AUTHOR=Matsuda Yoshiko , Imamura Ryoichi , Takahara Shiro 

TITLE=Evaluation of Antigen-Specific IgM and IgG Production during an In Vitro Peripheral Blood Mononuclear Cell Culture Assay

JOURNAL=Frontiers in Immunology

VOLUME=8

YEAR=2017

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2017.00794

DOI=10.3389/fimmu.2017.00794

ISSN=1664-3224

ABSTRACT=<p>The recent attention given to diseases associated with memory B-cell (mBC)-produced antibodies (Abs) suggests the need for a similar <italic>in vitro</italic> assay to evaluate the functions of mBCs. Here, we cultured peripheral blood mononuclear cells (PBMCs) with the intent to collect mBC-derived Abs <italic>in vitro</italic> and maintain their cell–cell contact-dependent interactions with helper T-cells. PBMCs were cultured with interleukin (IL)-21, CpG-oligodeoxynucleotides (ODN), phorbol myristate acetate (PMA), and phytohemagglutinin/leucoagglutinin (PHA-L) in 24-well flat-bottom plates (5 × 10<sup>5</sup> cells/well). A culture supernatant analysis of PBMCs from healthy donors (<italic>n</italic> = 10) indicated that antigen-specific IgM Ab levels in a PBMC culture supernatant might be better able to demonstrate the antigen sensitization status in a smaller peripheral blood sample, compared to IgG because Epstein–Barr virus-specific IgM mBCs circulate peripherally at a significantly higher frequency once antiviral humoral immunity has stabilized. Thus, our <italic>in vitro</italic> assay demonstrated the potential significance of antigen-specific IgM Ab production in the culture supernatants. Furthermore, an analysis of cultured PBMCs from allograft kidney recipients (<italic>n</italic> = 16) sensitized with <italic>de novo</italic> donor-specific human leukocyte antigen (HLA)-specific Abs (DSAs) showed that IgM-type HLA-specific Abs were detected mainly from the culture supernatants from PBMCs of patients with stable graft function, whereas IgG isotype HLA Abs were detectable only from patients with biopsy-proven antibody-mediated rejection. In other words, these IgG isotype Abs also represented an activated humoral immune response <italic>in vivo</italic>. Additionally, IgM- and IgG-expressing mBCs from healthy donors (<italic>n</italic> = 5) were cultured with IL-21, CpG-ODN, and a supernatant produced by stimulating CD19<sup>+</sup> B-cell-depleted PBMCs with PHA-L and PMA in 24-well flat-bottom plates (1 × 10<sup>5</sup> cells/well), and the resulting <italic>in vitro</italic> analysis provided some information regarding the biological processes of IgG and IgM mBCs in peripheral blood. Taken together, our findings suggest that antigen-specific Ab subtype analyses of supernatants from cultured PBMCs might more effectively and accurately reflect a patient’s Ab-associated pathological condition vs. than serum IgG and IgM levels.</p>