Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Nobels väg 16, S-171 77 Stockholm, Sweden.
J Immunol Methods. 2012 Nov 30;385(1-2):60-70. doi: 10.1016/j.jim.2012.08.008. Epub 2012 Aug 18.
Epstein Barr virus (EBV) is carried by almost all adults, mostly without clinical manifestations. Latent virus infection of B lymphocytes induces activation and proliferation that can be demonstrated in vitro. In healthy individuals, generation of EBV induced malignant proliferation is avoided by continuous immunological surveillance. The proliferation inducing set of the virally encoded genes is expressed exclusively in B cells in a defined differentiation window. It comprises nine EBV encoded nuclear proteins, EBNA 1-6, and three cell membrane associated proteins, LMP-1, 2A and 2B, designated as latency Type III. Outside this window the expression of the viral genes is limited. Healthy carriers harbor a low number of B lymphocytes in which the viral genome is either silent or expresses one virally encoded protein, EBNA-1, latency Type I. In addition, EBV genome carrying B cells can lack either EBNA-2 or LMP-1, latency Type IIa or Type IIb respectively. These cells have no inherent proliferation capacity. Detection of both EBNA-2 and LMP-1 can identify B cells with growth potential. We devised therefore a method for their simultaneous detection in cytospin deposited cell populations. Simultaneous detection of EBNA-2 and LMP-1 was reported earlier in tissues derived from infectious mononucleosis (IM), postransplantation lymphoproliferative disorders (PTLD) and from "humanized" mice infected with EBV. We show for the first time the occurrence of Type IIa and Type IIb cells in cord blood lymphocyte populations infected with EBV in vitro. Further, we confirm the variation of EBNA-2 and LMP-1 expression in several Type III lines and that they vary independently in individual cells. We visualize that in Type III LCL, induced for plasmacytoid differentiation by IL-21 treatment, EBV protein expression changes to Type IIa (EBNA-2 negative LMP-1 positive). We also show that when the proliferation of EBV infected cord blood lymphocyte culture is inhibited by the immunomodulator, PSK, the majority of the cells express latency Type IIa pattern. These results show that by modifying the differentiation state, the proliferating EBV positive B cells can be "curbed". Type IIa expression is a characteristic for EBV positive Reed-Sternberg (R/S) cells in EBV positive Hodgkin's lymphomas. For survival and proliferation, the R/S cells require the contribution of the in vivo microenvironment. Consequently, Type IIa lines could not be established from Hodgkin's lymphoma in vitro. We propose that these experimental cultures can be exploited for study of the Type IIa cells.
EB 病毒（EBV）几乎存在于所有成年人中，大多数情况下没有临床表现。潜伏的病毒感染 B 淋巴细胞会诱导激活和增殖，这种增殖可以在体外得到证明。在健康个体中，连续的免疫监测可以避免 EBV 诱导的恶性增殖。病毒编码基因的增殖诱导集仅在特定的分化窗口中在 B 细胞中表达。它包括九个 EBV 编码的核蛋白，EBNA1-6，和三个细胞膜相关蛋白，LMP-1、2A 和 2B，称为潜伏型 III。在这个窗口之外，病毒基因的表达受到限制。健康携带者的体内携带的 B 淋巴细胞数量较少，这些淋巴细胞的病毒基因组要么处于沉默状态，要么表达一种病毒编码蛋白，即潜伏型 I 的 EBNA-1。此外，携带 EBV 基因组的 B 细胞可以分别缺乏 EBNA-2 或 LMP-1，分别为潜伏型 IIa 或 IIb。这些细胞没有内在的增殖能力。检测到 EBNA-2 和 LMP-1 可以识别具有生长潜力的 B 细胞。因此，我们设计了一种用于同时检测细胞沉淀中 B 细胞的方法。以前在传染性单核细胞增多症（IM）、移植后淋巴组织增生性疾病（PTLD）和感染 EBV 的“人源化”小鼠的组织中已经报道了同时检测 EBNA-2 和 LMP-1 的方法。我们首次在体外感染 EBV 的脐带血淋巴细胞群体中发现了 IIa 型和 IIb 型细胞。此外，我们还证实了几种 III 型细胞系中 EBNA-2 和 LMP-1 表达的变化，并且它们在单个细胞中独立变化。我们发现，在 IL-21 诱导的浆细胞样分化的 III 型 LCL 中，EBV 蛋白表达转变为 IIa 型（EBNA-2 阴性 LMP-1 阳性）。我们还表明，当免疫调节剂 PSK 抑制 EBV 感染的脐带血淋巴细胞培养物的增殖时，大多数细胞表达潜伏型 IIa 模式。这些结果表明，通过改变分化状态，可以“抑制”增殖的 EBV 阳性 B 细胞。IIa 型表达是 EBV 阳性霍奇金淋巴瘤中 EBV 阳性 Reed-Sternberg（R/S）细胞的特征。为了生存和增殖，R/S 细胞需要体内微环境的贡献。因此，无法从体外的霍奇金淋巴瘤中建立 IIa 型细胞系。我们提出，这些实验培养物可以用于研究 IIa 型细胞。
