Mechanisms Of Tolerance Induction Research Articles

Nano-scale encapsulation enhances allograft survival and function of islets transplanted in a mouse model of diabetes.

The success of islet transplantation as a treatment for type 1 diabetes is currently hampered by post-transplantation loss of functional islets through adverse immune and non-immune reactions. We aimed to test whether early islet loss can be limited and transplant survival improved by the application of conformal nano-coating layers to islets. Our novel coating protocol used alternate layers of phosphorylcholine-derived polysaccharides (chitosan or chondroitin-4-sulphate) and alginate as coating materials, with the binding based on electrostatic complexation. The in vitro function of encapsulated mouse islets was studied by analysing islet secretory function and cell viability. The in vivo function was evaluated using syngeneic and allogeneic transplantation in the streptozotocin-induced mouse model of diabetes. Nano-scale encapsulated islets retained appropriate islet secretory function in vitro and were less susceptible to complement- and cytokine-induced apoptosis than non-encapsulated control islets. In in vivo experiments using a syngeneic mouse transplantation model, no deleterious responses to the coatings were observed in host animals, and the encapsulated islet grafts were effective in reversing hyperglycaemia. Allo-transplantation of the nano-coated islets resulted in preserved islet function post-implantation in five of seven mice throughout the 1month monitoring period. Nano-scale encapsulation offers localised immune protection for implanted islets, and may be able to limit early allograft loss and extend survival of transplanted islets. This versatile coating scheme has the potential to be integrated with tolerance induction mechanisms, thereby achieving long-term success in islet transplantation.

51-OR: The novel mechanisms of tolerance induction contribute to long term survival of heart grafts in MHC class I-mismatched host

51-OR: The novel mechanisms of tolerance induction contribute to long term survival of heart grafts in MHC class I-mismatched host

The role of pDC, recipient Treg, and donor Tregin HSC engraftment

Hematopoietic stem cell (HSC) transplantation (HSCT) has been utilized for treatment of many hematologic malignancies, genetic and metabolic disorders, and hemoglobinopathies such as sickle cell disease and thalassemia. It also induces donor-specific tolerance to organ and tissue transplants. The widespread success of HSCT is hampered by the toxicities of immunosuppression and development of graft-versus-host disease (GVHD). The mechanism of induction of transplantation tolerance (reciprocal donor/host) is still an elusive challenge in allogeneic HSCT. An understanding of the mechanisms for induction of tolerance and the critical cells involved in this process has resulted in novel cell-based therapies poised to be translated to clinical application. The focus of this review is those cells of interest. Bone marrow-derived plasmacytoid dendritic cells induce naïve T cells to differentiate to become antigen-specific regulatory T cells (Treg), creating a milieu for the induction of transplantation tolerance. Recently, CD8+/TCR- facilitating cells (FC), a novel cell population in mouse bone marrow, have been shown to potently enhance engraftment of allogeneic HSC without causing GVHD. The predominant subpopulation of FC resembles plasmacytoid precursor dendritic cells. FC induce antigen-specific Treg in vivo. Notably, FC address one major concern that has prevented the implementation of Treg cell therapy in the clinic: to expand Treg and have them remain tolerogenic in vivo. FC are novel in that they induce an antigen-specific regulatory milieu in vivo. The discovery of FC has opened new alternatives to expanded criteria in bone marrow transplantation that were previously restricted to human leukocyte antigen-matched recipients. The focus of this review is to cover what is currently known about the mechanism of FC action in inducing tolerance and preventing GVHD and host-versus-graft reactivity.

The role of pDC, recipient T(reg) and donor T(reg) in HSC engraftment: Mechanisms of facilitation.

Hematopoietic stem cell transplantation (HSCT) has been utilized for treatment of many hematologic malignancies, genetic and metabolic disorders, and hemoglobinopathies such as sickle cell disease and thalassemia. It also induces donor-specific tolerance to organ and tissue transplants. The widespread success of HSCT is hampered by the toxicities of immunosuppression and development of graft-versus-host disease (GVHD). The mechanism of induction of transplantation tolerance (reciprocal donor/host) is still an elusive challenge in allogeneic HSCT. An understanding of the mechanisms for induction of tolerance and the critical cells involved in this process has resulted in novel cell-based therapies poised to be translated to clinical application. The focus of this review is those cells of interest.Bone marrow-derived plasmacytoid dendritic cells induce naïve T cells to differentiate to become antigen-specific regulatory T cells (T(reg)), creating a milieu for the induction of transplantation tolerance. Recently, CD8(+)/TCR(-) facilitating cells (FC), a novel cell population in mouse bone marrow, have been shown to potently enhance engraftment of allogeneic HSC without causing GVHD. The predominant subpopulation of FC resembles plasmacytoid precursor dendritic cells. FC induce antigen-specific T(reg) in vivo. Notably, FC address one major concern that has prevented the implementation of T(reg) cell therapy in the clinic: to expand T(reg) and have them remain tolerogenic in vivo. FC are novel in that they induce an antigen-specific regulatory milieu in vivo. The discovery of FC has opened new alternatives to expanded criteria in bone marrow transplantation that were previously restricted to human leukocyte antigen-matched recipients. The focus of this review is to cover what is currently known about the mechanism of FC action in inducing tolerance and preventing GVHD and hostversus-graft reactivity.

Armed response: how dying cells influence T‐cell functions

Immune responses during infection, injury, and cancer proceed in the presence of tissue injury and cell death. Consequently, the system must deal with its own dead cells while it determines the appropriate response to the invader. As apoptotic cells are known to induce immune tolerance and necrotic cells can be potent stimulators of immunity, this decision becomes more complex. The key to understanding the immunologic choices made during cell death is to examine the mechanisms of tolerance induction by dying cells and then relate them to the mechanisms of immunity. Ideally, immunogenic cell death should be directed toward tumor cells and infected cells, whereas tolerogenic cell death should be associated with preventing unwanted immune responses to self. In this review, we discuss how the decision is made by focusing on the biochemical process of cell death and how its key components can influence both tolerance and immunity.

Enforced expression of the apoptosis inhibitor Bcl-2 ablates tolerance induction in DNA-reactive B cells through a novel mechanism

How self tolerance is maintained during B cell development in the bone marrow has been a focal area of study in immunology. Receptor editing, anergy and clonal deletion all play important roles in the regulation of autoimmunity in the immature population. The mechanisms of tolerance induction in the periphery, however, are less well characterized. Overexpression of the apoptosis inhibitor Bcl-2 rescues autoreactive B cells from deletion and can contribute to the development of autoimmune disease in certain genetic backgrounds. Using a peptide-induced autoimmunity model, we recently identified a peripheral tolerance checkpoint in antigen-activated B cells that have undergone class switching and somatic hypermutation. At this checkpoint, receptor editing, induced by antigen engagement, dampened the autoantibody response. In this study, we show that receptor editing fails to be induced in antigen-activated DNA-reactive B cells that overexpress Bcl-2 (Bcl-2 Tg). The failure to induce RAG and receptor editing is likely due, at least partially, to the lack of self antigen. First, the levels of circulating DNA and of apoptotic bodies in the spleen of Bcl-2 Tg mice are significantly lower than in control mice. Second, in Bcl-2 Tg mice, RAG can be induced in a population of antigen-activated B cells by providing exogenous soluble antigen. These data suggest that, in addition to its anti-apoptotic activity, Bcl-2 may indirectly inhibit tolerance induction in B cells acquiring anti-nuclear antigen reactivity after peripheral activation by limiting the availability of self antigen.

Identification of FOXO3A as a regulator of dendritic cell tolerogenicity in human and murine prostate cancer (165.22)

Abstract The limited success of cancer immunotherapies is often attributed to the loss of antigen-specific T cell function in situ. In this study, we demonstrate a novel mechanism of tolerance induction by tumor-associated dendritic cells (TADCs) in human and mouse prostate cancer. TADCs from human prostate cancer patients and TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice expressed elevated levels of FOXO3A which correlated with expression of suppressive genes that negatively regulate T cell function. Similar results were observed in both a B16 melanoma and a RENCA renal cell carcinoma model. Furthermore, TADC isolated from tumors in Foxo3a-/- mice had reduced tolerogenic function. Additionally, silencing Foxo3a with siRNAs abrogated the ability of TADCs to tolerize and induce suppressive activity by T cells. This was associated with diminished expression of tolerogenic mediators, indoleamine-2,3-dioxygenase, arginase, and TGF-beta and up-regulated expression of co-stimulatory molecules and pro-inflammatory cytokines. In TRAMP mice, provision of tumor-specific CD4+ T cell help abrogated TADC tolerogenicity which was associated with reduced FOXO3A expression. These findings demonstrate that FOXO3A plays a critical role in mediating TADC-induced immune suppression. Moreover, our results identify a novel target for preventing CTL tolerance and enhancing immune responses to cancer by modulating the immunosuppressive activity of TADC found in the tumor microenvironment.

CTLs' repertoire shaping in the thymus: A Monte Carlo simulation

Motivation: The human immune system evolved a multi-layered control mechanism to eliminate self-reactive cells. Of these so-called tolerance induction mechanisms, lymphocytes T education in the thymus gland represents the very first one. This complicated process is not fully understood and quantitative models able to help in this endeavor are lacking. Here, we present a stochastic computational model of the thymus which combines data-driven prediction methods and a novel method based on protein–protein potential measurements for assessing molecular binding among cell receptors, major histocompatibility complex (MHC) molecules, and self-peptides. Results: Of all possible specificities of immature T cells entering the thymus, only a small fraction is actually selected for maturation. Monte Carlo simulations of thymocytes selection in the thymus are performed varying the size of the self and a parameter determining the number of encounter with antigen-presenting cells (APCs). We score the fraction of self-reacting thymocytes leaving the thymus as mature naive T cells and show that self-reactivity is only marginally dependent on the number of self-molecules presented by APCs, while it is strongly affected by a parameter proportional to the time spent in the thymus. We study how this measure changes when we vary the number of MHC alleles and found an optimal number not too different from what we have in reality. The main result of this study is more methodological than biological as we show that immunoinformatics data and methods can be used in systemic level simulation of immune processes.

Experimental study of the mechanism of tolerance induction in dexamethasone-treated dendritic cells

SummaryBackgroundThe aim of this study was to investigate the mechanisms underlying tolerance induction of dexamethasone (Dex)-treated dendritic cells (DCs).Material/MethodsWell-grown DC2.4 cells were randomly assigned to receive control, 50 μg/L, 100 μg/L, or 200 μg/L of dexamethasone and then were cultured for 6 days. The expressions of CD80, CD86, galectin-9, and PD-L1 on the surface of DC2.4 cells were analyzed with flow cytometry and the level of IL-12 secreted by DC2.4 cells was determined by ELISA. The stimulating activity of DC2.4 cells on allogeneic T cells was assessed with mixed lymphocyte reaction. Dexamethasone-treated DC2.4 cells were co-cultured with allogeneic splenic lymphocytes and the Foxp3 expression in naive T lymphocytes was determined with flow cytometry.ResultsCompared with the control group, the expressions of CD80, CD86, galectin-9, and PD-L1 on the surface of DC2.4 cells exposed to different doses of dexamethasone showed no significant changes; however, dexamethasone treatment significantly reduced IL-12 secretion and inhibited DC2.4’s stimulation on the proliferation of allogeneic T lymphocytes. Moreover, dexamethasone-treated DC2.4 cells effectively promoted FOXP3 expression in naive T lymphocytes.ConclusionsDC2.4 is a stable cell line with high expressions of CD80, CD86, and PD-L1. Dexamethasone does not significantly change the cell phenotype of DC2.4 cells, but inhibits the secretion of IL-12 cytokine and attenuates DC2.4’s stimulation of the proliferation of allogeneic T cells. Dexamethasone-treated DC2.4 cells also effectively promote FOXP3 expression in naive T lymphocytes.

B-Cell Gene Therapy for Tolerance Induction: Host but Not Donor B-Cell Derived IL-10 is Necessary for Tolerance

Genetically modified B cells are excellent tolerogenic antigen-presenting cells (APCs) in multiple models of autoimmunity. However, the mechanisms of action are still not completely understood. In our models, we generate antigen-specific tolerogenic B cells by transducing naïve or primed B cells with an antigen–immunoglobulin G (peptide–IgG) construct. In order to be transduced, B cells require activation with mitogens such as LPS. We and others have found that LPS stimulation of B cells upregulates the production of IL-10, a key cytokine for maintaining immune tolerance. In the current study, we defined the role of B-cell produced IL-10 in tolerance induction by using IL-10 deficient B cells as donor APCs. We found that peptide–IgG transduced IL-10 KO B cells have the same effects as wt B cells in tolerance induction in an experimental autoimmune encephalomyelitis model. Moreover, we demonstrated that the tolerogenic effect of peptide–IgG B cells was completely abrogated in anti-IL-10 receptor antibody treated recipients. Taken together, our results suggest that tolerance induced by peptide–IgG B-cell gene therapy requires IL-10 from the host but not donor B cells. These data shed important insights into the mechanisms of tolerance induction mediated by B-cell gene therapy.

CD4+CD25-mTGF + T cells induced by nasal application of ovalbumin transfer tolerance in a therapeutic model of asthma

Intranasal administration of high amount of allergen was shown to induce tolerance and to reverse the allergic phenotype. However, mechanisms of tolerance induction via the mucosal route are still unclear. To characterize the therapeutic effects of intranasal application of ovalbumin (OVA) in a mouse model of bronchial inflammation as well as the cellular and molecular mechanisms leading to protection upon re-exposure to allergen. After induction of bronchial inflammation, mice were treated intranasally with OVA and re-exposed to OVA aerosols 10 days later. Bronchoalveolar lavage fluid (BALF), T cell proliferation and cytokine secretion were examined. The respective role of CD4(+)CD25(+) and CD4(+)CD25(-) T cells in the induction of tolerance was analysed. Intranasal treatment with OVA drastically reduced inflammatory cell recruitment into BALF and bronchial hyperresponsiveness upon re-exposure to allergen. Both OVA- specific-proliferation of T cells, T(h)1 and T(h)2 cytokine production from lung and bronchial lymph nodes were inhibited. Transfer of CD4(+)CD25(-) T cells, which strongly expressed membrane-bound transforming growth factor β (mTGFβ), from tolerized mice protected asthmatic recipient mice from subsequent aerosol challenges. The presence of CD4(+)CD25(+)(Foxp3(+)) T cells during the process of tolerization was indispensable to CD4(+)CD25(-) T cells to acquire regulatory properties. Whereas the presence of IL-10 appeared dispensable in this model, the suppression of CD4(+)CD25(-)mTGFβ(+) T cells in transfer experiments significantly impaired the down-regulation of airways inflammation. Nasal application of OVA in established asthma led to the induction of CD4(+)CD25(-)mTGFβ(+) T cells with regulatory properties, able to confer protection upon allergen re-exposure.

TCR Revision Generates Functional CD4+ T Cells

CD4(+)Vβ5(+) peripheral T cells in C57BL/6 mice respond to encounter with a peripherally expressed endogenous superantigen by undergoing either deletion or TCR revision. In this latter process, cells lose surface Vβ5 expression and undergo RAG-dependent rearrangement of endogenous TCRβ genes, driving surface expression of novel TCRs. Although postrevision CD4(+)Vβ5(-)TCRβ(+) T cells accumulate with age in Vβ5 transgenic mice and bear a diverse TCR Vβ repertoire, it is unknown whether they respond to homeostatic and antigenic stimuli and thus may benefit the host. We demonstrate in this study that postrevision cells are functional. These cells have a high rate of steady-state homeostatic proliferation in situ, and they undergo extensive MHC class II-dependent lymphopenia-induced proliferation. Importantly, postrevision cells do not proliferate in response to the tolerizing superantigen, implicating TCR revision as a mechanism of tolerance induction and demonstrating that TCR-dependent activation of postrevision cells is not driven by the transgene-encoded receptor. Postrevision cells proliferate extensively to commensal bacterial Ags and can generate I-A(b)-restricted responses to Ag by producing IFN-γ following Listeria monocytogenes challenge. These data show that rescued postrevision T cells are responsive to homeostatic signals and recognize self- and foreign peptides in the context of self-MHC and are thus useful to the host.

Authors' Reply: The Spleen as a Site for Hematopoiesis

Many studies, including those of Dor et al. (Letter to the Editor), have confirmed spleen as a source of hematopoietic stem/progenitor cells (1–3). In their studies, spleen transplantation is lauded as valuable therapy both in potential for hematopoiesis and ability to induce tolerance to allogeneic secondary tissue grafts (4). In those studies, allografting was dependent on myeloablative conditioning, and long-term graft acceptance or “tolerance” was dependent on spleen grafting. Because allografting represents an inflammatory response, hematopoiesis occurs under inflammatory conditions, providing tolerance by a mechanism so far undefined. Indeed, recent studies showing hematopoietic stem cells (HSC) as responsive to toll-like receptor signaling (5) serve to reinforce the importance of inflammation in the regulation of hematopoiesis. A distinct and harder question is whether spleen supports hematopoiesis in the steady state without inflammation. One hypothesis emerging from our former studies on spleen long-term cultures was that spleen supports steady-state hematopoiesis of distinct antigen presenting cells (APCs), which provide tissue-specific immunity. Spleen was identified as a source of self-renewing progenitors of a novel myeloid, dendritic-like cell type, consistent with spleen as a site for hematopoiesis (6). We subsequently demonstrated an equivalent in vivo subset having distinct immune capability with weak ability to stimulate CD4+ T cells and strong capacity for antigen crosspresentation to CD8+ T cells (7). Although their full functional significance is still under investigation, others have reported that bone marrow progenitors co-cultured with spleen stroma can induce differentiation of regulatory dendritic APC (8). Indeed, spleen as a site for development of regulatory APC may provide a mechanism for tolerance induction, in accordance with the experiments by Dor et al. (4). Faced with the question of whether spleen supports hematopoiesis from endogenous progenitors under steady-state conditions, we attempted spleen grafting of CD45-allotype distinct spleen into splenectomized hosts in the absence of myeloablative conditioning (2). Grafting across a CD45-allotype barrier can be weakly immunogenic in the absence of conditioning (9), and this may well account for our inability to show graft maintenance after 4 weeks (2). However, we did demonstrate myelopoiesis endogenous to spleen grafts within 2 weeks of grafting. We also demonstrated colonization of spleen by host HSC within 2 weeks, ahead of the inflammatory response evident at 4 weeks. We were unable to definitively demonstrate production of the novel splenic APC described previously, although this could be due to the limited number of cells available for analysis. We are currently pursuing other genetic and mutant animal models, and different spleen grafting procedures. Our studies to date involving early spleen grafting of splenectomized hosts support a role for spleen in myelopoiesis in the steady state. A role for the splenic microenvironment in directing hematopoiesis to development of tissue-specific APC has been supported to date by in vitro studies. Helen C. O'Neill1 Jonathan K. H. Tan2 1 Stem Cell and Immunology Lab, Division of Biomedical Sciences and Biochemistry, Research School of Biology, The Australian National University Canberra, Australia 2 Center for Innovation in Immunoregulative Technology and Therapeutics, Graduate School of Medicine, Kyoto University Kyoto, Japan

Anti-αβ-T-Cell Receptor Antibodies in the Setting of Laryngeal Transplantation

Inhibiting T-cell activation is critically important to the induction of transplantation tolerance. Monoclonal antibodies directed against the αβ-T-cell receptor have been shown to cause selective immunodepletion of this T-cell population and can provide long-term allograft acceptance. This article discusses the role of this promising immunosuppressive agent in scientific research and clinical utilization. Specifically, the article focuses on its efficacy and mechanism of tolerance induction in solid tissue and composite tissue allograft transplantation with a particular focus on laryngeal transplantation.

Immune regulation by apoptotic cell clearance

Apoptotic cell clearance by phagocytes is essential for the maintenance of self-tolerance under physiological conditions. Consistent with this, the intravenous injection of apoptotic cells can induce cell-associated antigen-specific immunosuppression or tolerance. The intravenous injection of apoptotic cells expressed a fragment of myelin oligodendrocyte glycoprotein (MOG)-induced MOG-specific T cell tolerance and suppressed the development of experimental autoimmune encephalomyelitis. However, the suppressive effects of the MOG-expressing apoptotic cells were largely eliminated by masking phosphatidylserine (PS) exposed on the apoptotic cells, suggesting that the PS-dependent engulfment of apoptotic cells is required for the tolerance induction. We found that this mechanism of tolerance induction requires the contribution of two cell populations in the splenic marginal zone (MZ). The MZ contains two types of macrophages: marginal metallophilic macrophages and MZ macrophages. These macrophages contribute to the rapid clearance of cell corpses in blood flow. In addition, we also found that CD8α(+), CD103(+) dendritic cells localizing in the MZ selectively phagocytose blood-borne dead cells and subsequently present dead cell-associated antigens to induce antigen-specific immunosuppression or tolerance.

Breast milk immune complexes are potent inducers of oral tolerance in neonates and prevent asthma development

Allergic asthma is a chronic lung disease resulting from an inappropriate T helper (Th)-2 response to environmental antigens. Early tolerance induction is an attractive approach for primary prevention of asthma. Here, we found that breastfeeding by antigen-sensitized mothers exposed to antigen aerosols during lactation induced a robust and long-lasting antigen-specific protection from asthma. Protection was more profound and persistent than the one induced by antigen-exposed non-sensitized mothers. Milk from antigen-exposed sensitized mothers contained antigen-immunoglobulin (Ig) G immune complexes that were transferred to the newborn through the neonatal Fc receptor resulting in the induction of antigen-specific FoxP3+ CD25+ regulatory T cells. The induction of oral tolerance by milk immune complexes did not require the presence of transforming growth factor-β in milk in contrast to tolerance induced by milk-borne free antigen. Furthermore, neither the presence of IgA in milk nor the expression of the inhibitory FcγRIIb in the newborn was required for tolerance induction. This study provides new insights on the mechanisms of tolerance induction in neonates and highlights that IgG immune complexes found in breast milk are potent inducers of oral tolerance. These observations may pave the way for the identification of key factors for primary prevention of immune-mediated diseases such as asthma.

CD4 + CD25 + regulatory T cells partially mediate the beneficial effects of FTY720, a sphingosine-1-phosphate analogue, during ischaemia/reperfusion-induced acute kidney injury

The synthetic sphingosine-1-phosphate (S1P) analogue, FTY720, attenuates ischaemia/reperfusion (I/R) injury by inducing peripheral lymphopaenia. Recent studies suggest that FTY720 may also exert protective effects by modulating dendritic cell (DC) function or directly affecting regulatory T cells (Tregs). The purpose of the present study was to examine whether the beneficial effect of FTY720 in I/R-induced acute kidney injury (AKI) involves modulation of DCs or Tregs. Mice underwent bilateral ischaemia, and FTY720 or vehicle was then administered. Biochemical values, histological kidney damage and tissue inflammation were assessed. Phenotype and function of DCs in blood/spleen or kidney were also examined by flow cytometry or mixed lymphocyte reaction (MLR) assay. Percent Tregs or FoxP3 mRNA expression was examined in kidney and spleen, and depletion and adoptive transfer of Tregs were also performed. Treatment with FTY720 attenuated I/R kidney injury and reduced inflammation. The beneficial effect of FTY720 was associated with expansion of peripheral CD11b( +) CD11c( +) DC and with maturation of spleen CD11c( +) DC, which showed impaired allostimulatory capacity. FTY720-treated animals also showed a higher frequency of CD4( +) CD25( +) Tregs and an upregulation of FoxP3 mRNA expression in spleen and kidney. In vitro experiments showed that FTY720 induced expansion of Tregs, possibly via conversion from non-Tregs to Tregs. Depletion and adoptive transfer of Tregs were associated with loss and recovery of the beneficial effects of FTY720. These results suggest that the beneficial effects of FTY720 in I/R injury may be partially mediated by DC modulation or by increasing Treg activity. Further studies that identify tolerance induction mechanisms will be useful for developing strategies for the prevention or treatment of AKI.

Clinical characteristics of oral tolerance induction of IgE-mediated and non-IgE-mediated food allergy using interferon gamma

Food allergies are classified as IgE-mediated food allergies (IFAs) and non-IgE-mediated food allergies (NFAs). Recently, oral immunotherapy (OIT) has been found to be successful for treating both IFA and NFA, especially using interferon (IFN) gamma. This study was designed to clarify the clinical characteristics of IFA and NFA and compare the therapeutic characteristics of OIT using subcutaneously administered IFN-gamma for both types of food allergy. In this study, 148 patients were categorized into the IFA and NFA group following food challenge, skin-prick test and food-specific IgE tests. The patients were then treated using protocols specific for IFA and NFA using subcutaneous IFN-gamma injection as a randomized controlled trial. The principle of complete allergy resolution at prior dose in the case of IFA was also evaluated. Only the patients with IFA and NFA treated with OIT using IFN-gamma achieved tolerance successfully. Tolerance was achieved from low-dose range in IFA and in high-dose range for NFA. Complete tolerance was not obtained without achieving complete allergy resolution at each dose of the allergen before increasing the dosage in IFA. Both IFA and NFA can be successfully treated with OIT using IFN-gamma but show different clinical and therapeutic characteristics. IFN-gamma is necessary for the tolerance induction but not for tolerance maintenance. Additional study for the mechanisms of tolerance induction by IFN-gamma is needed.

Tracking Antigen-Specific T-Cells during Clinical Tolerance Induction in Humans

Allergen immunotherapy presents an opportunity to define mechanisms of induction of clinical tolerance in humans. Significant progress has been made in our understanding of changes in T cell responses during immunotherapy, but existing work has largely been based on functional T cell assays. HLA-peptide-tetrameric complexes allow the tracking of antigen-specific T-cell populations based on the presence of specific T-cell receptors and when combined with functional assays allow a closer assessment of the potential roles of T-cell anergy and clonotype evolution. We sought to develop tools to facilitate tracking of antigen-specific T-cell populations during wasp-venom immunotherapy in people with wasp-venom allergy. We first defined dominant immunogenic regions within Ves v 5, a constituent of wasp venom that is known to represent a target antigen for T-cells. We next identified HLA-DRB1*1501 restricted epitopes and used HLA class II tetrameric complexes alongside cytokine responses to Ves v 5 to track T-cell responses during immunotherapy. In contrast to previous reports, we show that there was a significant initial induction of IL-4 producing antigen-specific T-cells within the first 3–5 weeks of immunotherapy which was followed by reduction of circulating effector antigen-specific T-cells despite escalation of wasp-venom dosage. However, there was sustained induction of IL-10-producing and FOXP3 positive antigen-specific T cells. We observed that these IL-10 producing cells could share a common precursor with IL-4-producing T cells specific for the same epitope. Clinical tolerance induction in humans is associated with dynamic changes in frequencies of antigen-specific T-cells, with a marked loss of IL-4-producing T-cells and the acquisition of IL-10-producing and FOXP3-positive antigen-specific CD4+ T-cells that can derive from a common shared precursor to pre-treatment effector T-cells. The development of new approaches to track antigen specific T-cell responses during immunotherapy can provide novel insights into mechanisms of tolerance induction in humans and identify new potential treatment targets.

Tolerance Induction in Response to Liver Inflammation

Background/Aims: The liver plays an important role in immunological tolerance due to its anatomical location, as it links the gastrointestinal tract and the systemic venous circulation. Therefore, immune reactions against dietary or bacterial antigens from the gut have to be avoided. However, immune responses resulting in elimination of harmful hepatotrophic pathogens have to be induced. We investigated mechanisms of tolerance induction in response to liver inflammation in a murine model of immune-mediated liver injury. Methods: Liver damage was induced by injection of the plant lectin concanavalin A (ConA). Cytokine levels were measured in plasma and liver tissue. The frequencies of intrahepatic and splenic cell subsets were measured by FACS analyses. Results: ConA hepatitis was mediated by activation of CD4+ T cells, NKT cells and Kupffer cells releasing IFN-γ and TNF-α. Tolerance developed towards ConA rechallenge within 8 days, lasted for several weeks and was characterized by significantly reduced plasma transaminase activities, decreased Th1/Th17 responses and an increased IL-10 release, the latter being produced by CD4+CD25+FoxP3+ regulatory T cells and Kupffer cells. Moreover, regulatory T cells from ConA-tolerant mice displayed a higher immunosuppressive potential in vitro and in vivo compared to those from non-tolerant animals. Interestingly, ConA hepatitis was aggravated in CCR5^–/– and CXCR3^–/– mice. Conclusion: These results suggest that ConA tolerance is mediated by induced IL10+ regulatory T cells, probably trafficking into the liver depending on the IFN-γ-inducible chemokine receptors CCR5 and CXCR3.