A disease-associated PTPN22 variant promotes systemic autoimmunity in murine models
Xuezhi Dai, Richard G. James, Tania Habib, Swati Singh, Shaun Jackson, Socheath Khim, Randall T. Moon, Denny Liggitt, Alejandro Wolf-Yadlin, Jane H. Buckner, David J. Rawlings
Xuezhi Dai, Richard G. James, Tania Habib, Swati Singh, Shaun Jackson, Socheath Khim, Randall T. Moon, Denny Liggitt, Alejandro Wolf-Yadlin, Jane H. Buckner, David J. Rawlings
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Research Article

A disease-associated PTPN22 variant promotes systemic autoimmunity in murine models

Abstract

Multiple autoimmune diseases, including type 1 diabetes, rheumatoid arthritis, Graves disease, and systemic lupus erythematosus, are associated with an allelic variant of protein tyrosine phosphatase nonreceptor 22 (PTPN22), which encodes the protein LYP. To model the human disease-linked variant LYP-R620W, we generated knockin mice expressing the analogous mutation, R619W, in the murine ortholog PEST domain phosphatase (PEP). In contrast with a previous report, we found that this variant exhibits normal protein stability, but significantly alters lymphocyte function. Aged knockin mice exhibited effector T cell expansion and transitional, germinal center, and age-related B cell expansion as well as the development of autoantibodies and systemic autoimmunity. Further, PEP-R619W affected B cell selection and B lineage–restricted variant expression and was sufficient to promote autoimmunity. Consistent with these features, PEP-R619W lymphocytes were hyperresponsive to antigen-receptor engagement with a distinct profile of tyrosine-phosphorylated substrates. Thus, PEP-R619W uniquely modulates T and B cell homeostasis, leading to a loss in tolerance and autoimmunity.

Authors

Xuezhi Dai, Richard G. James, Tania Habib, Swati Singh, Shaun Jackson, Socheath Khim, Randall T. Moon, Denny Liggitt, Alejandro Wolf-Yadlin, Jane H. Buckner, David J. Rawlings

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Figure 4

PEP-R619W enhances TCR signaling of in vitro–generated effector T cells.

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PEP-R619W enhances TCR signaling of in vitro–generated effector T cells....
(A) TCR-induced calcium flux of in vitro–generated effector T cells. Purified CD4+ T cells from T/T, T/C, and WT mice were stimulated with anti-CD3/CD28 for 3 days and rested in IL-2 medium for 2 days to generate effector T cells. Ca2+ mobilization was determined by flow cytometry. (B) Enhanced proliferation of T/C and T/T effector T cells. Cells were labeled with CFSE and cultured with anti-CD3; proliferation was determined by CFSE dilution. (C) IL-2 production in response to TCR engagement. In vitro–generated effector T cells were cultured with anti-CD3 with or without anti-CD28, and then IL-2 production was measured by ELISA. Error bars indicate SD of triplicate assays. **P < 0.01; ***P < 0.001. (D) PEP-R619W expression augments phosphorylation of TCR-dependent substrates. Cells were stimulated with anti-CD3/CD28 and cross-linked by secondary antibody for indicated times. Cell lysates were blotted using antibodies as indicated. (E) Phosphoproteome analysis. Effector T cells from T/T, PEP-deficient, and control mice were stimulated with anti-CD3/CD28. Following lysis and trypsinization, peptides were labeled with iTRAQ reporters and mixed. Tyrosine-phosphorylated peptides were isolated and analyzed using high-resolution mass spectrometry. Quantification of phosphopeptides corresponding to proteins previously described to regulate TCR signaling and/or found to be regulated by Ptpn22 are integrated into a schematic of the TCR pathway. Phosphopeptide abundance in PEP-R620W (left) and knockout (right) relative to control is depicted. Data are representative of at least 3 (A–D) and 2 (E) independent experiments.