Vasoactive intestinal peptide induces regulatory dendritic cells with therapeutic effects on autoimmune disorders

The induction of antigen-specific tolerance is critical for the prevention of autoimmunity and maintenance of immune tolerance. In addition to their classical role as sentinels of the immune response-inducing T cell reactivity, dendritic cells (DCs) play an important role in maintaining peripheral tolerance through the induction/activation of regulatory T cells (Tr). The possibility to generate tolerogenic DCs opens new therapeutic perspectives in autoimmune/inflammatory diseases. Therefore, the characterization of the endogenous factors that contribute to the development of tolerogenic DCs is highly relevant. In this study, we report on the use of the known immunosuppressive neuropeptide, the vasoactive intestinal peptide, as a new approach to induce tolerogenic DCs with capacity to generate Tr cells, to restore tolerance in vivo, and to reduce the progression of rheumatoid arthritis and experimental autoimmune encephalomyelitis.     

Selective delivery of beta cell antigen to dendritic cells in vivo leads to deletion and tolerance of autoreactive CD8+ T cells in NOD mice

Type 1 diabetes (T1D) is an autoimmune disease resulting from defects in central and peripheral tolerance and characterized by T cell-mediated destruction of islet beta cells. Cytotoxic CD8(+) T cells, reactive to beta cell antigens, are required for T1D development in the NOD mouse model of the disease, and CD8(+) T cells specific for beta cell antigens can be detected in the peripheral blood of T1D patients. It has been evident that in nonautoimmune-prone mice, dendritic cells (DCs) present model antigens in a tolerogenic manner in the steady state, e.g., in the absence of infection, and cause T cells to proliferate initially but then to be deleted or rendered unresponsive. However, this fundamental concept has not been evaluated in the setting of a spontaneous autoimmune disease. To do so, we delivered a mimotope peptide, recognized by the diabetogenic CD8(+) T cell clone AI4, to DCs in NOD mice via the endocytic receptor DEC-205. Proliferation of transferred antigen-specific T cells was initially observed, but this was followed by deletion. Tolerance was achieved because rechallenge of mice with the mimotope peptide in adjuvant did not induce an immune response. Thus, targeting of DCs with beta cell antigens leads to deletion of autoreactive CD8(+) T cells even in the context of ongoing autoimmunity in NOD mice with known tolerance defects. Our results provide support for the development of DC targeting of self antigens for treatment of chronic T cell-mediated autoimmune diseases.     

Interleukin-10-treated human dendritic cells induce a melanoma-antigen-specific anergy in CD8(+) T cells resulting in a failure to lyse tumor cells

Dendritic cells (DC) are critically involved in the initiation of primary immune processes, including tumor rejection. In our study, we investigated the effect of interleukin-10 (IL-10)-treated human DC on the properties of CD8(+) T cells that are known to be essential for the destruction of tumor cells. We show that IL-10-pretreatment of DC not only reduces their allostimulatory capacity, but also induces a state of alloantigen-specific anergy in both primed and naive (CD45RA+) CD8(+) T cells. To investigate the influence of IL-10-treated DC on melanoma-associated antigen-specific T cells, we generated a tyrosinase-specific CD8(+) T-cell line by several rounds of stimulation with the specific antigen. After coculture with IL-10-treated DC, restimulation of the T-cell line with untreated, antigen-pulsed DC demonstrated peptide-specific anergy in the tyrosinase-specific T cells. Addition of IL-2 to the anergic T cells reversed the state of both alloantigen- or peptide-specific anergy. In contrast to optimally stimulated CD8(+) T cells, anergic tyrosinase-specific CD8(+) T cells, after coculture with peptide-pulsed IL-10-treated DC, failed to lyse an HLA-A2-positive and tyrosinase-expressing melanoma cell line. Thus, our data demonstrate that IL-10-treated DC induce an antigen-specific anergy in cytotoxic CD8(+) T cells, a process that might be a mechanism of tumors to inhibit immune surveillance by converting DC into tolerogenic antigen-presenting cells.     

CD4(+) and CD8(+) anergic T cells induced by interleukin-10-treated human dendritic cells display antigen-specific suppressor activity

Interleukin-10 (IL-10)-treated dendritic cells (DCs) induce an alloantigen- or peptide-specific anergy in various CD4(+) and CD8(+) T-cell populations. In the present study, we analyzed whether these anergic T cells are able to regulate antigen-specific immunity. Coculture experiments revealed that alloantigen-specific anergic CD4(+) and CD8(+) T cells suppressed proliferation of syngeneic T cells in a dose-dependent manner. The same effect was observed when the hemagglutinin-specific CD4(+) T-cell clone HA1.7 or tyrosinase-specific CD8(+) T cells were cocultured with anergic T cells of the same specificity. Anergic T cells did not induce an antigen-independent bystander inhibition. Suppression was dependent on cell-to-cell contact between anergic and responder T cells, required activation by antigen-loaded DCs, and was not mediated by supernatants of anergic T cells. Furthermore, anergic T cells displayed an increased extracellular and intracellular expression of cytotoxic T-lymphocyte antigen (CTLA)-4 molecules, and blocking of the CTLA-4 pathway restored the T-cell proliferation up to 70%, indicating an important role of the CTLA-4 molecule in the suppressor activity of anergic T cells. Taken together, our experiments demonstrate that anergic T cells induced by IL-10-treated DCs are able to suppress activation and function of T cells in an antigen-specific manner. Induction of anergic T cells might be exploited therapeutically for suppression of cellular immune responses in allergic or autoimmune diseases with identified (auto) antigens.     

Regulatory dendritic cells protect against cutaneous chronic graft-versus-host disease mediated through CD4+CD25+Foxp3+ regulatory T cells

Chronic graft-versus-host disease (cGVHD) is a common cause of morbidity and mortality in allogeneic bone marrow transplantation (alloBMT). However, effective strategies for the treatment of cGVHD have not been established. In this study, we examined the therapeutic utility of modified dendritic cells (DCs) with a greater capacity to regulate immune responses than previously known tolerogenic DCs, regulatory DCs (DC(regs)), in the major histocompatibility complex-compatible, and multiple minor histocompatibility antigen-incompatible model of cGVHD in alloBMT. Treatment of the recipient mice after alloBMT with the recipient-type DC(regs) led to greater suppression of the incidence and severity of cutaneous cGVHD than rapamycin, whereas treatment with the recipient-type mature DCs promoted the pathogenesis. Analysis of the recipient mice suggested that the protective effect of the recipient-type DC(regs) involved the peripheral generation of alloreactive CD4(+)CD25(+)Foxp3(+)regulatory T (T(R)) cells from donor-derived CD4(+)CD25(-)Foxp3(-) T cells. Thus, immunotherapy with DC(regs) is a promising strategy for the treatment of cGVHD in alloBMT mediated through the induction of a dominant tolerance involving CD4(+)CD25(+)Foxp3(+) T(R) cells.     

Therapeutic treatment of experimental colitis with regulatory dendritic cells generated with vasoactive intestinal peptide

BACKGROUND & AIMS: Crohn's disease is a chronic debilitating disease characterized by severe T helper cell (Th)1-driven inflammation of the colon partially caused by a loss of immune tolerance against mucosal antigens. The use of regulatory dendritic cells (DCs) with the capacity to induce regulatory T cells has been proposed recently for the treatment of Crohn's disease in a strategy to restore immune tolerance. Vasoactive intestinal peptide is an immunomodulatory neuropeptide that induces regulatory DCs. The aim of this study was to investigate the therapeutic effect of vasoactive intestinal peptide-induced regulatory DCs (DC(VIP)) in a murine model of colitis. METHODS: We examined the therapeutic action of DC(VIP) in the colitis induced by intracolonic administration of trinitrobenzene sulfonic acid, evaluating diverse clinical signs of the disease including weight loss, diarrhea, colitis, and histopathology. We also investigated the mechanisms involved in the potential therapeutic effect of DC(VIP), such as inflammatory cytokines and chemokines, Th1-type response, and the generation of regulatory T cells. RESULTS: DC(VIP) injection significantly ameliorated the clinical and histopathologic severity of colitis, abrogating body weight loss, diarrhea, and inflammation, and increasing survival. The therapeutic effect was associated with down-regulation of both inflammatory and Th1-driven autoimmune response, by regulating a wide spectrum of inflammatory mediators directly through activated macrophages, and by generating interleukin-10-secreting regulatory T cells with suppressive capacity on autoreactive T cells. CONCLUSIONS: The possibility to generate/expand ex vivo regulatory DC(VIP) opens new therapeutic perspectives for the treatment of Crohn's disease in human beings, and may minimize the dependence on nonspecific immunosuppressive drugs used currently for autoimmune disorders.     

Conditional ablation of CD205+ conventional dendritic cells impacts the regulation of T-cell immunity and homeostasis in vivo

Dendritic cells (DCs) are composed of multiple subsets that play a dual role in inducing immunity and tolerance. However, it is unclear how CD205(+) conventional DCs (cDCs) control immune responses in vivo. Here we generated knock-in mice with the selective conditional ablation of CD205(+) cDCs. CD205(+) cDCs contributed to antigen-specific priming of CD4(+) T cells under steady-state conditions, whereas they were dispensable for antigen-specific CD4(+) T-cell responses under inflammatory conditions. In contrast, CD205(+) cDCs were required for antigen-specific priming of CD8(+) T cells to generate cytotoxic T lymphocytes (CTLs) mediated through cross-presentation. Although CD205(+) cDCs were involved in the thymic generation of CD4(+) regulatory T cells (Tregs), they maintained the homeostasis of CD4(+) Tregs and CD4(+) effector T cells in peripheral and mucosal tissues. On the other hand, CD205(+) cDCs were involved in the inflammation triggered by Toll-like receptor ligand as well as bacterial and viral infections. Upon microbial infections, CD205(+) cDCs contributed to the cross-priming of CD8(+) T cells for generating antimicrobial CTLs to efficiently eliminate pathogens, whereas they suppressed antimicrobial CD4(+) T-cell responses. Thus, these findings reveal a critical role for CD205(+) cDCs in the regulation of T-cell immunity and homeostasis in vivo.     

Dendritic cells with TGF-beta1 differentiate naive CD4+CD25- T cells into islet-protective Foxp3+ regulatory T cells

CD4(+)CD25(+)Foxp3(+) regulatory T cells (T regs) are important for preventing autoimmune diabetes and are either thymic-derived (natural) or differentiated in the periphery outside the thymus (induced). Here we show that beta-cell peptide-pulsed dendritic cells (DCs) from nonobese diabetic (NOD) mice can effectively induce CD4(+)CD25(+)Foxp3(+) T cells from na?ve islet-specific CD4(+)CD25(-) T cells in the presence of TGF-beta1. These induced, antigen-specific T regs maintain high levels of clonotype-specific T cell receptor expression and exert islet-specific suppression in vitro. When cotransferred with diabetogenic cells into NOD scid recipients, T regs induced with DCs and TGF-beta1 prevent the development of diabetes. Furthermore, in overtly NOD mice, these cells are able to significantly protect syngeneic islet grafts from established destructive autoimmunity. These results indicate a role for DCs in the induction of antigen-specific CD4(+)CD25(+)Foxp3(+) T cells that can inhibit fully developed autoimmunity in a nonlymphopoenic host, providing an important potential strategy for immunotherapy in patients with autoimmune diabetes.     

Induction of antigen-specific regulatory T lymphocytes by human dendritic cells expressing the glucocorticoid-induced leucine zipper

Dendritic cells (DCs) determine whether antigen presentation leads to immune activation or to tolerance. Tolerance-inducing DCs (also called regulatory DCs) act partly by generating regulatory T lymphocytes (Tregs). The mechanism used by DCs to switch toward regulatory DCs during their differentiation is unclear. We show here that human DCs treated in vitro with glucocorticoids produce the glucocorticoid-induced leucine zipper (GILZ). Antigen presentation by GILZ-expressing DCs generates CD25(high)FOXP3(+)CTLA-4/CD152(+) and interleukin-10-producing Tregs inhibiting the response of CD4(+) and CD8(+) T lymphocytes. This inhibition is specific to the antigen presented, and only proliferating CD4(+) T lymphocytes express the Treg markers. Interleukin-10 is required for Treg induction by GILZ-expressing DCs. It is also needed for the suppressive function of Tregs. Antigen-presenting cells from patients treated with glucocorticoids generate interleukin-10-secreting Tregs ex vivo. These antigen-presenting cells produce GILZ, which is needed for Treg induction. Therefore, GILZ is critical for commitment of DCs to differentiate into regulatory DCs and to the generation of antigen-specific Tregs. This mechanism may contribute to the therapeutic effects of glucocorticoids.     

Identification and expansion of highly suppressive CD8(+)FoxP3(+) regulatory T cells after experimental allogeneic bone marrow transplantation

FoxP3(+) confers suppressive properties and is confined to regulatory T cells (T(reg)) that potently inhibit autoreactive immune responses. In the transplant setting, natural CD4(+) T(reg) are critical in controlling alloreactivity and the establishment of tolerance. We now identify an important CD8(+) population of FoxP3(+) T(reg) that convert from CD8(+) conventional donor T cells after allogeneic but not syngeneic bone marrow transplantation. These CD8(+) T(reg) undergo conversion in the mesenteric lymph nodes under the influence of recipient dendritic cells and TGF-β. Importantly, this population is as important for protection from GVHD as the well-studied natural CD4(+)FoxP3(+) population and is more potent in exerting class I-restricted and antigen-specific suppression in vitro and in vivo. Critically, CD8(+)FoxP3(+) T(reg) are exquisitely sensitive to inhibition by cyclosporine but can be massively and specifically expanded in vivo to prevent GVHD by coadministering rapamycin and IL-2 antibody complexes. CD8(+)FoxP3(+) T(reg) thus represent a new regulatory population with considerable potential to preferentially subvert MHC class I-restricted T-cell responses after bone marrow transplantation.     

Release of dendritic cells from cognate CD4+ T-cell recognition results in impaired peripheral tolerance and fatal cytotoxic T-cell mediated autoimmunity

Resting dendritic cells (DCs) induce tolerance of peripheral T cells that have escaped thymic negative selection and thus contribute significantly to protection against autoimmunity. We recently showed that CD4(+)Foxp3(+) regulatory T cells (Tregs) are important for maintaining the steady-state phenotype of DCs and their tolerizing capacity in vivo. We now provide evidence that DC activation in the absence of Tregs is a direct consequence of missing DC-Treg interactions rather than being secondary to generalized autoimmunity in Treg-less mice. We show that DCs that lack MHC class II and thus cannot make cognate interactions with CD4(+) T cells are completely unable to induce peripheral CD8(+) T-cell tolerance. Consequently, mice in which interactions between DC and CD4(+) T cells are not possible develop spontaneous and fatal cytotoxic T lymphocyte-mediated autoimmunity.     

The differential production of cytokines by human Langerhans cells and dermal CD14(+) DCs controls CTL priming

We recently reported that human epidermal Langerhans cells (LCs) are more efficient than dermal CD14(+) DCs at priming naive CD8(+) T cells into potent CTLs. We hypothesized that distinctive dendritic cell (DC) cytokine expression profiles (ie, IL-15 produced by LCs and IL-10 expressed by dermal CD14(+) DCs) might explain the observed functional difference. Blocking IL-15 during CD8(+) T-cell priming reduced T-cell proliferation by ～ 50%. These IL-15-deprived CD8(+) T cells did not acquire the phenotype of effector memory cells. They secreted less IL-2 and IFN-γ and expressed only low amounts of CD107a, granzymes and perforin, and reduced levels of the antiapoptotic protein Bcl-2. Confocal microscopy analysis showed that IL-15 is localized at the immunologic synapse of LCs and naive CD8(+) T cells. Conversely, blocking IL-10 during cocultures of dermal CD14(+) DCs and naive CD8(+) T cells enhanced the generation of effector CTLs, whereas addition of IL-10 to cultures of LCs and naive CD8(+) T cells inhibited their induction. TGF-β1 that is transcribed by dermal CD14(+) DCs further enhanced the inhibitory effect of IL-10. Thus, the respective production of IL-15 and IL-10 explains the contrasting effects of LCs and dermal CD14(+) DCs on CD8(+) T-cell priming.     

Interleukin-11 induces Th2 polarization of human CD4(+) T cells

Exploration of the immunomodulatory activities of the multifunctional cytokine interleukin-11 (IL-11) has prompted several therapeutic applications. The immunomodulatory effects of IL-11 on human antigen-presenting cells and on T cells were investigated. IL-11 inhibited IL-12 production by activated CD14(+) monocytes, but not by mature dendritic cells (DCs) stimulated via CD40 ligation. Moreover, IL-11 did not affect either DC maturation, as demonstrated by phenotypic analysis and evaluation of cytokine production, or DC generation from progenitor cells in the presence of specific growth factors. Molecular analysis demonstrated the expression of IL-11 receptor messenger RNA in highly purified CD14(+) monocytes, CD19(+) B cells, CD8(+), and CD4(+) T cells, and CD4(+)CD45RA(+) naive T lymphocytes. In keeping with this finding, IL-11 directly prevented Th1 polarization of highly purified CD4(+)CD45RA(+) naive T cells stimulated with anti-CD3/CD28 antibodies, as demonstrated by significant increases of IL-4 and IL-5, by significantly decreased interferon-gamma production and by flow cytometry intracellular staining of cytokines. Coincubation of naive T cells with DCs, the most potent stimulators of Th1 differentiation, did not revert IL-11-mediated Th2 polarization. Furthermore, parallel experiments demonstrated that the activity of IL-11 was comparable with that induced by IL-4, the most effective Th2-polarizing cytokine. Taken together, these findings show that IL-11 inhibits Th1 polarization by exerting a direct effect on human T lymphocytes and by reducing IL-12 production by macrophages. Conversely, IL-11 does not exert any activity on DCs. This suggests that IL-11 could have therapeutic potential for diseases where Th1 responses play a dominant pathogenic role.     

Glucocorticoids transform CD40-triggering of dendritic cells into an alternative activation pathway resulting in antigen-presenting cells that secrete IL-10

Dendritic cell (DC) activation through CD40-CD40 ligand interactions is a key regulatory step for the development of protective T-cell immunity and also plays an important role in the initiation of T-cell responses involved in autoimmune diseases and allograft rejection. In contrast to previous reports, we show that the immunosuppressive drug dexamethasone (DEX) redirects rather than simply blocks this DC activation process. We found that DCs triggered through CD40 in the presence of DEX were unable to acquire high levels of costimulatory, adhesion, and major histocompatibility complex class I and II molecules and failed to express the maturation marker CD83, whereas antigen uptake was not affected. Moreover, DEX strikingly modified the CD40-activated DC cytokine secretion profile by suppressing the production of the proinflammatory cytokine interleukin (IL)-12 and potentiating the secretion of the anti-inflammatory cytokine IL-10. Accordingly, DEX-exposed CD40-triggered DCs displayed a decreased T-cell allostimulatory potential and a dramatically impaired ability to activate cloned CD4(+) T helper 1 (Th1) cells. Moreover, interaction between Th1 cells and these DCs rendered the T cells hyporesponsive to further antigen-specific restimulation. Collectively, our results demonstrate that DEX profoundly modulates CD40-dependent DC activation and suggest that the resulting alternatively activated DCs can be exploited for suppression of unwanted T-cell responses in vivo.     

Selective suicide of cross-presenting CD8+ dendritic cells by cytochrome c injection shows functional heterogeneity within this subset

Cross-presentation as a fundamental pathway of activating CD8(+) T cells has been well established. So far the application of this concept in vivo is limited, and the mechanisms that specialize CD8(+) dendritic cells (DCs) for this task are not fully understood. Here we take advantage of the specific cytosolic export feature of cross-presenting DCs together with the property of cytosolic cytochrome c (cyt c) in initiating Apaf-1-dependent apoptosis selectively in cross-presenting DCs. A single i.v. injection of cyt c in B6 mice produced a 2- to 3-fold reduction in splenic CD8(+) DCs but not in Apaf-1-deficient mice. Functional studies both in vivo and in vitro showed that cyt c profoundly abrogated OVA-specific CD8(+) T cell proliferation through its apoptosis-inducing effect on cross-presenting DCs. More importantly, in vivo injection of cyt c abolished the induction of cytotoxic T lymphocytes to exogenous antigen and reduced subsequent immunity to tumor challenge. In addition, only a proportion of CD8(+) DCs that express abundant IL-12 and Toll-like receptor 3 were efficient cross-presenters. Our data support the hypothesis that cross-presentation in vivo requires cytosolic diversion of endocytosed proteins, conferring cross-presentation specialization to a proportion of CD8(+) DCs. We propose that DCs incapable of such transfer, even within the CD8(+) DC subset, are unable to cross-present. Our model opens an avenue to specifically target cross-presenting DCs in vivo for manipulating cytotoxic T lymphocyte responses toward infections, tumors, and transplants.     

Mouse CD11c(+) B220(+) Gr1(+) plasmacytoid dendritic cells develop independently of the T-cell lineage

The developmental origin of dendritic cells (DCs) is controversial. In the mouse CD8alpha(+) and CD8alpha(-) DC subsets are often considered to be of lymphoid and myeloid origin respectively, although evidence on this point is conflicting. Very recently a novel CD11c(+) B220(+) DC subset has been identified that appears to be the murine counterpart to interferon alpha (IFNalpha)-producing human plasmacytoid DCs (PDCs). We show here that CD11c(+) B220(+) mouse PDCs, like human PDCs, are present in the thymus and express T lineage markers such as CD8alpha and CD4. However, the intrathymic development of PDCs can be completely dissociated from immature T lineage cells in mixed chimeras established with bone marrow cells from mice deficient for either Notch-1 or T-cell factor 1, two independent mutations that severely block early T-cell development. Our data indicate that thymic PDCs do not arise from a bipotential T/DC precursor.     

Tolerogenic maturation of liver sinusoidal endothelial cells promotes B7-homolog 1-dependent CD8+ T cell tolerance

Liver sinusoidal endothelial cells (LSEC) are unique organ-resident antigen-presenting cells capable of cross-presentation and subsequent tolerization of na?ve CD8(+) T cells. We investigated the molecular mechanisms underlying this tolerance induction in naive CD8(+) T cells. MHC class I-restricted antigen presentation by LSEC led to initial stimulation of na?ve CD8(+) T cells, which up-regulated CD69, CD25, CD44, and programmed death (PD)-1 and proliferated similar to dendritic cell (DC)-activated CD8(+) T cells. Importantly, cognate interaction with na?ve CD8(+) T cells triggered increased expression of co-inhibitory B7-H1 but not co-stimulatory CD80/86 molecules exclusively on LSEC but not DC. This matured phenotype of B7-H1(high) CD80/86(low) was critical for induction of CD8(+) T cell tolerance by LSEC: B7-H1-deficient LSEC, that failed to interact with PD-1 on stimulated T cells, were incapable of inducing CD8(+) T cell tolerance. Moreover, increased costimulation via CD28 interfered with tolerance induction, indicating that the noninducible low expression levels of CD80/86 on LSEC supported B7-H1-dependent tolerance induction. LSEC-tolerized CD8(+) T cells had a distinctive phenotype from na?ve and activated T cells with CD25(low), CD44(high), CD62L(high). They also expressed the homeostatic cytokine receptors CD127, CD122, and high levels of Bcl-2, indicating survival rather than deletion of tolerant CD8(+) T cells. On adoptive transfer into congenic animals, tolerized CD8(+) T cells failed to show specific cytotoxicity in vivo. CONCLUSION: Cognate interaction of LSEC with na?ve CD8(+) T cells elicits a unique tolerogenic maturation of LSEC and permissiveness of T cells for tolerogenic signals, demonstrating that LSEC-induced tolerance is an active and dynamic process.     

Human invariant Valpha24+ natural killer T cells acquire regulatory functions by interacting with IL-10-treated dendritic cells

Glycolipid-reactive Valpha24(+) invariant natural killer T (iNKT) cells have been implicated in regulating a variety of immune responses and in the induction of immunologic tolerance. Activation of iNKT cells requires interaction with professional antigen-presenting cells, such as dendritic cells (DCs). We have investigated the capacity of distinct DC subsets to modulate iNKT cell functions. We demonstrate that tolerogenic DCs (tolDCs), generated by treatment of monocyte-derived DC with interleukin (IL)-10, induced regulatory functions in human iNKT cells. tolDCs, compared with immunogenic DCs, had reduced capacity to induce iNKT-cell proliferation, but these cells produced large amounts of IL-10 and acquired an anergic phenotype. These anergic Valpha24(+) iNKT cells were able to potently inhibit allogeneic CD4(+) T-cell proliferation in vitro. Furthermore, the anergic Valpha24(+) iNKT cells could suppress DC maturation in vitro. We conclude that the interaction of iNKT cells with tolDCs plays an important role in the immune regulatory network, which might be exploited for therapeutic purposes.