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

•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.

BackgroundCNS 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.New methodUsing a viral encephalomyelitis model we developed a modified limiting dilution in vitro stimulation assay to convert CNS-derived virus specific Bmem into ASC.Comparison with existing methodsStimulation 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.ResultsFlow 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.ConclusionSimultaneous 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.