Harui Airi, Roth Michael D, Sanghvi Mihir, Vira Darshni, Mizuguchi Hiroyuki, Basak Saroj K
Pulmonary and Critical Care Medicine, David Geffen School of Medicine at UCLA, Los Angeles CA 90095-1690, USA.
J Immunol Methods. 2006 May 30;312(1-2):94-104. doi: 10.1016/j.jim.2006.02.014. Epub 2006 Mar 31.
The level of antigen loading can impact on the capacity for dendritic cells (DC) to activate T cell responses. Several different approaches to adenoviral (Ad)-based transduction were therefore assessed for their effect on both transgene expression and T cell activation. While a conventional E1(-)/E3Delta Ad vector (Ad/GFP) produced a concentration-dependent expression of GFP, a modified vector expressing Arginine-Glycine-Aspartic Acid (RGD) sequence on its fiber knob (Ad-RGD/GFP) enhanced transgene expression by 9-20-fold at each MOI. The addition of centrifugal force (2000xg) during DC transduction with Ad/GFP also increased expression up to 20-fold. However, combining centrifugation with the Ad-RGD/GFP vector produced no effect on transduction rate and only a 1.5- to 2-fold increase in GFP expression, suggesting overlapping mechanisms of action. Consistent with this, exogenous RGD peptide blocked transduction regardless of the vector used, or the addition of centrifugal force, and transduction was primarily limited to DC expressing the CD51 integrin receptor. Ad vectors expressing ovalbumin (OVA) were used to assess transduced DC for their capacity to activate OVA-specific T cells. We observed a significant relationship between transgene expression and the capacity for T cell activation regardless of whether transgene expression was increased by using a higher MOI, an RGD-modified vector, or by employing centrifugal force. Furthermore, combining these approaches produced synergistic effects on T cell activation. We conclude that RGD-modified vectors and centrifugation both enhance DC transduction by increasing entry via integrin receptors and that the capacity for T cell activation can be optimized by combining approaches to achieve the highest possible level of transgene expression.
抗原负载水平会影响树突状细胞（DC）激活T细胞反应的能力。因此，评估了几种基于腺病毒（Ad）转导的不同方法对转基因表达和T细胞激活的影响。虽然传统的E1（-）/E3Delta Ad载体（Ad/GFP）产生了浓度依赖性的GFP表达，但一种在其纤维钮上表达精氨酸-甘氨酸-天冬氨酸（RGD）序列的改良载体（Ad-RGD/GFP）在每个感染复数（MOI）下将转基因表达提高了9至20倍。在用Ad/GFP转导DC期间添加离心力（2000xg）也使表达增加了20倍。然而，将离心与Ad-RGD/GFP载体结合对转导率没有影响，仅使GFP表达增加了1.5至2倍，表明作用机制重叠。与此一致的是，外源性RGD肽无论使用何种载体或是否添加离心力都能阻断转导，并且转导主要限于表达CD51整合素受体的DC。使用表达卵清蛋白（OVA）的Ad载体评估转导的DC激活OVA特异性T细胞的能力。我们观察到，无论转基因表达是通过使用更高的MOI、RGD修饰的载体还是通过采用离心力来增加，转基因表达与T细胞激活能力之间都存在显著关系。此外，将这些方法结合对T细胞激活产生协同效应。我们得出结论，RGD修饰的载体和离心都通过增加经由整合素受体的进入来增强DC转导，并且通过结合各种方法以实现尽可能高的转基因表达水平，可以优化T细胞激活能力。
