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. 2013 Feb 28;121(9):1595-603.
doi: 10.1182/blood-2012-09-457465. Epub 2012 Dec 5.

Accumulation of peripheral autoreactive B cells in the absence of functional human regulatory T cells

Tuure Kinnunen  1 , Nicolas Chamberlain, Henner Morbach, Jinyoung Choi, Sangtaek Kim, Joseph Craft, Lloyd Mayer, Caterina Cancrini, Laura Passerini, Rosa Bacchetta, Hans D Ochs, Troy R Torgerson, Eric Meffre
Affiliations

Accumulation of peripheral autoreactive B cells in the absence of functional human regulatory T cells

Tuure Kinnunen et al. Blood. .

Abstract

Regulatory T cells (Tregs) play an essential role in preventing autoimmunity. Mutations in the forkhead box protein 3 (FOXP3) gene, which encodes a transcription factor critical for Treg function, result in a severe autoimmune disorder and the production of various autoantibodies in mice and in IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) patients. However, it is unknown whether Tregs normally suppress autoreactive B cells. To investigate a role for Tregs in maintaining human B-cell tolerance, we tested the reactivity of recombinant antibodies isolated from single B cells isolated from IPEX patients. Characteristics and reactivity of antibodies expressed by new emigrant/transitional B cells from IPEX patients were similar to those from healthy donors, demonstrating that defective Treg function does not impact central B-cell tolerance. In contrast, mature naive B cells from IPEX patients often expressed autoreactive antibodies, suggesting an important role for Tregs in maintaining peripheral B-cell tolerance. T cells displayed an activated phenotype in IPEX patients, including their Treg-like cells, and showed up-regulation of CD40L, PD-1, and inducibl T-cell costimulator (ICOS), which may favor the accumulation of autoreactive mature naive B cells in these patients. Hence, our data demonstrate an essential role for Tregs in the establishment and the maintenance of peripheral B-cell tolerance in humans.

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Figures

Figure 1
Figure 1
The central B-cell tolerance checkpoint is functional in IPEX patients. (A) Antibodies from new emigrant/transitional B cells from a healthy donor and IPEX 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. Horizontal lines define cutoff OD405 for positive reactivity. For each individual, the frequency of polyreactive (filled area) and nonpolyreactive (open area) clones is summarized in pie charts, with the total number of clones tested indicated in the centers. The frequencies of (B) polyreactive, (C) HEp-2–reactive, and (D) antinuclear new emigrant/transitional B cells in healthy controls and IPEX patients are shown.
Figure 2
Figure 2
Defective peripheral B-cell tolerance checkpoint in IPEX patients. (A) Antibodies from mature naive B cells from a healthy donor and IPEX patients were tested by ELISA for anti-HEp-2 cell reactivity. Dotted lines show ED38-positive control and solid lines show binding for each cloned recombinant antibody. Horizontal lines define cutoff OD405 for positive reactivity. For each individual, the frequency of HEp-2–reactive (filled area) and non-HEp-2–reactive (open area) clones is summarized in pie charts, with the total number of clones tested indicated in the centers. The frequencies of (B) HEp-2–reactive and (C) polyreactive mature naive B cells in healthy controls and IPEX patients (left) and their evolution between the new emigrant/transitional and mature naive B-cell compartments (right) are shown. (D) Mature naive B-cell clones from IPEX patients show various patterns of cytoplasmic HEp-2 staining. (E) The frequency of antinuclear clones is low (left) and is not increased between the new emigrant/transitional and mature naive B-cell compartments (right).
Figure 3
Figure 3
IPEX patients display normal serum BAFF concentrations. Serum BAFF concentrations (picograms per milliliter) in healthy donors (♢, n = 73) and IPEX patients (♦, n = 9) were measured by ELISA. Each diamond represents an individual, and the average is shown with a bar: healthy donors, 1019 ± 35; IPEX patients, 1177 ± 134.
Figure 4
Figure 4
Mature naive B cells from IPEX patients show an increased proliferative history. (A) Representative CD80, CD86, and CD69 expression on CD19+CD27− naive B cells in IPEX patients (solid gray) compared with healthy donors (bold). (B) Mean fluorescence intensities (MFI) of CD80, CD86, and CD69 expression on naive B cells from healthy donors (n = 7) and IPEX patients (n = 7). (C) Evaluation of the number of cell divisions undergone in vivo by KREC analysis on new emigrant and mature naive B cells of healthy donors (n = 41) and IPEX patients (n = 6).
Figure 5
Figure 5
Increased frequency of CD4+FOXP3+Helios+ T cells in IPEX patients. (A) Representative CD25 and FOXP3 (left) and Helios and FOXP3 staining (right) on CD3+CD4+ cells from a healthy donor and a representative IPEX patient. CD3+CD4+CD25+CD127loFOXP3+ (B) and CD3+CD4+FOXP3+Helios+ (C) T-cell frequencies in 38 healthy donors and 5 IPEX patients. (D) Expression levels of CD25, CD127, and CD45RO on FOXP3−Helios− and FOXP3+Helios+ CD4+ T cells from a representative IPEX patient (solid gray) and a healthy donor (bold) gated as shown in panel A. The mean fluorescence intensities (MFI) of (E) CD25, (F) CD127, and (G) CD45RO expression is shown for Helios−FOXP3− and Helios+FOXP3+ CD4+ T cells from healthy donors and IPEX patients.
Figure 6
Figure 6
IPEX patients harbor activated T cells including Tfh-like cells. Increased expression of CD40L, ICOS, and PD-1 but not CD69 on total (A) CD3+CD4+ cells and (B) CD3+CD4+FOXP3+ cells in IPEX patients (solid gray) compared with healthy donors (bold). (C) Increased PD-1 expression on circulating CD4+CXCR5+ cells reveals Tfh-like cells in IPEX patients.
Figure 7
Figure 7
PBMCs from IPEX patients reflect the presence of Tfh-like cells. (A) Quantitative real-time PCRs validate the increased transcription of Tfh genes in IPEX patients. Gene expression was assessed by comparing 11 healthy donors and 5 IPEX patients. Error bars represent the mean ± SEM. (B) Gene expression is displayed relative to CD4 expression and demonstrates PD1, ICOS, and IL10 up-regulation in IPEX patients.
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