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. 2018 Mar 21;29(3):813-823.
doi: 10.1021/acs.bioconjchem.7b00624. Epub 2017 Nov 30.

Conjugation of Transforming Growth Factor Beta to Antigen-Loaded Poly(lactide- Co-Glycolide) Nanoparticles Enhances Efficiency of Antigen-Specific Tolerance

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Conjugation of Transforming Growth Factor Beta to Antigen-Loaded Poly(lactide- Co-Glycolide) Nanoparticles Enhances Efficiency of Antigen-Specific Tolerance

Liam M Casey et al. Bioconjug Chem. .
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Abstract

Current strategies for treating autoimmunity involve the administration of broad-acting immunosuppressive agents that impair healthy immunity. Intravenous (i.v.) administration of poly(lactide- co-glycolide) nanoparticles (NPs) containing disease-relevant antigens (Ag-NPs) have demonstrated antigen (Ag)-specific immune tolerance in models of autoimmunity. However, subcutaneous (s.c.) delivery of Ag-NPs has not been effective. This investigation tested the hypothesis that codelivery of the immunomodulatory cytokine, transforming growth factor beta 1 (TGF-β), on Ag-NPs would modulate the immune response to Ag-NPs and improve the efficiency of tolerance induction. TGF-β was coupled to the surface of Ag-NPs such that the loadings of Ag and TGF-β were independently tunable. The particles demonstrated bioactive delivery of Ag and TGF-β in vitro by reducing the inflammatory phenotype of bone marrow-derived dendritic cells and inducing regulatory T cells in a coculture system. Using an in vivo mouse model for multiple sclerosis, experimental autoimmune encephalomyelitis, TGF-β codelivery on Ag-NPs resulted in improved efficacy at lower doses by i.v. administration and significantly reduced disease severity by s.c. administration. This study demonstrates that the codelivery of immunomodulatory cytokines on Ag-NPs may enhance the efficacy of Ag-specific tolerance therapies by programming Ag presenting cells for more efficient tolerance induction.

Conflict of interest statement

The authors declare the following competing financial interest(s): R.M.P., S.D.M., and L.D.S. have financial interests in Cour Pharmaceuticals Development Co.

Figures

Figure 1.
Figure 1.
Surface-coupled TGF-β is bioactively delivered by PLG nanoparticles. (A) Schematic depiction of the NP production and TGF-β conjugation procedure used to create the NPs in this study. TGF-β was conjugated to blank PLG, Ag-encapsulated, and Ag-polymer conjugate NPs. (B) Naïve CD4+CD25 T cells were cultured in anti-CD3-coated plates for 4 days in the presence of 2 μg/mL anti-CD28, 10 ng/mL IL-2, and 2 ng/mL of soluble TGF-β, blank PLG NPs, or 300 μg/mL of PLG-TGF-β NPs (166 ng TGF-β/mg). T cells were characterized by flow cytometry for CD25 and intracellular Foxp3.
Figure 2.
Figure 2.
TGF-β and antigen are bioactively codelivered by PLG particles to induce antigen-specific Tregs. (A) Characterization of Ag-NPs before and after TGF-β conjugation. Antigen-loaded NPs were prepared by encapsulating OVA peptide [PLG(OVA) NPs] or by incorporating PLG-Ag conjugates with unmodified PLG (acPLG NPs). (B–D) BMDCs were treated for 3 h with 300 μg/mL of Ag-NPs. After 3 h, media was exchanged to remove excess particles. The BMDCs were then cocultured for 4 days with naïve OT-II CD4+CD25 T cells at a 1:1 ratio. Cells that were not treated with NPs received 100 ng/mL of OVA and 2 ng/mL of TGF-β. (B) Representative plots of flow cytometry data gated on live CD4+ T cells. (C) Treg induction was compared between solubly derived TGF-β and NP-bound TGF-β codelivered with PLG(OVA) NPs (3 μg OVA/mg) and acPLG-OVA NPs (2 μg OVA/mg). (D) Treg induction was evaluated with titrated amounts of TGF-β conjugated to acPLG-OVA NPs (OVA loading constant at 8 μg/mg). Statistical differences were determined using a two-tailed unpaired t test. Error bars indicate SD.
Figure 3.
Figure 3.
NP treatment decreases BMDC costimulatory molecule expression and cytokine secretion. BMDCs were treated with soluble OVA or with NPs for 3 h. Excess NPs were washed away and cells were incubated for 4 days. (A and C) BMDCs were unstimulated (immature) or (B and D) stimulated (mature) with 10 ng/mL of IFNγ and 10 ng/mL LPS after 3 h NP treatment. (A and B) BMDCs were evaluated by flow cytometry on day 4. Histograms show live cell surface molecule expression with red line histograms representing the indicated treatment and blue filled histograms representing untreated BMDCs. (C and D) The cytokines present in the BMDC media on day 4 were evaluated by ELISA. Statistical differences were determined by one-way ANOVA with Tukey’s multiple comparisons test. Within data subsets, no significant difference was observed between conditions labeled with matching letters (p > 0.05). Error bars indicate SD.
Figure 4.
Figure 4.
Ag-NPs cause Ag-specific changes in T cell populations in vivo. OT-II mice were untreated or injected with 1.25 mg of NPs (8 μg Ag/mg, 166 ng TGF-β/mg). (A–C) Mice treated i.v. received a single tail vein injection. (D–F) Mice treated s.c. received 4 injections distributed over the shoulders and hind limbs. After 7 days, the livers, spleens, and pooled brachial, axillary, and inguinal lymph nodes were excised and analyzed by flow cytometry for CD4+ T cells and CD25+Foxp3+ Tregs. Statistical differences were determined by Fisher’s Least Significant Difference (LSD) test. Within data subsets, no significant difference was observed between conditions labeled with matching letters (p > 0.05). Error bars indicate SEM.
Figure 5.
Figure 5.
TGF-β codelivery improves the tolerogenic efficiency of Ag-loaded NPs in EAE model. (A,B) SJL mice were prophylactically injected i.v. with subtherapeutic doses of Ag-NPs (A, 1.25 mg or B, 0.0625 mg at 8 μg Ag/mg and 166 TGF-β/mg). After 7 days, mice were immunized with PLP/CFA on day 0 to induce EAE (n = 5). (C,D) SJL mice (n = 7) were immunized (day 0) and injected with 1.25 mg of Ag-NPs (8 μg Ag/mg, 166 ng TGF-β/mg) on day 13 (the third day of disease presentation). Six days after injection, 3 mice per cohort were euthanized and (D) the number of liver CD25+Foxp3+ Tregs was quantified using flow cytometry. Statistical differences between treatments for the indicated time periods were determined by (A and C) the Kruskal–Wallis test (one-way ANOVA nonparametric test) with Dunn’s multiple comparisons test (p < 0.05) or(B) the Mann–Whitney (two-tailed nonparametric) test (p < 0.05). Statistical differences between cell numbers (D) were determined using Fisher’s Least Significant Difference (LSD) test. Within data subsets, no significant difference was observed between conditions labeled with matching letters (p > 0.05). Error bars indicate SD.
Figure 6.
Figure 6.
Subcutaneous delivery of TGF-β-coupled Ag-NPs reduces the severity of relapse symptoms in EAE. SJL mice were injected s.c. with 1.25 mg of Ag-NPs (8 μg Ag/mg, 166 ng TGF-β/mg) 7 days before immunization with PLP/CFA to induce EAE (n = 5). Statistical differences between treatments for the indicated time periods were determined by the Kruskal–Wallis test (one-way ANOVA nonparametric test) with Dunn’s multiple comparisons test (p < 0.05).

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