term lipopolysaccharide (LPS) stimulation to modulate the stage of differentiation of bone marrow-derived DCs. For this purpose, DCs obtained from DBA1/lacJ mice were stimulated for four (4hLPS/DCs) or 24 (24hLPS/DCs) hours with LPS, using DCs without stimulation (0hLPS/DCs) as a control. Flow cytometry analysis of 4hLPS/DCs showed intermediate CD40 and MHC class II expression, lower than that of 24hLPS/DCs (fully mature), and greater than that of 0hLPS/DCs (immature). A functional assay showed that 4hLPS/DCs displayed increased endocytotic ability compared to 24hLPS/DCs, indicating a semi-mature state. 4hLPS/DCs were greater producers of IL-10 protein and TGFbeta1 mRNA than 24hLPS/DCs and immature DCs, displaying a cytokine production pattern that is characteristic of tolerogenic DCs. An assay for antigen-presenting capacity demonstrated that 4hLPS/DCs induced secretion of IL-2 from an OTH4 T cell hybridoma, indicating a functional presenting activity. Finally, the tolerogenic phenotype of 4hLPS/DCs was demonstrated by their ability to interfere with the progression of bovine type II collagen (bII)-induced arthritis (CIA) when they were loaded with bCII antigen and injected into mice with established CIA. We conclude that the stimulation of murine bone marrow-derived DCs with LPS for four hours generates semi-mature DCs with tolerogenic capability.     

PI16 is expressed by a subset of human memory Treg with enhanced migration to CCL17 and CCL20

It is well known that adoptive transfer of donor-derived tolerogenic dendritic cells (DCs) helps to induce immune tolerance. RelB, one of NF-κB subunits, is a critical element involved in DC maturation. In the present study, our results showed tolerogenic DCs could be acquired via silencing RelB using small interfering RNA. Compared with imDCs, the tolerogenic DCs had more potent ability to inhibit mixed lymphocyte reaction (MLR) and down-regulate Th1 cytokines and prompt the production of Th2 cytokines. They both mediated immune tolerance via the increased of T cell apoptosis and generation of regulatory T cells. Administration of donor-derived tolerogenic DCs significantly prevented the allograft rejection and prolonged the survival time in a murine heart transplantation model. Our results demonstrate donor-derived, RelB-shRNA induced tolerogenic DCs can significantly induce immune tolerance in vitro and in vivo.     

Low-affinity, Smith antigen-specific B cells are tolerized by dendritic cells and macrophages

Polyclonal B cell activation promotes immunity without the loss of tolerance. Our data show that during activation of the innate immune system, B cell tolerance to Smith Ag Sm is maintained by dendritic cells (DCs) and macrophages (MPhi). TLR4-activated myeloid DCs and MPhi, but not plasmacytoid or lymphoid DCs, repressed autoreactive B cells through the secretion of soluble mediators, including IL-6. Although IL-6 promotes plasma cell differentiation of B cells acutely stimulated by Ag, we show that it repressed cells that were chronically exposed to self-Ag. This mechanism of tolerance was not limited to Smith Ag-specific B cells as hen egg lysozyme- and p-azophenylarsonate-specific B cells were similarly affected. Our data define a tolerogenic role for MPhi and DCs in regulating autoreactive B cells during activation of the innate immune system.     

Role of dendritic cells in the induction of regulatory T cells

Dendritic cells (DCs) play a key role in initiating immune responses and maintaining immune tolerance. In addition to playing a role in thymic selection, DCs play an active role in tolerance under steady state conditions through several mechanisms which are dependent on IL-10, TGF-β, retinoic acid, indoleamine-2,3,-dioxygenase along with vitamin D. Several of these mechanisms are employed by DCs in induction of regulatory T cells which are comprised of Tr1 regulatory T cells, natural and inducible foxp3+ regulatory T cells, Th3 regulatory T cells and double negative regulatory T cells. It appears that certain DC subsets are highly specialized in inducing regulatory T cell differentiation and in some tissues the local microenvironment plays a role in driving DCs towards a tolerogenic response. In this review we discuss the recent advances in our understanding of the mechanisms underlying DC driven regulatory T cell induction.     

Dynamics of dendritic cell phenotype and interactions with CD4+ T cells in airway inflammation and tolerance

An emerging concept is that different types of dendritic cells (DCs) initiate different immune outcomes, such as tolerance vs inflammation. In this study, we have characterized the DCs from the lung draining lymph nodes of mice immunized for allergic airway inflammation or tolerance and examined their interactions with CD4(+) T cells. The DC population derived from tolerized mice was predominantly CD11c(+), B220(+), Gr-1(+), CD11b(-), and MHC class II(low), which resembled plasmacytoid-type DCs whereas DCs from the inflammatory condition were largely CD11c(+), B220(-), Gr-1(-), CD11b(+), and MHC class II(high) resembling myeloid-type DCs. The DCs from the tolerogenic condition were poor inducers of T cell proliferation. DCs from both conditions induced T cell IL-4 production but the T cells cultured with tolerogenic DCs were unresponsive to IL-4 as indicated by inhibition of STAT6 activation and expression of growth factor-independent 1, which has been recently shown to be important for STAT6-activated Th2 cell expansion. Our data suggest that airway tolerance vs inflammation is determined by the DC phenotype in lung draining lymph nodes.     

Cytotoxic dendritic cells generated from cancer patients

Known for years as professional APCs, dendritic cells (DCs) are also endowed with tumoricidal activity. This dual role of DC as killers and messengers may have important implications for tumor immunotherapy. However, the tumoricidal activity of DCs has mainly been investigated in animal models. Cancer cells inhibit antitumor immune responses using numerous mechanisms, including the induction of immunosuppressive/ tolerogenic DCs that have lost their ability to present Ags in an immunogenic manner. In this study, we evaluated the possibility of generating tumor killer DCs from patients with advanced-stage cancers. We demonstrate that human monocyte-derived DCs are endowed with significant cytotoxic activity against tumor cells following activation with LPS. The mechanism of DC-mediated tumor cell killing primarily involves peroxynitrites. This observed cytotoxic activity is restricted to immature DCs. Additionally, after killing, these cytotoxic DCs are able to activate tumor Ag-specific T cells. These observations may open important new perspectives for the use of autologous cytotoxic DCs in cancer immunotherapy strategies.     

Functional role of P-selectin glycoprotein ligand 1/P-selectin interaction in the generation of tolerogenic dendritic cells

Dendritic cells (DCs) have a key role in both the generation of the immune response and the induction of tolerance to self-Ags. In this work, the possible role of P-selectin glycoprotein ligand 1 (PSGL-1) on the tolerogenic activity of human DCs was explored. We found that the engagement of PSGL-1 by P-selectin on DCs induced the expression of c-Fos, IDO, IL-10, and TGF-beta genes. Remarkably, stimulation of DCs through PSGL-1 with P-selectin enhanced their capability to generate CD4(+)CD25(+)Foxp3(+) regulatory T cells, which expressed high levels of TGF-beta1 mRNA, synthesized IL-10, and suppressed the proliferation of autologous CD4(+)CD25(-) T cells. Accordingly, we found that DCs from PSGL-1(-/-) mice expressed higher levels of MHC class II molecules, and exhibited an enhanced immunogenicity compared with wild-type mice. In addition, the percentage of CD4(+)CD25(+)Foxp3(+) regulatory T cells in the thymus of PSGL-1-deficient animals was significantly reduced. Our data reveal an unexpected role of PSGL-1 on the tolerogenic function of DCs, and the regulation of the immune response.     

On/off TLR signaling decides proinflammatory or tolerogenic dendritic cell maturation upon CD1d-mediated interaction with invariant NKT cells

Invariant NKT (iNKT) cells play an effector/adjuvant function during antimicrobial and antitumoral immunity and a regulatory role to induce immune tolerance and prevent autoimmunity. iNKT cells that differentially modulate adaptive immunity do not bear a unique phenotype and/or specific cytokine secretion profile, thus opening questions on how a single T cell subset can exert opposite immunological tasks. In this study, we show that iNKT cells perform their dual roles through a single mechanism of action relying on the cognate interaction with myeloid dendritic cells (DCs) and leading to opposite effects depending on the presence of other maturation stimuli simultaneously acting on DCs. The contact of murine purified iNKT cells with immature autologous DCs directly triggers the tolerogenic maturation of DCs, rendering them able to induce regulatory T cell differentiation and prevent autoimmune diabetes in vivo. Conversely, the interaction of the same purified iNKT cells with DCs, in the presence of simultaneous TLR4 stimulation, significantly enhances proinflammatory DC maturation and IL-12 secretion. The different iNKT cell effects are mediated through distinct mechanisms and activation of different molecular pathways within the DC: CD1d signaling and activation of the ERK1/2 pathway for the tolerogenic action, and CD40-CD40L interaction and NF-κB activation for the adjuvant effect. Our data suggest that the DC decision to undergo proinflammatory or tolerogenic maturation results from the integration of different signals received at the time of iNKT cell contact and could have important therapeutic implications for exploiting iNKT cell adjuvant/regulatory properties in autoimmune diseases, infections, and cancer.     

Dendritic cells as key targets for immunomodulation by Vitamin D receptor ligands

Vitamin D receptor (VDR) ligands, in addition to controlling calcium metabolism, exert important effects on the growth and differentiation of many cell types and possess pronounced pro-tolerogenic immunoregulatory activities. VDR ligands can act directly on T cells, but antigen-presenting cells (APCs), and in particular dendritic cells (DCs), appear to be primary targets for their tolerogenic properties. The capacity of VDR ligands to target APCs and T cells is mediated by VDR expression in both cell types and by the presence of common targets in their signal transduction pathways, such as the nuclear factor NF-kB that is down-regulated in APCs and in T cells. VDR ligands can induce in vitro and in vivo tolerogenic DCs able to enhance CD4(+)CD25(+) suppressor T cells that, in turn, inhibit Th1 cell responses. These mechanisms of action can explain some of the immunoregulatory properties of VDR ligands, and are potentially relevant for the treatment of Th1-mediated autoimmune diseases and allograft rejection.     

Steady state dendritic cells present parenchymal self-antigen and contribute to, but are not essential for, tolerization of naive and Th1 effector CD4 cells

Bone marrow-derived APC are critical for both priming effector/memory T cell responses to pathogens and inducing peripheral tolerance in self-reactive T cells. In particular, dendritic cells (DC) can acquire peripheral self-Ags under steady state conditions and are thought to present them to cognate T cells in a default tolerogenic manner, whereas exposure to pathogen-associated inflammatory mediators during the acquisition of pathogen-derived Ags appears to reprogram DCs to prime effector and memory T cell function. Recent studies have confirmed the critical role of DCs in priming CD8 cell effector responses to certain pathogens, although the necessity of steady state DCs in programming T cell tolerance to peripheral self-Ags has not been directly tested. In the current study, the role of steady state DCs in programming self-reactive CD4 cell peripheral tolerance was assessed by combining the CD11c-diphtheria toxin receptor transgenic system, in which DC can be depleted via treatment with diphtheria toxin, with a TCR-transgenic adoptive transfer system in which either naive or Th1 effector CD4 cells are induced to undergo tolerization after exposure to cognate parenchymally derived self-Ag. Although steady state DCs present parenchymal self-Ag and contribute to the tolerization of cognate naive and Th1 effector CD4 cells, they are not essential, indicating the involvement of a non-DC tolerogenic APC population(s). Tolerogenic APCs, however, do not require the cooperation of CD4(+)CD25(+) regulatory T cells. Similarly, DC were required for maximal priming of naive CD4 cells to vaccinia viral-Ag, but priming could still occur in the absence of DC.     

Estriol generates tolerogenic dendritic cells in vivo that protect against autoimmunity

Chronic inflammation contributes to numerous diseases, and regulation of inflammation is crucial for disease control and resolution. Sex hormones have potent immunoregulatory abilities. Specifically, estrogen influences immune cells and inflammation, which contributes to the sexual dimorphism of autoimmunity and protection against disease seen during pregnancy in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Although long thought to act primarily on T cells, recent evidence demonstrated that myeloid cells, such as dendritic cells (DCs), are essential in mediating estrogen's protective effects. Estriol (E3), a pregnancy-specific estrogen, has therapeutic efficacy in MS and EAE, and we evaluated whether E3 could act exclusively through DCs to protect against the inflammatory autoimmune disease EAE. Levels of activation markers (CD80 and CD86) and inhibitory costimulatory markers (PD-L1, PD-L2, B7-H3, and B7-H4) were increased in E3 DCs. E3 DCs had decreased proinflammatory IL-12, IL-23, and IL-6 mRNA expression, increased immunoregulatory IL-10 and TGF-β mRNA expression, and a decreased ratio of IL-12/IL-10 protein production. Importantly, transfer of E3 DCs to mice prior to active induction of EAE protected them from developing EAE through immune deviation to a Th2 response. This protection was apparent, even in the face of in vitro and in vivo inflammatory challenge. In summary, our results showed that E3 generates tolerogenic DCs, which protect against the inflammatory autoimmune disease EAE. Targeted generation of tolerogenic DCs with immunomodulatory therapeutics, such as E3, has potential applications in the treatment of numerous autoimmune and chronic inflammatory diseases.     

Functional plasticity of dendritic cell subsets as mediated by CD40 versus B7 activation

Murine dendritic cells (DCs) can present Ag in an immunogenic or tolerogenic fashion, the distinction depending on either the occurrence of specialized DC subsets or the maturation or activation state of the DC. Although DC subsets may be programmed to direct either tolerance or immunity, it is not known whether appropriate environmental stimulation can result in complete flexibility of a basic program. Using splenic CD8(-) and CD8(+) DCs that mediate the respective immunogenic and tolerogenic presentation of self peptides, we show that both the in vivo and in vitro activities of either subset can be altered by ligation of specific surface receptors. Otherwise immunogenic CD8(-) DCs become tolerogenic upon B7 ligation by soluble CTLA-4, a maneuver that initiates immunosuppressive tryptophan catabolism. In contrast, CD40 ligation on tolerogenic CD8(+) DCs makes these cells capable of immunogenic presentation. Thus, environmental conditioning by T cell ligands may alter the default function of DC subsets to meet the needs of flexibility and redundancy.     

Dendritic cells transduced with SOCS-3 exhibit a tolerogenic/DC2 phenotype that directs type 2 Th cell differentiation in vitro and in vivo

Dendritic cells (DCs) have been suggested to direct a type of Th differentiation through their cytokine profile, e.g., high IL-12/IL-23 for Th1 (named DC1/immunogenic DCs) and IL-10 for Th2 (DC2/tolerogenic DCs). Suppressor of cytokine signaling (SOCS)-3 is a potent inhibitor of Stat3 and Stat4 transduction pathways for IL-23 and IL-12, respectively. We thus hypothesize that an enhanced SOCS-3 expression in DCs may block the autocrine response of IL-12/IL-23 in these cells, causing them to become a DC2-type phenotype that will subsequently promote Th2 polarization of naive T cells. Indeed, in the present study we found that bone marrow-derived DCs transduced with SOCS-3 significantly inhibited IL-12-induced activation of Stat4 and IL-23-induced activation of Stat3. These SOCS-3-transduced DCs expressed a low level of MHC class II and CD86 on their surface, produced a high level of IL-10 but low levels of IL-12 and IFN-gamma, and expressed a low level of IL-23 p19 mRNA. Functionally, SOCS-3-transduced DCs drove naive myelin oligodendrocyte glycoprotein-specific T cells to a strong Th2 differentiation in vitro and in vivo. Injection of SOCS-3-transduced DCs significantly suppressed experimental autoimmune encephalomyelitis, a Th1 cell-mediated autoimmune disorder of the CNS and an animal model of multiple sclerosis. These results indicate that transduction of SOCS-3 in DCs is an effective approach to generating tolerogenic/DC2 cells that then skew immune response toward Th2, thus possessing therapeutic potential in Th1-dominant autoimmune disorders such as multiple sclerosis.