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

eb病毒（EBV）是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/淋巴细胞增生性疾病（LPD）的持续再激活和随后的发展。这种γ-疱疹病毒可以感染T/NK细胞，因此与包括慢性活动性EBV感染（CAEBV）和噬血细胞淋巴组织细胞病（HLH）在内的淋巴网状疾病有关。这些情况现在被归类为EBV^+ t细胞LPD。目前对CAEBV的研究表明：1) t细胞被激活并同源扩增，2)激活后的t细胞携带EBV基因组拷贝数增加，3)细胞因子表达不平衡高表达。这些结果表明，活化的CAEBV t细胞可能代表了恶性肿瘤的克隆进化，而不是对EBV感染的反应状态。我们还证明了定量监测EBV- dna在预测移植后lpd发展方面的有效性。1)EBV载量和CAEBV t细胞中细胞因子的表达利用t细胞抗原受体（TCR） Vβ/Jβ区域基因的反相PCR方法显示CAEBV患者的t细胞寡克隆扩增。T细胞通过细胞分选器（EPICS-XL）分离成HLA-DR^+和HLA-DR^-CD3^+细胞，分别构建DNA和cDNA。采用实时荧光定量PCR （TaqMan）检测EBV-DNA和细胞因子mRNA。CD3^+HLA-DR^+细胞的病毒拷贝数高于CD3^+HLA-DR^-细胞。细胞因子定量PCR结果显示，活化t细胞中Th1 （IFNγ、IL-2）、Th2 （IL-10、TGF-β）细胞因子高表达。2)移植后EBV-DNA监测的临床意义采用实时荧光定量PCR对12例造血干细胞移植后患者的EBV-DNA进行序列定量分析。EBV-DNA的逐渐升高提示移植后LPD的发展。

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.