Re Francesca, Srinivasan Ramaprasad, Igarashi Takehito, Marincola Franco, Childs Richard
H. S. Raffaele Scientific Institute, Milan, Italy.
Exp Hematol. 2004 Feb;32(2):210-7. doi: 10.1016/j.exphem.2003.10.014.
Green fluorescent protein (GFP) has been used to monitor and select cells transduced with vectors encoding other transgenes of interest. We investigated the immunogenic nature of GFP in humans and further explored whether this xenoprotein could be used as a functional adjuvant to enhance T-cell immunity to the melanoma tumor antigen MART1.
Peripheral blood lymphocytes from healthy donors were stimulated by autologous dendritic cells expressing GFP, then cloned by limiting dilution and tested for antigen specificity following coculture with GFP-expressing or GFP-negative targets. In a parallel experiment, lymphocytes from HLA A 0201+ healthy donors were stimulated with four different Melan-A/MART1(27-35) peptide-pulsed stimulators: 1) MART1 peptide-pulsed DCs, 2) MART1 peptide-pulsed DCs loaded with GFP protein, 3) MART1 peptide-pulsed GFP adenovirus-transduced DCs, and 4) MART1 peptide-pulsed null adenovirus-transduced DCs. The percentage of CD3+/CD8+ MART1 peptide-specific T cells was determined by intracellular cytokine staining for gamma-IFN.
Multiple CD4+ and CD8+ T cell clones were expanded which secreted gamma-IFN and demonstrated high levels of cytotoxicity to GFP-expressing targets as assessed by ELISA and Cr51 release respectively. We next investigated the impact of GFP expression on DCs used to stimulate cytotoxic T cells specific for a tumor-associated peptide. The percentage of MART1- specific CD8+ T cells that were generated was higher when MART1-pulsed GFP adenovirus-transduced DCs were used as stimulators (28%) compared to MART1-pulsed DCs alone (11%, p = 0.01), MART1-pulsed null adenovirus-transduced DCs (11.7%, p = 0.02), or MART1-pulsed DCs loaded with GFP protein (12.2%).
These findings further support GFP's immunogenicity and suggest this xenoprotein might further be used to enhance the expansion of tumor-specific T cells.
绿色荧光蛋白（GFP）已被用于监测和筛选用编码其他感兴趣转基因的载体转导的细胞。我们研究了GFP在人体内的免疫原性，并进一步探讨了这种异种蛋白是否可用作功能性佐剂来增强对黑色素瘤肿瘤抗原MART1的T细胞免疫。
用表达GFP的自体树突状细胞刺激健康供体的外周血淋巴细胞，然后通过有限稀释进行克隆，并在与表达GFP或GFP阴性靶细胞共培养后检测抗原特异性。在一项平行实验中，用四种不同的Melan-A/MART1(27 - 35)肽脉冲刺激物刺激来自HLA A 0201+健康供体的淋巴细胞：1）MART1肽脉冲的树突状细胞，2）负载GFP蛋白的MART1肽脉冲的树突状细胞，3）MART1肽脉冲的GFP腺病毒转导的树突状细胞，4）MART1肽脉冲的空腺病毒转导的树突状细胞。通过细胞内细胞因子γ-干扰素染色测定CD3+/CD8+ MART1肽特异性T细胞的百分比。
多个CD4+和CD8+ T细胞克隆得以扩增，它们分泌γ-干扰素，分别通过ELISA和Cr51释放评估，对表达GFP的靶细胞表现出高水平的细胞毒性。接下来，我们研究了GFP表达对用于刺激肿瘤相关肽特异性细胞毒性T细胞的树突状细胞的影响。与单独使用MART1脉冲的树突状细胞（11%，p = 0.01）、MART1脉冲的空腺病毒转导的树突状细胞（11.7%，p = 0.02）或负载GFP蛋白的MART1脉冲的树突状细胞（12.2%）相比，当使用MART1脉冲的GFP腺病毒转导的树突状细胞作为刺激物时，产生的MART1特异性CD8+ T细胞百分比更高（28%）。
这些发现进一步支持了GFP的免疫原性，并表明这种异种蛋白可能进一步用于增强肿瘤特异性T细胞的扩增。
