We found previously that antigenic peptides coupled to splenic leukocytes can be used as treatments in preclinical models of autoimmune disease, allergy and transplantation12,18,27,28
. This technique is currently being evaluated in the clinic for treatment of multiple sclerosis (S.D.M., unpublished data). However, the complexity and cost of producing antigen-coupled cells under GMP conditions may impede clinical translation. We undertook the current study to determine whether inert microparticles could be used as surrogates for apoptotic leukocytes as antigen ‘carriers’. Inert microparticles coupled to peptides, unlike Ag-SP, can be produced in large amounts under GMP conditions. We demonstrate that both polystyrene and biodegradable PLG microparticles are highly efficient substitutes for apoptotic cells, as they are taken up in a MARCO scavenger receptor–dependent fashion and are capable of inducing long-term antigen-specific T-cell abortive activation and anergy.
To avoid complications of immunosuppression, the ability to tolerize T cells specific for autoantigens and alloantigens remains the desired treatment for a myriad of immune-mediated diseases. However, the translatability of the tolerance approaches demonstrated to be efficacious in animal studies has been lacking. Intravenous administration of anti-CD3 to induce tolerance in type 1 diabetes has resulted in questionable efficacy and safety concerns29
. Attempts to potentially overcome some of these barriers through oral administration are under way, and some positive data have been obtained in small study involving 15 patients30
. Initial studies using anti-CD3 to treat type 1 diabetes also showed similar positive outcomes29
; therefore, larger patient studies will be needed to clearly define any clinical benefit of oral over intravenous anti-CD3 treatment.
A number of antigen-specific approaches to generate tolerance have previously been tested in autoimmune diseases. Intradermal administration of CGP77116, an altered peptide ligand of MBP83–99
, worsened symptoms in three patients with multiple sclerosis because in at least two of the patients there were6
increased immune responses to MBP83–99
. Attempts to induce ‘high-zone tolerance’31 by i.v. (MBP8298) infusion of a large bolus of peptide recently failed a phase 3 clinical trial in patients with multiple sclerosis32
. Similarly, in type 1 diabetes, s.c. injection of the 65-kDa isoform of glutamic acid decarboxylase in alum had no effect on disease progression33
. Mucosal antigen delivery has also shown promise in animal models of multiple sclerosis and type 1 diabetes34
, but larger clinical trials testing oral and nasal administration of insulin have been ineffective in the prevention or reversal of new-onset type 1 diabetes35,36
Two DNA vaccines are also in development for treating patients with multiple sclerosis. BHT3009 encodes full-length MBP. In a phase 2 study, BHT3009 was administered at weeks 0, 2 and 4 and then monthly by s.c. injection. Although this treatment did not alter the relative risk for relapse or first time to relapse, there was a reduction in new CNS lesions37
. The reduction in lesion formation was most prominent in patients that had high concentrations of antibodies targeting MBP, whereas patients with low anti-MBP titers had a similar rate of lesion formation as placebo-treated controls. Although further studies are needed to confirm the potential utility of BHT3009, the existence of a patient population that may not be responsive to BHT3009 supports the need for other therapeutic modalities.
A second DNA vaccine, ATX-MS-1467, expresses peptides that are thought to mimic processed myelin antigens and therefore act similarly to glatiramer acetate (GLAT), a random-length polymer of four amino acids (glutamic acid, lysine, alanine and tyrosine) found in MBP, which has been shown to compete with myelin peptides for access to the peptide binding cleft in the MHC complex38
, promote TH
2 cell responses and induce IL-10–producing Treg
. These effects are not antigen specific, and as such, it may be predicted that the efficacy of ATX-MS-1467 may be similar to that of GLAT, resulting in a 50% reduction in multiple sclerosis relapses in responsive patients38
Our data showing the requirement for peptides to be covalently linked to 500-nm particles and confirming the importance of i.v. administration for efficient tolerance induction12
reinforce the importance of delivering the antigen-linked particles to the splenic marginal zone. As a possible explanation of the particle size requirement, it has been reported that compared to 20-nm diameter particles, 200-nm and 1,000-nm diameter particles have a higher propensity to bind to MARCO in vitro40
. We show that MARCO-expressing MZM, but not SIGLEC-1–expressing metallophilic macrophages, take up peptide-linked particles. To our knowledge, MARCO has not previously been ascribed a role in T-cell tolerance, although this receptor was previously implicated in the pathogenesis of systemic lupus erythematosus; in systemic lupus erythematosus, it is postulated that failure of MARCO to remove apoptotic debris has a potential role in autoantibody formation41,42
. MARCO is also important for the uptake of bacteria43
, silica particles44
and polymer-based micro-particles40
. We speculate that MARCO functions through its ability to take up antigen-linked particles and assist in macrophage antigen presentation and/or antigen transfer to local dendritic cells. MARCO may also inhibit inflammatory responses by preventing dendritic cell migration45
or by other unknown anti-inflammatory mechanisms46
. However, although macrophage production of IL-10 is thought to be crucial for tolerance to apoptotic cells12
, IL-10 neutralization failed to completely inhibit the tolerance induced by antigenic peptides coupled to microparticles. In addition, it seems that the MARCO pathway of tolerance induction is limited to microparticle-bound peptide, as Marco−/−
mice were effectively tolerized to soluble peptide and peptides coupled to apoptotic splenic leukocytes.
-PSB infusion triggered antigen-specific T-cell extravasation into the lymphoid compartment. This extravasation was notable, as T cells isolated from the peripheral blood but not the lymph nodes within 48 h after treatment remained highly reactive to self antigen. This is probably the result of the inability of T cells to interact with tolerogenic antigen-presenting cells and/or Treg
cells while in the laminar flow of the blood. In addition, PLP139–151
-PSB infusion resulted in suboptimal PLP139–151
-specific T-cell proliferation, cytokine production and activation in general. T-cell anergy was also induced, as nonresponsiveness could be largely reversed through the addition of exogenous IL-2 (refs. 26,47
). Functional inactivation of Treg
cells partially prevented the tolerance induced by antigenic peptide-coupled microparticles, which is in line with our previous studies using splenic leukocytes12
. Upregulation of PD-L1 expression on splenic macrophages was implicated in our previous work, but we found only slight upregulation of PD-L1 expression on macrophages in mice treated with antigenic peptides coupled to microparticles (data not shown). Future experiments are required to further delineate the role of PD-L1 and CTLA-4 in antigen-coupled particle tolerance17
Clinical translation of tolerance-based therapies for the treatment of autoimmune disease requires the ability to suppress pre-existing autoreactive effector T cells and/or establish tolerance of naive autoreactive T cells that may be activated after exposure to endogenous autoantigens released from damaged target organs (epitope spreading)10
. Here we show that i.v. administration of PLP139–151
-PSB can prophylactically prevent R-EAE, inhibit established R-EAE and suppress relapse caused by epitope spreading.
This study supports the use of antigen-coupled microparticles as a tool for tolerance induction. This option will probably have broad therapeutic utility, with preliminary studies in airway allergy27
yielding promising results. From a clinical development perspective, identification of specific autoantigens and T-cell epitopes to be targeted in various autoimmune diseases, and a determination of the roles of scavenger receptors in various diseases, remain challenges for further investigations.