Tumor-derived autophagosome vaccine: mechanism of cross-presentation and therapeutic efficacy

Abstract

Purpose: We previously reported that autophagy in tumor cells plays a critical role in cross-presentation of tumor antigens and that autophagosomes are efficient antigen carriers for cross-priming of tumor-reactive CD8(+) T cells. Here, we sought to characterize further the autophagosome-enriched vaccine named DRibble (DRiPs-containing blebs), which is derived from tumor cells after inhibition of protein degradation, and to provide insights into the mechanisms responsible for their efficacy as a novel cancer immunotherapy.

Experimental design: DRibbles were characterized by Western blot and light or transmission electron microscopy. The efficiency of cross-presentation mediated by DRibbles was first compared with that of whole-tumor cells and pure proteins. The mechanisms of antigen cross-presentation by DRibbles were analyzed, and the antitumor efficacy of the DRibble vaccine was tested in 3LL Lewis lung tumors and B16F10 melanoma.

Results: The DRibbles sequester both long-lived and short-lived proteins, including defective ribosomal products (DRiP), and damage-associated molecular pattern molecules exemplified by HSP90, HSP94, calreticulin, and HMGB1. DRibbles express ligands for CLEC9A, a newly described C-type lectin receptor expressed by a subset of conventional and plasmacytoid dendritic cells (DC), and cross-presentation was partially CLEC9A dependent. Furthermore, this autophagy-assisted antigen cross-presentation pathway involved both caveolae- and clathrin-mediated endocytosis and endoplasmic reticulum-associated degradation machinery. It depends on proteasome and TAP1, but not lysosome functions of antigen-presenting cells. Importantly, DCs loaded with autophagosome-enriched DRibbles can eradicate 3LL Lewis lung tumors and significantly delay the growth of B16F10 melanoma.

Conclusions: These data documented the unique characteristics and potent antitumor efficacy of the autophagosome-based DRibble vaccine. The efficacy of DRibble cancer vaccine will be further tested in clinical trials.

Figure 1

Colocalization of the long-lived M-GFP-OVA, short-lived R-GFP-OVA, and GFP-ubiquitin with tomato-LC3 fusion protein in punctuates after proteasome and lysosome inhibition. (A). Colocalization of M-GFP-OVA and Tomato-LC3 in punctuates. (B). Colocalization of R-GFP-OVA and Tomato-LC3 in punctuates. (C). Colocalization of GFP-Ubiquitin and Tomato-LC3 in punctuates. HEK 293T cells that are stably expressing Tomato-LC3 were transfected with plasmids that express M-GFP-OVA, R-GFP-OVA, or GFP-ubiquitin separately and cultured for 48 hours. Cells were then treated with 100 nM bortezomib and 10 mM NH4Cl for 24 hours. Untreated cells were used as control. Arrows denote the spots of colocalization.

Figure 2

DRibbles were highly efficient antigen carriers for cross-presentation of antigens to naïve CD8+ T cells. DRibbles were collected from HEK 293T cells that expressed M-GFP-OVA protein or the human melanoma antigen gp100. DCs were loaded with the indicated amount of antigen-expressing DRibbles, irradiated antigen-expressing cells, or pure OVA/gp100 protein. CFSE-labeled OT-I, or pmel-1 transgenic T cells were added. Activation of T cells was assessed by dilution of CFSE in the labeled T cells at day 4 or 6. (A). DCs pulsed with DRibbles were superior to DCs pulsed with irradiated 6 whole tumor cells at activating OT-I and pmel-1 CD8⁺ T cells. DRibbles from 3×10⁶ HEK 293T cells that expressed OVA or gp100, or equivalent number of antigen-expressing irradiated cells were loaded onto DCs. (B). DCs pulsed with DRibbles were more efficient (on a μg basis) than pure proteins in stimulating OT-I CD8⁺ T cells. Data are representative of 3 independent experiments with similar results.

Figure 3

The involvement of CLEC9A in cross-presentation of DRibbles in vitro. (A). The majority of DRibbles expressed a ligand for the CLEC9A receptor. DRibbles harvested from HEK 293T cells were incubated with the soluble FcmILZ-CTLD9A fusion protein (filled histogram) for 1 hour. The soluble FcmILZ OX40 ligand fusion protein was used as negative control (open histogram). PE-conjugated anti-human Fc Fab was used to binding by flow cytometry. (B). CLEC9A was detected on the surface of the majority of splenic DCs from mice that had received sequential injection of plasmid DNA encoding Flt3 ligand and GM-CSF. All events were gated on CD11c+ population. The open histogram shows the isotype control. The filled histogram shows expression of CLEC9 A. (C) (bar graph)&(D) (histogram). Cross-presentation of DRibble-OVA, but not soluble OVA protein, was significantly reduced by incubating DRibbles with soluble FcmILZ CTLD9A. DRibbles were pretreated with soluble FcmILZ-CTLD9A overnight at 4°C and then used to pulse DC for 6 hours. Percentages of divided OT-I T cells are shown as mean±SEM. Data are representative of results from 2-4 independent experiments.

Figure 4

Multiple pathways are involved in cross-presentation of DRibble antigens. (A). Inhibition of caveolae (filipin III) and/or clathrin-mediated (chlorpromazine) endocytosis diminished cross-presentation of ovalbumin by DRibble-pulsed DCs. DCs loaded with SIINFEKL peptides were used as a control. Division of CFSE-labeled OT-I T cells was monitored by flow cytometry. (B). Blockade of ERAD-mediated retrotranslocation, proteosome activity, or knockout of TAP1, but not blockade of lysosomal activity markedly reduced cross-presentation of OVA- DRibble. The results shown in the bar graph are expressed as the percentage±SEM of OT-I T cells that had undergone at least one division 4 days after coculture with DCs loaded with antigens. Data are representative of three independent experiments.

Figure 5

DRibbles harvested from tumor cells were enriched in autophagosomes and induced highly efficient cross-priming of CD8+ T cells in vitro. (A). DRibbles were induced from a variety of tumor cell lines, including human non-small lung cancer cells (a), mouse Lewis lung cancer 3LL cells (b), human melanoma FEMX cells (c), and mouse melanoma B16F10 (d). Images were taken with a light microscope. Small vesicles indicated by the arrowheads were distinctive morphologically from large cells. (B). Transmission electron micrographs of autophagosome-rich DRibbles harvested from mouse Lewis lung 3LL tumor cells. a. A large tumor cell is surrounded by a number of vesicles. b. Multiple vesicles with a variety of morphology in DRibbles. Their sizes ranged between 100 nm and 1μm. c. Fine structure of autophagosomes with the typical double-membrane structure and differential light density. d. Display of autophagosomes with different morphology. e. An autophagosome with a clear vacuole inside. f. A phagophore in the process of packaging a small vesicle. TEM images were collected at original magnifications of 1,000-37,000x.

Figure 6

Vaccination with DCs pulsed with 3LL tumor cell-derived DRibbles showed significant anti-tumor effect in mice bearing 3LL tumors. (A). DRibbles derived from 3LL cells that expressed OVA stimulated proliferation of transgenic OT-I T cells in vitro. (B and C). The anti-tumor efficacy of DRibble vaccine in mice bearing s.c. tumors. The vaccination with DRibble-loaded DCs plus poly [I:C] and IFN-γ mediated tumor regression (B) and cured 5 out of 6 mice with 6-day 3LL s.c. lung tumors (C). Data are representative of three independent experiments. (D). Therapeutic efficacy of DCs loaded with DRibbles, lysates, or whole tumor cells. This is representative of two experiments with similar results.