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Abstract

Exosomes (EXOs) derived from tumor cells have been used to stimulate antitumor immune responses. It has been demonstrated that EXO released by tumor cells engineered to express cytokines are of enhanced stimulatory effect on CD8⁺ cytotoxic T-lymphocyte (CTL) responses and antitumor immunity. J558 is a mouse myeloma cell line expressing tumor antigen P1A. In this study, we purified EXO_TNF-a, EXO_IL-2, and EXO_IFN-γ released by three cytokine-gene (TNF-α, IL-2 and IFN-γ)-engineered J558 (J558_TNF-a, J558_IL-2 and J558_IFN-γ) tumor cell lines from their culture supernatants, respectively, by differential ultracentrifugation. These EXOs showed a “saucer” or round shape with a diameter between 50 and 90 nm by electron microscopy and contained EXO-associated proteins, such as LAMP-1 and AIP1, but not lysate-associated protein galectin, by Western blot analysis. EXO displayed expression of molecules (H-2K^d, CD54, and P1A) similarly to, but to a lesser extent to, J558 tumor cells. We then compared the stimulatory effect of these EXOs on P1A-specific CD8⁺ CTL responses and antitumor immunity 6 days subsequent to intravenous (i.v.) EXO immunization (30 μg/each BALB/c mouse). We demonstrated that EXO_TNF-α immunization was able to induce more efficient P1A-specific CD8⁺ T-cell response accounting for 0.62% of the total CD8⁺ T-cell population, using PE-H-2K^d/P1A peptide and FITC-CD8 staining by flow cytometric analysis then EXO_IL-2 (0.31%) and EXO_IFN-γ (0.22%) immunization (P < 0.05), respectively, at day 6 after immunization. EXO_IL-2 and EXO_IFN-γ vaccine (i.v. 30 μg/each mouse) only protected 3 of 8 (38%) and 2 of 8 (25%) mice from tumor growth after subcutaneous (s.c.) challenging of immunized mice with J558 tumor cells (0.5 × 10⁶ cells/each mouse), whereas EXO_TNF-α immunization protected all 8 of 8 (100%) mice from tumor growth (P < 0.05). Taken together, we demonstrate that EXO_TNF-a released by engineered J558_TNF-a tumor cells more efficiently stimulate tumor antigen P1A-specific CD8⁺ CTL responses and antitumor immunity than EXO_IL-2 and EXO_IFN-γ released by engineered J558_IL-2 and J558_IFN-γ tumor cells. Therefore, TNF-α-expressing tumor cell-released EXO may represent a more effective EXO-based vaccine in the induction of antitumor immunity.

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Tumor Necrosis Factor Gene-Engineered J558 Tumor Cell–Released Exosomes Stimulate Tumor Antigen P1A-Specific CD8 + CTL Responses and Antitumor Immunity

Abstract

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