T-cell analysis on chronic active Epstein-Barr virus (EBV) infection and EBV-associated lymphoproliferative diseases

EB病毒(Epstein-Barr Virus，EBV)是引起急性传染性单核细胞增多症(IM)的病原体，可导致免疫缺陷患者B细胞淋巴瘤/淋巴增生性疾病(LPD)的持续激活和继发。这种γ疱疹病毒可以感染T/NK细胞，从而与包括慢性活动性EB病毒感染和噬血细胞淋巴组织细胞增多症在内的淋巴网状疾病有关。目前对CAEBV的研究表明：1)T细胞以克隆方式被激活和扩增，2)激活的T细胞携带EBV基因组的拷贝数增加，3)细胞因子的表达不平衡。这些结果提示CAEBV激活的T细胞可能代表了肿瘤的克隆性进化，而不是对EBV感染的反应状态。我们还证明了定量监测EB病毒DNA在预测移植后EB病毒载量和细胞因子表达方面的作用。利用T细胞抗原受体Vβ/Jβ区基因的反向聚合酶链式反应方法显示CAEBV患者T细胞的寡克隆性增殖。用细胞分选仪(EPICS-XL)将T细胞分离为人类白细胞抗原-DR^+或人类白细胞抗原-DR^-CD3^+细胞，分别构建DNA和cDNA。用实时荧光定量聚合酶链式反应(TaqMan)检测EBV-DNA和细胞因子mRNA的表达。CD3+HLADR^+细胞的病毒拷贝数高于CD3+HLADR^-细胞。细胞因子定量聚合酶链式反应显示Th1(干扰素γ，IL-2)和Th2(IL-10，转化生长因子-β)细胞因子在活化的T细胞中高表达。EBV-DNA进行性升高提示移植后LPD的发生。

Epstein-Barr virus (EBV), the causative agent of acute infectious mononucleosis (IM) leads to the persistent reactivation and the subsequent development of B-cell lymphoma/lymphoproliferative disease (LPD) in immunodeficient patients. This γ-herpes virus can infect T/NK cells and thus be associated with lymphoreticular diseases including chronic active EBV infection (CAEBV) and hemophagocytic lymphohistiocytosis (HLH). These conditions have now been classified into EBV^+T-cell LPD.The present study on CAEBV revealed the followings ; 1) T-cells are activated and expanded ologoclonally, 2) The activated T-cells carried increased copy number of EBV genome, 3) and expressed unbalanced high expression of cytokines. These results suggest that activated T-cells of CAEBV may represent the clonal evolution of malignancy, rather than the reactive condition against EBV infection. We also demonstrated the usefulness of quantitative monitoring of EBV-DNA in predicting the development of posttransplant LPD.1) EBV load and cytokine expression in CAEBV T-cellsThe inverse PCR methods using T-cell antigen receptor (TCR) Vβ/Jβ region genes revealed oligoclonal expansion of T-cells in CAEBV patients. T cells were fractionated into HLA-DR^+ or HLA-DR^-CD3^+ cells by a cell sorter (EPICS-XL), and DNA and cDNA were constructed from each population, respectively. EBV-DNA and cytokine mRNA were quantified by real-time PCR (TaqMan). CD3^+HLA-DR^+ cells contained higher copy numbers of virus than CD3^+HLA-DR^- cells. Quantitative PCR for cytokines indicated high expressions of Th1 (IFNγ, IL-2) and Th2 (IL-10, TGF-β) cytokines in activated T-cells.2) Clinical usefulness of posttransplant EBV-DNA monitoringEBV-DNA was sequentially quantified by real-time PCR in 12 patients after hematopoietic stem cell transplantation. Progressive increase of EBV-DNA indicated the development of posttransplant LPD.