Induction Of Antigen-specific Tolerance Research Articles

Engineering Antigen-Specific Tolerance to an Artificial Protein Hydrogel.

Artificial protein hydrogels are an emerging class of biomaterials with numerous prospective applications in tissue engineering and regenerative medicine. These materials are likely to be immunogenic due to their frequent incorporation of novel amino acid sequence domains, which often serve a functional role within the material itself. We engineered injectable "self" and "nonself" artificial protein hydrogels, which were predicted to have divergent immune outcomes in vivo on the basis of their primary amino acid sequence. Following implantation in mouse, the nonself gels raised significantly higher antigel antibody titers than the corresponding self gels. Prophylactic administration of a fusion antibody targeting the nonself hydrogel epitopes to DEC-205, an endocytic receptor involved in Treg induction, fully suppressed the elevated antibody titer against the nonself gels. These results suggest that the clinical immune response to artificial protein biomaterials, including those that contain highly antigenic sequence domains, can be tuned through the induction of antigen-specific tolerance.

Tolerogenic Nano-/Microparticle Vaccines for Immunotherapy.

Autoimmune diseases, allergies, transplant rejections, generation of antidrug antibodies, and chronic inflammatory diseases have impacted a large group of people across the globe. Conventional treatments and therapies often use systemic or broad immunosuppression with serious efficacy and safety issues. Tolerogenic vaccines represent a concept that has been extended from their traditional immune-modulating function to induction of antigen-specific tolerance through the generation of regulatory T cells. Without impairing immune homeostasis, tolerogenic vaccines dampen inflammation and induce tolerogenic regulation. However, achieving the desired potency of tolerogenic vaccines as preventive and therapeutic modalities calls for precise manipulation of the immune microenvironment and control over the tolerogenic responses against the autoantigens, allergens, and/or alloantigens. Engineered nano-/microparticles possess desirable design features that can bolster targeted immune regulation and enhance the induction of antigen-specific tolerance. Thus, particle-based tolerogenic vaccines hold great promise in clinical translation for future treatment of aforementioned immune disorders. In this review, we highlight the main strategies to employ particles as exciting tolerogenic vaccines, with a focus on the particles' role in facilitating the induction of antigen-specific tolerance. We describe the particle design features that facilitate their usage and discuss the challenges and opportunities for designing next-generation particle-based tolerogenic vaccines with robust efficacy to promote antigen-specific tolerance for immunotherapy.

Transdermally delivered tolerogenic nanoparticles induced effective immune tolerance for asthma treatment

Induction of antigen-specific immune tolerance for the treatment of allergic or autoimmune diseases is an attractive strategy. Herein, we investigated the protective effect of a transdermal microneedle patch against allergic asthma by stimulating allergen-specific immune tolerance. We fabricated biodegradable tolerogenic nanoparticles (tNPs) that are loaded with a model allergen ovalbumin (OVA) and an immunomodulator rapamycin, and filled the tNPs into microneedle tips by centrifugation to form sustained-release microneedles. After intradermal immunization, the microneedles successfully delivered the cargos into the skin and sustainedly released them for over 96 h. Importantly, the microneedles induced allergen-specific regulatory T cells (Treg), decreased the levels of pro-inflammatory cytokines and antibodies while increased anti-inflammation cytokines, finally leading to restored immune homeostasis. The lung tissue analysis illustrated that the sustained-release microneedles significantly reduced the infiltration of eosinophils, decreased the accumulation of mucus and collagen, and significantly relived asthma symptoms. Our results suggested that the sustained-release microneedle-based transdermal delivery system can induce antigen-specific immune tolerance with improved compliance and efficacy, providing a new therapeutic strategy for the treatment of allergic and autoimmune diseases.

Safe and efficient oral allergy immunotherapy using one-pot-prepared mannan-coated allergen nanoparticles

Allergen immunotherapy (AIT) is the only curative treatment for allergic diseases. However, AIT has many disadvantages related to efficiency, safety, long-term duration, and patient compliance. Dendritic cells (DCs) have an important role in antigen-specific tolerance induction; thus, DC-targeting strategies to treat allergies such as glutaraldehyde crosslinked antigen to mannoprotein (MAN) have been established. However, glutaraldehyde crosslinking may reduce the antigen presentation efficiency of DCs. To overcome this, we developed a MAN-coated ovalbumin (OVA) nanoparticle (MDO), which uses intermolecular disulfide bond to crosslink OVA and MAN. MDO effectively targeted DCs resulting in tolerogenic DCs, and promoted higher antigen presentation efficiency by DCs compared with OVA or glutaraldehyde crosslinked nanoparticles. In vitro and in vivo experiments showed that DCs exposed to MDO induced Treg cells. Moreover, MDO had low reactivity with anti-OVA antibodies and did not induce anaphylaxis in allergic mice, demonstrating its high safety profile. In a mouse model of allergic asthma, MDO had significant preventative and therapeutic effects when administered orally or subcutaneously. Therefore, MDO represents a promising new approach for the efficient and safe treatment of allergies.

One-pot preparation of mannan-coated antigen nanoparticles using human serum albumin as a matrix for tolerance induction

Nanoparticles (NPs) for allergen immunotherapy have garnered attention for their high efficiency and safety compared with naked antigen proteins. In this work, we present mannan-coated protein NPs, incorporating antigen proteins for antigen-specific tolerance induction. The heat-induced formation of protein NPs is a one-pot preparation method and can be applied to various proteins. Here, the NPs were formed spontaneously via heat denaturation of three component proteins: an antigen protein, human serum albumin (HSA) as a matrix protein, and mannoprotein (MAN) as a targeting ligand for dendritic cells (DCs). HSA is non-immunogenic, therefore suitable as a matrix protein, while MAN coats the surface of the NP. We applied this method to various antigen proteins and found that the self-disperse after heat denaturation was a requirement for incorporation into the NPs. We also established that the NPs could target DCs, and the incorporation of rapamycin into the NPs enhanced the induction of a tolerogenic phenotype of DC. The MAN coating provided steric hindrance and heat denaturation destroyed recognition structures, successfully preventing anti-antigen antibody binding, indicating the NPs may avoid anaphylaxis induction. The MAN-coated NPs proposed here, prepared by a simple method, have the potential for effective and safe allergies treatment for various antigens.

Live Cell Imaging by Förster Resonance Energy Transfer Fluorescence to Study Trafficking of PLGA Nanoparticles and the Release of a Loaded Peptide in Dendritic Cells.

Our previous study demonstrated that a selected β-lactoglobulin-derived peptide (BLG-Pep) loaded in poly(lactic-co-glycolic acid) (PLGA) nanoparticles protected mice against cow's milk allergy development. However, the mechanism(s) responsible for the interaction of the peptide-loaded PLGA nanoparticles with dendritic cells (DCs) and their intracellular fate was/were elusive. Förster resonance energy transfer (FRET), a distance-dependent non-radioactive energy transfer process mediated from a donor to an acceptor fluorochrome, was used to investigate these processes. The ratio of the donor (Cyanine-3)-conjugated peptide and acceptor (Cyanine-5) labeled PLGA nanocarrier was fine-tuned for optimal (87%) FRET efficiency. The colloidal stability and FRET emission of prepared NPs were maintained upon 144 h incubation in PBS buffer and 6 h incubation in biorelevant simulated gastric fluid at 37 °C. A total of 73% of Pep-Cy3 NP was internalized by DCs as quantified using flow cytometry and confirmed using confocal fluorescence microscopy. By real-time monitoring of the change in the FRET signal of the internalized peptide-loaded nanoparticles, we observed prolonged retention (for 96 h) of the nanoparticles-encapsulated peptide as compared to 24 h retention of the free peptide in the DCs. The prolonged retention and intracellular antigen release of the BLG-Pep loaded in PLGA nanoparticles in murine DCs might facilitate antigen-specific tolerance induction.

Polysarcosine-based lipid formulations for intracranial delivery of mRNA.

Messenger RNA (mRNA) is revolutionizing the future of therapeutics in a variety of diseases, including neurological disorders. Lipid formulations have shown to be an effective platform technology for mRNA delivery and are the basis for the approved mRNA vaccines. In many of these lipid formulations, polyethylene glycol (PEG)-functionalized lipid provides steric stabilization and thus plays a key role in improving the stability both ex vivo and in vivo. However, immune responses towards PEGylated lipids may compromise the use of those lipids in some applications (e.g., induction of antigen specific tolerance), or within sensitive tissues (e.g., central nervous system (CNS)). With respect to this issue, polysarcosine (pSar)-based lipopolymers were investigated as an alternative to PEG-lipid in mRNA lipoplexes for controlled intracerebral protein expression in this study. Four polysarcosine-lipids with defined sarcosine average molecular weight (Mn=2k, 5k) and anchor diacyl chain length (m=14, 18) were synthesized, and incorporated into cationic liposomes. We found that the content, pSar chain length and carbon tail lengths of pSar-lipids govern the transfection efficiency and biodistribution. Increasing carbon diacyl chain length of pSar-lipid led up to 4- and 6-fold lower protein expression in vitro. When the length of either pSar chain or lipid carbon tail increased, the transfection efficiency decreased while the circulation time was prolonged. mRNA lipoplexes containing 2.5% C14-pSar2k resulted in the highest mRNA translation in the brain of zebrafish embryos through intraventricular injection, while C18-pSar2k-liposomes showed a comparable circulation with DSPE-PEG2k-liposomes after systemic administration. To conclude, pSar-lipid enable efficient mRNA delivery, and can substitute PEG-lipids in lipid formulations for controlled protein expression within the CNS.

Manufacturing and validation of Good Manufacturing Practice–compliant regulatory dendritic cells for infusion into organ transplant recipients

Background aimsRegulatory (or “tolerogenic”) dendritic cells (DCregs) are a highly promising, innovative cell therapy for the induction or restoration of antigen-specific tolerance in immune-mediated inflammatory disorders. These conditions include organ allograft rejection, graft-versus-host disease following bone marrow transplantation and various autoimmune disorders. DCregs generated for adoptive transfer have potential to reduce patients’ dependence on non-specific immunosuppressive drugs that can induce serious side effects and enhance the risk of infection and certain types of cancer. Here, our aim was to provide a detailed account of our experience manufacturing and validating comparatively large numbers of Good Manufacturing Practice–grade DCregs for systemic (intravenous) infusion into 28 organ (liver) transplant recipients and to discuss factors that influence the satisfaction of release criteria and attainment of target cell numbers. ResultsDCregs were generated in granulocyte-macrophage colony stimulating factor and interleukin (IL)-4 from elutriated monocyte fractions isolated from non-mobilized leukapheresis products of consenting healthy adult prospective liver transplant donors. Vitamin D3 was added on day 0 and 4 and IL-10 on day 4 during the 7-day culture period. Release and post-release criteria included cell viability, purity, phenotype, sterility and functional assessment. The overall conversion rate of monocytes to DCregs was 28 ± 8.2%, with 94 ± 5.1% product viability. The mean cell surface T-cell co-inhibitory to co-stimulatory molecule (programmed death ligand-1:CD86) mean fluorescence intensity ratio was 3.9 ± 2.2, and the mean ratio of anti-inflammatory:pro-inflammatory cytokine product (IL-10:IL-12p70) secreted upon CD40 ligation was 60 ± 63 (median = 40). The mean total number of DCregs generated from a single leukapheresis product (n = 25 donors) and from two leukapheresis products (n = 3 donors) was 489 ± 223 × 106 (n = 28). The mean total number of DCregs infused was 5.9 ± 2.8 × 106 per kg body weight. DCreg numbers within a target cell range of 2.5–10 × 106/kg were achieved for 25 of 27 (92.6%) of products generated. ConclusionsHigh-purity DCregs meeting a range of quality criteria were readily generated from circulating blood monocytes under Good Manufacturing Practice conditions to meet target cell numbers for infusion into prospective organ transplant recipients.

Treg-based immunotherapy for antigen-specific immune suppression and stable tolerance induction: a perspective.

FoxP3-expressing regulatory T cells (Tregs), whether naturally generated in the immune system or unnaturally induced from conventional T cells (Tconvs) in the laboratory, have much therapeutic value in treating immunological diseases and establishing transplantation tolerance. Natural Tregs (nTregs) can be selectively expanded in vivo by administration of low-dose IL-2 or IL-2 muteins for immune suppression. For adoptive Treg cell therapy, nTregs can be expanded in vitro by strong antigenic stimulation in the presence of IL-2. Synthetic receptors such as CAR can be expressed in nTregs to equip them with a particular target specificity for suppression. In addition, antigen-specific Tconvs can be converted in vitro to functionally stable Treg-like cells by a combination of antigenic stimulation, FoxP3 induction, and establishment of the Treg-type epigenome. This review discusses current and prospective strategies for Treg-based immune suppression and the issues to be resolved for achieving stable antigen-specific immune suppression and tolerance induction in the clinic by targeting Tregs.

Induction of antigen-specific tolerance by hepatic AAV immunotherapy regardless of T cell epitope usage or mouse strain background

Invivo induction of antigen (Ag)-specific regulatory Tcells (Treg) is considered the holy grail of therapeutic strategies for restoring tolerance in autoimmunity. Unfortunately, in the autoimmune disease multiple sclerosis, an effective and durable therapy targeting the diverse repertoire of emerging Ags without compromising the patient's natural immunity has remained elusive. To address this deficiency, we have developed an Ag-specific adeno-associated virus (AAV) immunotherapy that will restore tolerance in a Treg-dependent manner. Using multiple strains of mice with different genetic and immunological backgrounds, we demonstrate that a liver directed AAV vector expressing a single transgene can prevent experimental autoimmune encephalomyelitis from developing and effectively mitigate pre-existing or established disease that was induced by one or more auto-reactive myelin oligodendrocyte glycoprotein-derived peptides. Overall, the results suggests that AAV can efficiently restore Ag-specific immune tolerance to an immunogenic protein that is neither restricted by the major histocompatibility complex haplotype, nor by the specific antigenic epitope(s) presented. These findings may pave the way for developing a comprehensive Ag-specific immunotherapy that does not require prior knowledge of the specific immunogenic epitopes and that may prove to be universally applicable to all MS patients, and adaptable for other autoimmune diseases.

Particle-Based therapies for antigen specific treatment of type 1 diabetes

Type 1 diabetes mellitus (T1D) is the leading metabolic disorder in children worldwide. Over time, incidence rates have continued to rise with 20 million individuals affected globally by the autoimmune disease. The current standard of care is costly and time-consuming requiring daily injections of exogenous insulin. T1D is mediated by autoimmune effector responses targeting autoantigens expressed on pancreatic islet β-cells. One approach to treat T1D is to skew the immune system away from an effector response by taking an antigen-specific approach to heighten a regulatory response through a therapeutic vaccine. An antigen-specific approach has been shown with soluble agents, but the effects have been limited. Micro or nanoparticles have been used to deliver a variety of therapeutic agents including peptides and immunomodulatory therapies to immune cells. Particle-based systems can be used to deliver cargo into the cell and microparticles can passively target phagocytic cells. Further, surface modification and controlled release of encapsulated cargo can enhance delivery over soluble agents. The induction of antigen-specific immune tolerance is imperative for the treatment of autoimmune diseases such as T1D. This review highlights studies that utilize particle-based platforms for the treatment of T1D.

Small Extracellular Vesicles Loaded with Immunosuppressive miRNAs Leads to an Inhibition of Dendritic Cell Maturation

In particular conditions, inhibition of an immune response is required to prevent tissue damage. Among these conditions are diseases caused by an over-reactive immune response, such as autoimmune or allergic disorders, or imminent organ rejection after transplantation. To avoid tissue damage, drug-mediated systemic immune suppression is an option, but it comes with high costs in the form of susceptibility to viral and bacterial infections. Thus, the induction of antigen-specific tolerance is preferable. Extracellular vesicles (EVs) are capable of delivering antigen together with immunosuppressive signals and may be used to specifically induce antigen-specific tolerance. However, naturally occurring EVs are heterogeneous and not all of them show immunosuppressive character. In our trials to engineer cell culture derived EVs to increase their tolerogenic potential, we equipped them with immunosuppressive miRNA mimics. Small EVs (sEVs) were isolated and purified from the human monocytic THP-1 cell line or from healthy donor peripheral blood mononuclear cells, and electroporated with miR-494 and miR-146a mimics. The acquired immunosuppressive potential of the modified sEVs was demonstrated by their ability to alter the major histocompatibility complex molecules and co-stimulatory receptors present on dendritic cells (DCs). To avoid allogeneic responses, the same cells that produced the sEVs served also as recipient cells. In contrast to the treatment with unmodified sEVs, the tolerogenic sEVs impeded lipopolysaccharide-induced maturation and kept DCs in a more immature developmental stage. Our experiments show that simple manipulations of sEVs using immunosuppressive cargo can lead to the inhibition of DC maturation.

Symposium 2-2 - Challenges in elucidating the mechanisms of fetal recognition and tolerance by T cells

The fetus is a semi-graft expressing paternally derived antigens that are foreign to the mother, however, it is not rejected by the mother because of the establishment of feto-maternal tolerance. Maternal cytotoxic T cells (CTLs) and CD4+ T helper cells (CD4+Tconv) recognize fetal antigens at the T cell receptor (TCR), while regulatory T cells (Treg) suppress immune responses. To investigate the role of antigen-specific Tregs and CTLs in human pregnancy, we analyzed the TCR variations from single T cells from decidua. In normal pregnancies, the number of Tregs with specific TCRs which were presumed to be paternal antigen-specific Tregs increased in a biased manner. In preeclampsia, antigen specific Tregs were decreased. This suggests that induction of antigen-specific immune tolerance by Tregs was inadequate in preeclampsia. Next, to comprehensively search for pathologically relevant molecules in Tregs and CD4+Tconv that could be targets for immunological therapy, we performed single-cell transcriptome analysis of T cells. The results suggest that Th17 among CD4+Tconvs is increased in PE, but the differentiation pathway may be different from that of Tregs. We would like to mention the results of this study and the prospects for immunological approaches to PE therapy.

In Vitro Model of Suppression of the Alloantigen Response by Tolerogenic Dendritic Cells Transfected with Personalized DNA Constructs Encoding HLA Epitopes.

A search for efficient graft rejection modulation techniques for the promotion of durable engraftment remains to be a matter of close study all over the world. Despite the variety of immunosuppressive drugs, the schemes currently used show a lack of selectivity and have a number of side effects. Here we investigated an approach for the induction of antigen-specific tolerance in a human "stimulator-responder" model in vitro, using dendritic cells (DCs) transfected with designed DNA constructs encoding the stimulator's major histocompatibility complex (MHC) epitopes. The object of the study is peripheral blood mononuclear cells (PBMCs) from 10 healthy donors. To induce antigen-specific tolerance, personalized DNA constructs were created for five responder-stimulator pairs, based on the sequences of donors' and recipients' MHCs. DNA sequencing was performed to select epitopes for incorporation into genetic constructs. A mixed lymphocyte culture assay was used (i) to assess the proliferative response in both directions for all possible stimulator-responder pairs (90 reactions) and (ii) to assess the tolerogenic properties of the generated transfected DCs (5 reactions). A significant increase in the amounts of FoxP3+ CD4+CD25+ cells and in IL-10 production was shown in culture of donor mononuclear cells after co-cultivation with the responder's dendritic cells transfected with donor-specific plasmids. The tolerogenic cultures generated using tolerogenic DCs transfected with MHC epitopes had a significantly greater ability to inhibit the proliferation of autologous MNCs in response to an allogeneic MHC stimulus. The produced DCs transfected with DNA constructs against HLA stimulating epitopes exhibited tolerogenic properties and may be used to develop antigen-specific tolerance. Thus, we proposed a perspective approach to the induction of antigen-specific tolerance, which should subsequently be studied for use in clinical practice.

Allergen specific immunotherapy with plasmid DNA encoding OVA-immunodominant T cell epitope fused to Tregitope in a murine model of allergy

BackgroundPeptide-based immunotherapy (PIT) was introduced as an attractive approach in allergen-specific immunotherapy (AIT). However, PIT clinical trials have shown variable results, and immune response to peptides is not precisely predictable. On the other hand, induction of antigen-specific tolerance may be augmented when allergens are combined with the regulatory T cell epitope (Tregitope). This study aimed to evaluate the therapeutic administration of a plasmid DNA encoding Tregitope and ovalbumin (OVA) immunodominant epitope in the murine model of allergy. MethodsFollowing the induction of allergic rhinitis by ovalbumin, vaccinated group received three doses of recombinant plasmid containing Signal peptide-Tregitope-OVA T cell epitope. After the final OVA challenge, clinical symptoms, histopathological changes, OVA-specific IgE level, and cytokine secretion pattern of spleen cells were examined. ResultsOur data are showing that AIT with the recombinant DNA vaccine significantly suppressed airway inflammation; reduced eosinophilic infiltration in the nasal mucosa; decreased expression level of IL-4 and IL-17 in spleen cells, while IFN-γ, IL-10, and TGF-β expression were increased. Moreover, OVA-specific IgE levels were also decreased. ConclusionThese results suggest that Tregitope-immunodominant T cell epitope fusion can act as a safe and effective approach in DNA-based allergen-specific immunotherapy.

Drug design for induction of antigen-specific immune tolerance

Drug design for induction of antigen-specific immune tolerance

Tuning Surface Charges of Peptide Nanofibers for Induction of Antigen-Specific Immune Tolerance: An Introductory Study

Induction of antigen-specific immune tolerance has emerged as the next frontier in treating autoimmune disorders, including atherosclerosis and graft-vs-host reactions during transplantation. Nanostructures are under investigation as a platform for the coordinated delivery of critical components, i.e., the antigen epitope combined with tolerogenic agents, to the target immune cells and subsequently induce tolerance. In the present study, the utility of supramolecular peptide nanofibers to induce antigen-specific immune tolerance was explored. To study the influence of surface charges of the nanofibers towards the extent of the induced immune response, the flanking charge residues at both ends of the amphipathic fibrillization peptide sequences were varied. Dexamethasone, an immunosuppressive glucocorticoid drug, and the ovalbumin-derived OVA323-339 peptide that binds to I-A(d) MHC Class II were covalently linked at either end of the peptide sequences. It was shown that the functional extensions did not alter the structural integrity of the supramolecular nanofibers. Furthermore, the surface charges of the nanofibers were modulated by the inclusion of charged residues. Dendritic cell culture assays suggested that nanofiber of less negative ζ-potential can augment the antigen-specific tolerogenic response. Our findings illustrate a molecular approach to calibrate the tolerogenic response induced by peptide nanofibers, which pave the way for better design of future tolerogenic immunotherapies.

Can Immune Tolerance Be Re-established in Neuromyelitis Optica?

Neuromyelitis optica (NMO) is a chronic inflammatory disease of the central nervous system that primarily affects the optic nerves and spinal cord of patients, and in some instances their brainstem, diencephalon or cerebrum as spectrum disorders (NMOSD). Clinical and basic science knowledge of NMO has dramatically increased over the last two decades and it has changed the perception of the disease as being inevitably disabling or fatal. Nonetheless, there is still no cure for NMO and all the disease-modifying therapies (DMTs) are only partially effective. Furthermore, DMTs are not disease- or antigen-specific and alter all immune responses including those protective against infections and cancer and are often associated with significant adverse reactions. In this review, we discuss the pathogenic mechanisms of NMO as they pertain to its DMTs and immune tolerance. We also examine novel research therapeutic strategies focused on induction of antigen-specific immune tolerance by administrating tolerogenic immune-modifying nanoparticles (TIMP). Development and implementation of immune tolerance-based therapies in NMO is likely to be an important step toward improving the treatment outcomes of the disease. The antigen-specificity of these therapies will likely ameliorate the disease safely and effectively, and will also eliminate the clinical challenges associated with chronic immunosuppressive therapies.

The DNA co-vaccination using Sm antigen and IL-10 as prophylactic experimental therapy ameliorates nephritis in a model of lupus induced by pristane.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies such as anti-Sm. Studies in patients with SLE and murine models of lupus reveal that the most critical anti-Sm autoantibodies are predominantly direct against D1(83-119), D2, and B´/B epitopes. The present study aimed to analyze the induction of antigen-specific tolerance after prophylactic immunization with a DNA vaccine encoding the epitopes: D183-119, D2, B´/B, and B´/BCOOH in co-vaccination with IFN-γ or IL-10 in a murine model of lupus induced by pristane. To obtain endotoxin-free DNA vaccines, direct cloning techniques using pcDNA were performed: D183-119, D2, B´/B, B´/BCOOH, IFN-γ, or IL-10. Lupus was induced by 0.5 mL of pristane via intraperitoneal in BALB/c female mice. Immunoprecipitation with K562 cells was metabolically labeled with 35S and ELISA to detect serum antibodies or mice IgG1, IgG2a isotypes. ELISA determined IL-10 and IFN-γ from splenocytes supernatants. Proteinuria was assessed monthly, and lupus nephritis was evaluated by immunofluorescence, and electron microscopy. The prophylactic co-vaccination with D2/IL-10 reduced the expression of kidney damage observed by electron microscopy, direct immunofluorescence, and H & E, along with reduced level of anti-nRNP/Sm antibodies (P = 0.048). The prophylactic co-vaccination of IL-10 with D2 in pristane-induced lupus ameliorates the renal damage maybe by acting as prophylactic DNA tolerizing therapy.

Lymph Node-Targeted Synthetically Glycosylated Antigen Leads to Antigen-Specific Immunological Tolerance.

Inverse vaccines that tolerogenically target antigens to antigen-presenting cells (APCs) offer promise in prevention of immunity to allergens and protein drugs and treatment of autoimmunity. We have previously shown that targeting hepatic APCs through intravenous injection of synthetically glycosylated antigen leads to effective induction of antigen-specific immunological tolerance. Here, we demonstrate that targeting these glycoconjugates to lymph node (LN) APCs under homeostatic conditions leads to local and increased accumulation in the LNs compared to unmodified antigen and induces a tolerogenic state both locally and systemically. Subcutaneous administration directs the polymeric glycoconjugate to the draining LN, where the glycoconjugated antigen generates robust antigen-specific CD4+ and CD8+ T cell tolerance and hypo-responsiveness to antigenic challenge via a number of mechanisms, including clonal deletion, anergy of activated T cells, and expansion of regulatory T cells. Lag-3 up-regulation on CD4+ and CD8+ T cells represents an essential mechanism of suppression. Additionally, presentation of antigen released from the glycoconjugate to naïve T cells is mediated mainly by LN-resident CD8+ and CD11b+ dendritic cells. Thus, here we demonstrate that antigen targeting via synthetic glycosylation to impart affinity for APC scavenger receptors generates tolerance when LN dendritic cells are the cellular target.