Differential regulation of self-reactivity discriminates between IgG+ human circulating memory B cells and bone marrow plasma cells

Abstract

Long-term humoral immunity is maintained by the formation of high-affinity class-switched memory B cells and long-lived antibody-secreting plasma cells. In healthy humans, a substantial fraction of IgG-positive memory B cells express self-reactive and polyreactive IgG antibodies that frequently develop by somatic mutations. Whether self- and polyreactive IgG-secreting B cells are also tolerated in the long-lived plasma cell pool is not known. To address this question, we cloned and expressed the Ig genes from 177 IgG-producing bone marrow plasma cells of four healthy donors. All antibodies were highly mutated but the frequency of self- and polyreactive IgG antibodies was significantly lower than that found in circulating memory B cells. The data suggest that in contrast to the development of memory B cells, entry into the bone marrow plasma cell compartment requires previously unappreciated selective regulation by mechanisms that limit the production of self- and polyreactive serum IgG antibodies.

Fig. 1.

Ig gene features of IgG-positive plasma cells. Ig gene repertoire and Ig gene features of IgG plasma cell antibodies from four HDs (HD15, HD16, HD20, and HD21) are shown in comparison with historical data from IgG memory B cell antibodies (5, 7). VH/JH gene family use and IgH CDR3 amino acid-positive charges and length (A) and Vκ/Jκ and Vλ/Jλ gene family use (B) and IgG subclass distribution (C) are shown. Pie charts show the data for individual HDs with the absolute number of sequences analyzed indicated in the pie chart center. Bar graphs compare the data obtained from IgG plasma cell antibodies (n = 238; κ, n = 147; λ, n = 97) to historical data from IgG memory B cell antibodies (n = 254; κ, n = 172; λ, n = 83) (5, 7). Error bars indicate SD. (D) Dots represent the absolute number of somatic mutations (nucleotide exchanges compared with the nearest germline gene segment) in individual IgH and IgL Vκ and Vλ genes in IgG plasma cell antibodies from all four HDs. Horizontal lines indicate averages.

Fig. 2.

Low frequency of self-reactive IgG-positive plasma cells compared with memory B cells. IgG plasma cell antibodies from four HDs were tested for self-reactivity with HEp-2 cells. (A) HEp-2 cell lysate ELISA. Black lines represent individual plasma cell antibodies. Horizontal lines indicate cutoff OD₄₀₅ for positive reactivity determined by comparison with low-positive control serum (red line). Data are representative of at least two independent experiments. (B) Dot plots compare the frequency of HEp-2 lysate-reactive plasma cell antibodies determined in A to the frequency of HEp-2 lysate-reactive IgG-positive memory B cells in individual HDs (5, 7). Horizontal lines represent mean values of reactivity for all HDs; gray boxes indicate SD; n indicates the number of tested antibodies. (C) Representative HEp-2 cell IFA staining patterns of antibodies cloned from IgG-positive plasma cells. (Scale bar, 50 μm.) (D) Bar graphs compare the frequency of nonreactive (white) and HEp-2 self-reactive IgG plasma cell antibodies and memory B-cell antibodies with nuclear (black), nuclear plus cytoplasmic (dark gray), and cytoplasmic (light gray) IFA staining patterns (5, 7). Error bars indicate the SD; n indicates the number of tested antibodies. P values were calculated by Fisher’s exact test.

Fig. 3.

Low frequency of polyreactive IgG-positive plasma cells compared with memory B cells. (A) IgG plasma cell antibodies (black lines) from HDs were tested for polyreactivity by ELISA with ss/dsDNA, insulin, and LPS. Dotted lines represent the high-positive control antibody ED38 (35); horizontal lines show cutoff OD₄₀₅ for positive reactivity determined by comparison with the negative control antibody mGO53 (green line) (2) and low-positive control eiJB40 (red line) (2). Pie charts summarize the frequency of polyreactive clones for individual HDs. The numbers of tested antibodies are indicated in the pie chart centers. Data are representative of at least two independent experiments. (B) Dot plots compare the frequency of polyreactive plasma cell antibodies to the frequency of IgG-positive memory B cells in individual HDs (5, 7). Horizontal lines represent mean values of reactivity for all HDs; gray boxes indicate SD; n indicates the number of tested antibodies.