Human Germinal Center B Cells Differ from Naïve and Memory B Cells in CD40 Expression and CD40L-Induced Signaling Response

Cytometry A. 2019 Apr;95(4):442-449. doi: 10.1002/cyto.a.23737. Epub 2019 Mar 5.


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 naïve, 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 naïve and memory B cells. Despite this, GC B cells responded to CD40L engagement with lower phosphorylation of NFκB p65 during the first 30 min following CD40L activation. Before CD40L stimulation, GC B cells expressed higher levels of suppressor protein IκBα than naïve and memory B cells. Following CD40 activation, IκBα was rapidly degraded and reached equivalently low levels in naïve, 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 NFκB p65 phosphorylation, whereas comparable IκBα 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. © 2019 International Society for Advancement of Cytometry.

Keywords: CD40 signaling; germinal center B cells; human tonsils; mass cytometry; phospho-flow.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • B-Lymphocytes / cytology
  • B-Lymphocytes / metabolism
  • B-Lymphocytes / physiology*
  • CD40 Antigens / metabolism*
  • CD40 Ligand / metabolism*
  • CD40 Ligand / physiology
  • Cells, Cultured
  • Germinal Center / cytology*
  • Germinal Center / immunology
  • Germinal Center / metabolism
  • Humans
  • Immunologic Memory*
  • NF-kappa B / metabolism
  • Phosphorylation
  • Signal Transduction / immunology


  • CD40 Antigens
  • NF-kappa B
  • CD40 Ligand