Gene Edited Human CD4+ T Cells Reveal that Loss of PTPN22 Function Enhances Mildly Autoreactive T Cell Responses

Abstract

Abstract A genetic variant in the gene PTPN22 (R620W, rs2476601) is strongly associated with increased risk for multiple autoimmune diseases. PTPN22 is a non-receptor tyrosine phosphatase that negatively regulates proximal T cell receptor (TCR) signaling. PTPN22 deficiency increases TCR signaling in mouse and human T cells. Mouse models of the variant similarly exhibit increased TCR signaling, while primary T cells from human carriers of the variant exhibit decreased signaling. Here, we utilize gene editing in primary human T cells to address these discordant findings and explore how the risk variant impacts TCR signaling. Using Crispr/Cas9 nucleases and DNA repair templates, we established a robust platform for precise homology-directed-repair based gene editing of PTPN22, allowing the generation of PTPN22 risk edited, control edited (silent modification), and knock-out T cells from the same donor. In contrast to control edited cells, PTPN22 risk edited T cells exhibit increased activation in response to TCR engagement with higher expression of activation markers including CD40L and CD25, mimicking PTPN22 KO cells. Further, using lentiviral delivery of transgenic TCRs (derived from autoreactive T cells of T1D patients), we demonstrate that loss of PTPN22 leads to enhanced TCR signaling in weakly autoreactive, but not strongly autoreactive T cells, causing increased phosphorylation of S6 kinase, enhanced proliferation, and greater Th1 skewing. Taken together, these findings suggest that direct effects of the PTPN22 rs2476601 variant represents a loss of function in T cells, increasing TCR signaling in mildly autoreactive T cells, potentially expanding the pool of autoreactive T cells and skewing them toward an inflammatory phenotype.