Enhanced and prolonged cross-presentation following endosomal escape of exogenous antigens encapsulated in biodegradable nanoparticles

Abstract

CD8(+) T-cell responses are critical in the immunological control of tumours and infectious diseases. To prime CD8(+) T cells against these cell-associated antigens, exogenous antigens must be cross-presented by professional antigen-presenting cells (APCs). While cross-presentation of soluble antigens by dendritic cells is detectable in vivo, the efficiency is low, limiting the clinical utility of protein-based vaccinations. To enhance the efficiency of presentation, we generated nanoparticles from a biodegradable polymer, poly(D,L-lactide-co-glycolide) (PLGA), to deliver antigen into the major histocompatibility complex (MHC) class I antigen presentation pathway. In primary mouse bone marrow-derived dendritic cells (BMDCs), the MHC class I presentation of PLGA-encapsulated ovalbumin (OVA) stimulated T cell interleukin-2 secretion at 1000-fold lower concentration than soluble antigen and 10-fold lower than antigen-coated latex beads. The microparticles also served as an intracellular antigen reservoir, leading to sustained MHC class I presentation of OVA for 72 hr, decreasing by only 20% after 96 hr, a time at which the presentation of soluble and latex bead-associated antigens was undetectable. Cytosol extraction demonstrated that antigen delivery via PLGA particles increased the amount of protein that escaped from endosomes into the cytoplasm, thereby increasing the access of exogenous antigen to the classic MHC class I loading pathway. These data indicate that the unique properties of PLGA particle-mediated antigen delivery dramatically enhance and sustain exogenous antigen presentation by MHC class I, potentially facilitating the clinical use of these particles in vaccination.

Figure 1

PLGA microparticles enhance exogenous antigen cross-presentation by human DC-like KG-1.K^b cells. KG-1.K^b cells were cocultured with PLGA/OVA particles (open diamonds) or soluble OVA (open circles) at the concentrations indicated for 16 hr. The amount of particles added was calculated to yield concentrations of OVA equivalent to that of soluble OVA. Following extensive washing, cells were stained with Alexa 647-conjugated 25-D1·16 mAb, which recognizes the SIINFEKL peptide (OVA_257−264) in association with K^b. The cross-presentation of exogenous antigen was detected as a shift in the fluorescence intensity of samples incubated with OVA from the level seen when incubated with an irrelevant protein (BSA).

Figure 2

Microparticle-encapsulated antigens generate potent CD8⁺ T cell responses in vitro. Immature BMDCs from C57BL/6 mice were incubated with varying concentrations of OVA-containing PLGA particles (open diamonds), OVA-coated latex beads (solid squares), and soluble OVA (open circles) for 3 hr followed by particle separation from cells to remove excess antigen. 30 ng/ml LPS was then added for an additional 18 hr to induce DC maturation. After maturation, B3Z T cells were added at an effector:APC ratio of 1: 1. The IL-2 content of culture supernatants, isolated after 6 hr incubation, was assayed by IL-2 ELISA. Results show the mean and SD of triplicate measurements, and are representative of two different experiments with DCs from different mice.

Figure 3

Cross-presentation by two B-cell lines (PeCr2.K^b and C1R.K^b) and TAP-deficient T2.K^b cells in comparison to human DC-like KG-1.K^b cells. Indicated cell lines were cocultured with latex bead/OVA, PLGA/OVA particles, soluble OVA, or control latex bead/BSA, PLGA/BSA particles, or soluble BSA for 16 hr. Following extensive washing, cells were stained with Alexa 647-conjugated 25-D1·16 mAb, which recognizes the SIINFEKL peptide (OVA_257−264) in association with K^b. Blue histograms represent signal from BSA and red for signal from OVA.

Figure 4

(a) Endosomal escape of FITC–OVA after being phagocytosed as soluble form, coated onto latex beads, or encapsulated in PLGA particles. KG-1 cells (open bars) or C1R B cells (closed bars) were incubated with indicated antigen forms for 16 hr, followed by cytosol extraction for fluorimetry assay. Percent in cytosol was calculated from total antigen initially added to cells. (b) Confocal images of murine BM-derived DC showing endosomal escape of antigen. Left panel shows FITC–OVA PLGA particles, middle panel shows LysoTracker Red stained endosomal/lysosomal compartments, right panel is overlay which includes a superimposed DIC image to outline the cell membrane. Arrows in overlay panel show non-colocalized FITC microparticles.

Figure 5

PLGA particles sustain the level of antigen in DCs. Mouse BMDCs were cultured in mGM-CSF and mIL-4 for 4 days, and then were exposed to PLGA/FITC–OVA, soluble FITC–OVA, and latex beads/FITC–OVA for three hrs. At various timepoints (0, 24 and 48 hr), cells were harvested to assay antigen internalization and degradation by flow cytometry and fluorescence microscopy.

Figure 6

PLGA particles prolong cross-presentation by C57BL/6 mouse BMDCs. DCs were incubated with soluble OVA (100 µg/ml), PLGA particles containing 100 µg/ml OVA (PLGA/OVA), latex beads containing 100 µg/ml OVA (latex beads/OVA), or SIINFEKL peptide (1 µm), matured and then cocultured with B3Z T cells at a 1: 1 ratio. At various time points, supernatants were assayed for IL-2 production. Data shows mean ± SD (n = 3).