Metes D, Storkus W, Zeevi A, Patterson K, Logar A, Rowe D, Nalesnik M A, Fung J J, Rao A S
Thomas E. Starzl Transplantation Institute and the Department of Surgery, School of Medicine, University of Pittsburgh, PA 15213, USA.
Transplantation. 2000 Nov 27;70(10):1507-15. doi: 10.1097/00007890-200011270-00019.
Although readily accomplished from immunocompetent Epstein-Barr virus- (EBV) seropositive individuals, the effective ex vivo generation of EBV-specific cytotoxic T lymphocytes (CTL) from the peripheral blood mononuclear cells (PBMC) of EBV-seronegative subjects has proven to be a challenge. The focus of our study was to ascertain optimized culture conditions required for the ex vivo generation of EBV-reactive autologous CTL from the PBMC of EBV-seronegative volunteers.
Freshly isolated PBMC obtained from immunocompetent EBV-seronegative and -seropositive individuals were used to generate EBV-specific autologous CTL lines using both conventional and a novel, modified ex vivo culture technique.
In contrast to responses observed in EBV-seropositives after two to three rounds of ex vivo stimulation, gamma-irradiated autologous lymphoblastoid cell lines (LCL) were incapable of eliciting an effective anti-EBV cytotoxic response when freshly-isolated PBMC from EBV-seronegative individuals were used as responders. Under these culture conditions, CD4+ T cells with preferential expression of the Th2-type cytokine IL-4 were predominantly expanded in the PBMC obtained from EBV-seronegative individuals. However, the addition of recombinant human (rh) IL-12 during the primary phase of ex vivo stimulation resulted in augmentation of EBV-specific cytolysis of autologous LCL by CD8+ T cells. Furthermore, there was down-regulation in the secretion of IL-4 and up-regulation in that of the Th1-type cytokine IFN-gamma by responder CD4+ and CD8+ T cells.
Taken together these data suggest that the addition of rhIL-12 during the primary phase of ex vivo stimulation of freshly isolated PBMC from EBV-seronegative individuals results in skewing of the immune response predominantly towards a CD4+ Th1-type (IFN-gamma) with the generation of an efficacious CTL-mediated anti-EBV reactivity. This novel ex vivo approach for generating effective autologous EBV-specific CTL could be adopted to treat refractory post-transplant lymphoproliferative disorders, which may be encountered in EBV-seropositive-->EBV-seronegative organ transplant recipients. Additionally, these ex vivo generated anti-EBV T cells could also be infused perioperatively to enhance prophylactically immunity against EBV infection in high-risk EBV-seronegative organ allograft recipients.
尽管从具有免疫活性的爱泼斯坦-巴尔病毒（EBV）血清学阳性个体中很容易实现，但从EBV血清学阴性受试者的外周血单个核细胞（PBMC）中有效地体外产生EBV特异性细胞毒性T淋巴细胞（CTL）已被证明是一项挑战。我们研究的重点是确定从EBV血清学阴性志愿者的PBMC中体外产生EBV反应性自体CTL所需的优化培养条件。
使用从具有免疫活性的EBV血清学阴性和阳性个体中新鲜分离的PBMC，通过传统和一种新型改良的体外培养技术来产生EBV特异性自体CTL系。
与在两轮至三轮体外刺激后在EBV血清学阳性个体中观察到的反应相反，当将来自EBV血清学阴性个体的新鲜分离的PBMC用作反应细胞时，经γ射线照射的自体淋巴母细胞系（LCL）无法引发有效的抗EBV细胞毒性反应。在这些培养条件下，优先表达Th2型细胞因子IL-4的CD4 + T细胞在从EBV血清学阴性个体获得的PBMC中主要扩增。然而，在体外刺激的初始阶段添加重组人（rh）IL-12导致CD8 + T细胞对自体LCL的EBV特异性细胞溶解增强。此外，反应性CD4 +和CD8 + T细胞分泌的IL-4减少，Th1型细胞因子IFN-γ分泌增加。
这些数据综合表明，在从EBV血清学阴性个体新鲜分离的PBMC的体外刺激初始阶段添加rhIL-12会导致免疫反应主要偏向CD4 + Th1型（IFN-γ），并产生有效的CTL介导的抗EBV反应性。这种用于产生有效的自体EBV特异性CTL的新型体外方法可用于治疗难治性移植后淋巴增殖性疾病，这可能在EBV血清学阳性→EBV血清学阴性器官移植受者中遇到。此外，这些体外产生的抗EBV T细胞也可在围手术期输注，以增强高危EBV血清学阴性器官同种异体移植受者对EBV感染的预防性免疫。
