Activation-induced cytidine deaminase (AID) is required for B-cell tolerance in humans

Abstract

Impaired immune functions leading to primary immunodeficiencies often correlate with paradoxical autoimmune complications; patients with hyper-IgM syndromes who are deficient in activation-induced cytidine deaminase (AID), which is required for class-switch recombination and somatic hypermutation, are prone to develop autoimmune diseases. To investigate the impact of AID-deficiency on early B-cell tolerance checkpoints in humans, we tested by ELISA the reactivity of recombinant antibodies isolated from single B cells from AID-deficient patients. New emigrant/transitional and mature naive B cells from AID-deficient patients express an abnormal Ig repertoire and high frequencies of autoreactive antibodies, demonstrating that AID is required for the establishment of both central and peripheral B-cell tolerance. In addition, B-cell tolerance was further breached in AID-deficient patients as illustrated by the detection of anti-nuclear IgM antibodies in the serum of all patients. Thus, we identified a major and previously unsuspected role for AID in the removal of developing autoreactive B cells in humans.

Fig. 1.

AID-deficient new emigrant/transitional B cells display an unusual IgH repertoire. (A) VH gene-usage frequencies in new emigrant/transitional B cells are represented for 11 healthy control subjects and seven AID-deficient patients. Sequences from 353 healthy control and 189 AID-deficient single transitional B cells were pooled. (B) The increased VH4-34 gene usage in AID-deficient new emigrant/transitional B cells is further analyzed. The frequencies of long IgH CDR3s (>14 aa) and IgH CDR3s containing two or more positively charged aa are represented in C and D, respectively. Each diamond represents an individual; the average is shown with a bar. Statistically significant differences are indicated.

Fig. 2.

Defective central B-cell tolerance checkpoint in AID-deficient patients. (A) Antibodies from new emigrant/transitional B cells from a healthy donor and AID-deficient patients were tested by ELISA for reactivity against dsDNA, insulin, and lipopolysaccharide (LPS). Dotted lines show ED38-positive control and solid lines show binding for each cloned recombinant antibody (20, 26). Horizontal lines define cutoff OD₄₀₅ for positive reactivity. The frequencies of polyreactive (B) and antinuclear (C) new emigrant/transitional B cells are compared between controls and CD40L- and AID-deficient patients, and statistically significant differences are indicated. (D) Autoreactive antibodies from AID-deficient new emigrant B cells show various patterns of HEp-2 staining.

Fig. 3.

AID gene expression in human immature B cells. AID gene expression was assessed by quantitative PCR in unstimulated human precursors and immature B cells purified from fetal liver, bone marrow (BM), and spleen (A), or in immature B cells stimulated for 2 d with CpG and/or F(ab′)2 anti-IgM (B). Tonsillar GC B cells were used as positive control for AID gene expression. Error bars represent the mean ± SEM.

Fig. 4.

Defective peripheral B-cell tolerance checkpoint in AID-deficient patients. The frequency of mature naive B-cell clones from healthy controls and AID-deficient patients expressing IgH CDR3s containing two or more positively charged aa (Left) or VH4-34 gene (Right) is represented in A, and the average is shown with a bar. (B) Antibodies from mature naive B cells from a healthy donor and AID-deficient patients were tested by ELISA for anti–HEp-2 cell reactivity. Dotted lines show ED38-positive control (20, 26). Horizontal lines define cutoff OD₄₀₅ for positive reactivity. Frequencies of HEp-2–reactive (C) and polyreactive (D) clones in the mature naive fraction of CD40L and AID-deficient patients are higher than in controls. Mature naive B cells from AID-deficient patients contain ANA-expressing clones with diverse HEp-2 staining patterns (E). The frequency of ANA-expressing B cells in healthy controls and AID-deficient patients (Top) and its evolution between the new emigrant/transitional and mature naive B-cell compartments (Bottom) are represented in F. ANAs that bind the kinetoplast of C. luciliae (white arrows) are shown in G.

Fig. 5.

Low Treg cell frequency and increased serum BAFF concentrations and autoantibodies in AID-deficient patients. Treg cell frequencies among peripheral CD4⁺ T cells were assessed by analyzing the proportion of CD25⁺Foxp3⁺ cells. (A) Dot plots representative of a healthy control, a CD40L-deficient patient, and two AID-deficient patients. (B) CD127 expression was further analyzed by gating as indicated. (C) Treg cell frequencies from all patients were significantly lower than those in healthy controls (P < 0.0001 for CD40L-, AID-deficient, and XLA patients). (D) Significantly elevated serum BAFF concentrations (in pg/mL) in CD40L-, AID-deficient, and XLA patients were measured by ELISA (P < 0.0001 for each group of patients). (E) AID-deficient patients display secreted autoreactive IgM antibodies in their serum. Sera from six healthy donors, a patient with systemic Lupus erythematosus, four CD40L-deficient patients, and eight AID-deficient patients were tested for HEp-2–reactive (Top) and C. luciliae-reactive (Bottom) IgM and IgG antibodies.