Abstract
The recent attention given to diseases associated with memory B-cell (mBC)-produced antibodies (Abs) suggests the need for a similar in vitro assay to evaluate the functions of mBCs. Here, we cultured peripheral blood mononuclear cells (PBMCs) with the intent to collect mBC-derived Abs in vitro 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 × 105 cells/well). A culture supernatant analysis of PBMCs from healthy donors (n = 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 in vitro 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 (n = 16) sensitized with de novo 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 in vivo. Additionally, IgM- and IgG-expressing mBCs from healthy donors (n = 5) were cultured with IL-21, CpG-ODN, and a supernatant produced by stimulating CD19+ B-cell-depleted PBMCs with PHA-L and PMA in 24-well flat-bottom plates (1 × 105 cells/well), and the resulting in vitro 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.
Highlights
Antigen-specific antibodies (Abs) are produced by memory B-cell-derived plasma cells (PCs)
Our in vitro IgM memory B-cell (mBC) assay demonstrated that cross-linking-induced IgM-B-cell receptors (BCRs) stimulation is indispensable for the class-switching of IgM to IgG, and that growth factors and cytokines produced by activated T-cells, macrophages, and dendritic cells promoted this process. These results suggest that circulating IgM mBCs can undergo class-switching to become IgG mBCs and could thereby cause tissue injury following an antigen–BCR reaction, as BCR cross-linking-mediated stimulation increases the generation of antigen-specific IgG mBCs in the peripheral blood (Figure S1 in Supplementary Material)
We found that IgG DSAs were only detectable in the culture supernatants from the cells of kidney allograft recipients with biopsy-proven ABMR, whereas IgM Abs were generally detectable in supernatants from the cells of patients with stable graft function
Summary
Antigen-specific antibodies (Abs) are produced by memory B-cell (mBC)-derived plasma cells (PCs). Some reports indicate that no available immunosuppressive agent can control PCs growth and survival. An understanding of Ab-associated disease first requires an understanding of the biological processes that underlie the growth and survival of mBCs. Briefly, B-cells initially develop in the bone marrow. Highly self-reactive immature B-cells are deleted, and the remaining cells exit the bone marrow to the peripheral circulation. During the transitional stage of B-cell differentiation, cells that express self-antigen-reactive B-cell receptors (BCRs) are subjected to clone deletion, BCR editing, anergy, and immunological ignorance [1]
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