An optimized assay for the enumeration of antigen-specific memory B cells in different compartments of the human body

Object: In the framework of our current study we set out to develop an optimized assay for the quantification of antigen-specific B cells in different compartments of the human body. Methods: Mononuclear cells (MNC) derived from the peripheral blood, bone marrow (BM), or human tonsils were incubated with different combinations of stimuli. The stimulated cells and culture supernatants were then applied to IgG-ELISPOT and ELISA read-out assays and tetanus toxoid (TT)-specific B cell responses were quantified. Results: We found that a combination of CD40L, CpG, and IL21 was optimal for the induction of 'TT-specific IgG-producing cells from memory B cell (mBc) precursors. This cocktail of stimuli led to a proliferation-dependent induction of CD19(intermediate)CD38(high)CD138(high)IgD(negative) terminally differentiated plasma cells. Applying our optimized methodology we were also able to quantify mBc specific for cytomegalovirus and influenza virus A. Most importantly, the same method proved useful for the comparison of mBc frequencies between different compartments of the body and, accordingly, we were able to demonstrate that TT-specific mBc preferably reside within tonsillar tissue. Conclusion: Here, we optimized an assay for the quantification of antigen-specific B cells in different human tissues demonstrating, for example, that TT-specific mBc preferably reside in human tonsils but not in the BM or the peripheral blood. We suggest that our approach can be used for the enumeration of mBc specific for a wide variety of Ag (microbial, tumor-related, auto-antigens), which will lead to significant improvements regarding our knowledge about the biology of humoral immunity. (C) 2010 Published by Elsevier B.V.