Cutting edge: the PTPN22 allelic variant associated with autoimmunity impairs B cell signaling

Abstract

PTPN22 is a gene encoding the protein tyrosine phosphatase Lyp. A missense mutation changing residue 1858 from cytosine to thymidine (1858C/T) is associated with multiple autoimmune disorders. Studies have demonstrated that Lyp has an inhibitory effect on TCR signaling; however, the presence of autoantibodies in all of the diseases associated with the 1858T variant and recent evidence that Ca(2+) flux is altered in B cells of 1858T carriers indicate a role for Lyp in B cell signaling. In this study we show that B cell signal transduction is impaired in individuals who express the variant. This defect in signaling is characterized by a deficit in proliferation, a decrease in phosphorylation of key signaling proteins, and is reversed by inhibition of Lyp. These findings suggest that the PTPN22 1858T variant alters BCR signaling and implicate B cells in the mechanism by which the PTPN22 1858T variant contributes to autoimmunity.

FIGURE 1

B cell proliferation is diminished in 1858C/T subjects. A, Total (CD19⁺; top), naive (CD19⁺CD27^-; middle), and memory (CD19⁺CD27⁺; bottom) B cells were purified and activated with anti-IgG plus IgM F(ab′)₂ (IgM+IgG) or PMA/ionomycin (P+I). Proliferation was measured at 72 h. Each dot represents a unique individual. B, B cells were activated as above and the CD27⁺ and CD27^- populations were assessed by FACS for cleaved caspase-3 (n = 4 per group). Unt, Untreated.

FIGURE 2

Total phosphorylated tyrosine is significantly decreased in memory B cells of PTPN22 1858C/T subjects. Pan-tyrosine phosphorylation was assessed in B cells by intracellular flow cytometry. A, The percentage of unstimulated CD27⁺ (memory) and CD27^- (naive) B cells with basal tyrosine phosphorylation is shown. B, B cells were activated for 2 min (2′) with anti-IgM/IgG. The log₁₀-fold change in the mean fluorescence intensity of phosphorylation relative to the unstimulated control for CD27^- naive and CD27⁺ memory B cells is shown. Black bars represent C/C samples (n = 6) and gray bars represent C/T samples (n = 6).

FIGURE 3

B cells from subjects harboring the 1858 T variant display BCR-mediated signaling defects. B cells were isolated and then activated with anti-IgM for the indicated time in minutes (′), followed by cell lysis. Western blotting was performed with the following Abs: phospho (P)-Akt-Ser473, phospho-PLCγ2-Tyr759, Syk, phospho-Syk-Tyr525/526, phospho-p38 MAPK-Thr180/Tyr182, and Lyp. A, Two control subjects were compared, one homozygous for 1858C/C and the other heterozygous for 1858C/T. B, Two control subjects were compared, one homozygous for 1858C/C and the other homozygous for the1858T/T variant. Blots are representative of three independent experiments. C, B cells were stimulated with soluble anti-IgM plus IgG F(ab′)₂ for the indicated times. Total phospho-PLCγ2 (Tyr759) was assessed by intracellular flow cytometry in the CD27⁺ memory population of 1858 C/C and 1858 C/T subjects (n = 6). Mean fluorescence intensity (MFI) values for phospho-PLCγ2 are shown.

FIGURE 4

Inhibition of Lyp by I-C11 enhances B cell signaling in PTPN22 C/T subjects. A, Freshly isolated B cells were treated with vehicle or 5 μM I-C11 followed by stimulation (stim) for 5 min with anti-IgM and intra-cellular staining for phospho-PLCγ2. B, Total B cells were treated as in A followed by stimulation with anti-kappa and intracellular staining for phospho-PLCγ2. Each dot represents the fold change in mean fluorescence intensity (MFI) relative to the unstimulated control for a unique individual. C, B cells were treated with vehicle or I-C11 (10 μM) and stimulated as in A. Whole cell lysates were immunoblotted using anti-phospho-Syk (p-Syk; Tyr525/526) and then reprobed with anti-Syk antisera (lower panel). Band intensities were quantified by densitometry (ImageQuant TL software). The ratio of phospho-Syk (P-Syk) to total Syk protein is shown below each lane. Adjacent panels depicted in C are from the same gel. Data shown are representative of three experiments.