Mechanisms Of Tolerance Induction Research Articles

Anti-PD-L1 antibody reverses the immune tolerance induced by multiple MUC1-MBP vaccine immunizations by increasing the CD80/PD-L1 ratio, resulting in DC maturation, and decreasing Treg activity in B16-MUC1 melanoma-bearing mice.

In this study, we explored the possible mechanism of tumor tolerance induced by multiple repeated immunizations with a tumor vaccine (MUC1-MBP fusion protein plus CpG2006). We first analyzed the mechanism of tolerance by immunizing tumor-bearing mice 2, 5, or 8 times and found that compared with five immunizations with the M-M vaccine, eight immunizations increased tumor volume and weight and Treg levels, while the proportions of Th1 and Tc1 cells in the spleen and lymph nodes were decreased. In particular, the M-M vaccine induced PD-L1 expression in CD11c+DCs and decreased their CD80/PD-L1 ratio. Therefore, the mechanism of tolerance induction by multiple immunizations with the M-M vaccine was investigated by focusing on the CD80/PD-L1 ratio, and an anti-PD-L1 antibody (αPD-L1) and the M-M vaccine were used in combination to treat melanoma. The results showed that αPD-L1 increased the CD80/PD-L1 ratio and enhanced the maturation of cDC1s by blocking PD-L1 on DCs, which potentially increased the activity of Th1 and Tc1 cells. Furthermore, the combination of the M-M vaccine with αPD-L1 decreased the activity and proportion of Tregs, which reversed the immune tolerance induced by eight immunizations with the vaccine. This study reveals the mechanism of the combination of M-M and αPD-L1 and provides a new combination strategy for improving the therapeutic effect of the M-M vaccine, laying a theoretical basis for the clinical application of the vaccine.

Potential of growth-promoting bacteria in maize (Zea mays L.) varies according to soil moisture

Climate change has caused irregularities in water distribution, which affect the soil drying-wetting cycle and the development of economically important agricultural crops. Therefore, the use of plant growth-promoting bacteria (PGPB) emerges as an efficient strategy to mitigate negative impacts on crop yield. We hypothesized that the use of PGPB (in consortium or not) had potential to promote maize (Zea mays L.) growth under a soil moisture gradient in both non-sterile and sterile soils. Thirty PGPB strains were characterized for direct plant growth-promotion and drought tolerance induction mechanisms and were used in two independent experiments. Four soil water contents were used to simulate a severe drought (30% of field capacity [FC]), moderate drought (50% of FC), no drought (80% of FC) and, finally, a water gradient comprising the three mentioned soil water contents (80%, 50%, and 30% of FC). Two bacteria strains (BS28–7 Arthrobacter sp. and BS43 Streptomyces alboflavus), in addition to three consortia (BC2, BC4 and BCV) stood out in maize growth performance in experiment 1 and were used in experiment 2. Overall, under moderate drought, inoculation with BS43 surpassed the control treatment in root dry mass and nutrient uptake. Considering the water gradient treatment (80–50–30% of FC), the greatest total biomass was found in the uninoculated treatment when compared to BS28–7, BC2, and BCV. The greatest development of Z. mays L. was only observed under constant water stress conditions in the presence of PGPB. This is the first report that demonstrated the negative effect of individual inoculation of Arthrobacter sp. and the consortium of this strain with Streptomyces alboflavus on the growth of Z. mays L. based on a soil moisture gradient; however, future studies are needed for further validation.

Biomarkers and mechanisms of tolerance induction in food allergic patients drive new therapeutic approaches.

Immunotherapy for food-allergic patients has been effective in inducing desensitization in some populations, but long-term tolerance has remained an elusive target. A challenge facing our field is how to differentiate immune markers that are impacted by immunotherapy from those that are critical biomarkers of tolerance. Data from recent clinical trials have identified several biomarkers and mechanisms for achieving tolerance. These biomarkers include younger age, lower food-specific IgE, lower food component-specific IgE, specific linear epitope profiles, and subsets of food-specific CD4+ T cells. Additional biomarkers under investigation for their relevance in tolerance induction include TCR repertoires, gastrointestinal and skin microbiome, and local tissue immunity. This mini-review highlights recent advances in understanding biomarkers and mechanisms of tolerance induction in food immunotherapy and how these are influencing clinical trial development.

Biophysical Characterization of Tolerogenic Lipid-Based Nanoparticles Containing Phosphatidylcholine and Lysophosphatidylserine

Autoimmune conditions, allergies, and immunogenicity against therapeutic proteins are initiated by the unwanted immune response against self and non-self proteins. The development of tolerance induction approaches can offer an effective treatment modality for these clinical conditions. We recently showed that oral administration of lipidic nanoparticles containing phosphatidylcholine (PC) and lysophosphatidylserine (Lyso-PS) converted an immunogen to a tolerogen and induced immunological tolerance towards several antigens. While the biophysical properties such as lamellar characteristics of this binary lipid system are critical for stability, therapeutic delivery, and mechanism of tolerance induction, such information has not been thoroughly investigated. In the current study, we evaluated the lamellar phase properties of PC/Lyso-PS system using orthogonal biophysical methods such as fluorescence (steady-state, anisotropy, PSvue, and Laurdan), dynamic light scattering, and differential scanning calorimetry. The results showed that Lyso-PS partitioned into the PC bilayers and led to changes in the particles’ lamellar phase properties, lipid-packing, and lipid-water dynamics. Additionally, the biophysical characteristics of PC/Lyso-PS system are different from the well-studied PC/double-chain phosphatidylserine (PS) system. Notably, the incorporation of Lyso-PS significantly reduced the hydrodynamic diameter of PC particles. Results from the in vivo uptake study and intestinal loop assay utilizing flow cytometry analysis also indicated that the uptake of Lyso-PS-containing nanoparticles by immune cells in the gut and Peyer's patches is significantly higher than that of double-chain PS due to the differential transport through microfold cells. It was also found that the acyl chain mismatch between PC and Lyso-PS is critical for the miscibility and particle stability. Collectively, the results suggest that these biophysical characteristics likely influence the in vivo behaviors and contribute to the oral tolerance property of PC/Lyso-PS system.

Impact of Post-Transplant High-Dose Cyclophosphamide on Reconstitution in Bone Marrow Recipients

Introduction: Hematopoetic stem cell transplantation (HSCT) is the only curative therapy for many patients with hematologic malignancies. Occurrence of complications and mortality after allo HSCT is still high and it's strongly associated with immune reconstitution. Despite the wide-spread of Post-Transplant High-Dose Cyclophosphamide (PTCy) immune reconstitution and immunological safety of this method is still poorly understood.Aim: to evaluate immune reconstitution profile in patients who was underwent HSCT with a bone marrow as a stem cell source and with or without PTCy.Patients and methods: In National Research Center for Hematology 41patients after allogeneic HSCT were analyzed in 2 groups; patients with PTCy (n=17) and without PTCy (n=24). The total cohort had 12 males and 29 females, and had a median age of 33 years (range 19-61). All patients were diagnosed with a hematological malignancy. 14 patients were underwent myeloablative conditioning and 27 patients - non-myeloalative prior HSCT.The GVHD prophylaxis consisted of ATG-PTCy-CsA-MMF (n=10), ATG-PTCy (n=5), Mono-PTCy (n=1), ATG-CsA-MMF-MTX (n=20), CsA-MTX (n=2), ATG-CsA-MTX (n=2), CsA-MMF-MTX(n=1). The patients who only had received ATG were included on the analysis.Immune reconstitution profile was tested via serial flow cytometry analysis of peripheral blood on day +14, +30, +60 and +90 after allo-HSCT. Anti-CD3 FITC, anti-CD16PE, anti-CD56 PE, anti-CD45 Per-CP-CYTM5.5, anti-CD4 PE-CyTM7, anti-CD19 APC, anti-CD8 APC-Cy7; anti-CD62L FITC (BD Biosciences, USA); anti-CD14PE, anti-CD16PE, anti-HLA-DR APC (eBiosciences, USA) were used to define white blood cell subsets.Results: Reported detailed data is presented in Table 1. In bone marrow recipients the number of CD4+- cells was significantly lower on day +14 and +30, NK-cells on day +30 and CD8+-cells on day +60 in case of using PTCy versus immunosuppression without PTCy.The number of classical monocytes on day +30 and patrol monocytes on day +30 and +90 was significantly lower in case of using PTCy versus a standard protocol.Conclusion:Lymphocyte and monocyte reconstitution was impaired for the PTCy groups in the immediate post-HSCT period but quickly recovered. The mechanism of tolerance induction with PTCy on day+ 3, + 4 is not limited only to deletion of alloreactive T-cell clones, but also affects other leukocyte subpopulations (B cells, monocytes, granulocytes). The use of PTCy on day+ 3, + 4 is an immunologically safe method for prevention of GVHD.Table 1. Short-term reconstitution in BM recipients with and without PTCy [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

ОСОБЕННОСТИ ДИНАМИКИ CD4+CD25+ СУБПОПУЛЯЦИЙ Т-ЛИМФОЦИТОВ ПАЦИЕНТОВ С УДОВЛЕТВОРИТЕЛЬНОЙ РАННЕЙ ФУНКЦИЕЙ ПОЧЕЧНОГО ТРАНСПЛАНТАТА

Background. Experimental models indicate a certain role of T-regulatory lymphocytes in the induction of immunological tolerance. However, it is necessary to study their function and phenotype in more detail for the purposes of applying the mechanisms of tolerance induction. Objective. To assess the dynamics of CD3+CD4+CD25+ and CD3+CD4+CD25+highCD127+low indicators in renal transplant recipients with satisfactory early allograft function. Material and Methods. Kidney transplantation was performed in 197 recipients. We assessed CD3+CD4+CD25+ and CD3+CD4+CD25+highCD127+low level before the transplantation and on the 1st, 3rd, 7th, 30th and 90th days after the transplantation. The following groups of recipients were identified: PGF – those with satisfactory primary graft function, PGD – those with primary graft dysfunction, TR – those with transplant rejection in the early postoperative period. The comparison group (CG) consisted of healthy volunteers. Results. A significant increase in the relative and absolute level of CD3+CD4+CD25+highCD127+low from day 30 to 90 after surgery was revealed in recipients with a primary functioning graft. At the same time, CD3+CD4+CD25+ indicators in the recipients of the studied groups did not have significant differences throughout the study. Conclusions. CD3+CD4+CD25+highCD127+low monitoring can be used to identify patients with high tolerogenic potential.

One Hundred Ten Years of Allergen Immunotherapy: A Broad Look Into the Future

Allergen immunotherapy (AIT) is the only disease-modifying treatment option for patients with type 1-mediated allergic diseases such as allergic rhinitis/rhinoconjunctivitis with/without allergic asthma. Although many innovations have been developed since the first clinical report of Noon etal in 1911, the improvement of clinical efficacy and tolerability of this treatment is still an important unmet need. Hence, much progress has been made in the characterization of the cell types, cytokines, and intracellular signaling events involved in the development, maintenance, and regulation of allergic reactions, and also in the understanding of the mechanisms of tolerance induction in AIT. This comprehensive review aims to summarize the current innovative approaches in AIT, but also gives an outlook on promising candidates of the future. On the basis of an extensive literature review, integrating a clinical point of view, this article focuses on recent and future innovations regarding biologicals, allergen-derived peptides, recombinant allergens, "Toll"-like receptor agonists and other adjuvants, and novel application routes being developed for future AIT.

Immutol regulates CD4+Tregs, CD8+Tregs and pDCs via IDO signaling pathway to induce immune tolerance in rat heart allograft transplant

Indoleamine 2,3-dioxygenase (IDO) can promote tryptophan metabolism to kynurenine and modulate regulatory T cells (Tregs), thereby maintains lower efficiency to induce tolerance. Our aim is to investigate the mechanism of tolerance induction by a IDO metabolite named Immutol. MethodsWe established rat heterotopic heart transplantation models and treated them with Immutol, cyclosporine A (CsA) and 1-methyl-DL-tryptophan (1-MT) in vivo. The drugs were administered via gavage to all but the control group one day before surgery. CsA was gavaged continually for 20 days and Immutol for 60 days; after withdrawal of the drugs, the recipients were observed for at least 10 months. Immune cells were analyzed by flow cytometry. The IDO signaling pathway was evaluated by Western blotting, RT-PCR and immunochemical staining. Enzyme-linked immunosorbent assays (ELISAs) were used to detect changes in cytokines. ResultsCsA or Immutol alone prolonged survival but did not induce tolerance after withdrawal. Immutol+CsA inhibited acute rejection, and the grafts survived more than 400 d, with tolerance detected in most rats (13/15). Increased protein IDO and kynurenine could regulate the accumulation of CD4+Tregs, CD8+Tregs and pDC to induce immune tolerance. I-MT specifically blocked IDO, weakened the expression of IDO and kynurenine, and produced grafts rejection. Additionally, Tregs could down-regulate immune responses through production of the immunosuppressive cytokines IL-10 and TGF-beta, thus induce immune tolerance. CD8+ Tregs produce IFN-γ, and tolerance is dependent on both IFN-γ and IDO. ConclusionImmutol combined with CsA can control acute rejection and induce tolerance in rats with cardiac allografts after withdrawal. Immutol may become a novel drug for future clinical use.

Immunogenicity of TNF-Inhibitors

Tumor necrosis factor inhibitors (TNFi) have significantly improved treatment outcome of rheumatic diseases since their incorporation into treatment protocols two decades ago. Nevertheless, a substantial fraction of patients experiences either primary or secondary failure to TNFi due to ineffectiveness of the drug or adverse reactions. Secondary failure and adverse events can be related to the development of anti-drug antibodies (ADA). The earliest studies that reported ADA toward TNFi mainly used drug-sensitive assays. Retrospectively, we recognize this has led to an underestimation of the amount of ADA produced due to drug interference. Drug-tolerant ADA assays also detect ADA in the presence of drug, which has contributed to the currently reported higher incidence of ADA development. Comprehension and awareness of the assay format used for ADA detection is thus essential to interpret ADA measurements correctly. In addition, a concurrent drug level measurement is informative as it may provide insight in the extent of underestimation of ADA levels and improves understanding the clinical consequences of ADA formation. The clinical effects are dependent on the ratio between the amount of drug that is neutralized by ADA and the amount of unbound drug. Pharmacokinetic modeling might be useful in this context. The ADA response generally gives rise to high affinity IgG antibodies, but this response will differ between patients. Some patients will not reach the phase of affinity maturation while others generate an enduring high titer high affinity IgG response. This response can be transient in some patients, indicating a mechanism of tolerance induction or B-cell anergy. In this review several different aspects of the ADA response toward TNFi will be discussed. It will highlight the ADA assays, characteristics and regulation of the ADA response, impact of immunogenicity on the pharmacokinetics of TNFi, clinical implications of ADA formation, and possible mitigation strategies.

Mechanisms of Tolerance Induction through T Regulatory Cells during Chemotherapy-Mediated Immunogenic Cell Death in Acute Myeloid Leukemia

Background: Although the recent advances have allowed the discovery of new and less toxic forms of treatment for acute myeloid leukemia (AML), chemotherapy remains as one of the most used treatments. Some chemotherapeutic agents can induce a type of cell death - immunogenic cell death (ICD) - that can promote modifications in cancer cells, which activates immune system against leukemia cells. Even though this can help the immune system fighting the cancer, chemotherapy is also responsible for inducing tolerogenic mechanisms, that enable cancer cells to escape from immune system control. We previously demonstrated that enzyme indoleamine-2,3-dioxygenase-1 (IDO1) is involved in T regulatory cells (Tregs) induction in AML during ICD induced by chemotherapy. Dying leukemia cells release ATP which binds P2X7 receptor on dendritic cells (DCs), resulting in IDO1 up-regulation. In the same time, ATP can be catabolized by CD39 e CD73 ectonucleotidases expressed on DCs in adenosine with suppressive properties, thus resulting in the stabilization of Tregs phenotype and function. In the present work, we have investigated the role of ATP catabolism in Tregs induction after chemotherapy treatment in AML. Materials and methods: For in vitro experiments,CD14 and CD3 positive cells were obtained from peripheral blood mononuclear cells of healthy donors. HL-60 leukemia cell line cells, untreated or treated with daunorubicin (DNR; 500 ng/ml) or cytarabine (Ara-C; 20 µg/ml) were loaded in immature DCs, previously differentiated from monocytes. After 24 h, immature DCs and DCs matured by treated HL-60 cells or DCs treated with ATP in presence or absence of P2X7 inhibitor, were analyzed for phenotype and used to activate T cells. After 5 days, a detailed phenotype characterization of induced Tregs was performed by flow cytometry. For in vivo experiments, Balbc/J wt mice were subcutaneously injected with 2x106 WEHI-3B leukemia cells. DNR (3 mg/kg), Ara-C (50 mg/kg) or sterile PBS vehicle (placebo) were administered at post inoculum days 7 and 9, then the tumor infiltrating lymphocytes were purified from tumor mass and stained by flow cytometry. Results: Firstly, we in vitro characterized immune phenotype of DCs loaded with HL-60 cells, previously treated with DNR, Ara-C or medium alone. We identified DNR as a potent maturation stimulus as compared to DCs loaded with untreated cells or unloaded immature DCs. In particular, both DNR and Ara-C induced a significant up-regulation of CD83, CD80 and CD86, but only DNR induced a significant up-regulation of CCR7, which is required for DC migration to lymphonodes. Then, the effect of chemotherapy on CD39 and CD73 expression in DCs was also evaluated. DNR induced a significant up-regulation of CD73-the rate limiting step of ATP catabolism, whereas upon Ara-C treatment there was a trend in the up-regulation of both CD39 and CD73. Interestingly, DCs treated with ATP alone achieved similar maturation markers level as DNR, but showed neither CCR7 nor CD39/CD73 up-regulation, which suggests that ATP alone is not sufficient to activate the ATP catabolism pathway. The analysis of Tregs induced after 5 days of co-culture with DCs treated as previously described showed a significant up-regulation of "fitness markers", in particular OX40/CD39 after DNR and OX40/PD-1 after DNR and Ara-C treatment as compared to unloaded immature DCs. Mouse models confirmed the capacity of DNR and Ara-C to induce DCs maturation and activation of ATP catabolism pathways by significantly inducing CD39 and CD73 up-regulation. Moreover, chemotherapy treatment resulted in the stabilization of Tregs suppressive phenotype as demonstrated by the up-regulation of OX40 and ICOS fitness markers in comparison to placebo treatment. Conclusions: Our data suggest that different mechanisms operate in tumor microenvironment. IDO may induce Tregs in an ATP-dependent manner, whereas the ATP catabolism may be involved in the stabilization of Tregs suppressive phenotype. In this context, the specific role of the main bioproduct of ATP catabolism, adenosine, is under investigation. Our results can help to understand the mechanisms of tolerance induction after chemotherapy in AML. Disclosures Cavo: takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; novartis: Honoraria.

A hypomorphic Ephb2 gene variant contributes to autoimmune diabetes in NOD mice associated with altered Mapk3 activities in B-lymphocytes

Abstract β-cell destruction in type 1 diabetes (T1D) is considered a T-cell mediated autoimmune disease, but based on studies in the NOD mouse model, B-lymphocytes also play a critical pathogenic role. They do so by serving as a preferential subset of pathogenic APC that expand diabetogenic T cells. Immunological tolerance induction mechanisms that normally eliminate or inactivate autoreactive B-lymphocytes are defective in NOD mice and also in T1D patients. We previously mapped a faulty NOD B-lymphocyte tolerance phenotype to a 6.22Mb region on Chromosome 4. With a combination of differential gene expression and ingenuity pathway analyses (IPA) we identified a hypomorphic Ephb2 allelic variant within this region on Chromosome 4 as a potential candidate gene interacting with human T1D susceptibility genes in a common pathway modulating Mapk3 activities. Ephrin-B1 ligand engagement of Ephb2 attenuated B-cell receptor (BCR) induced Mapk3 phosphorylation in vitro. We tested the candidacy of Ephb2 by introducing into NOD mice a transgenic higher expression variant of Ephb2 derived from the B6 strain (NOD-Ephb2B6). Elevated expression of Ephb2 afforded significant T1D protection due to changes in hematopoietic cells. Despite having enhanced proliferative capacity, B-lymphocytes from NOD-Ephb2B6 mice had diminished diabetogenic activity associated with attenuated Mek-Erk signaling. Introduction of the Ephb2 transgene into NOD BCR transgenic models expressing autoreactive peripherin (Per) specific Ig molecules also inhibited the accelerated T1D development characterizing these stocks. Thus, signaling via Ephb2 in B-lymphocytes can fine-tune BCR signaling that ultimately determine their diabetogenic activities.

Subcellular antigen localization in commensal E. coli is critical for T cell activation and induction of specific tolerance.

Oral tolerance to soluble antigens is critically important for the maintenance of immunological homeostasis in the gut. The mechanisms of tolerance induction to antigens of the gut microbiota are still less well understood. Here, we investigate whether the subcellular localization of antigens within non-pathogenic E. coli has a role for its ability to induce antigen-specific tolerance. E. coli that express an ovalbumin (OVA) peptide in the cytoplasm, at the outer membrane or as secreted protein were generated. Intestinal colonization of mice with non-pathogenic E. coli expressing OVA at the membrane induced the expansion of antigen-specific Foxp3+ Tregs and mediated systemic immune tolerance. In contrast, cytoplasmic OVA was ignored by antigen-specific CD4+ T cells and failed to induce tolerance. In vitro experiments revealed that surface-displayed OVA of viable E. coli was about two times of magnitude more efficient to activate antigen-specific CD4+ T cells than soluble antigens, surface-displayed antigens of heat-killed E. coli or cytoplasmic antigen of viable or heat-killed E. coli. This effect was independent of the antigen uptake efficiency in dendritic cells. In summary, our results show that subcellular antigen localization in viable E. coli strongly influences antigen-specific CD4+ cell expansion and tolerance induction upon intestinal colonization.

Antigen Uptake by Langerhans Cells Is Required for the Induction of Regulatory T Cells and the Acquisition of Tolerance During Epicutaneous Immunotherapy in OVA-Sensitized Mice.

The skin is a major immunologic organ that may induce protection, sensitization or tolerance. Epicutaneous immunotherapy (EPIT) has been proposed as an attractive strategy to actively treat food allergy and has been shown to induce tolerance in sensitized mice through the induction of Foxp3+ regulatory T cells (Tregs), especially CD62L+ Tregs. Among immune cells in the skin, dendritic cells are key players in antigen-specific immune activation or regulation. The role of different populations of skin DCs in tolerance induction remains to be elucidated. Using OVA-sensitized BALB/c mice, we demonstrated that the application of a patch containing OVA-A647 to the skin resulted in allergen uptake by Langerhans cells (LCs) and CD11b+ dermal cDC2 and subsequent migration into skin draining lymph nodes. These 2 populations induced Foxp3 expression in CD4+ cells in vitro. Only LCs induced LAP+ cells and CD62L+ Tregs. Using Langerin-eGFP-DTR mice, we analyzed the role of LCs in the mechanisms of tolerance induction by EPIT in vivo. Following complete depletion of LCs, a dramatic decrease in the number of OVA+ DCs and OVA+ CD11b+ dermal cDC2 was observed in skin draining lymph nodes 48 h after epicutaneous application. Likewise, 2 weeks of EPIT in non-depleted mice induced Foxp3+ Tregs, especially CD62L+, and LAP+ Tregs in skin draining lymph nodes and spleen, whereas no induction of Tregs was observed in LC-depleted mice. Following 8 weeks of treatment, EPIT-treated mice showed significant protection against anaphylaxis accompanied by a significant increase of Foxp3+ Tregs, especially CD62L+ Tregs, which was not seen in the absence of LCs. In summary, although both LCs and CD11b+ dermal cDC2s could induce regulatory T cells, the absence of LCs during EPIT impaired treatment efficacy, indicating their crucial role in skin-induced tolerance.

Subcutaneous immunotherapy suppresses Th2 inflammation and induces neutralizing antibodies, but sublingual immunotherapy suppresses airway hyperresponsiveness in grass pollen mouse models for allergic asthma.

Both subcutaneous and sublingual allergen immunotherapy (SCIT and SLIT) have been shown to effectively suppress allergic manifestations upon allergen exposure, providing long-term relief from symptoms in allergic disorders including allergic asthma. Clinical studies directly comparing SCIT and SLIT report a different kinetics and magnitude of immunological changes induced during treatment. Comparative studies into the mechanisms underlying immune suppression in SCIT and SLIT are lacking. We aimed to establish an experimental model for grass pollen (GP) SCIT and SLIT that would allow a head-to-head comparison of the two treatments. BALB/c mice were sensitized with GP extract, followed by SCIT and SLIT treatments with various GP dosages. Subsequently, we challenged mice with GP and measured airway responsiveness (AHR), GP-specific immunoglobulins, ear swelling tests (EST), eosinophilic inflammation in bronchoalveolar lavage fluid (BALF), and T cell cytokine release after restimulation of lung cells (IL-5, IL-10, and IL-13). We find that SLIT treatment was able to suppress allergen-induced AHR, while allergic inflammation was not effectively suppressed even at the highest GP dose in this model. In contrast, SCIT treatment induced higher levels of GP-specific IgG1, while SLIT was superior in inducing a GP-specific IgG2a response, which was associated with increased Th1 activity in lung tissue after SLIT, but not SCIT treatment. Interestingly, SCIT was able to suppress Th2-type cytokine production in lung cell suspensions, while SLIT failed to do so. In conclusion, GP-SCIT suppresses Th2 inflammation and induced neutralizing antibodies, while GP-SLIT suppresses the clinically relevant lung function parameters in an asthma mouse model, indicating that the two application routes depend on partially divergent mechanisms of tolerance induction. Interestingly, these data mirror observations in clinical studies, underscoring the translational value of these mouse models.

The mechanism of preventing EAE via gene therapy

Abstract Similar to Multiple Sclerosis, EAE is a complex disease in which immune tolerance is lost and antigen specific (Ag-sp) Tregs are known to play a role. We have developed a gene immunotherapy that has been shown to induce MOG specific Tregs, restore tolerance, and effectively prevent, and/or reverse, significant disease. However, the spatiotemporal profile of the induced Tregs is unclear. To identify the tissue specific populations of AAV8.MOG-induced specific Tregs, we used a transgenic Foxp3 reporter mouse with a MOG35–55/I-Ab tetramer. Two weeks prior to EAE induction, mice were tolerized via a single injection of vector. At the peak of disease, tissues were harvested and fluorescently labeled for phenotypic identification. FACS analysis revealed a higher proportion of Ag-sp Tregs in the cervical and inguinal lymph nodes (LN) and spleen, as compared to controls. In contrast, a higher proportion of Ag-sp effector cells were seen in control mice as compared to treated. Furthermore, a significant number of infiltrating effector T cells were isolated from the spinal cords of control mice, while very few T cells were recovered from treated mice. This aligns with cytokine data that was collected showing there to be higher production of IFN-γ in the spinal cord and spleen of control mice. These results suggest that AAV.MOG induced Ag-sp Treg populations accumulate in the peripheral organs, and may prevent effector cells from migrating to the spinal cord. This work provides new insights on tolerance induction mechanisms. These insights will be useful for the development of effective cell based therapies capable of treating autoimmune diseases.

Mechanisms of Tolerance Induction by Dendritic Cells In Vivo.

Dendritic cells (DCs) are a heterogeneous population playing a pivotal role in immune responses and tolerance. DCs promote immune tolerance by participating in the negative selection of autoreactive T cells in the thymus. Furthermore, to eliminate autoreactive T cells that have escaped thymic deletion, DCs also induce immune tolerance in the periphery through various mechanisms. Breakdown of these functions leads to autoimmune diseases. Moreover, DCs play a critical role in maintenance of homeostasis in body organs, especially the skin and intestine. In this review, we focus on recent developments in our understanding of the mechanisms of tolerance induction by DCs in the body.

Tolerance through Education: How Tolerogenic Dendritic Cells Shape Immunity.

Dendritic cells (DCs) are central players in the initiation and control of responses, regulating the balance between tolerance and immunity. Tolerogenic DCs are essential in the maintenance of central and peripheral tolerance by induction of clonal T cell deletion and T cell anergy, inhibition of memory and effector T cell responses, and generation and activation of regulatory T cells. Therefore, tolerogenic DCs are promising candidates for specific cellular therapy of allergic and autoimmune diseases and for treatment of transplant rejection. Studies performed in rodents have demonstrated the efficacy and feasibility of tolerogenic DCs for tolerance induction in various inflammatory diseases. In the last years, numerous protocols for the generation of human monocyte-derived tolerogenic DCs have been established and some first phase I trials have been conducted in patients suffering from autoimmune disorders, demonstrating the safety and efficiency of this cell-based immunotherapy. This review gives an overview about methods and protocols for the generation of human tolerogenic DCs and their mechanisms of tolerance induction with the focus on interleukin-10-modulated DCs. In addition, we will discuss the prerequisites for optimal clinical grade tolerogenic DC subsets and results of clinical trials with tolerogenic DCs in autoimmune diseases.

Epicutaneous Tolerance Induction to a Bystander Antigen Abrogates Colitis and Ileitis in Mice.

Although inflammatory bowel disease (IBD) is a failure in maintaining tolerance to the intestinal microbiota, few studies have investigated the use of immunologic tolerance as a treatment approach for IBD. We hypothesized that induction of immune tolerance at a distal site could suppress intestinal inflammation through a process of bystander regulation. Epicutaneous tolerance was induced by topical application of ovalbumin (OVA) using a Viaskin patch for 48 hours. In some experiments, a single feed of ovalbumin was used to drive epicutaneous tolerance-induced regulatory T cells (Tregs) to the intestine. The mechanism of tolerance induction was tested using neutralizing antibodies against TGF-β, IL-10, and Treg depletion using Foxp3-DTR mice. The capacity of skin-draining Tregs, or epicutaneous tolerance, to prevent or treat experimental IBD was tested using T-cell transfer colitis, dextran sodium sulfate (DSS) colitis, and ileitis in SAMP-YITFc mice. Weight loss, colonic inflammatory cytokines and histology were assessed. Epicutaneous exposure to ovalbumin induced systemic immune tolerance by a TGF-β-dependent, but IL-10 and iFoxp3 Treg-independent mechanism. Skin draining Tregs suppressed the development of colitis. Epicutaneous tolerance to a model antigen prevented intestinal inflammation in the dextran sodium sulfate and SAMP-YITFc models and importantly could halt disease in mice already experiencing weight loss in the T-cell transfer model of colitis. This was accompanied by a significant accumulation of LAP and Foxp3 Tregs in the colon. This is the first demonstration that epicutaneous tolerance to a model antigen can lead to bystander suppression of inflammation and prevention of disease progression in preclinical models of IBD.

Mechanisms of Tolerance Induction by Hematopoietic Chimerism: The Immune Perspective.

SummaryHematopoietic chimerism is one of the effective approaches to induce tolerance to donor‐derived tissue and organ grafts without administration of life‐long immunosuppressive therapy. Although experimental efforts to develop such regimens have been ongoing for decades, substantial cumulative toxicity of combined hematopoietic and tissue transplants precludes wide clinical implementation. Tolerance is an active immunological process that includes both peripheral and central mechanisms of mutual education of coresident donor and host immune systems. The major stages include sequential suppression of early alloreactivity, establishment of hematopoietic chimerism and suppressor cells that sustain the state of tolerance, with significant mechanistic and temporal overlap along the tolerization process. Efforts to devise less toxic transplant strategies by reduction of preparatory conditioning focus on modulation rather than deletion of residual host immunity and early reinstitution of regulatory subsets at the central and peripheral levels. Stem Cells Translational Medicine 2017;6:700–712

Tracking of TCR-Transgenic T Cells Reveals That Multiple Mechanisms Maintain Cardiac Transplant Tolerance in Mice.

Solid organ transplantation tolerance can be achieved following select transient immunosuppressive regimens that result in long-lasting restraint of alloimmunity without affecting responses to other antigens. Transplantation tolerance has been observed in animal models following costimulation or coreceptor blockade therapies, and in a subset of patients through induction protocols that include donor bone marrow transplantation, or following withdrawal of immunosuppression. Previous data from our lab and others have shown that proinflammatory interventions that successfully prevent the induction of transplantation tolerance in mice often fail to break tolerance once it has been stably established. This suggests that established tolerance acquires resilience to proinflammatory insults, and prompted us to investigate the mechanisms that maintain a stable state of robust tolerance. Our results demonstrate that only a triple intervention of depleting CD25+ regulatory T cells (Tregs), blocking programmed death ligand-1 (PD-L1) signals, and transferring low numbers of alloreactive T cells was sufficient to break established tolerance. We infer from these observations that Tregs and PD-1/PD-L1 signals cooperate to preserve a low alloreactive T cell frequency to maintain tolerance. Thus, therapeutic protocols designed to induce multiple parallel mechanisms of peripheral tolerance may be necessary to achieve robust transplantation tolerance capable of maintaining one allograft for life in the clinic.