Alpha-alumina nanoparticles induce efficient autophagy-dependent cross-presentation and potent antitumour response

Journal name:
Nature Nanotechnology
Volume:
6,
Pages:
645–650
Year published:
DOI:
doi:10.1038/nnano.2011.153
Received
Accepted
Published online

Abstract

Therapeutic cancer vaccination is an attractive strategy because it induces T cells of the immune system to recognize and kill tumour cells in cancer patients. However, it remains difficult to generate large numbers of T cells that can recognize the antigens on cancer cells using conventional vaccine carrier systems1, 2. Here we show that α-Al2O3 nanoparticles can act as an antigen carrier to reduce the amount of antigen required to activate T cells in vitro and in vivo. We found that α-Al2O3 nanoparticles delivered antigens to autophagosomes in dendritic cells, which then presented the antigens to T cells through autophagy. Immunization of mice with α-Al2O3 nanoparticles that are conjugated to either a model tumour antigen or autophagosomes derived from tumour cells resulted in tumour regression. These results suggest that α-Al2O3 nanoparticles may be a promising adjuvant in the development of therapeutic cancer vaccines.

At a glance

Figures

  1. Conjugation of OVA to [alpha]-Al2O3 nanoparticles resulted in efficient cross-presentation of the OVA antigen in vitro.
    Figure 1: Conjugation of OVA to α-Al2O3 nanoparticles resulted in efficient cross-presentation of the OVA antigen in vitro.

    a, Schematic showing the structure of the α-Al2O3–OVA conjugate. b,c, TEM images of α-Al2O3 nanoparticles (60 nm) before (b) and after (c) conjugation with OVA protein. Inset in b: high-resolution TEM image of an α-Al2O3 nanoparticle. d, Representative bright-field (left), fluorescence (middle) and overlaid (right) images of DCs after incubation with FITC-labelled α-Al2O3 (60 nm)–OVA for 0.5 h (upper) and 24 h (lower). e, Surface expression of major histocompatibility complex class I peptide complexes (Kb -SIINFEKL) on DCs without antigen (shadow) and the DCs pulsed with 10 µg ml−1 OVA (red), or α-Al2O3 (60 nm)–OVA containing 0.1 µg ml−1 OVA (green).

  2. DCs pulsed with [alpha]-Al2O3-OVA efficiently cross-presented OVA antigen to naive OT-I T cells in vitro and in vivo.
    Figure 2: DCs pulsed with α-Al2O3–OVA efficiently cross-presented OVA antigen to naive OT-I T cells in vitro and in vivo.

    ac, Flow cytometric analysis showing that DCs loaded with α-Al2O3–OVA induced the proliferation (a) and secretion of IFN-γ and IL-2 (b,c) by OT-I CD8+ T cell more efficiently than DCs loaded with either TiO2–OVA or α-Fe2O3–OVA. d, DCs loaded with α-Al2O3–OVA were more effective at stimulating naive OT-I T cells in vitro than DCs loaded with OVA immunocomplexes or OVA plus TLR4 agonist. e, Subcutaneous injection of α-Al2O3–OVA activated OT-I CD8+ T cells more efficiently than OVA, anatase TiO2–OVA, α-Fe2O3–OVA or the mixture of OVA/alum in vivo. *P < 0.05. Error bars show standard error of the mean.

  3. Autophagy is required for [alpha]-Al2O3 nanoparticle-mediated cross-presentation of OVA to naive T cells.
    Figure 3: Autophagy is required for α-Al2O3 nanoparticle-mediated cross-presentation of OVA to naive T cells.

    a, Confocal images of untreated DCs, and DCs loaded with α-Al2O3–OVA and stained with antibody against LC3 (red) (upper panels). Lower panels show DCs expressing tdtomato-LC3 or tdtomato-p62 fusion proteins (red) after loading with FITC-labelled α-Al2O3–OVA. b, TEM analysis showing that internalized α-Al2O3–OVA were mainly inside endosomes/phagosomes, autophagosomes and autolysosomes of DCs. cf, Flow cytometric analysis showing that cross-presentation of α-Al2O3–OVA by DCs, but not OVA, was blocked by treatment with 3-MA or wortmannin (c) and by knockdown of the autophagy initiation genes, Beclin 1 or Atg 12 (e), and was reduced by Brefeldin A treatment (f). Results confirmed by western blot analysis (d). Ammonium chloride treatment enhanced cross-presentation of OVA by DCs, but not α-Al2O3–OVA (c).

  4. [alpha]-Al2O3 nanoparticles increased the efficiency of cross-presentation and antitumour response of cancer vaccines.
    Figure 4: α-Al2O3 nanoparticles increased the efficiency of cross-presentation and antitumour response of cancer vaccines.

    a,b, Vaccination with α-Al2O3–OVA induced a high frequency of OVA-specific IFN-γ producing CD8+ T cells in spleens of mice (a) and eliminated the established B16-OVA tumours (b). c,d, Scanning electron microscopy images of isolated autophagosomes derived from 3LL tumour cells (c) and of α-Al2O3–autophagosome conjugates (d). Inset in c: TEM image of an autophagosome. e, Flow cytometry profiles showing that DCs loaded with α-Al2O3–autophagosomes (bottom) more efficiently cross-primed naive OT-I T cells than DCs loaded with naked autophagosomes (top) in vitro. f, With assistance of the anti-OX40 antibody, α-Al2O3–autophagosome demonstrated high therapeutic efficacy in mice bearing 3LL lung tumours. *P < 0.05.

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Affiliations

  1. Department of Physics, Portland State University, Portland, Oregon, USA

    • Haiyan Li &
    • Jun Jiao
  2. Laboratory of Cancer Immunobiology, Earle A. Chiles Research Institute, Providence Portland Medical Center, Portland, Oregon, USA

    • Yuhuan Li &
    • Hong-Ming Hu

Contributions

H.L. performed the experiments and wrote the manuscript. Y.L. performed some experiments. J.J. and H-M.H. directed this work and wrote the manuscript.

Competing financial interests

H.L., J.J. and H-M.H. have filed a patent application titled 'Alumina nanoparticle bioconjugates and methods of stimulating immune response using said bioconjugates'. Y.L. has no competing financial interests.

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