Activation-induced cytidine deaminase mediates central tolerance in B cells

Abstract

The Aicda gene product, activation-induced cytidine deaminase (AID), initiates somatic hypermutation, class-switch recombination, and gene conversion of Ig genes by the deamination of deoxycytidine, followed by error-prone mismatch- or base-excision DNA repair. These processes are crucial for the generation of genetically diverse, high affinity antibody and robust humoral immunity, but exact significant genetic damage and promote cell death. In mice, physiologically significant AID expression was thought to be restricted to antigen-activated, mature B cells in germinal centers. We now demonstrate that low levels of AID in bone marrow immature and transitional B cells suppress the development of autoreactivity. Aicda(-/-) mice exhibit significantly increased serum autoantibody and reduced capacity to purge autoreactive immature and transitional B cells. In vitro, AID deficient immature/transitional B cells are significantly more resistant to anti-IgM-induced apoptosis than their normal counterparts. Thus, early AID expression plays a fundamental and unanticipated role in purging self-reactive immature and transitional B cells during their maturation in the bone marrow.

Fig. 1.

Developmental effects of Aicda on B-lineage cells. The consequences of Aicda on the generation and maturation of specific hematopoietic cell lineages was determined by competitive hematopoietic reconstitution. Chimeric mice containing approximately 1:1 ratios of competitor stem cell [LSK (18); Fig. S2] populations were generated, and the frequencies of CD45.1⁺Aicda^+/+ (filled diamonds, solid line) and CD45.2⁺Aicda^−/− (open circles, solid line) cells in each hematopoietic compartment (Fig. S2), normalized to the frequency of Aicda^+/+ or Aicda^−/− LSK cells, are shown. Control reconstitutions with CD45.1⁺ (diamonds) and CD45.2⁺ (circles) Aicda^+/+ BM cells (broken lines) demonstrate no hematopoietic advantage for either CD45 marker allele. Data are shown as mean ± SEM (n = 7–9).

Fig. 2.

Increased serum autoAb in Aicda^−/− mice. DNA and cell-reactive autoAb levels in the sera of Aicda^−/− mice were compared with congenic B6, autoimmune C4^−/− (22), and Ab-deficient Rag1^−/− animals. Anti-dsDNA Igκ Ab (A) and self-reactive Igκ Ab (B) (reactive to NIH 3T3 cells) in sera of B6 (●), Aicda^−/− (○), C4^−/− (▲), and Rag1^−/− (◆) mice were determined by ELISA. (C) The presence of anti-DNA autoAb (Igκ) in diluted sera (1:800) of B6 and Aicda^−/− mice was compared in a C. luciliae immunofluorescence assay. Representative micrographs are shown; original magnification of ×400. (D) The presence of self-reactive autoAb (Igκ) in diluted sera (1:400) of B6 and Aicda^−/− mice was screened on NIH 3T3 cells. Representative photomicrographs are shown; original magnification of ×200. (E) Self-reactive IgM Ab (reactive to NIH 3T3 cells) in sera of B6 (●), Aicda^−/− (○), and Aicda^−/−Cd154^−/− (○) mice were determined by ELISA. Before the assay, serum IgM in each sample was determined and adjusted to 400 μg IgM/mL. In B and E, relative values (normalized to C4^−/− serum) are shown. In A, B, and E, points represents individual mice.

Fig. 3.

Effect of Aicda on the development of autoreactive B cells. B-cell development in BM of B6, Aicda^−/−, 3H9, and 3H9.Aicda^−/− mice was compared. (A) Representative flow data for IgM/IgD expression by BM cells of B6, Aicda^−/−, 3H9, and 3H9.Aicda^−/− mice are shown. Specific developmental compartments [immature (imm), T1 and T2, and mature B (38, 43)] were designated for B6; identical gatings were used for all samples. Numbers within squares represent frequency of cells in those squares. (B) Absolute cell number of the indicated B-cell compartments in of B6 (Aicda^+/+, filled bars; Aicda^−/− open bars) and 3H9 (Aicda^+/+, filled bars; Aicda^−/− open bars) mice are shown. Histograms represent mean values ± SEM (n = 7–10).

Fig. 4.

Effects of Aicda on autoAb production in 3H9 mice. (A) Expression of 3H9 VDJ knock-in alleles (relative to Cd14) in genomic DNA of immature/T1 (im/T1), mature follicular (MF) B cells, and thymocytes (THY) from 3H9 (filled bars) and 3H9.Aicda^−/− (open bars) mice were determined by quantitative PCR. The ratio of 3H9/Cd14 in the 3H9 cell compartments was normalized (1.0) for comparison with the analogous ratios for 3H9.Aicda^−/− mice. Histograms represent the mean ± SD of individual mice (n = 4–9) in three experiments. (B) The presence of ds DNA autoAb (Igκ) in 3H9.Aicda^−/− and 3H9 mice was compared in C. luciliae immunofluorescence assays. Reactivity in diluted sera (1:800) from four 3H9 and four 3H9.Aicda^−/− mice were compared individually (42).

Fig. 5.

SHM in 3H9^+/+ immature/T1 B cells. Frequencies of mutated 3H9 VDJ rearrangements (A), and distributions of the numbers of point mutations in each sequence (B) from genomic DNA of BM immature/T1 B cells from 3H9 (n = 45), 3H9.Aicda^−/− (n = 51), or PP GC B cells from 3H9 mice (n = 20) are shown.

Fig. 6.

Aicda^−/− immature/T1 B cells are resistant to BCR-induced apoptosis. The susceptibility of immature/T1 B cells from Aicda^+/+ and Aicda^−/− BM to anti-IgM Ab-induced apoptosis was compared in cell culture (31). BM cells from Aicda^+/+ and Aicda^−/− mice were cultured in triplicate in media containing various concentrations of anti-IgM (anti-μ) Ab for 24 h. The numbers of Aicda^+/+ (filled symbols) and Aicda^−/− (open symbols) immature/T1 B cells in each treatment are shown. Data shown represent the means ± SD from three independent experiments.