Generating CTLs against the subdominant Epstein-Barr virus LMP1 antigen for the adoptive immunotherapy of EBV-associated malignancies
Generating CTLs against the subdominant Epstein-Barr virus LMP1 antigen for the adoptive immunotherapy of EBV-associated malignancies
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DOI：
10.1182/blood-2002-05-1514
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发表时间：
2003-03-01
期刊：
影响因子：
20.3
通讯作者：
Rooney, CM
中科院分区：
文献类型：
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作者：
Gottschalk, S;Edwards, OL;Rooney, CM
EB病毒（EBV）编码的LMP 1蛋白在EBV阳性霍奇金病中表达，是细胞毒性T淋巴细胞（CTL）治疗的潜在靶点。然而，LMP 1特异性CTL频率低，并且迄今为止LMP 1特异性CTL的产生需要T细胞克隆。LMP 1的毒性已经阻止了树突状细胞（DC）用于CTL刺激的使用，并且我们推断无活性的无毒LMP 1突变体（DeltaLMP-1）可以在DC中表达：并且将使得能够激活和扩增多克隆LMP 1特异性CTL。检测表达LMP 1或DeltaLMP 1的重组腺病毒载体对DC的杀伤能力。LMP 1表达在48小时内是有毒的，而DeltaLMP 1表达的高水平是以最小的毒性实现的。表达DeltaLMP 1的DC能够重新激活和扩增来自3名健康EB病毒血清阳性供体的LMP 1特异性CTL。使用HLA-A2限制性LMP 1肽YLQQN-WWTL（YLQ）通过干扰素-γ（IFN-γ）酶联免疫斑点试验（ELISPOT）检测LMP 1特异性T细胞。在供体外周血单核细胞（PBMC）中检测不到YLQ特异性T细胞（小于0.001%）;然而，刺激后频率增加至0.5%至3.8%。CTL对自体靶细胞的裂解依赖于LMP 1的表达水平。相反，使用淋巴母细胞系，EBV特异性CTL中YLQ特异性CTL的再活化和扩增的频率较低，并且没有观察到LMP 1特异性细胞毒活性。因此，DeltaLMP 1在DC中的表达是无毒的，并且能够产生LMP 1特异性CTL，用于LMP 1阳性恶性肿瘤如EBV阳性霍奇金病患者的未来过继免疫治疗方案。在这些恶性肿瘤中靶向LMP 1可能会提高目前过继免疫治疗方法的疗效。
The Epstein-Barr virus (EBV)-encoded LMP1 protein is expressed in EBV-positive Hodgkin disease and is a potential target for cytotoxic T-lymphocyte (CTL) therapy. However, the LMP1-specific CTL frequency is low, and so far the generation of LMP1-specific CTLs has required T-cell cloning. The toxicity of LMP1 has prevented the use of dendritic cells (DCs) for CTL stimulation, and we reasoned that an inactive, nontoxic LMP1 mutant (DeltaLMP-1) could be expressed in DCs: and would enable the activation and expansion of polyclonal LMP1-specific CTLs. Recombinant adenoviral vectors expressing LMP1 or DeltaLMP1 were tested for their ability to transduce DCs. LMP1 expression was toxic within 48 hours whereas high levels of DeltaLMP1 expression were achieved with minimal toxicity. DeltaLMP1-expressing DCs were able to reactivate and expand LMP1-specific CTLs from 3 healthy EBV-seropositive donors. LMP1-specific T cells were detected by interferon-gamma (IFN-gamma) enzyme-linked immunospot assay (ELISPOT) assays using the HLA-A2-restricted LMP1 peptide, YLQQN-WWTL (YLQ). YLQ-specific T cells were undetectable (less than 0.001%) in donor peripheral blood mononuclear cells (PBMCs); however, after stimulation the frequency increased to 0.5% to 3.8%. Lysis of autologous target cells by CTLs was dependent on the level of LMP1 expression. in contrast, the frequency of YLQ-specific CTLs in EBV-specific CTLs reactivated and expanded, using lymphoblastoid cell lines was low and no LMP1-specific cytotoxic activity was observed. Thus, DeltaLMP1 expression in DCs is nontoxic and enables the generation of LMP1-specific CTLs for future adoptive immunotherapy protocols for patients with LMP1-positive malignancies such as EBV-positive Hodgkin disease. Targeting LMP1 in these malignancies may improve the efficacy of current adoptive immunotherapy approaches.