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

Abstract

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.

Keywords: Autoimmune disease; Drug delivery; Immune tolerance; Nanoparticle.

Figure 1. Treatment with PLG(Ag) prevents and treats the progression of R-EAE

(A) Clinical scores for SJL/J mice treated with PLG(OVA_323–339) (red) or PLG(PLP_139–151) (black) and immunized with PLP_139–151 7 days later. (B) Clinical scores for mice were treated with PLG-OVA_323–339 (red) or PLG-PLP_139–151 (black) and immunized with PLP_139–151 7 days later. (C) Clinical scores for SJL/J mice immunized with PLP_178–191 and treated with PLG(OVA_323–339) (red), PLG(PLP_139–151) (black), or PLG(PLP_139–151+PLP_178–191) (green) on day 18 (black arrow). Groups were examined for clinical paralysis for an additional 8 days. The arrow indicates the day on which various particles were administered to mice previously immunized with PLP_178–191/CFA. (D) iLNs harvested from PLG(PLP_139–151) or PLG(OVA_323–339) treated or naïve mice and ex vivo proliferation of lymphocytes in response to 10 µM PLP_139–151 (black) or OVA_323–339 (red). Each data point represents the mean counts per minute (³H-TdR incorporation) for each mouse. Supernatants from the cultures were examined for (E) IL-17A, (F) GM-CSF, and (G) IFN-γ. Data are representative of at least 2–3 independent experiments. Differences between disease courses of different treatment groups were analysed for statistical significance using the Mann-Whitney U test (Figure 1A–C). Differences in proliferation and cytokine production were analysed for statistical significance using a 2-way ANOVA followed by the Sidak test for multiple comparisons (Figure 1D–G). Error bars represent statistical variation between individual animals within each group, 3 mice per group. An asterisk is indicative of statistical significance where * indicates p < 0.05, ** indicates p < 0.01, and *** indicates < 0.001.

Figure 2. Tolerance induced by PLG(Ag) treatment does not require the spleen

Splenectomy does not abrogate prophylactic tolerance induction with PLG(PLP_139–151) in R-EAE in vivo (A,B), recall proliferation (C,D), IFN-γ (E,F), or IL-17A (G,H) secretion ex vivo. Differences between disease courses of different treatment groups were analysed for statistical significance using the Mann-Whitney U test (Figure 2A–B). Differences in proliferation and cytokine production upon restimulation with PLP_139–151 were analysed for statistical significance using a 2-way ANOVA followed by the Sidak test for multiple comparisons (Figure 2C–H). Error bars represent assay standard deviation of pooled samples (C–H) and standard error of the mean (A–B). N=3–5 mice per group (A–B). An asterisk is indicative of statistical significance where * indicates p < 0.05, ** indicates p < 0.01, and *** indicates < 0.001.

Figure 3. PLG(Ag) distribute predominantly to the liver

(A) PLG·silver(PLP_139–151) biodistribution to the liver, lungs, and spleen at multiple times after I.V. administration as measured by ICP-MS and expressed as a percentage of the total mass of silver administered per mouse. (B) PLG_PEMA particles (green) co-localized primarily with F4/80⁺ cells (red) in the liver (white arrows), counter-stained with DAPI (blue), 18 hours following I.V. infusion. Accumulation of PLP_139–151-specific CD90.1⁺ CD4⁺ transgenic T cells in liver (C), spleen (D), lung (E), and inguinal lymph nodes (iLN) (F) was quantified by flow cytometry 18 hours post administration. Differences in biodistribution of PLG(Ag) particles were analysed for statistical significance using a 2-way ANOVA followed by the Sidak test for multiple comparisons (Figure 3A). Differences in T cell accumulation in various organs were analysed for statistical significance using a one-tailed t test (Figure 3C–F). Error bars represent standard error of the mean (A–F). N=3 mice per group (A–F). An asterisk is indicative of statistical significance where * indicates p < 0.05.

Figure 4. Tolerance induced by PLG(Ag) treatment is abrogated by blockade of PD-1

MFI for (A) F4/80⁺MHCII^lowCD11b^low Kupffer cells and (B) MHCII^highCD11c⁺CD11b^lowCD103⁺ dendritic cells in the liver 18 hours post administration in mice that had received adoptive transfer of PLP_139–151-specific CD90.1⁺ CD4⁺ transgenic T cells. (C) Clinical scores for mice treated with mouse anti-PD-1 monoclonal antibody (αPD-1, clone RMP1–14) 24 h prior to particle administration (day -7 relative to disease induction). (D) Restored recall PLP_139–151 specific proliferation and (E) IL-17A and (F) IFN-γ secretion. (D–F) follow the same legend as in (C). Differences in MFI of PD-L1 on Kupffer cells and dendritic cells were analysed for statistical significance using the Kruskal-Wallis test (Figure 4A–B). Differences between disease courses of different treatment groups were analysed for statistical significance using the Mann-Whitney U test (Figure 4C). Differences in proliferation and cytokine production were analysed for statistical significance using a 2-way ANOVA followed by the Sidak test for multiple comparisons (Figure 4D–F). Error bars represent standard error of the mean (A–F). N=2–4 mice per group (A–B), and N=5–8 mice per group (C–F). An asterisk is indicative of statistical significance where * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p< 0.001.