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. 2011 Sep;121(9):3635-44.
doi: 10.1172/JCI45790. Epub 2011 Aug 1.

The PTPN22 allele encoding an R620W variant interferes with the removal of developing autoreactive B cells in humans

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The PTPN22 allele encoding an R620W variant interferes with the removal of developing autoreactive B cells in humans

Laurence Menard et al. J Clin Invest. 2011 Sep.

Abstract

Protein tyrosine phosphatase nonreceptor type 22 (PTPN22) gene polymorphisms are associated with many autoimmune diseases. The major risk allele encodes an R620W amino acid change that alters B cell receptor (BCR) signaling involved in the regulation of central B cell tolerance. To assess whether this PTPN22 risk allele affects the removal of developing autoreactive B cells, we tested by ELISA the reactivity of recombinant antibodies isolated from single B cells from asymptomatic healthy individuals carrying one or two PTPN22 risk allele(s) encoding the PTPN22 R620W variant. We found that new emigrant/transitional and mature naive B cells from carriers of this PTPN22 risk allele contained high frequencies of autoreactive clones compared with those from non-carriers, revealing defective central and peripheral B cell tolerance checkpoints. Hence, a single PTPN22 risk allele has a dominant effect on altering autoreactive B cell counterselection before any onset of autoimmunity. In addition, gene array experiments analyzing mature naive B cells displaying PTPN22 risk allele(s) revealed that the association strength of PTPN22 for autoimmunity may be due not only to the impaired removal of autoreactive B cells but also to the upregulation of genes such as CD40, TRAF1, and IRF5, which encode proteins that promote B cell activation and have been identified as susceptibility genes associated with autoimmune diseases. These data demonstrate that early B cell tolerance defects in autoimmunity can result from specific polymorphisms and precede the onset of disease.

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Figures

Figure 1
Figure 1. Altered central B cell tolerance checkpoint in healthy individuals carrying PTPN22 risk allele(s).
(A) Antibodies from new emigrant/transitional B cells from healthy donors who did not carry the PTPN22 risk allele (HD-CC) or carried one (HD-CT) or two (HD-TT) PTPN22 risk allele(s) were tested by ELISA for reactivity against ssDNA, dsDNA, insulin and LPS. Polyreactive antibodies reacted against all 4 antigens. Dotted lines show ED38-positive control (5). Horizontal lines show cutoff OD405 for positive reactivity. For each individual, the frequency of polyreactive and non-polyreactive clones is summarized in pie charts, with the number of antibodies tested indicated in the center. The frequencies of polyreactive (B) and anti-nuclear (C) new emigrant/transitional B cells are compared between healthy donors carrying or not carrying PTPN22 risk allele(s), and statistically significant differences are indicated. Each diamond represents an individual, and the horizontal bars show the average. (D) Autoreactive antibodies expressed by new emigrant (ne) B cells from PTPN22 risk allele carriers mostly show various cytoplasmic patterns of HEp-2 staining. Original magnification, ×40.
Figure 2
Figure 2. The PTPN22 risk allele interferes with the peripheral B cell tolerance checkpoint.
(A) Antibodies from mature naive B cells from healthy donors who did not carry the PTPN22 risk allele (HD-CC) or carried one (HD-CT) or two (HD-TT) PTPN22 risk allele(s) were tested by ELISA for anti–HEp-2 cell reactivity. Dotted lines show ED38-positive control (5, 29). Horizontal lines show cutoff OD405 for positive reactivity. For each individual, the frequency of HEp-2–reactive and nonreactive clones is summarized in pie charts, with the number of antibodies tested indicated in the center. Frequencies of HEp-2–reactive and polyreactive clones in the mature naive fraction of healthy donors carrying or not PTPN22 risk allele(s) are summarized in B and C, respectively.
Figure 3
Figure 3. Defective central B cell tolerance checkpoint in T1D patients.
Antibodies from new emigrant B cells from non-carrier T2D and T1D (CC) and carrier T1D (CT or TT) patients were tested by ELISA for reactivity with ssDNA, dsDNA, insulin, and LPS. Dotted lines show ED38-positive control. Horizontal lines show cutoff OD405 for positive reactivity. For each individual, the frequency of polyreactive (black) and non polyreactive (white) clones is summarized in pie charts, with the number of antibodies tested indicated in the center.
Figure 4
Figure 4. Healthy donors carrying the PTPN22 risk allele(s) display elevated frequencies of autoreactive B cells similar to those in T1D and RA patients.
The frequencies of polyreactive new emigrant/transitional (A) and HEp-2–reactive mature naive (B) B cells are compared between healthy donors and T1D patients carrying (T1D-CT+TT) or not (T1D-CC) PTPN22 risk allele(s) as well as non-carrier T2D patients (T2D-CC). Similar analyses were performed in C and D with RA patients carrying (RA-CT+TT) or not (RA-CC) PTPN22 risk allele(s). Statistically significant differences are indicated. All T1D and RA patients display high frequencies of autoreactive clones similar to those from healthy donors carrying PTPN22 risk allele(s).
Figure 5
Figure 5. Mature naive B cells from healthy donors carrying PTPN22 risk allele(s) upregulate the expression of many susceptibility genes associated with autoimmune diseases.
Gene array comparisons of mature naive B cells from healthy donors (HD) carrying or not PTPN22 risk allele(s) using the Affymetrix Human Genome U133 Plus 2.0 Array. Selected transcripts differentially expressed and well known to be involved in B cell biology are presented. One or more probes are shown for each gene. Up- and downregulated transcripts are indicated in red and green, respectively. The magnitude of expression is depicted by the color bar. Additional differentially expressed genes are shown in Supplemental Figure 4.
Figure 6
Figure 6. Mature naive B cells from healthy donors carrying PTPN22 risk allele(s) display a phenotype reflecting gene array profiling data.
(A) Quantitative real-time PCR validates the increased transcription of many genes found upregulated in gene array profiling experiments. Gene expression was assessed by comparing 31 non-carrier healthy donors and 16 individuals carrying PTPN22 risk allele(s). Error bars represent the mean ± SEM. (B) Flow cytometry analyses reveal the upregulation of CD40 and SLAMF6 but not CD19 on the surface of mature naive B cells from individuals carrying PTPN22 risk allele(s). Each diamond represents the mean fluorescence of the indicated molecules expressed on the surface of mature naive B cells from individuals carrying or not PTPN22 risk allele(s). Representative overlays for the indicated markers are shown in C.
Figure 7
Figure 7. Enhanced CD40 responses in naive B cells from PTPN22 risk allele carriers.
Representative dot plots (A) and combined average expression (B) of CD25 and CD69 on naive B cells from healthy donors carrying or not the PTPN22 risk allele are shown after stimulation in vitro with various indicated concentrations of multimeric soluble recombinant CD40L (left) or F(ab′)2 anti-IgM (right) for 2 days. Values in A represent the percentage of positive cells in each quadrant. Results for HDC10 (non-carrier of the PTPN22 risk allele) and HDP07 (carrier of one PTPN22 risk allele) are shown. Data are representative of 4 independent experiments, and statistical differences are indicated (*P < 0.05, **P < 0.01).

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