CHU de Nancy, Unité de Thérapie Cellulaire et Tissus, Vandoeuvre-lès-Nancy, France; CNRS, UMR 7365 et FR 3209, Faculté de Médecine, Université de Lorraine, Vandoeuvre-lès-Nancy, France.
CHU de Nancy, Epidémiologie et Evaluation Cliniques, Vandoeuvre-lès-Nancy, France.
Cytotherapy. 2014 Jan;16(1):122-34. doi: 10.1016/j.jcyt.2013.07.008. Epub 2013 Oct 1.
Epstein-Barr virus (EBV) infection is a major cause of morbidity following hematopoietic stem cell transplantation. EBV-infected B cells may not respond to rituximab treatment and may lead to a life-threatening post-transplantation lymphoproliferative disorder. Adoptive cellular immunotherapy using EBV-lymphoblastoid cell lines (LCL) as stimulating antigen has proved effective in restoring specific immunity. However, EBV presents several immunodominant antigens, and developing a swift and effective clinical-grade immunotherapy relies on the definition of a Good Manufacturing Practices (GMP) universal stimulating antigen.
Peripheral blood mononuclear cells (PBMCs) from six donors with a cellular immune response against EBV were immunoselected after stimulation with a new EBV antigen associated with an EBNA3 peptide pool.
After immunoselection, a mean of 0.53 ± 0.25 × 10⁶ cells was recovered consisting of a mean of 24.77 ± 18.01% CD4⁺-secreting interferon (IFN)-γ and 51.42 ± 26.92% CD8⁺-secreting IFN-γ. The T memory stem cell sub-population was identified. EBV-specific T cells were expanded in vitro, and their ability to secrete IFN-γ and to proliferate after re-stimulation with EBV antigen was confirmed. A specific lysis was observed against autologous target cells pulsed with EBV peptide pools (57.6 ± 11.5%) and against autologous EBV-LCL (18.3 ± 7.3%). A mean decrease of 94.7 ± 3.3% in alloreactivity against third-party donor mononuclear cells with EBV-specific T cells was observed compared with PBMCs before selection.
Our results show that a combination of peptide pools including EBNA3 is needed to generate EBV-specific T cells with good specific cytotoxicity and devoid of alloreactivity, but as yet GMP grade is not fully achieved.
EB 病毒（EBV）感染是造血干细胞移植后发病的主要原因。受 EBV 感染的 B 细胞可能对利妥昔单抗治疗无反应，并且可能导致危及生命的移植后淋巴组织增生性疾病。使用 EBV-淋巴母细胞系（LCL）作为刺激抗原的过继细胞免疫疗法已被证明可有效恢复特异性免疫。然而，EBV 具有多个免疫优势抗原，并且开发快速有效的临床级免疫疗法依赖于定义符合良好生产规范（GMP）的通用刺激抗原。
用与 EBNA3 肽池相关的新 EBV 抗原刺激后，从对 EBV 具有细胞免疫反应的 6 名供者的外周血单个核细胞（PBMC）中进行免疫选择。
免疫选择后，平均可回收 0.53±0.25×106 个细胞，其中包含平均 24.77±18.01%分泌干扰素（IFN）-γ的 CD4+细胞和 51.42±26.92%分泌 IFN-γ的 CD8+细胞。鉴定出 T 记忆干细胞亚群。体外扩增 EBV 特异性 T 细胞，并确认其再次用 EBV 抗原刺激后分泌 IFN-γ和增殖的能力。观察到针对用 EBV 肽池脉冲处理的自体靶细胞（57.6±11.5%）和自体 EBV-LCL（18.3±7.3%）的特异性溶解。与选择前的 PBMC 相比，用 EBV 特异性 T 细胞观察到针对同种异体供体单核细胞的总体反应性平均降低 94.7±3.3%。
我们的结果表明，需要包含 EBNA3 的肽池组合来产生具有良好特异性细胞毒性且无同种异体反应性的 EBV 特异性 T 细胞，但尚未完全达到 GMP 级别。
