The Majority of Human Memory B Cells Recognizing RhD and Tetanus Resides in IgM+ B Cells

Abstract

B cell memory to T cell–dependent (TD) Ags are considered to largely reside in class-switched CD27+ cells. However, we previously observed that anti-RhD (D) Igs cloned from two donors, hyperimmunized with D+ erythrocytes, were predominantly of the IgM isotype. We therefore analyzed in this study the phenotype and frequency of D- and tetanus toxoid–specific B cells by culturing B cells in limiting dilution upon irradiated CD40L-expressing EL4.B5 cells and testing the culture supernatant. Most Ag-specific B cells for both TD Ags were found to reside in the IgM-expressing B cells, including CD27− B cells, in both hyperimmunized donors and nonhyperimmunized volunteers. Only shortly after immunization a sharp increase in Ag-specific CD27+IgG+ B cells was observed. Next, B cells were enriched with D+ erythrocyte ghosts and sorted as single cells. Sequencing of IGHV, IGLV, IGKV, and BCL6 genes from these D-specific B cell clones demonstrated that both CD27−IgM+ and CD27+IgM+ B cells harbored somatic mutations, documenting their Ag-selected nature. Furthermore, sequencing revealed a clonal relationship between the CD27−IgM+, CD27+IgM+, and CD27+IgG+ B cell subsets. These data strongly support the recently described multiple layers of memory B cells to TD Ags in mice, where IgM+ B cells represent a memory reservoir which can re-enter the germinal center and ensure replenishment of class-switched memory CD27+ B cells from Ag-experienced precursors.