An optimized method for enumerating CNS derived memory B cells during viral-induced inflammation

Journal of Neuroscience Methods
Krista D DiSanoCornelia C Bergmann

Abstract

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 ...Continue Reading

Citations

Jun 26, 2021·Frontiers in Immunology·Krista D DiSanoAndrew R Pachner

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Methods Mentioned

BETA
ELISA
flow cytometry
dissection
FCS
FACS

Software Mentioned

Prism
GraphPad
FlowJo

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