![]() (a) Illustration of bicistronic vector (BV) showing iMC and TSA (LacZ) separated by the picornavirus F2A sequence. FITC, fluorescein isothiocyanate IFN-γ, interferon-γ MHCI, major histocompatibility complex class I PBL, peripheral blood lymphocytes pfu, plaque-forming unit vp, virus particles.ĭevelopment of iMC/LacZ bicistronic vector. All results are representative of two or more independent experiments. Panel shows one Ad-LacZ representative dot plot of flow cytometry data and the average+ % intratumoral CD8+ cells (n = 3). (d) β-gal-tet staining of CD8+ cells from c indicate the presence of tumor antigen-specific T-cells in the tumors of Ad-LacZ treated mice (1 × 1010 vp/mouse). Tumors were disaggregated by digestion and strained to make single-cell suspensions before staining with anti-CD8 antibodies and MHCI tetramers. Panel shows one Ad-LacZ representative dot plot of flow cytometry data and the overall combined percentage of CD8+ cells in the tumors for three mice per group. (c) Intratumoral lymphocyte analysis shows an increased number of CD8+ T-cells resident within the tumors of Ad-LacZ treated mice (1.6 × 108 pfu per mouse). Panel shows one representative well of each treatment group and the average number of spots of four mice per group. (b) IFN-γ ELISpot of splenocytes extracted from mice 9 days after treatment as in a. Panel indicates a representative dot plot of flow cytometry data from the Ad-LacZ group and the average % tetramer+ peripheral blood CD8+ cells of 15 mice per group (two mice in naive group). (a) H2-Kb-ICPMYARV tetramer (β-gal-tet) and CD8 staining of PBLs extracted from mice 8 days after footpad injection with Ad-LacZ (1 × 1010 vp/mouse). ![]() Further, it demonstrates the feasibility of in vivo molecular enhancement of DCs necessary for effective antitumor immune responses.Īd-LacZ induces a potent antigen-specific CD8+ T-cell response. This study demonstrates the spatial-temporal importance of concurrent DC activation and TSA presentation. However, when delivered simultaneously with TSA in a single bicistronic vector (BV), iMC is able to significantly enhance antigen-specific cytotoxic T-cell (CTL) responses and inhibit established tumor growth. Addition of iMC delivered in a separate vector is insufficient to enhance this effect. In this study, we demonstrate that simple TSA delivery via adenoviral vectors results in strong antitumor immunity. We describe here a method to co-deliver both tumor-specific antigen (TSA) and an iMyD88/CD40 adjuvant (iMC), to DCs that combines toll-like receptor (TLR) and CD40 signaling. Although ex vivo vaccine approaches continue to be proving grounds for new DC manipulation techniques, the intrinsic limits of ex vivo therapy, including high cost, minimal standardization, cumbersome delivery, and poor accessibility, incentivizes the development of vaccines compatible with in vivo DC targeting. Recent modest successes in ex vivo dendritic cell (DC) immunotherapy have motivated continued innovation in the area of DC manipulation and activation.
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