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.

FIGURE 1.

Frequency of Ag-specific B cells is similar in IgM⁺ and class-switched IgM⁻ B cells, but IgM⁺ exceeds class-switched B cells in absolute numbers. B cells were sorted based on their IgM⁺ or IgM⁻ surface phenotype. The production of Ag-specific Abs was assessed after 10 d of culture with irradiated EL4B5 cells by either erythrocyte agglutination (rhesus D Ag) or ELISA (TT) in three anti-D and two anti-TT hyperimmunized donors. (A) The frequency of Ag-specific B cells was estimated by limiting dilution analysis and is expressed as a number of Ag-specific B cells per million of total PB B cells of that given subset. Each symbol represents a donor. (B) The relative number of IgM⁺ and IgM⁻ B cells within the Ag-specific B cells was calculated for each donor taking the frequency and subset size of each B cell population into account.

FIGURE 2.

The largest proportion of Ag-specific B cells resides in IgM-expressing CD27⁻ and CD27⁺ B cell populations. The frequency of Ag-specific B cells in five anti-D and two anti-TT hyperimmunized donors was determined by limiting dilution after sorting CD27⁻IgM⁺, CD27⁺IgM⁺, and CD27⁺IgG⁺ B cell subsets onto EL4B5 cells, allowing them to grow for 10 d before testing supernatants for specific Abs as described in Fig. 1. (A) Frequency of Ag-specific B cells given as a number of Ig-producing Ag-specific B cells per million cells within subset. Black lines indicate geometric means. (B) The frequencies reported are per million total B cells or (C) as relative number of cells from the different subsets. Note that no IgG⁺ anti-TT B cells were identified from the anti-TT3 donor.

FIGURE 3.

Ig production from single B cells is equally good in CD27⁻ and CD27⁺ B cells. B cells were sorted as one cell per well and cultured as described in the legend to Fig. 1. Supernatants were tested for IgM and IgG production by ELISA, and the number of Ab-producing B cells are given as a mean percentage (bars), together with SDs from 14 different experiments.

FIGURE 4.

IgM-expressing TT-specific B cells are present also in nonhyperimmunized donors. (A) The frequency of TT-specific B cells, in four nonhyperimmunized donors who had not received a TT booster for at least 10 y, was estimated by limiting dilution, as in Figs. 1–3. The frequency is given as the number of B cells per million within a subset. (B) Frequency of TT-specific B cells per million peripheral B cells in two control donors before and 14 d after receiving TT booster immunization.

FIGURE 5.

Both D-specific CD27⁻IgM⁺ and CD27⁺IgM⁺ B cells contain somatic mutations, indicative of a post-GC origin. The number of mutations per Ab for the variable H chain (V_H, μ or γ) and variable L chain (V_L, κ or λ) are grouped for their either anti-D specificity (D) or control (C), as well as their CD27 expression. ***p < 0.0001 when comparing anti-D–specific and control B cells within B cell subsets using a one-tailed Student t test.

FIGURE 6.

Analysis of anti-D–specific B cell IGV_H and IGV_L sequences. The schematic diagram illustrates the clonal relationship in the H (A) and L (B) chains of anti-D–specific B cells isolated from the RhD4 hyperimmunized donor. Each small vertical stick represents a point mutation, and each horizontal line indicates a unique B cell sequence, whereas the clonally related B cells and shared mutations are grouped in different colors. Each cell ID and the B cell subset are given. Additionally, the V(D)J region usage and the CDR3 amino acidic length are also displayed to point out the closeness of the different B cell clones. VH and VK/VL genes were sequenced for all 36 cells, except for four VH sequences and one VK/VL sequence that could not be amplified. VH gene usage was in 15 cases restricted to use VH4–34, with 13 residing in both IgM-expressing B cell subsets and of which 11 showed clonal relatedness. These 11 sequences also restrictedly used the same VL1–39 gene. Two B cell clones were found to be clonally related by their VL3–19 gene sequence only, because in one clone the Ig VH sequence was not amplified.

FIGURE 7.

Clonal relationship of anti-D–specific B cells. V_H and V_L chain genes were sequenced for all, except for four VH sequences and one Vκ/λ sequence that could not be amplified. A schematic representation is shown of the IGV_H and IGV_L sequence relationship among D-specific B cell clones belonging to the CD27⁻IgM⁺ (n = 8), CD27⁺IgM⁺ (n = 13), and CD27⁺IgG⁺ (n = 15) subsets derived from the RhD4 hyperimmunized donor. Lines connect the B cell clones whose Ig V_H and/or V_L regions showed the same sequence, harboring the same or a different number of somatic point mutations as indicated by the numbers within the circle. Colors match the scheme used in Fig. 6.