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

Journal name:
Nature Nanotechnology
Year published:
Published online


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


  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.


  1. Pardoll, D. M. Cancer vaccines. Nature Med. 4, 525531 (1998).
  2. Finn, O. J. Cancer vaccines: between the idea and the reality. Nature Rev. Immunol. 3, 630641 (2003).
  3. Heath, W. R. & Carbone, F. R. Cross-presentation, dendritic cells, tolerance and immunity. Annu. Rev. Immunol. 19, 4764 (2001).
  4. Heijst, J. W. J. et al. Recruitment of antigen-specific CD8+ T cells in response to infection is markedly efficient. Science 325, 12651269 (2009).
  5. Burgdorf, S. & Kurts, C. Endocytosis mechanisms and the cell biology of antigen-presentation. Curr. Opin. Immunol. 20, 8995 (2008).
  6. Vyas, J. M., Van der Veen, A. G. & Ploegh, H. L. The known unknowns of antigen processing and presentation. Nature Rev. Immunol. 8, 607618 (2008).
  7. Cresswell, P., Ackerman, A. L., Giodini, A., Peaper, D. R. & Wearsch, P. A. Mechanisms MHC class I restricted antigen processing and cross-presentation. Immunol. Rev. 207, 145157 (2005).
  8. Guy, B. The perfect mix: recent progress in adjuvant research. Nature Rev. Micro. 5, 505517 (2007).
  9. Pulendran, B. & Ahmed, R. Immunological mechanisms of vaccination. Nature Immunol. 12, 509517 (2011).
  10. Marrack, P., McKee, A. S. & Munks, M. W. Towards an understanding of the adjuvant action of aluminium. Nature Rev. Immunol. 9, 287293 (2009).
  11. Coffman, R. L., Sher, A. & Seder, R. A. Vaccine adjuvants: putting innate immunity to work. Immunity 33, 492503 (2010).
  12. Hermanson, G. T. Bioconjugate Techniques 2nd edn, 1202 (Academic Press, 2008).
  13. Matteoni, R. & Kreis, T. E. Translocation and clustering of endosomes and lysosomes depends on microtubules. J. Cell Biol. 105, 12531265 (1987).
  14. Porgador, A., Yewdell, J. W., Deng, Y., Bennink, J. R. & Germain, R. N. Localization, quantitation, and in situ detection of specific peptide-MHC class I complexes using a monoclonal antibody. Immunity 6, 715726 (1997).
  15. Regnault, A. et al. Fcγ receptor-mediated induction of dendritic cell maturation and major histocompatibility complex class I-restricted antigen presentation after immune complex internalization. J. Exp. Med. 189, 371380 (1999).
  16. Kratzer, R., Mauvais, F. X., Burgevin, A., Barilleau, E. & van Endert, P. Fusion proteins for versatile antigen targeting to cell surface receptors reveal differential capacity to prime immune responses. J. Immunol. 184, 68556864 (2010).
  17. Reddy, S. T. et al. Exploiting lymphatic transport and complement activation in nanoparticle vaccines. Nature Biotechnol. 25, 11591164 (2007).
  18. Pelka, K. & Latz, E. Getting closer to the dirty little secret. Immunity 34, 455458 (2011).
  19. Flach, T. L. et al. Alum interaction with dendritic cell membrane lipids is essential for its adjuvanticity. Nature Med. 17, 479487 (2011).
  20. Bjørkøy, G. et al. p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on Huntington-induced cell death. J. Cell. Biol. 171. 603614 (2005).
  21. Kirkin, V., McEwan, D. G., Novak, I. & Dikic, I. A role for ubiquitin in selective autophagy. Mol. Cell. 34, 259269 (2009).
  22. Crotzer, V. L. & Blum, J. S. Autophagy and its role in MHC-mediated antigen presentation. J. Immunol. 182, 33353341 (2009).
  23. Li, Y. H. et al. Efficient cross-presentation depends on autophagy in tumor cells. Cancer Res. 68, 68896895 (2008).
  24. Uhl, M. et al. Autophagy within the antigen donor cell facilitates efficient antigen cross-priming of virus-specific CD8+ T cells. Cell. Death Differ. 16, 9911005 (2009).
  25. English, L. et al. Autophagy enhances the presentation of endogenous viral antigens on MHC class I molecules during HSV-1 infection. Nature Immunol. 10, 480487 (2009).
  26. Jagannath, C. et al. Autophagy enhances the efficacy of BCG vaccine by increasing peptide presentation in mouse dendritic cells. Nature Med. 15, 267276 (2009).
  27. Nishida, Y. et al. Discovery of Atg5/Atg7-independent alternative macroautophagy. Nature 461, 654658 (2009).
  28. Savina, A. et al. NOX2 controls phagosomal pH to regulate antigen processing during crosspresentation by dendritic cells. Cell 126, 205218 (2006).
  29. de Gruijl, T. D., van den Eertwegh, A. J. M., Pinedo, H. M. & Scheper, R. J. Whole-cell cancer vaccination: from autologous to allogeneic tumor- and dendritic cell-based vaccines. Cancer Immunol. Immunother. 57, 15691577 (2008).
  30. Jensen, S. M. et al. Signaling through OX40 enhances antitumor immunity. Semin. Oncol. 37, 524532 (2010).

Download references

Author information


  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


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.

Corresponding authors

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary information (1,986 KB)

    Supplementary information

Additional data