Journal
CYTOMETRY PART A
Volume 95A, Issue 4, Pages 442-449Publisher
WILEY
DOI: 10.1002/cyto.a.23737
Keywords
CD40 signaling; phospho-flow; mass cytometry; germinal center B cells; human tonsils
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Funding
- NIH/NCI [R00 CA143231-03, R25 CA136440-04]
- Norwegian Cancer Society
- Centre for Cancer Biomedicine
- Vanderbilt-Ingram Cancer Center (VICC) [P30 CA68485]
- VICC Ambassadors and Hematology Helping Hand Fund awards
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CD40 expression is required for germinal center (GC) formation and function, but the kinetics and magnitude of signaling following CD40 engagement remain poorly characterized in human B cells undergoing GC reactions. Here, differences in CD40 expression and signaling responses were compared across differentiation stages of mature human tonsillar B cells. A combination of mass cytometry and phospho-specific flow cytometry was used to quantify protein expression and CD40L-induced signaling in primary human naive, GC, and memory B cells. Protein expression signatures of cell subsets were quantified using viSNE and Marker Enrichment Modeling (MEM). This approach revealed enriched expression of CD40 protein in GC B cells, compared to naive and memory B cells. Despite this, GC B cells responded to CD40L engagement with lower phosphorylation of NF kappa B p65 during the first 30 min following CD40L activation. Before CD40L stimulation, GC B cells expressed higher levels of suppressor protein I kappa B alpha than naive and memory B cells. Following CD40 activation, I kappa B alpha was rapidly degraded and reached equivalently low levels in naive, GC, and memory B cells at 30 min following CD40L. Quantifying CD40 signaling responses as a function of bound ligand revealed a correlation between bound CD40L and degree of induced NF kappa B p65 phosphorylation, whereas comparable I kappa B alpha degradation occurred at all measured levels of CD40L binding. These results characterize cell-intrinsic signaling differences that exist in mature human B cells undergoing GC reactions. (c) 2019 International Society for Advancement of Cytometry
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