Resident macrophages of pancreatic islets have a seminal role in the initiation of autoimmune diabetes of NOD mice.

Treatment of C57BL/6 or NOD mice with a monoclonal antibody to the CSF-1 receptor resulted in depletion of the resident macrophages of pancreatic islets of Langerhans that lasted for several weeks. Depletion of macrophages in C57BL/6 mice did not affect multiple parameters of islet function, including glucose response, insulin content, and transcriptional profile. In NOD mice depleted of islet-resident macrophages starting at 3 wk of age, several changes occurred: (i) the early entrance of CD4 T cells and dendritic cells into pancreatic islets was reduced, (ii) presentation of insulin epitopes by dispersed islet cells to T cells was impaired, and (iii) the development of autoimmune diabetes was significantly reduced. Treatment of NOD mice starting at 10 wk of age, when the autoimmune process has progressed, also significantly reduced the incidence of diabetes. Despite the absence of diabetes, NOD mice treated with anti-CSF-1 receptor starting at 3 or 10 wk of age still contained variably elevated leukocytic infiltrates in their islets when examined at 20-40 wk of age. Diabetes occurred in the anti-CSF-1 receptor protected mice after treatment with a blocking antibody directed against PD-1. We conclude that treatment of NOD mice with an antibody against CSF-1 receptor reduced diabetes incidence and led to the development of a regulatory pathway that controlled autoimmune progression.

An antigen-encapsulating nanoparticle platform for TH1/17 immune tolerance therapy.

Tolerogenic nanoparticles (NPs) are rapidly being developed as specific immunotherapies to treat autoimmune disease. However, many NP-based therapies conjugate antigen (Ag) directly to the NP posing safety concerns due to antibody binding or require the co-delivery of immunosuppressants to induce tolerance. Here, we developed Ag encapsulated NPs comprised of poly(lactide-co-glycolide) [PLG(Ag)] and investigated the mechanism of action for Ag-specific tolerance induction in an autoimmune model of T helper type 1/17 dysfunction - relapse-remitting experimental autoimmune encephalomyelitis (R-EAE). PLG(Ag) completely abrogated disease induction in an organ specific manner, where the spleen was dispensable for tolerance induction. PLG(Ag) delivered intravenously distributed to the liver, associated with macrophages, and recruited Ag-specific T cells. Furthermore, programmed death ligand 1 (PD-L1) was increased on Ag presenting cells and PD-1 blockade lessened tolerance induction. The robust promotion of tolerance by PLG(Ag) without co-delivery of immunosuppressive drugs, suggests that these NPs effectively deliver antigen to endogenous tolerogenic pathways.

Exploiting apoptosis for therapeutic tolerance induction.

Immune tolerance remains the most promising yet elusive strategy for treating immune-mediated diseases. An experimental strategy showing promise in phase 1 clinical studies is the delivery of Ag cross-linked to apoptotic leukocytes using ethylene carbodiimide. This approach originated from demonstration of the profound tolerance-inducing ability of i.v. administered Ag-coupled splenocytes (Ag-SP) in mice, which has been demonstrated to treat T cell-mediated disorders including autoimmunity, allergy, and transplant rejection. Recent studies have defined the intricate interplay between the innate and adaptive immune systems in Ag-SP tolerance induction. Innate mechanisms include scavenger receptor-mediated uptake of Ag-SP by host APCs, Ag representation, and the required upregulation of PD-L1 expression and IL-10 production by splenic marginal zone macrophages leading to Ag-specific T cell regulation via the combined effects of cell-intrinsic anergy and regulatory T cell induction. In this paper, we discuss the history, advantages, current mechanistic understanding, and clinical potential of tolerance induction using apoptotic Ag-coupled apoptotic leukocytes.

Tolerance induction using nanoparticles bearing HY peptides in bone marrow transplantation.

Allogeneic cell therapies have either proven effective or have great potential in numerous applications, though the required systemic, life-long immunosuppression presents significant health risks. Inducing tolerance to allogeneic cells offers the potential to reduce or eliminate chronic immunosuppression. Herein, we investigated antigen-loaded nanoparticles for their ability to promote transplant tolerance in the minor histocompatibility antigen sex-mismatched C57BL/6 model of bone marrow transplantation. In this model, the peptide antigens Dby and Uty mediate rejection of male bone marrow transplants by female CD4+ and CD8+ T cells, respectively, and we investigated the action of nanoparticles on these T cell subsets. Antigens were coupled to or encapsulated within poly(lactide-co-glycolide) (PLG) nanoparticles with an approximate diameter of 500 nm. Delivery of the CD4-encoded Dby epitope either coupled to or encapsulated within PLG particles prevented transplant rejection, promoted donor-host chimerism, and suppressed proliferative and IFN-γ responses in tolerized recipients. Nanoparticles modified with the Uty peptide did not induce tolerance. The dosing regimen was investigated with Dby coupled particles, and a single dose delivered the day after bone marrow transplant was sufficient for tolerance induction. The engraftment of cells was significantly affected by PD-1/PDL-1 costimluation, as blockade of PD-1 reduced engraftment by ∼50%. In contrast, blockade of regulatory T cells did not impact the level of chimerism. The delivery of antigen on PLG nanoparticles promoted long-term engraftment of bone marrow in a model with a minor antigen mismatch in the absence of immunosuppression, and this represents a promising platform for developing a translatable, donor-specific tolerance strategy.

Oligodendrocyte death results in immune-mediated CNS demyelination.

Although multiple sclerosis is a common neurological disorder, the origin of the autoimmune response against myelin, which is the characteristic feature of the disease, remains unclear. To investigate whether oligodendrocyte death could cause this autoimmune response, we examined the oligodendrocyte ablation Plp1-CreER(T);ROSA26-eGFP-DTA (DTA) mouse model. Approximately 30 weeks after recovering from oligodendrocyte loss and demyelination, DTA mice develop a fatal secondary disease characterized by extensive myelin and axonal loss. Strikingly, late-onset disease was associated with increased numbers of T lymphocytes in the CNS and myelin oligodendrocyte glycoprotein (MOG)-specific T cells in lymphoid organs. Transfer of T cells derived from DTA mice to naive recipients resulted in neurological defects that correlated with CNS white matter inflammation. Furthermore, immune tolerization against MOG ameliorated symptoms. Overall, these data indicate that oligodendrocyte death is sufficient to trigger an adaptive autoimmune response against myelin, suggesting that a similar process can occur in the pathogenesis of multiple sclerosis.

Targeted immunomodulation using antigen-conjugated nanoparticles.

The growing prevalence of nanotechnology in the fields of biology, medicine, and the pharmaceutical industry is confounded by the relatively small amount of data on the impact of these materials on the immune system. In addition to concerns surrounding the potential toxicity of nanoparticle (NP)-based delivery systems, there is also a demand for a better understanding of the mechanisms governing interactions of NPs with the immune system. Nanoparticles can be tailored to suppress, enhance, or subvert recognition by the immune system. This 'targeted immunomodulation' can be achieved by delivery of unmodified particles, or by modifying particles to deliver drugs, proteins/peptides, or genes to a specific site. In order to elicit the desired, beneficial immune response, considerations should be made at every step of the design process: the NP platform itself, ligands, and other modifiers, the delivery route, and the immune cells that will encounter the conjugated NPs can all impact host immune responses.

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.

Mouse models of multiple sclerosis: experimental autoimmune encephalomyelitis and Theiler's virus-induced demyelinating disease.

Experimental autoimmune encephalomyelitis (EAE) and Theiler's Murine Encephalitis Virus-Induced Demyelinating Disease (TMEV-IDD) are two clinically relevant murine models of multiple sclerosis (MS). Like MS, both are characterized by mononuclear cell infiltration into the CNS and demyelination. EAE is induced by either the administration of myelin protein or peptide in adjuvant or by the adoptive transfer of encephalitogenic T cell blasts into naïve recipients. The relative merits of each of these protocols are compared. Depending on the type of question being asked, different mouse strains and peptides are used. Different disease courses are observed with different strains and different peptides in active EAE. These variations are also addressed. Additionally, issues relevant to clinical grading of EAE in mice are discussed. In addition to EAE induction, useful references for other disease indicators such as DTH, in vitro proliferation, and immunohistochemistry are provided. TMEV-IDD is a useful model for understanding the possible viral etiology of MS. This section provides detailed information on the preparation of viral stocks and subsequent intracerebral infection of mice. Additionally, virus plaque assay and clinical disease assessment are discussed. Recently, recombinant TMEV strains have been created for the study of molecular mimicry which incorporate various 30 amino acid myelin epitopes within the leader region of TMEV.

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.

Have we overestimated the benefit of human(ized) antibodies?

The infusion of animal-derived antibodies has been known for some time to trigger the generation of antibodies directed at the foreign protein as well as adverse events including cytokine release syndrome. These immunological phenomena drove the development of humanized and fully human monoclonal antibodies. The ability to generate human(ized) antibodies has been both a blessing and a curse. While incremental gains in the clinical efficacy and safety for some agents have been realized, a positive effect has not been observed for all human(ized) antibodies. Many human(ized) antibodies trigger the development of anti-drug antibody responses and infusion reactions. The current belief that antibodies need to be human(ized) to have enhanced therapeutic utility may slow the development of novel animal-derived monoclonal antibody therapeutics for use in clinical indications. In the case of murine antibodies, greater than 20% induce tolerable/negligible immunogenicity, suggesting that in these cases humanization may not offer significant gains in therapeutic utility. Furthermore, humanization of some murine antibodies may reduce their clinical effectiveness. The available data suggest that the utility of human(ized) antibodies needs to be evaluated on a case-by-case basis, taking a cost-benefit approach, taking both biochemical characteristics and the targeted therapeutic indication into account.