Polyclonal B cell activation for accurate analysis of pre-existing antigen-specific memory B cells

Authors

  • G. E. Karahan,

    1. Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, the Netherlands
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  • M. Eikmans,

    1. Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, the Netherlands
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  • J. D. H. Anholts,

    1. Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, the Netherlands
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  • F. H. J. Claas,

    1. Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, the Netherlands
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  • S. Heidt

    Corresponding author
    1. Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, the Netherlands
    • Correspondence: S. Heidt, Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Albinusdreef 2, 2300 RC Leiden, the Netherlands.

      E-mail: S.Heidt@lumc.nl

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Summary

The enzyme-linked immunospot (ELISPOT) assay is a widely used tool for enumeration of antigen-specific memory B cells in several disciplines, such as vaccination, cancer immunotherapy and transplantation. For the accurate estimation of antigen-specific memory B cell frequencies, a well-defined B cell activation protocol is pivotal. In this study, we aimed to characterize a polyclonal B cell activation protocol to facilitate optimal monitoring of antigen-specific memory B cell frequencies. Total, naive and memory B cells were activated polyclonally with an α-CD40 monoclonal antibody, cytosine–phosphate–guanine (CPG) oligodeoxynucleotide (ODN) 2006, interleukin (IL)-2, IL-10 and IL-21. Polyclonal activation of B cells resulted in equal cell death ratios in naive and memory B cells. When tested in an antigen-specific system, immunoglobulin (Ig)G spots were detected only in the memory fraction. There was no change in B cell polyclonality due to in-vitro activation. Our data show that the current polyclonal activation protocol may be used reliably to estimate the frequency of memory B cells in ELISPOT assays.

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