The establishment of early B cell tolerance in humans: lessons from primary immunodeficiency diseases

Abstract

Patients with primary immunodeficiency (PID) provide rare opportunities to study the impact of specific gene mutations on the regulation of human B cell tolerance. Alterations in B cell receptor and Toll-like receptor signaling pathways result in a defective central checkpoint and a failure to counterselect developing autoreactive B cells in the bone marrow. In contrast, CD40L- and MHC class II-deficient patients only displayed peripheral B cell tolerance defects, suggesting that decreased numbers of regulatory T cells and increased concentration of B cell activating factor (BAFF) may interfere with the peripheral removal of autoreactive B cells. The pathways regulating B cell tolerance identified in PID patients are likely to be affected in patients with rheumatoid arthritis, systemic lupus erythematosus, and type 1 diabetes who display defective central and peripheral B cell tolerance checkpoints. Indeed, risk alleles encoding variants altering BCR signaling, such as PTPN22 alleles associated with the development of these diseases, interfere with the removal of developing autoreactive B cells. Hence, insights into B cell selection from PID patients are highly relevant to the understanding of the etiology of autoimmune conditions.

Figure 1

Early B cell tolerance checkpoints in healthy donors. Single CD34⁻CD19⁺CD10⁺IgM⁻ early immature B cells and CD34⁻CD19⁺CD10⁺IgM⁺ immature B cells from bone marrow and CD19⁺CD10⁺IgM⁺⁺CD27⁻ new emigrant/transitional and CD19⁺CD10⁻IgM⁺CD27⁻ mature naive B cells from peripheral blood of healthy controls were isolated by flow cytometry based on the indicated surface markers. IgH and IgL chain genes from single purified B cells were cloned, and the monoclonal antibodies were expressed in vitro. The frequency of HEp-2 reactive antibodies (top panel) was determined by HEp-2 cell ELISA and indirect immunofluorescence on HEp-2 cells. The frequency of polyreactive antibodies (bottom panel) was determined by ELISA with ssDNA, dsDNA, insulin, and lipopolysaccharide as antigens. Polyreactive antibodies recognized at least two structurally diverse antigens and often all four. Each diamond represents an individual; the average is shown with a bar. The central and peripheral B cell tolerance checkpoints are indicated.

Figure 2

Central B cell tolerance requires proper BCR and TLR signaling. The frequencies of polyreactive (A) and antinuclear (B) new emigrant/transitional B cells are compared between controls (open diamonds), subjects with the PTPN22 “T” risk allele, patients with diverse PID, rheumatoid arthritis (RA), and type 1 diabetes (T1D) (black diamonds). Alteration in either BCR or TLR signaling results in a failure to counterselect developing autoreactive B cells in the bone marrow and results in increased frequencies of polyreactive new emigrant/transitional B cells. IRAK4- and MYD88-deficient new emigrant/transitional B cells were especially enriched in antinuclear clones, as shown in B.

Figure 3

Specific defective peripheral B cell tolerance checkpoint in CD40L- and MHC class II-deficient patients. The frequencies of HEp-2 reactive (A), polyreactive (B), and antinuclear (C) mature naive B cells are compared between controls (open diamonds), subjects with the PTPN22 “T” risk allele, patients with diverse PID, rheumatoid arthritis (RA), and type 1 diabetes (T1D) (black diamonds). Defects in CD40L expression or antigen presentation through MHC class II molecules specifically either interfere with the removal or fail to prevent the accumulation of autoreactive B cells in the periphery. All other subjects who presented central B cell tolerance defects also display large numbers of autoreactive B cells in their mature naive B cell compartment.

Figure 4

Decreased T_reg cell frequency in CD40L-, AID-deficient, and XLA patients. T_reg cell frequencies among peripheral CD4⁺ T cells were assessed by analyzing the proportion of CD25⁺Foxp3⁺ cells. Dot plots representative for a healthy control and CD40L-, AID-deficient, and XLA patients are displayed in (A). (B) T_reg cell frequencies from all patients were significantly lower than those in healthy controls (P < 0.0001 for CD40L-, AID-deficient, and XLA patients).

Figure 5

Elevated serum BAFF concentrations in CD40L-, AID-deficient, and XLA patients. BAFF concentrations (pg/mL) in the serum of healthy donor controls (open diamonds), CD40L-, and AID-deficient patients as well as XLA patients (black diamonds) were measured by ELISA. Each diamond represents an individual, and the average is shown with a bar.