Wnt Signaling Downregulation Mediates T Cell Apoptosis Following OKT3 ‐Induced T Cell Activation in Preclinical Models

ABSTRACT OKT3 is a murine monoclonal antibody (mAb) directed against human CD3 expressed on T cells and is known to induce T cell activation followed by activation‐induced cell death. This study investigated the role of canonical Wnt signaling in OKT3‐mediated T cell activation and apoptosis using preclinical models. In primary human PBMCs, OKT3 treatment downregulated the expression of canonical Wnt signaling‐related genes, whereas the Wnt pathway activator CHIR99021 upregulated their expression and reversed the OKT3‐induced suppression. OKT3 stimulation upregulated T cell activation markers (CD69, CD25), particularly in CD8 + T cells, and induced apoptosis as assessed by flow cytometry. Co‐treatment with CHIR99021 attenuated OKT3‐induced CD25 expression and late apoptosis, suggesting that Wnt signaling modulates T cell activation and cell death. In a humanized mouse model, OKT3 administration led to marked depletion of hCD3 + , hCD4 + , and hCD8 + T cells in the peripheral blood. Transcriptomic profiling using RNA‐seq of liver‐infiltrating human immune cells revealed significant upregulation of genes involved in T cell activation, apoptosis, cytokine signaling, and oxidative stress, accompanied by widespread downregulation of Wnt signaling genes (CTNNB1, LRP6, DVL1, FZD family). Therefore, these findings suggest that OKT3 induces T cell responses characterized by activation and subsequent apoptosis, in association with Wnt signaling. This study provides mechanistic insight into OKT3‐mediated immunomodulation and supports preclinical evaluation of T cell‐targeting monoclonal antibodies.