DiSano Krista D, Stohlman Stephen A, Bergmann Cornelia C
Department of Neurosciences NC30, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, United States; School of Biomedical Sciences, Kent State University, Kent, OH 44242, United States.
Department of Neurosciences NC30, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, United States.
J Neurosci Methods. 2017 Jun 15;285:58-68. doi: 10.1016/j.jneumeth.2017.05.011. Epub 2017 May 8.
CNS inflammation resulting from infection, injury, or neurodegeneration leads to accumulation of diverse B cell subsets. Although antibody secreting cells (ASC) within the inflamed CNS have been extensively examined, memory B cell (Bmem) characterization has been limited as they do not secrete antibody without stimulation. Moreover, unlike human Bmem, reliable surface markers for murine Bmem remain elusive.
Using a viral encephalomyelitis model we developed a modified limiting dilution in vitro stimulation assay to convert CNS-derived virus specific Bmem into ASC.
Stimulation methods established for lymphoid tissue cells using prolonged stimulation with viral lysate resulted in substantial ASC loss and minimal Bmem to ASC conversion of CNS-derived cells. By varying stimulation duration, TLR activators, and culture supplements, we achieved optimal conversion by culturing cells with TLR7/8 agonist R848 in the presence of feeder cells for 2days.
Flow cytometry markers CD38 and CD73 characterizing murine Bmem from lymphoid tissue showed more diverse expression patterns on corresponding CNS-derived B cell subsets. Using the optimized TLR7/8 stimulation protocol, we compared virus-specific IgG Bmem versus pre-existing ASC within the brain and spinal cord. Increasing Bmem frequencies during chronic infection mirrored kinetics of ASC. However, despite initially similar Bmem and ASC accumulation, Bmem prevailed in the brain, but were lower than ASC in the spinal cord during persistence.
Simultaneous enumeration of antigen-specific Bmem and ASC using the Bmem assay optimized for CNS-derived cells enables characterization of temporal changes during microbial or auto-antigen induced neuroinflammation.
由感染、损伤或神经退行性变引起的中枢神经系统炎症会导致多种B细胞亚群的积累。尽管对炎症性中枢神经系统内的抗体分泌细胞（ASC）已进行了广泛研究，但记忆B细胞（Bmem）的特征描述却很有限，因为它们在未受刺激时不分泌抗体。此外，与人类Bmem不同，小鼠Bmem可靠的表面标志物仍然难以捉摸。
利用一种病毒性脑脊髓炎模型，我们开发了一种改良的有限稀释体外刺激试验，以将中枢神经系统来源的病毒特异性Bmem转化为ASC。
针对淋巴组织细胞建立的刺激方法，即使用病毒裂解物进行长时间刺激，导致大量ASC损失，且中枢神经系统来源细胞的Bmem向ASC的转化率极低。通过改变刺激持续时间、Toll样受体（TLR）激活剂和培养补充剂，我们在饲养细胞存在的情况下，用TLR7/8激动剂R848培养细胞2天，实现了最佳转化。
表征来自淋巴组织的小鼠Bmem的流式细胞术标志物CD38和CD73，在相应的中枢神经系统来源的B细胞亚群上显示出更多样化的表达模式。使用优化的TLR7/8刺激方案，我们比较了大脑和脊髓内病毒特异性IgG Bmem与预先存在的ASC。慢性感染期间Bmem频率的增加反映了ASC的动力学。然而，尽管最初Bmem和ASC的积累相似，但在持续感染期间，Bmem在大脑中占优势，但在脊髓中低于ASC。
使用针对中枢神经系统来源细胞优化的Bmem检测方法同时计数抗原特异性Bmem和ASC，能够表征微生物或自身抗原诱导的神经炎症期间的时间变化。
