Tumor specific CD cytolytic T lymphocytes

Tumor-specific CD8+ cytolytic T lymphocytes (CTLs) recognizing tumor antigenic peptide/major histocompatibility complexes-I (pMHC-I) presented on tumor cells play an important role in antitumor immunity [12]. However, immune tolerance including central (natural) or peripheral (acquired) tolerance becomes a major obstacle in antitumor immunity [13]. Central tolerance refers to the tolerance established in the thymus by deleting high affinity autoreactive CD8+ T cells and differentiating some autoreactive T lymphocytes into natural polyclonal CD4+CD25+Foxp3+ or CD8+CD25+Foxp3+ regulatory T (nTreg) cells, which act as sentinels to suppress potential T cell autoreactivity [14]. Peripheral tolerance is driven by induced Treg (iTreg) cells such as CD4+Fop3+ Treg and CD4+Foxp3− type-1 Treg (Tr1) or type-3 helper T (Th3) cells developed in peripheral lymphoid tissues or organs. These cells have critical role in mucosal immune tolerance and controlling allergic inflammation, and become major obstacles in antitumor immunity [14]. Heterologous pexidartinib sale that share a significant sequence homology with self-antigens have been shown to be effective in overcoming peripheral tolerance [15], triggering autoimmune diseases, such as mouse experimental autoimmune encephalomyelitis and arthritis, respectively, by using bovine myelin basic protein [16] and bovine, porcine or human collagen [17]. Tumor antigens detected in sera of cancer patients are often self-tolerated proteins inducing central and peripheral immune tolerance [18], [19]. To break the tolerance, heterologous DNA vaccines composed of both human HER2 and rat neu DNA sequences have been shown to stimulate stronger antibody responses and more efficient antitumor immunity than either HER2 or neu DNA vaccine in transgenic (Tg) mice with HER2-specific self-immune tolerance [3], [20]. However, the HER2-specific therapeutic antitumor immunity derived from immune cell-based vaccines engineered to express heterologous human HER2 and rat neu has yet been investigated. We previously demonstrated that a recombinant adenoviral vector-mediated neu gene-engineered dendritic cell (DC) vaccine is superior to neu DNA vaccination by stimulating stronger neu-specific humoral and cellular immune responses in wild-type mice [21]. We also developed a novel ovalbumin (OVA)-specific T cell-based vaccine OVA-TEXO, using ConA-stimulated polyclonal CD4+ T cells with the uptake of OVA-specific dendritic cell (DCOVA)-released exosomes (EXO) via DC’s CD54 and T cell’s LFA-1 interaction [22], [23]. Since the OVA-TEXO cells expressed exosomal pMHC-I and CD80 and T cell’s CD40L and IL-2, they could thus directly stimulated OVA-specific CD8+ T cell responses [22], [23]. We showed that the OVA-TEXO vaccine induced more efficient OVA-specific CTL responses and antitumor immunity than the DCOVA vaccine because it counteracts CD4+25+Foxp3+ Treg suppression [22], [23]. Recently, we developed a HER2-specific DC (DCHER2)-released EXO-targeted T cell-based vaccine, HER2-TEXO, and demonstrated that the HER2-TEXO vaccine was capable of stimulating HER2-specific CTL responses. However, it induced only partially protective immunity in double-Tg HLA-A2/HER2 mice with self-immune tolerance to HER2 [24]. Therefore, it would be interesting to determine, whether heterologous human/rat HER2-specific T cell vaccine enhances the circumvention of the HER2 tolerance in our double Tg HLA-A2/HER2 mice. In this study, we constructed by recombinant DNA technology a recombinant adenoviral vector AdVHuRt carrying a fused HuRt cDNA encoding human NH2-HER21-407 (Hu) and rat COOH-neu408-690 (Rt) fusion protein. With the help of this construct, we developed a heterologous human/rat HER2-specific EXO-targeted T cell-based vaccine HuRt-Texo. This was achieved by using polyclonal CD4+ T cells with the uptake of human HER2/rat neu-specific EXO released by AdVHuRt-transfected DCs. We then assessed enhancement of HER2-specific CD8+ CTL responses and antitumor immunity in wild-type BALB/c and double Tg HLA-A2/HER2 mice immunized with HuRt-TEXO. In addition, we also examined the cytolytic effect of HuRt-TEXO-stimulated CD8+ T cells against trastuzumab-resistant HER2-positive breast cancer cells, BT474, in vitro, and in BT474 breast cancer-bearing athymic nude mice in vivo.