Dendritic cell-derived exosomes induce monocyte antigen-presentation and immune amplification in neoantigen vaccine therapy

Abstract

Mature dendritic cells release exosomes; however, the immunological role of exosomes in dendritic cell vaccine therapy remains unclear. We examined the immunogenicity of neoantigen peptide-pulsed dendritic cell-derived exosomes (Neo-P DEX) and investigated their role in vaccine therapy. The quality of DEX derived from dendritic cell cultures was confirmed via electron microscopy, western blotting, flow cytometry, and CD63 ELISA. When DEX released from neoantigen-pulsed DCs was applied to monocytes, they showed dendritic cell-like properties such as surface antigen expression. Furthermore, monocytes receiving Neo-P DEX activated neoantigen-reactive T lymphocytes. Fluorescence-activated cell sorting (FACS) analysis showed that plasma exosomes after neoantigen-pulsed DC vaccine may contain more DEX compared to before the vaccine, suggesting that DEX released after DC vaccination may be involved in the amplification of tumor-specific immune responses by translocating to monocytes in the patient body and transforming them into antigen-presenting dendritic cells. This study suggests that dendritic cell exosomes may act as endogenous neoantigen vaccines or immune amplifiers.

Keywords: dendritic cell; dexosomes; immune amplification; neoantigen; neoepitope; vaccine.

Figure 1

Quality analysis of dexosomes (DEX). (A) DEX concentration from mature DCs (Pts 1–7) pulsed with neoantigen-peptide (CD63 ELISA); the concentration of CD63+ exosomes extracted from 10 mL DC culture supernatant was measured after suspension in 200 μL PBS. (B) Western blot analysis of DEX (neoantigen-peptide pulsed DC culture supernatant derived from Pts 1 to 7). (C) Electron micrograph of DEX derived from Pt 6. (D) Dexosome FACS analysis; 2 ng of DEX derived from Pts 1 to 7 were bound to CD9 capture microbeads and analyzed for HLA class I, HLA-DR, CD40, CD86, and ICAM-1 by FACS analysis (white in each panel indicates the isotype control for each antibody). DC, Dendritic Cell; DEX, Dendritic Cell-Derived Exosomes; PCR, Polymerase Chain Reaction; INF, interferon; Pt, patient.

Figure 2

DEX uptake into monocytes. (A) DEX uptake into monocytes (fusion image of fluorescently stained DEX in monocytes). Evaluation of DEX uptake into monocytes by fluorescence microscopy. DEX from Pt 1 was fluorescently labeled, the unbound dye was removed, and the labeled DEX were incubated with monocytes derived from the patient peripheral blood for 24 h and observed under a fluorescence microscope. Most monocytes were stained red, indicating that DEX were bound to or incorporated into the cells. (B, C) Images of migration of fluorescently labeled DEX within monocytes by confocal microscopy: Composite image of fluorescently labeled DEX (red, left) and CD14 antibody-fluorescently labeled monocytes (green, center) with nuclei stained blue with DAPI (right). Pt, patient.

Figure 3

Changes in surface antigens of monocytes upon addition of DEX. (A–D) DEX (2 ng/mL) from Pts 1, 3, 4, and 7 were added to CD14+ selected monocytes from the same patients and incubated for 24 h, followed by FACS analysis of HLA-Class-I, HLA-DR, CD40, and CD86 expression (A–D, respectively). Pt, patient; DEX, Dendritic Cell-Derived Exosomes.

Figure 4

DEX-added monocytes or immature DCs activate neoantigen-reactive CD8T cells. (A) Experimental schema. (B, D) Activation of antigen-reactive CD8 T lymphocytes by DEX-loaded monocytes from Pts 1 and 3: DEX was added to their respective monocytes, and CD8 T cells from PBMC collected and cryopreserved after three vaccinations were co-cultured with the monocytes. The monocytes were cultured for 48 h to assess ELISpot responses. Controls included monocytes alone, CD8+ T cells alone, and CD8 T cells reacted with monocytes pulsed with peptide without DEX (b, data from Pt 1; d, data from Pt 3). Notably, only monocytes crossed with neoantigen peptide-pulsed DEX-induced stimulation of CD8+ T cell responses. (C, E) DEX-added monocyte-derived imDCs, along with CD8 T lymphocytes, significantly activated CD8 T lymphocytes in the group treated with neoantigen-added DC-derived exosomes. Pt, patient; DEX, Dendritic Cell-Derived Exosomes.

Figure 5

Characterization of plasma exosomes following administration of neoantigen peptide-pulsed DC vaccine. (A) Western blot analysis of plasma exosomes (top: CD9, bottom: CD63, Pts1–7). (B) Plasma exosome concentration: exosomes were extracted from 10 mL of plasma and suspended in 200 μL PBS, and their concentration was measured by CD63 ELISA. (C, D) Surface antigen analysis of plasma exosomes (HLA-A, B, C, HLA-DR, CD40, and CD86). Plasma exosomes after vaccination showed increased positivity and relative intensity of expression of HLA-DR, CD40, and CD86 compared with plasma exosomes before vaccination (C, data from Pt 1; D, data from Pt 5). The negative control was isotype-matched fluorescent-labeled nonspecific antibody binding. (E, F) Activation of T lymphocytes by exosomes in plasma after vaccine administration: Treatment with plasma exosomes obtained after vaccination in Pt 1 (E) and Pt 5 (F) increased IFN-γ production in PBMCs. Pt, patient.