Exosomes derived from rAAV/AFP-transfected dendritic cells elicit specific T cell-mediated immune responses against hepatocellular carcinoma

Abstract

Background: Dendritic cell (DC)-derived exosomes (Dexs) have been proved to induce and enhance antigen-specific T cell responses in vivo, and previous clinical trials have shown the feasibility and safety of Dexs in multiple human cancers. However, there is little knowledge on the efficacy of Dexs against hepatocellular carcinoma (HCC) until now.

Methods: In this study, human peripheral blood-derived DCs were loaded with recombinant adeno-associated viral vector (rAAV)-carrying alpha-fetoprotein (AFP) gene (rAAV/AFP), and high-purity Dexs were generated. Then naive T cells were stimulated with Dexs to investigate the specific T cell-mediated immune responses against HCC.

Results: Our findings showed that Dexs were effective to stimulate naive T cell proliferation and induce T cell activation to become antigen-specific cytotoxic T lymphocytes (CTLs), thereby exhibiting antitumor immune responses against HCC. In addition, Dex-sensitized DC precursors seemed more effective to trigger major histocompatibility complex class I (MHC I)-restricted CTL response and allow DCs to make full use of the minor antigen peptides, thereby maximally activating specific immune responses against HCC.

Conclusion: It is concluded that Dexs, which combine the advantages of DCs and cell-free vectors, are promising to completely, or at least in part, replace mature DCs (mDCs) to function as cancer vaccines or natural antitumor adjuvant.

Keywords: cytotoxic T lymphocyte; dendritic cell; exosome; hepatocellular carcinoma; immune response.

Figure 1

The morphologic and phenotypic features of mDCs. Note: (A) mDCs post-transfection with rAAV/AFP were obtained by LPS stimulation on day 7, and the typical mDC morphology was observed under a light microscope (×40); (B) mDCs were stained with CD11C, CD209, CD54, CD86, CD80, and HLA-DR or isotype-matched control antibodies and analyzed by flow cytometry. Abbreviations: AFP, alpha-fetoprotein; HLA, human leukocyte antigen; LPS, lipopolysaccharide; mDC, mature dendritic cell; rAAV, recombinant adeno-associated viral vector.

Figure 2

Validation of successful Dex isolation. Notes: (A) Dex ultrastructure displayed by transmission electron microscopy (red arrows). Scale bar =200 nm; (B) the size distribution profile of Dex using the Malvern NanoSight NS300 system, showing a size peak of 91 nm in mature Dex; (C) the expression of the exosome-negative marker calnexin and positive markers, Alix, CD81, CD9, and CD63. In addition, the functional protein expressions of MHC-I, MHC-II, CD86, and AFP were detected in mDCs and Dex lysates by Western blotting. A total of 30 µg protein from the lysates of mDCs and Dex was loaded into each lane. Data were normalized to the GAPDH levels. The bands for targeted proteins were analyzed using the software Gel-Pro analyzer version 4.1. Abbreviations: AFP, alpha-fetoprotein; Dex, dendritic cell-derived exosome; mDC, mature dendritic cell; MHC, major histocompatibility complex.

Figure 3

DC- and Dex-induced naive T cell proliferation. Note: (A and E) DC-rAAV/AFP; (B and F) Dex; (C and G) DC-Dex; (D and H) non-transfected DCs; (A–D) CFSE staining; (E–H) flow cytometry. Abbreviations: AFP, alpha-fetoprotein; CFSE, 5,6-carboxyfluorescein diacetate succinimidyl ester; DC, dendritic cell; Dex, dendritic cell-derived exosome; rAAV, recombinant adeno-associated viral vector.

Figure 4

Flow cytometry detects surface markers on immune effector cells. Notes: Cells in the four groups of DC-rAAV/AFP, Dex, DC-Dex and non-transfected DCs were co-incubated with naive T cells for 10 days. Expression of surface proteins was tested by flow cytometric analysis. Data from one experiment representative of three performed. *P<0.05; #P<0.05. Abbreviations: AFP, alpha-fetoprotein; DC, dendritic cell; Dex, dendritic cell-derived exosome; HLA, human leukocyte antigen; rAAV, recombinant adeno-associated viral vector.

Figure 5

IFN-γ-secreting CTLs are quantified by the ELISPOT assay. Notes: CTLs induced by DC-rAAV/AFP, Dex, DC-Dex, and non-transfected DCs were co-cultured with 1×10⁴ HepG2 cells at an E/T ratio of 20:1. Compared with those in the non-transfected DCs group, the CTLs in the DC-rAAV/AFP, Dex, and DC-Dex groups showed a significantly greater frequency of IFN-γ secretion (**P<0.01), while the PHA group serves as a positive control. Abbreviations: AFP, alpha-fetoprotein; CTL, cytotoxic T lymphocyte; DC, dendritic cell; Dex, dendritic cell-derived exosome; ELISPOT, enzyme-linked immune absorbent spot; IFN, interferon; rAAV, recombinant adeno-associated viral vector.

Figure 6

Flow cytometry detects the IL-2, IL-4, IL-6, IL-10, TNF, IFN-γ, and IL-17A expressions in the effector cells and HepG2 cell co-culture supernatant. Note: *P<0.05 vs Dex group; #P<0.05 vs non-transfected DC group. Abbreviations: DC, dendritic cell; Dex, dendritic cell-derived exosome; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor.

Figure 7

The killing action of Dex-induced CTL against HCC HepG2 and SMMC7721 cell lines. Notes: (A) Flow cytometry; (B) bar chart. A, DC-rAAV/AFP; B, Dex; C, DC-Dex; D, non-transfected DCs; E, HepG2/SMMC-7721 cells. *P<0.05. (C) flow cytometry detects the cell surface marker CD107a expression on the surface of DC- and Dex-induced CTLs. A, DC-rAAV/AFP; B, Dex; C, DC-Dex; D, non-transfected DC. Abbreviations: AFP, alpha-fetoprotein; DC, dendritic cell; Dex, dendritic cell-derived exosome; HCC, hepatocellular carcinoma; rAAV, recombinant adeno-associated viral vector; CTL, cytotoxic T lymphocyte.