Tolerogenic APC Research Articles

Correction: A Tolerogenic Artificial APC Durably Ameliorates Experimental Autoimmune Encephalomyelitis by Directly and Selectively Modulating Myelin Peptide-Autoreactive CD4+ and CD8+ T Cells.

Correction: A Tolerogenic Artificial APC Durably Ameliorates Experimental Autoimmune Encephalomyelitis by Directly and Selectively Modulating Myelin Peptide-Autoreactive CD4+ and CD8+ T Cells.

A Tolerogenic Artificial APC Durably Ameliorates Experimental Autoimmune Encephalomyelitis by Directly and Selectively Modulating Myelin Peptide-Autoreactive CD4+ and CD8+ T Cells.

In this study, a tolerogenic artificial APC (TaAPC) was developed to directly and selectively modulate myelin-autoreactive CD4+ and CD8+ T cells in the myelin oligodendrocyte glycoprotein (MOG)35-55 peptide-induced experimental autoimmune encephalomyelitis in C57BL/6J mice. Cell-sized polylactic-coglycolic acid microparticles were generated to cocouple target Ags (MOG40-54/H-2Db-Ig dimer, MOG35-55/I-Ab multimer), regulatory molecules (anti-Fas and PD-L1-Fc), and "self-marker" CD47-Fc and encapsulate inhibitory cytokine (TGF-β1). Four infusions of the TaAPCs markedly and durably inhibited the experimental autoimmune encephalomyelitis progression and reduced the local inflammation in CNS tissue. They circulated throughout vasculature into peripheral lymphoid tissues and various organs, but not into brain, with retention of 36 h and exerted direct effects on T cells in vivo and in vitro. Two infusions of the TaAPCs depleted 65-79% of MOG35-55-specific CD4+ and 46-62% of MOG40-54-specific CD8+ T cells in peripheral blood, spleen, and CNS tissues in an Ag-specific manner and regulatory molecule-dependent fashion; induced robust T cell apoptosis; inhibited the activation and proliferation of MOG peptide-reactive T cells; reduced MOG peptide-reactive Th1, Th17, and Tc17 cells; and expanded regulatory T cells. They also inhibited IFN-γ/IL-17A secretion and elevated IL-10/TGF-β1 production in splenocytes but not in CNS tissue. More importantly, the TaAPCs treatment did not obviously suppress the overall immune function of host. To our knowledge, this study provides the first experimental evidence for the capability of TaAPCs to directly modulate autoreactive T cells by surface presentation of multiple ligands and paracrine release of cytokine, thus suggesting a novel Ag-specific immunotherapy for the T cell-mediated autoimmune diseases.

Tregitopes: The active ingredient in IVIG-mediated tolerance induction? (P5224)

Abstract In the course of screening IgG sequences for T cell epitopes, we identified Treg epitope peptides, now called Tregitopes, contained in conserved framework regions of Fab and Fc. Tregitopes may provide one explanation for the expansion and stimulation of Treg following intravenous immunoglobulin (IVIg) therapy. Their distinguishing characteristics include in silico signatures of high-affinity binding to multiple human HLA class II DR, and conservation across IgG isotypes as well as mammalian species. Tregitopes induce expansion of CD4+/CD25hi/FoxP3+ T cells and suppress immune responses to co-incubated antigens in vitro. Comparing human Tregitopes (hTregitopes) to murine sequences, we identified class II-restricted murine Tregitope homologs (mTregitopes). We provide evidence that when APCs present Tregitopes to natural (n) Treg, APC expression of MHC II, CD80, and CD86 are decreased. Moreover, Tregitopes increase proportions of IL-10-producing nTreg. We propose the mechanism of Tregitope-mediated tolerance induction to be as follows: 1) APC present Tregitopes to nTreg, 2) nTreg are activated, proliferate and produce IL-10, 3) nTreg provide tolerogenic feedback signals to APC, modulating the APC phenotype, and 4) nTreg and tolerogenic APC together suppress antigen-specific T cell responses. The discovery of Tregitopes in IgG and other autologous proteins may lead to the development of new insights as to the role of Tregs in autoimmune diseases.

A single population of tolerogenic APC induce two types of CD4+ Treg cells by two different mechanisms. (P1054)

Abstract Anterior chamber associated immune deviation,ACAID, is an animal model developed to study immune privileged mechanisms of the eye. The in vitro correlate of ACAID is traditionally used to study the cellular and molecular mechanism of the Treg cell development. Tolerogenic antigen-presenting cells (TolAPC) are required to express F4/80 for induction of efferent CD8+ Treg cells. We wondered if F480 is required for the development of afferent CD4+ Treg cells subpopulations. CD4+ Treg cells from cultures containing F4/80 deficient TolAPC cells partially inhibited an in vivo proliferative response to antigen. The depletion of CD25+ T cells after (but not before) exposure to TolAPC in vitro removed all inhibitory activity of the T cells in vivo. Thus, CD4+CD25+ Treg cells generated in vitro are not dependent on F4/80 but do require PDL-1 expression by the TolAPC . Since the F4/80 dependent CD4+ CD25- Treg cells express LAP-1 and PD-1, but not FoxP3, and the F4/80 independent CD4+ CD25+ Treg cells express FoxP3, PD-1, PD-L1, but not LAP-1, they resemble the mucosally-defined Th3 and inducible (i)Treg cells, respectively. Flow cytometry analyses showed that rather than two subpopulations of TolAPC, a single population of F4/80+PD-L1+TolAPC was generated by TGFβ and antigen pretreatment. Together, these data suggest that two subpopulations of afferent CD4+ Treg cells are induced by a single population of TolAPC in vitro.

Tregitope mechanism of action and applications for tolerance induction. (116.8)

Abstract Tregitopes are T cell epitopes naturally located in immunoglobulins that bind to multiple MHC Class II alleles and induce regulatory T cell (Treg) responses. Harnessing tolerogenic effects of Tregitopes provides a novel tool to suppress unwanted immune responses and maintain antigen-specific tolerance, thus changing treatment paradigms in autoimmunity. We have now demonstrated that APCs present Tregitopes to natural (n) Treg, engage feedback mechanisms promoting a tolerogenic APC phenotype, induce Treg expansion, and modulate antigen-specific effector T cell responses. Proportions of APC expressing MHC II, CD80, and CD86 are suppressed, consistent with reported effects of IVIG (Bayry et al. Blood, 2003, 101:758) and of the IgG-derived peptide hCDR1 (Sela et al. Immunology, 2009, 128:395). Moreover, we observed significant increases in proportions of IL-10-producing CD4+CD25+ FoxP3-expressing nTreg in the presence of Tregitopes. These studies are an exciting first step towards understanding the basic mechanism of Tregitope tolerance induction that we propose to be as follows: 1) APC present Tregitopes to nTreg, 2) nTreg are activated to proliferate and produce IL-10, 3) nTreg provide tolerogenic feedback signals to APC, modulating the APC phenotype, and 4) nTreg and tolerogenic APC together suppress antigen-specific T cell responses. These data suggest a role for Tregitopes in the mechanism of action for IVIG, and provide insight critical for future therapeutic applications.

Tolerance induction towards cardiac allografts under costimulation blockade is impaired in CCR7‐deficient animals but can be restored by adoptive transfer of syngeneic plasmacytoid dendritic cells

Deficiency of transplant recipients for the chemokine receptor CCR7 was originally described to slightly increase the survival time of vascularized solid organ grafts, probably due to a reduced priming of alloreactive T cells. Using a model of allotolerance induction by donor-specific splenocyte transfusion (DST) in combination with anti-CD40L mAb-mediated costimulation blockade (CSB), we show here a striking failure of CCR7-deficient (CCR7(-/-) ) recipients to tolerate cardiac allografts. Furthermore, in addition to the recently described lack of Treg, CCR7(-/-) mice were found to harbor significantly reduced numbers of plasmacytoid dendritic cells (pDCs) within peripheral as well as mesenteric lymph nodes (LNs), but not the bone marrow or spleen. pDCs had previously been suggested to function as tolerogenic APC during allograft transplantation, and a single transfer of syngeneic WT pDCs, but not conventional DCs, was indeed sufficient to rescue graft survival in DST+CSB-treated CCR7(-/-) recipients in a dose-dependent manner. We therefore conclude that the nearly complete absence of pDCs within LNs of CCR7(-/-) mice prevents the successful induction of DST+CSB-mediated allotolerance, leading to the observed acute rejection of cardiac allografts under tolerizing conditions.

Dendritic Cells Secrete the Immunosuppressive HLA-G Molecule upon CTLA4-Ig Treatment: Implication in Human Renal Transplant Acceptance

CTLA4-Ig (Belatacept) is a new recombinant molecule that interferes with the signal of T lymphocyte activation and prevents acute rejection after renal transplantation. HLA-G acts as a naturally tolerogenic molecule in humans. In this study, we analyzed whether HLA-G contributes to CTLA4-Ig-mediated graft acceptance. Our results demonstrate that patients treated with CTLA4-Ig displayed significantly higher soluble HLA-G (sHLA-G) plasma concentrations (72 +/- 14 ng/ml) than patients treated with calcineurin inhibitors (5 +/- 1 ng/ml) or healthy donors (5 +/- 5 ng/ml). Notably, sHLA-G purified from plasma of CTLA4-Ig-treated patients was biologically active as it inhibited allogeneic T cell proliferation in vitro. Dendritic cells (DC) were identified as one of the cellular sources of sHLA-G in CTLA4-Ig-treated patients. Supporting this observation, we showed that DC generated in vitro in presence of CTLA4-Ig released sHLA-G in response to allostimulation. These CTLA4-Ig-treated DC acted as tolerogenic APC through sHLA-G secretion as they suppressed T cell alloproliferation, which could be restored by using a neutralizing anti-HLA-G Ab. These data define a novel pathway by which CTLA4-Ig immunomodulates allogenic response through posttranscriptional regulation of HLA-G expression in DC. CTLA4-Ig-mediated HLA-G release appears as a critical factor in T cell alloresponse inhibition, thereby contributing to the immunosuppressive effect and graft acceptance.

The ability of dendritic cells to prevent diabetes development in NOD mice depends on their production of high levels of IL‐10 versus low levels of IL‐12

NOD mice, similar to diabetic patients, exhibit various defects in different types of immune cells, including dendritic cells (DC) that produce pro‐inflammatory cytokines, and regulatory T cells. Recognition of TLR2 can lead to immune evasion of various microorganisms via induction of tolerogenic APC and regulatory cells. The goal of this study was to induce tolerogenic NOD DC able to prevent diabetes. DC were stimulated for 24 hrs with two TLR2 agonists, lipoteichoic acid (LTA) from S. aureus and Lactobacillus. DC stimulated with LTA produced 50 times more IL‐12 than IL‐10, and mediated acceleration of diabetes development when transferred into NOD mice. In contrast, high doses of Lactobacilli (LC) favored the production of IL‐10 versus IL‐12. Transfer of DC treated with high‐dose LC, not low‐dose LC, into NOD mice delayed diabetes onset and decreased diabetes incidence. Interestingly, DC treated with high‐dose LC produced 5 times more IL‐10 than those treated with low‐dose LC. Finally, significant increases in IL‐10 levels were detected in most of the samples from LC DC‐injected NOD mice. These data indicate that the levels of IL‐10 versus IL‐12 produced by DC cultured with various stimuli correlates with the ability of those cells to prevent diabetes in NOD mice. IL‐10 may play a significant role in diabetes prevention either directly, by suppressing pro‐inflammatory responses, and/or by inducing regulatory cells.

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.

Modulation of Ovalbumin-Induced Airway Inflammation and Hyperreactivity by Tolerogenic APC

Allergic asthma is mediated in part by unregulated Th2 inflammation in response to an allergen. Induction of peripheral tolerance by inoculation of Ags into the anterior chamber of the eye (ocular tolerance) before sensitization blocks Th2 responses. Thus, we proposed that induction of ocular tolerance to the allergen might modulate an ongoing allergen-induced Th2 pathogenesis in the lung. We initiated ocular tolerance in previously immunized mice in a classic mouse model of OVA-induced pulmonary allergic inflammation. In the model of ocular tolerance, the need for inoculation of Ag into the anterior chamber can be bypassed by i.v. inoculation of in vitro-generated tolerogenic (TGF-beta2-treated, Ag-pulsed) APC (tol-APC). We observed that with i.v. inoculation, such tolerogenic APC, but not control APC, reduced eosinophil and lymphocyte pulmonary infiltration in experimental mice. Similarly, production of Th2 cytokines (IL-4, -5, and -13), but not IFN-gamma, was reduced. Importantly, airway hyperresponsiveness and mucus production were significantly reduced after treatment with the tol-APC. We also show that in vitro suppression of IL-13 production from OVA-sensitized effector T cells was mediated by CD8+, not CD4+, T regulatory cells. Thus, i.v. inoculation of the tol-APC induced peripheral tolerance that suppressed Th2-mediated pathogenesis in the lungs of presensitized mice. The ability of the tol-APC to induce peripheral tolerance and suppress existing Th2 immune inflammation may lead to novel therapies for pulmonary allergic inflammation and its related pathology.

License to Heal: Bidirectional Interaction of Antigen-Specific Regulatory T Cells and Tolerogenic APC

Naturally occurring CD4(+)CD25(+) regulatory T (T(R)) cells, a component of the innate immune response, which play a key role in the maintenance of self-tolerance, have become the focus of numerous studies over the last decade. These cells inhibit the immune response in an Ag-nonspecific manner, interacting with other T cells. Much less is known about adaptive T(R) cells, which develop in response to chronic antigenic stimulation, and act directly on professional and nonprofessional APC, rendering them tolerogenic and able to elicit the differentiation of CD8(+) and CD4(+) T cells with suppressive activity. In this review, we will discuss data pertaining to the bidirectional interaction between Ag-specific T(R) with APC and their clinical relevance.

Utilizing regulatory T cells to control alloreactivity

Effective resetting of the immune system cannot be achieved by non-specific immunosuppression. Instead, novel strategies aim at harnessing the body's natural tolerance mechanisms to rectify an Ag-specific response without disturbing other immune functions. Fine-tuning of the balance between Ag-specific effector and regulatory T (Tr) cells is a promising strategy that requires detailed understanding of the differentiation and expansion pathways of the relevant Tr cell subsets. Here we review recent developments regarding the control of alloreactivity by induction and expansion of Tr cells. T-cell activation in the presence of tolerogenic APC and cytokines leads to the induction of Tr cells, which can mediate tolerance through cytokine-dependent and/or contact-dependent mechanisms. Better understanding of the mechanisms of immune regulation mediated by Tr cells may enable fine-tuning of specific immune responses and pave the way for novel therapeutic approaches.

Cutting edge: in vitro-generated tolerogenic APC induce CD8+ T regulatory cells that can suppress ongoing experimental autoimmune encephalomyelitis.

APC exposed to TGFbeta2 and Ag (tolerogenic APC) promote peripheral Ag-specific tolerance via the induction of CD8(+) T regulatory cells capable of suppressing Th1 and Th2 immunity. We postulated that tolerogenic APC might reinstate tolerance toward self-neuronal Ags and ameliorate ongoing experimental autoimmune encephalomyelitis (EAE). Seven days after immunization with myelin basic protein (MBP), mice received MBP-specific tolerogenic APC, and EAE was evaluated clinically. To test for the presence and the phenotype of T regulatory cells, CD4 and/or CD8 T cells from tolerogenic APC-treated mice were transferred to naive mice before their immunization with MBP. The MBP-specific tolerogenic APC decreased both the severity and incidence of ongoing EAE. Tolerance to self-neuronal Ags was induced in naive recipient mice via adoptive transfer of CD8(+), but not CD4(+) T cells. Rational use of in vitro-generated tolerogenic APC may lead to novel therapy for autoimmune disease.

Tolerogenic APC generate CD8+ T regulatory cells that modulate pulmonary interstitial fibrosis.

Transforming growth factor-beta2-treated Ag-pulsed APC mimic APC from the immune privileged eye, and provide signals that generate regulatory T (Tr) cells and mediate peripheral tolerance. We postulated that TGF-beta2-treated Ag-pulsed APC (tolerogenic APC (tol-APC)) might also orchestrate regulation of immune mediated pathogenesis in nonimmune privileged tissues such as the lung. We used an adoptive transfer model of autoimmune pulmonary interstitial fibrosis called hapten immune pulmonary interstitial fibrosis (ADT-HIPIF) in this study. Mice that received 2,4,6-trinitrobenzene sulfonic acid-sensitized cells and challenged (intratracheally) with the hapten developed pulmonary interstitial fibrosis. However, transfer (i.v.) of TGF-beta2-treated 2,4,6-trinitrobenzene sulfonic acid-pulsed bone marrow-derived APC (tol-APC) to experimental mice 1 day after intratracheal challenge reduced the collagen deposition in the interstitium of the lung that usually follows challenge. Furthermore, ADT-HIPIF mice that received tol-APC developed Ag-specific efferent CD8+ Tr cells. Adoptive transfer of the Tr cells to another set of presensitized mice mediated suppression of the efferent phase of Th1 immune response and the subsequent immune dependent pulmonary interstitial fibrosis. Thus, tol-APC induced efferent CD8+ Tr cells in immune mice, and the regulation of the immune response limited the development of autoimmune pulmonary fibrosis in sensitized and pulmonary-challenged mice. Because ADT-HIPIF shares etiological and pathological characteristics with a variety of human immune inflammatory conditions in the lung that eventuate into interstitial fibrosis, these studies provide insight into potential therapy to alter the course of pulmonary fibrosis in humans.

Deletion of naive CD8 T cells requires persistent antigen and is not programmed by an initial signal from the tolerogenic APC.

Activation of naive CD8 T cells in vivo requires the recognition of cognate peptide-MHC complexes on APCs. Depending upon the activation status of the APC, such recognition will promote either a vigorous immune response or T cell tolerance and deletion. Recent studies suggest that the initial signals provided by APCs are sufficient to program the proliferation of naive CD8 T cells and their differentiation into effector cells. In this study, we sought to determine whether an initial encounter with tolerogenic APCs was sufficient to program deletion of naive CD8 T cells. Surprisingly, we find that regardless of whether naive CD8 T cells were stimulated by activated or quiescent APCs, transfer of the activated T cells into an Ag-free host was sufficient to ensure survival. Thus, although the extent of clonal expansion and development of effector function is determined by the activation status of the stimulatory APC, peripheral clonal deletion requires persistent Ag and is not determined by the initial stimulatory event.

CD4 cell priming and tolerization are differentially programmed by APCs upon initial engagement.

Bone marrow-derived APCs present both parenchymal-self and pathogen-derived Ags in a manner that elicits either T cell tolerization or immunity, respectively. To study the parameters that confer tolerogenic vs immunogenic APC function we used an adoptive transfer system in which naive TCR transgenic hemagglutinin (HA)-specific CD4(+) T cells are either tolerized upon encountering HA expressed constitutively as a parenchymal self-Ag (self-HA) or primed to express effector function upon encountering transiently expressed vaccinia-derived HA (viral-HA). When the duration of viral-HA presentation was extended for the period required to elicit tolerization toward self-HA, CD4 cell tolerization to viral-HA did not occur. Furthermore, CD4 cells exhibited both phenotypic as well as functional differences during early stages of tolerization and priming, suggesting that these divergent differentiation processes are programmed soon after the initial APC-CD4 cell interaction. When mice expressing self-HA were infected with an irrelevant vaccinia, CD4 cell tolerization still occurred, indicating that priming vs tolerization cannot be explained by pathogen-induced third parties (i.e., non-APCs) that act directly on CD4 cells. Taken together, these results suggest that CD4 cell tolerization to parenchymal self-Ags and priming to pathogen-derived Ags are initiated by functionally distinct APCs.

Distinct mRNA microarray profiles of tolerogenic dendritic cells

Dendritic cells are crucial to the activation as well as suppression of the immune response. Previous repots have illustrated that APC interacting with antigen-specific T suppressor cells become tolerogenic, inducing T helper anergy. To characterize the molecular changes occurring in tolerogenic APC, the mRNA profile of KG-1 dendritic cells exposed to allospecific T helper and T suppressor cells were analyzed. This study now provides evidence that immature dendritic cells stimulated by T suppressor cells differentiate into mature dendritic cells with a distinct phenotype. The identification of Ts induced pathways of dendritic cell differentiation is critical to the development of new therapeutic strategies.

UVB-irradiated dendritic cells are impaired in their APC function and tolerize primed Th1 cells but not naive CD4+ T cells

We have shown that low-dose UVB radiation converts Langerhans cells (LC) from immunogenic to tolerogenic APC. Therefore, we questioned whether low-dose UVB irradiation of bone marrow-derived dendritic cells (DC) alters their APC function, thereby inducing tolerance in T cells. To address this issue, cocultures of DC; and naive, allogeneic T cells; naïve, OVA-specific TCR-transgenic T cells from DO11.10 mice; or primed, antigen-specific T cells using the Th1 clone AE7 were analyzed. First, we found low-dose UVB-irradiated DC (UVB-DC) to dose-dependently (50-200 J/m2) inhibit T-cell proliferation of naive and primed T cells. In addition, supernatants harvested from cocultures of UVB-DC and naive T cells showed markedly reduced levels of IL-2 and IFN-gamma and to a lesser degree of IL-4 and IL-10, suggesting a preferential down-regulation of Th1 responses by UVB-DC. FACS analysis of UVB-DC revealed no changes in surface expression of MHC, costimulatory, and adhesion molecules. To test tolerance induction, allo- or antigen-specific T cells isolated from cocultures with unirradiated DC and UVB-DC were restimulated with unirradiated DC or IL-2. It is interesting that UVB-DC induced antigen-specific tolerance in the Th1 clone AE7. In contrast, UVB-DC induced a partial inhibition of allogeneic T-cell proliferation but no tolerance with similar unresponsiveness to restimulation with IL-2 and unirradiated DC irrespective of their haplotype. Similar observations were made when naive, TCR-transgenic T cells from DO11.10 mice were used. In conclusion, UVB-DC are impaired in their APC function and tolerize the primed antigen-specific Th1 clone AE7 but not naive allo- or OVA-specific T cells.

Mechanisms of tolerance induction by a gene-transferred peptide-IgG fusion protein expressed in B lineage cells.

A gene therapy model has been designed to induce tolerance to multiple epitopes expressed in-frame on a soluble IgG fusion protein scaffold. Tolerance to the lambda repressor cI sequence p1-102 or its immunodominant epitopes (p12-26, p73-88) can be elicited when bone marrow (BM) or LPS blasts are transduced and injected into naive or even primed recipients. To explore the mechanism of tolerance, class II(-/-) (knockout, KO) BM cells were transduced with p1-102-IgG and transferred to irradiated recipients. These cells failed to induce tolerance to challenge with p1-102 epitopes, whereas transduced +/+ BM cells did. This supports the importance of class II MHC on the tolerogenic APC rather than secretion and representation in tolerogenesis. When BM cells from muMT KO mice were transfected with p12-26-IgG and injected into irradiated mice, these transduced BM cells also failed to induce tolerance to an immunodominant epitope. These results suggest the direct involvement of B cells in tolerance to p1-102 epitopes. IL-10 KO BM cells infected with a p12-26-IgG construct were still tolerogenic. Importantly, anti-CTLA-4 injections reversed tolerance in primed, but not in naive, recipients of transduced LPS blasts. These data emphasize the importance of MHC class II presentation, B cell involvement, and CTLA-4 engagement in induction and/or maintenance of tolerance.

Induction of tolerance by IL-10-treated dendritic cells.

Dendritic cells (DC) form a specialized system for presenting Ag to naive or quiescent T cells and consequently play a central role in the induction of T and B cell immunity. In this study we used DC generated from peripheral progenitors to analyze the effect of IL-10 on the accessory function of human DC. We demonstrate that immature DC, harvested on days 9 to 11 and exposed to IL-10 for the last 2 days of culture, show a strongly reduced capacity to stimulate a CD4+ T cell response in an allogeneic MLR in a dose-dependent manner. In contrast, fully mature DC are completely resistant to the effects of IL-10. These results were obtained in both an alloantigen-induced MLR and an anti-CD3 mAb-induced response of primed and naive (CD45RA+) CD4+ T cells. FACS analysis revealed inhibition of the up-regulation of the costimulatory molecules CD58 and CD86 and the specific DC marker CD83 in DC pretreated with IL-10. These data suggest that IL-10 inhibited the development of fully mature DC. Furthermore, DC precultured with IL-10, but not controls, induced a state of alloantigen-specific anergy in CD4+ T cells and of peptide-specific anergy in the influenza hemagglutinin-specific T cell clone HA1.7. Analysis of the supernatants of these anergic T cells revealed a reduced production of IL-2 and IFN-gamma compared with that in control cells. Collectively, these data suggest that IL-10 converts immature DC into tolerogenic APC, which might be a useful tool in the therapy of patients with autoimmune or allergic diseases.