Xu Haiyan, Dong Panpan, Ma Xuyi, Song Dan, Xue Dong, Xu Renfang, Lu Hao, He Xiaozhou
Urology Department, Third Affiliated Hospital of Soochow University, Changzhou 213003, China.
Soochow University, Suzhou 215007, China.
Immunol Lett. 2017 Jul;187:1-6. doi: 10.1016/j.imlet.2017.04.013. Epub 2017 Apr 23.
Previous studies have suggested that B lymphocytes can be polyclonally activated by human cytomegalovirus (HCMV), and individuals infected by HCMV exhibit characteristic features of an autoimmunity disease. B cell-activating factor (BAFF) plays important roles in the survival and differentiation of B cells; however, few studies have examined the potential role of BAFF on B cells infected by HCMV.
HCMV virus strain (HCMV AD-169) was concentrated by normal methods and used to infect microbead-purified tonsil CD19+ B cells. Cells and supernatants were collected at the 1st, 3rd, 5th, and 7th day of co-culture, respectively. Cellular phenotypes, including expression of BAFF and its cognate receptors (BAFF-R, TACI, and BCMA) were detected by flow cytometry (FCM); cells apoptosis rates were also examined by FCM; and IgG titers in supernatants was detected by ELISA. In parallel, neutralizing anti-BAFF-R antibody was applied to observe the effect of BAFF/BAFF-R signaling on apoptosis and the IgG secretion ability of B cells stimulated by HCMV.
LogTCID of 3rd and 4th generation of HCMV was -3.54 and -3.28, respectively. FCM results showed that the purity of CD19 B cells was >98%. BAFF-R was highly expressed and upregulated on HCMV-infected B cells (93.5%-99.3%), compared with B cells prior to HCMV infection and uninfected group; while BAFF-R expression gradually decreased with time and to the lowest level at 5th day (81%) in the control medium-only group. In contrast, expression of TACI and BCMA gradually increased during culture in both HCMV-infected and medium-only control B cells. Furthermore, the apoptosis rate of HCMV-infected and medium-only control B cells did not vary significantly during culture, but IgG secretion ability of HCMV-infected B cells significantly increased over time while no changes were observed with the medium-only control. Importantly, the apoptosis rate of B cells significantly increased when BAFF/BAFF-R signal was blocked prior to HCMV infection (P<0.05), although no significant changes of IgG levels were observed (P>0.05).
BAFF-R was consistently expressed on B cells infected by HCMV. Enhancement of BAFF/BAFF-R signaling decreased the apoptosis rate and extended the survival of B cells.
先前的研究表明，人类巨细胞病毒（HCMV）可多克隆激活B淋巴细胞，感染HCMV的个体表现出自身免疫性疾病的特征。B细胞激活因子（BAFF）在B细胞的存活和分化中起重要作用；然而，很少有研究探讨BAFF对感染HCMV的B细胞的潜在作用。
采用常规方法浓缩HCMV病毒株（HCMV AD-169），用于感染微珠纯化的扁桃体CD19+B细胞。分别在共培养的第1、3、5和7天收集细胞和上清液。通过流式细胞术（FCM）检测细胞表型，包括BAFF及其同源受体（BAFF-R、TACI和BCMA）的表达；也通过FCM检测细胞凋亡率；通过ELISA检测上清液中的IgG滴度。同时，应用中和抗BAFF-R抗体观察BAFF/BAFF-R信号对HCMV刺激后B细胞凋亡和IgG分泌能力的影响。
第3代和第4代HCMV的LogTCID分别为-3.54和-3.28。FCM结果显示，CD19+B细胞的纯度>98%。与HCMV感染前的B细胞和未感染组相比，BAFF-R在感染HCMV的B细胞上高表达且上调（93.5%-99.3%）；而在仅含培养基的对照组中，BAFF-R表达随时间逐渐降低，在第5天降至最低水平（81%）。相反，在HCMV感染的B细胞和仅含培养基的对照B细胞培养过程中，TACI和BCMA的表达均逐渐增加。此外，HCMV感染的B细胞和仅含培养基的对照B细胞在培养过程中的凋亡率无显著差异，但HCMV感染的B细胞的IgG分泌能力随时间显著增加，而仅含培养基的对照组未观察到变化。重要的是，在HCMV感染前阻断BAFF/BAFF-R信号时，B细胞的凋亡率显著增加（P<0.05），尽管未观察到IgG水平的显著变化（P>0.05）。
BAFF-R在感染HCMV的B细胞上持续表达。增强BAFF/BAFF-R信号可降低凋亡率并延长B细胞的存活时间。
