Combinatorial delivery of immunosuppressive factors to dendritic cells using dual-sized microspheres

Abstract

Microparticulate systems are beginning to show promise for delivery of modulatory agents for immunotherapeutic applications which modulate dendritic cell (DC) functions. Co-administration of multiple factors is an emerging theme in immune modulation which may prove beneficial in this setting. Herein, we demonstrate that localized, controlled delivery of multiple factors can be accomplished through poly (lactic-co-glycolic acid) (PLGA) microparticle systems fabricated in two size classes of phagocytosable and unphagocytosable microparticles (MPs). The immunosuppressive ability of combinatorial multi-factor dual MP systems was evaluated by investigating effects on DC maturation, DC resistance to LPS-mediated maturation and proliferation of allogeneic T cells in a mixed lymphocyte reaction. Phagocytosable MPs (~2 μm) were fabricated encapsulating either rapamycin (RAPA) or all-trans retinoic acid (RA), and unphagocytosable MPs (~30 μm) were fabricated encapsulating either transforming growth factor beta-1 (TGF-β1) or interleukin-10 (IL-10). Combinations of these MP classes reduced expression of stimulatory/costimulatory molecules (MHC-II, CD80 and CD86) in comparison to iDC and soluble controls, but not necessarily to single factor MPs. Dual MP-treated DCs resisted LPS-mediated activation, in a manner driven by the single factor phagocytosable MPs used. Dendritic cells treated with dual MP systems suppressed allogeneic T cell proliferation, generally demonstrating greater suppression by combination MPs than single factor formulations, particularly for the RA/IL-10 MPs. This work demonstrates feasibility of simultaneous targeted delivery of immunomodulatory factors to cell surface receptors and intracellular locations, and indicates that a combinatorial approach can boost immunoregulatory responses for therapeutic application in autoimmunity and transplantation.

Figure 1

Microparticle size determined using dynamic light scattering techniques for (A) Phagocytosable MPs (average diameter ~ 2.3 μm) and, (B) Un-phagocytosable (average diameter ~ 30 μm).

Figure 2

In vitro release profiles of factors from phagocytosable microparticles: (A) Rapamycin, (B) Retinoic acid; and unphagocytosable microparticles: (C) TGF-β1 and (D) IL-10 in 2% Tween-20 PBS solution. Error bars on graphs represent standard deviation based on n = 3 for each MP type.

Figure 3

Dual microparticle (MP) systems result in reduced levels of surface expression of MHC II and costimulatory markers (CD80, CD86) relative to immature dendritic cells (iDCs). Combinations of immunomodulatory phagocytosable MPs (RAPA MP, RA MP) and un-phagocytosable MPs (TGF-β1 MP, IL-10 MP) were investigated. Unloaded MPs and soluble equivalent doses were included as controls. The composite maturation index (Panels A – D) represents an unweighted average of the percent positive cells for activation markers in a treatment group, normalized to the iDC group. Pair-wise significant differences from: iDC and Unloaded MP groups are denoted by the * symbol; iDC, Unloaded MP and either one of the single factor MP groups are denoted by the ** symbol; iDC, Unloaded MP and both single factor MP groups are denoted by the ¥ symbol (p≤0.05).

Figure 4

Dual MP systems modulate levels of IL-12 secretion by dendritic cells (DCs). Combinations of immunomodulatory phagocytosable MPs (RAPA MP, RA MP) and un-phagocytosable MPs (TGF-β1 MP, IL-10 MP) were investigated. Unloaded MPs, unloaded MPs + Sol LPS, iDC and Sol LPS conditions were included as controls. Supernatants were by ELISA. Data shown represent mean cytokine concentration ± standard error (n ≥ 7). Pair-wise significant differences from iDC and Unloaded MP groups are denoted by the * symbol (p≤0.05).

Figure 5

Dendritic cells treated with dual microparticle (MP) systems resist LPS stimulation. Combinations of immunomodulatory phagocytosable MPs (RAPA MP, RA MP) and un-phagocytosable MPs (TGF-β1 MP, IL-10 MP) were investigated. Unloaded MPs, soluble equivalent doses and addition of soluble LPS were included as controls. MPs or soluble treatments were incubated with DCs for 48 h then washed to remove unbound MPs and soluble factors. Subsequently, soluble LPS was added to culture wells at a concentration of 1 μg/ml. After 24 h incubation period, DCs were lifted and surface markers of activation - MHC II, CD80, CD86 were quantified by flow cytometry. The composite maturation index represents an average of the activation markers. Pair-wise significant differences from Sol LPS, Unloaded MPs + Sol LPS and either one of the single factor MP + Sol LPS groups are denoted by the ** symbol (p≤0.05).

Figure 6

Dendritic cells treated with dual MP systems differentially suppress T cell activation. Briefly, MPs were incubated with C57Bl6 DCs for 2 d followed by washing to remove unbound MPs. Subsequently, freshly-isolated Balbc CD4+ T cells were added to culture wells and co-cultured in a mixed lymphocyte reaction. Allogeneic T cell proliferation was then measured. Data shown represent the mean ± standard error (n ≥ 5). Pair-wise significant differences are denoted by: the * symbol for comparisons to the iDC group; the ** symbol for comparisons to the iDC and Unloaded MPs groups; the £ symbol for comparisons to the iDC, Unloaded MPs and either one of the single factor groups; the ¥ symbol for comparisons to the iDC, Unloaded MPs and both of the single factor groups; and the € symbol for comparisons to the all other groups (p≤0.05).

Figure 7

Dendritic cells treated with dual MP systems do not produce IFN-γ or IL-4, Th1/Th2 skewing cytokines, but do induce anti-inflammatory cytokine TGF-β1 secretion in a mixed lymphocyte setting. Briefly, MPs were incubated with C57Bl6 DCs for 2 d followed by washing to remove unbound MPs. Subsequently, freshly-isolated Balbc CD4+ T cells were added to culture wells and co-cultured in a mixed lymphocyte reaction (MLR). Following the MLR, supernatants were collected and analyzed for (A) IFN-γ, (B) IL-4, and (C) TGF-β1 cytokines by ELISA. Data shown represent mean cytokine concentration ± standard error (n ≥ 7). Pair-wise significant differences are denoted by: the * symbol for comparisons to the iDC group and the ** symbol for comparisons to the iDC and Unloaded MPs groups (p≤0.05).