An optimized method for enumerating CNS derived memory B cells during viral-induced inflammation.
An optimized method for enumerating CNS derived memory B cells during viral-induced inflammation.
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DOI：
10.1016/j.jneumeth.2017.05.011
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发表时间：
2017-06-15
影响因子：
3
通讯作者：
Bergmann CC
中科院分区：
文献类型：
--
作者：
DiSano KD;Stohlman SA;Bergmann CC
表征外周B细胞表型的记忆B细胞标志物在受感染的中枢神经系统（CNS）中显示更多样化的表达模式。ELISPOT分析前2天的TLR 7/8刺激实现了CNS衍生的记忆B细胞向ASC的最佳转化，同时最大限度地减少了细胞损失。 体外刺激允许同时评估抗体分泌细胞和记忆B细胞同种型、抗原特异性和CNS炎症期间的时间变化。由感染、损伤或神经变性引起的CNS炎症导致不同B细胞亚群的积累。虽然炎症中枢神经系统内的抗体分泌细胞（ASC）已被广泛研究，但记忆B细胞（B细胞）的表征受到限制，因为它们在没有刺激的情况下不分泌抗体。此外，与人Bcl 3不同，鼠Bcl 3的可靠表面标志物仍然难以捉摸。使用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CNS衍生的病毒特异性Bcl 3转化为ASC。使用病毒裂解物长时间刺激淋巴组织细胞建立的刺激方法导致大量ASC损失和CNS衍生细胞的最小BCLs至ASC转化。通过改变刺激持续时间、TLR激活剂和培养补充剂，我们通过在饲养细胞存在下用TLR 7/8激动剂R848培养细胞2天来实现最佳转化。流式细胞术标记物CD 38和CD 73表征来自淋巴组织的小鼠BCLs在相应的CNS衍生的B细胞亚群上显示出更多样化的表达模式。使用优化的TLR 7/8刺激方案，我们比较了病毒特异性IgG Bcl 3与脑和脊髓内预先存在的ASC。在慢性感染期间增加的Bcirrhosis频率反映了ASC的动力学。然而，尽管最初类似的Bcl 3和ASC积累，Bcl 3在大脑中占主导地位，但在持续性期间低于ASC在脊髓中。使用针对CNS衍生的细胞优化的Bclad测定法同时计数抗原特异性Bclad和ASC使得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。
Memory B cell markers characterizing peripheral B cell phenotypes show more diverse expression patterns in the infected central nervous system (CNS). TLR7/8 stimulation for 2 days prior to ELISPOT analysis achieves optimal conversion of CNS-derived memory B cells to ASC while minimizing cell loss. In vitro stimulation allows simultaneous assessment of antibody secreting cell and memory B cell isotype, antigen specificity, and temporal alterations during CNS inflammation. 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 2 days. 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.
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DOI：
10.4049/jimmunol.0804233
发表时间：
2009-09-01
期刊：
Journal of immunology (Baltimore, Md. : 1950)
影响因子：
--
作者：
Henn AD;Rebhahn J;Brown MA;Murphy AJ;Coca MN;Hyrien O;Pellegrin T;Mosmann T;Zand MS
通讯作者：
Zand MS
DOI：
10.1084/jem.20062571
发表时间：
2007-09-03
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
Anderson SM;Tomayko MM;Ahuja A;Haberman AM;Shlomchik MJ
通讯作者：
Shlomchik MJ
影响因子：
7.3
作者：
Dang AK;Tesfagiorgis Y;Jain RW;Craig HC;Kerfoot SM
通讯作者：
Kerfoot SM
影响因子：
12.7
作者：
BURKE, DS;NISALAK, A;LAORPONGSE, T
通讯作者：
LAORPONGSE, T
影响因子：
30.5
作者：
Dogan, Ismail;Bertocci, Barbara;Weill, Jean-Claude
通讯作者：
Weill, Jean-Claude