Woon Teck Yap

Microparticles bearing encephalitogenic peptides induce T-cell tolerance and ameliorate experimental autoimmune encephalomyelitis

Aberrant T-cell activation underlies many autoimmune disorders, yet most attempts to induce T-cell tolerance have failed. Building on previous strategies for tolerance induction that exploited natural mechanisms for clearing apoptotic debris, we show that antigen-decorated microparticles (500-nm diameter) induce long-term T-cell tolerance in mice with relapsing experimental autoimmune encephalomyelitis. Specifically, intravenous infusion of either polystyrene or biodegradable poly(lactide-co-glycolide) microparticles bearing encephalitogenic peptides prevents the onset and modifies the course of the disease. These beneficial effects require microparticle uptake by marginal zone macrophages expressing the scavenger receptor MARCO and are mediated in part by the activity of regulatory T cells, abortive T-cell activation and T-cell anergy. Together these data highlight the potential for using microparticles to target natural apoptotic clearance pathways to inactivate pathogenic T cells and halt the disease process in autoimmunity.

A Biodegradable Nanoparticle Platform for the Induction of Antigen-Specific Immune Tolerance for Treatment of Autoimmune Disease

Targeted immune tolerance is a coveted therapy for the treatment of a variety of autoimmune diseases, as current treatment options often involve nonspecific immunosuppression. Intravenous (iv) infusion of apoptotic syngeneic splenocytes linked with peptide or protein autoantigens using ethylene carbodiimide (ECDI) has been demonstrated to be an effective method for inducing peripheral, antigen-specific tolerance for treatment of autoimmune disease. Here, we show the ability of biodegradable poly(lactic-co-glycolic acid) (PLG) nanoparticles to function as a safe, cost-effective, and highly efficient alternative to cellular carriers for the induction of antigen-specific T cell tolerance. We describe the formulation of tolerogenic PLG particles and demonstrate that administration of myelin antigen-coupled particles both prevented and treated relapsing-remitting experimental autoimmune encephalomyelitis (R-EAE), a CD4 T cell-mediated mouse model of multiple sclerosis (MS). PLG particles made on-site with surfactant modifications surpass the efficacy of commercially available particles in their ability to couple peptide and to prevent disease induction. Most importantly, myelin antigen-coupled PLG nanoparticles are able to significantly ameliorate ongoing disease and subsequent relapses when administered at onset or at peak of acute disease, and minimize epitope spreading when administered during disease remission. Therapeutic treatment results in significantly reduced CNS infiltration of encephalitogenic Th1 (IFN-γ) and Th17 (IL-17a) cells as well as inflammatory monocytes/macrophages. Together, these data describe a platform for antigen display that is safe, low-cost, and highly effective at inducing antigen-specific T cell tolerance. The development of such a platform carries broad implications for the treatment of a variety of immune-mediated diseases.

Biodegradable antigen-associated PLG nanoparticles tolerize Th2-mediated allergic airway inflammation pre- and postsensitization

Significance Allergic diseases are characterized by inappropriate inflammatory responses to benign environmental antigens (Ags). Primary clinical approaches to allergic disease consist of symptom control or administration of soluble Ag to skew the immune response to alternate phenotypes or induce immune tolerance. However, such approaches carry a significant risk of adverse events and require a long treatment course to achieve efficacy. In this paper, we describe the use of i.v.-administered nanoparticles as carriers of whole-protein Ag to induce tolerance safely and effectively in the absence of nonspecific immunosuppression for the prevention and treatment of a mouse model of allergic asthma. Specific immunotherapy (SIT) is the most widely used treatment for allergic diseases that directly targets the T helper 2 (Th2) bias underlying allergy. However, the most widespread clinical applications of SIT require a long period of dose escalation with soluble antigen (Ag) and carry a significant risk of adverse reactions, particularly in highly sensitized patients who stand to benefit most from a curative treatment. Thus, the development of safer, more efficient methods to induce Ag-specific immune tolerance is critical to advancing allergy treatment. We hypothesized that antigen-associated nanoparticles (Ag-NPs), which we have used to prevent and treat Th1/Th17-mediated autoimmune disease, would also be effective for the induction of tolerance in a murine model of Th2-mediated ovalbumin/alum-induced allergic airway inflammation. We demonstrate here that antigen-conjugated polystyrene (Ag-PS) NPs, although effective for the prophylactic induction of tolerance, induce anaphylaxis in presensitized mice. Antigen-conjugated NPs made of biodegradable poly(lactide-co-glycolide) (Ag-PLG) are similarly effective prophylactically, are well tolerated by sensitized animals, but only partially inhibit Th2 responses when administered therapeutically. PLG NPs containing encapsulated antigen [PLG(Ag)], however, were well tolerated and effectively inhibited Th2 responses and airway inflammation both prophylactically and therapeutically. Thus, we illustrate progression toward PLG(Ag) as a biodegradable Ag carrier platform for the safe and effective inhibition of allergic airway inflammation without the need for nonspecific immunosuppression in animals with established Th2 sensitization.

Heparin-chitosan nanoparticle functionalization of porous poly(ethylene glycol) hydrogels for localized lentivirus delivery of angiogenic factors

Hydrogels have been extensively used for regenerative medicine strategies given their tailorable mechanical and chemical properties. Gene delivery represents a promising strategy by which to enhance the bioactivity of the hydrogels, though the efficiency and localization of gene transfer have been challenging. Here, we functionalized porous poly(ethylene glycol) hydrogels with heparin-chitosan nanoparticles to retain the vectors locally and enhance lentivirus delivery while minimizing changes to hydrogel architecture and mechanical properties. The immobilization of nanoparticles, as compared to homogeneous heparin and/or chitosan, is essential to lentivirus immobilization and retention of activity. Using this gene-delivering platform, we over-expressed the angiogenic factors sonic hedgehog (Shh) and vascular endothelial growth factor (Vegf) to promote blood vessel recruitment to the implant site. Shh enhanced endothelial recruitment and blood vessel formation around the hydrogel compared to both Vegf-delivering and control hydrogels. The nanoparticle-modified porous hydrogels for delivering gene therapy vectors can provide a platform for numerous regenerative medicine applications.

Quantification of particle-conjugated or particle-encapsulated peptides on interfering reagent backgrounds

Particle-based technologies are increasingly being used in diagnostics and therapeutics. The particles employed in these applications are usually composed of polymers such as poly(lactide-co-glycolide) (PLG) and functionalized with peptides or proteins. Peptide or protein conjugation to particles is frequently achieved using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), while dimethyl sulfoxide (DMSO) is used to retrieve surface-attached or encapsulated peptides or proteins by solubilizing the particles. We examined strategies based on bicinchoninic acid (BSA), Coomassie Plus, and 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) assays for the quantification of surface-attached or encapsulated peptides or proteins. We determined that the CBQCA assay is a highly sensitive and accurate substitute for radioactivity-based assays that is suitable for measuring multiple particle-bound or particle-encapsulated peptides or proteins in the presence of EDC or PLG in DMSO, compounds that interfere with the more commonly used BSA and Coomassie Plus assays. Our strategy enables the accurate quantification of peptides or proteins loaded onto or into particles-an essential component of particle-based platform design for diagnostics and therapeutics.

Erratum: Microparticles bearing encephalitogenic peptides induce T-cell tolerance and ameliorate experimental autoimmune encephalomyelitis (Nature Biotechnology (2012) 30 (1217-1224))

In the version of this article initially published, there were two errors in the discussion of epigenetic marks on page 854. In sentence 4, paragraph 2 of the section “Genetic and epigenetic changes predictive of malignancy,” dimethylated and trimethylated H3K9 were said incorrectly to be “polycomb” marks. “Polycomb” has been deleted from the sentence, and the following two sentences inserted for clarification: “In ES cells these genes are held in a ‘transcription ready’ state by two marks, a repressive H3K27me mark and an active mark, H3K4me64. Changes in the balance of repressive versus active marks can alter the activity of these genes, hypothetically keeping cells in a proliferative state.” Further down in the paragraph, DNMT3L was described incorrectly as catalytic. The original text, “... related to activation of the de novo methyltransferase DNMT3L68” has been revised to “...and maintain expression of the de novo methyltransferase–like protein DNMT3L68. Expression of DNMT3L appears to be a common feature in pluripotent cells including ES, EC and embryonic germ cells.” The errors have been corrected in the HTML and PDF versions of the article.