Analysis of Antitumor Immunity Using Dendritic Cells Treated with Various Protein-transduction Domain (PTD)-containing Protein Antigen

蛋白质转导结构域（PTDs）已越来越多地用于将试剂，如蛋白质，寡核苷酸传递到各种细胞类型的体外和体内。我们之前已经证明，含有全蛋白抗原（Ags）的HIV tat - ptd转导的树突状细胞（DC）通过加工并将ag表位呈递到MHC I类和II类上，刺激ag特异性CD8+和CD4+ T细胞。虽然产生的CTL活性足以阻止表达ag的肿瘤小鼠的移植，但用TAT-PTD ag转导的DC治疗荷瘤小鼠，在一些动物中导致肿瘤消退，但不是所有动物。最近，其他几个ptd被报道具有比TAT-PTD更高的转导效率。为了在肿瘤疫苗接种研究中评估PTD诱导更有效的免疫应答，我们设计了几种含有PTD的重组融合OVA蛋白，其中包括被称为最有效的PTD的聚精氨酸（R9）。我们的研究结果表明，R9-PTD在研究的PTDs中表现出更高的DC转导效率，并且这种转导效率与体内和体外ag特异性CD4+、CD8+ T细胞活化的效力密切相关。在（表达ova的）荷瘤小鼠中两次接种r9 - ptd - ova转导DC，通过强大的增强作用诱导更高的抗肿瘤免疫，并且在与LPS或OK432共同注射时引起肿瘤肿块的完全排斥。这种方法应该在临床上适用，并为目前使用的方法（如肽治疗）提供理论和实践优势。

Protein transduction domains (PTDs) have been used increasingly to deliver reagents, such as proteins, oligonucleotides to a variety of cell types in vitro and in vivo. We have previously shown that HIV TAT-PTD-containing whole protein antigens (Ags)-transduced dendritic cells (DC) stimulated Ag-specific CD8+ and CD4+ T cells by processing and presenting Ag-epitopes onto MHC class I and class II. Although the CTL activity generated was sufficient to prevent engraftment of mice with Ag-expressing tumors, treatment of tumor-bearing mice with TAT-PTD Ag-transduced DC resulted in tumor regression in some, but not all animals. Recently, several other PTDs were reported their higher transduction efficiencies than TAT-PTD. To evaluate the PTD for induction of more efficient immune responses in tumor vaccination study, we engineered several recombinant fusion OVA proteins which contain PTDs including polyarginine (R9), known as the most efficacious PTD. Our results demonstrated that R9-PTD showed higher transduction efficiency to DC among PTDs studied, and that efficacy was closely correlated with the potency of Ag-specific CD4+, CD8+ T cell activations in vitro and in vivo. Twice vaccination with R9-PTD-OVA-transduced DC in (OVA-expressing) tumor-bearing mice induced higher antitumor immunity by strong boosting effects, and elicited complete rejection of tumor mass when co-injection with LPS or OK432. This approach should be clinically applicable, and offers theoretical and practical advantages to those that are in current use, such as peptide therapy.