Weck K E, Kim S S, Virgin HW I V, Speck S H
Department of Pathology and Center for Immunology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Virol. 1999 Apr;73(4):3273-83. doi: 10.1128/JVI.73.4.3273-3283.1999.
B cells have previously been identified as the major hematopoietic cell type harboring latent gammaherpesvirus 68 (gammaHV68) (N. P. Sunil-Chandra, S. Efstathiou, and A. A. Nash, J. Gen. Virol. 73:3275-3279, 1992). However, we have shown that gammaHV68 efficiently establishes latency in B-cell-deficient mice (K. E. Weck, M. L. Barkon, L. I. Yoo, S. H. Speck, and H. W. Virgin, J. Virol. 70:6775-6780, 1996), demonstrating that B cells are not required for gammaHV68 latency. To understand this dichotomy, we determined whether hematopoietic cell types, in addition to B cells, carry latent gammaHV68. We observed a high frequency of cells that reactivate latent gammaHV68 in peritoneal exudate cells (PECs) derived from both B-cell-deficient and normal C57BL/6 mice. PECs were composed primarily of macrophages in B-cell-deficient mice and of macrophages plus B cells in normal C57BL/6 mice. To determine which cells in PECs from C57BL/6 mice carry latent gammaHV68, we developed a limiting-dilution PCR assay to quantitate the frequency of cells carrying the gammaHV68 genome in fluorescence-activated cell sorter-purified cell populations. We also quantitated the contribution of individual cell populations to the total frequency of cells carrying latent gammaHV68. At early times after infection, the frequency of PECs that reactivated gammaHV68 correlated very closely with the frequency of PECs carrying the gammaHV68 genome, validating measurement of the frequency of viral-genome-positive cells as a measure of latency in this cell population. F4/80-positive macrophage-enriched, lymphocyte-depleted PECs harbored most of the gammaHV68 genome and efficiently reactivated gammaHV68, while CD19-positive, B-cell-enriched PECs harbored about a 10-fold lower frequency of gammaHV68 genome-positive cells. CD4-positive, T-cell-enriched PECs contained only a very low frequency of gammaHV68 genome-positive cells, consistent with previous analyses indicating that T cells are not a reservoir for gammaHV68 latency (N. P. Sunil-Chandra, S. Efstathiou, and A. A. Nash, J. Gen. Virol. 73:3275-3279, 1992). Since macrophages are bone marrow derived, we determined whether elicitation of a large inflammatory response in the peritoneum would recruit additional latent cells into the peritoneum. Thioglycolate inoculation increased the total number of PECs by about 20-fold but did not affect the frequency of cells that reactivate gammaHV68, consistent with a bone marrow reservoir for latent gammaHV68. These experiments demonstrate gammaHV68 latency in two different hematopoietic cell types, F4/80-positive macrophages and CD19-positive B cells, and argue for a bone marrow reservoir for latent gammaHV68.
B细胞先前已被确定为携带潜伏性γ疱疹病毒68（γHV68）的主要造血细胞类型（N.P.苏尼尔 - 钱德拉、S.埃夫斯塔西乌和A.A.纳什，《普通病毒学杂志》73:3275 - 3279，1992年）。然而，我们已经表明γHV68能够在B细胞缺陷小鼠中高效建立潜伏状态（K.E.韦克、M.L.巴孔、L.I.柳、S.H.斯佩克和H.W.维尔金，《病毒学杂志》70:6775 - 6780，1996年），这表明γHV68潜伏并不需要B细胞。为了理解这种二分法，我们确定除了B细胞之外，其他造血细胞类型是否携带潜伏性γHV68。我们观察到，在源自B细胞缺陷和正常C57BL/6小鼠的腹腔渗出细胞（PEC）中，重新激活潜伏性γHV68的细胞频率很高。在B细胞缺陷小鼠中，PEC主要由巨噬细胞组成，而在正常C57BL/6小鼠中，PEC由巨噬细胞和B细胞组成。为了确定C57BL/6小鼠的PEC中哪些细胞携带潜伏性γHV68，我们开发了一种有限稀释PCR检测方法，以定量荧光激活细胞分选纯化细胞群体中携带γHV68基因组的细胞频率。我们还定量了各个细胞群体对携带潜伏性γHV68的细胞总频率的贡献。在感染后的早期，重新激活γHV68的PEC频率与携带γHV68基因组的PEC频率密切相关，这验证了将病毒基因组阳性细胞频率作为该细胞群体中潜伏状态的一种测量方法。富含F4/80阳性巨噬细胞且淋巴细胞耗尽的PEC携带了大部分γHV68基因组，并能高效重新激活γHV68，而富含CD19阳性B细胞的PEC携带γHV68基因组阳性细胞的频率约低10倍。富含CD4阳性T细胞的PEC仅含有极低频率的γHV68基因组阳性细胞，这与先前的分析一致，表明T细胞不是γHV68潜伏的储存库（N.P.苏尼尔 -
钱德拉、S.埃夫斯塔西乌和A.A.纳什，《普通病毒学杂志》73:3275 - 3279，1992年）。由于巨噬细胞来源于骨髓，我们确定在腹膜中引发大规模炎症反应是否会将额外的潜伏细胞募集到腹膜中。硫代乙醇酸盐接种使PEC总数增加了约20倍，但不影响重新激活γHV68的细胞频率，这与γHV68潜伏的骨髓储存库一致。这些实验证明了γHV68在两种不同的造血细胞类型，即F4/80阳性巨噬细胞和CD19阳性B细胞中的潜伏状态，并支持γHV68潜伏的骨髓储存库的观点。
