Anti-CD3 Fab Fragments Enhance Tumor Killing by Human γδ T Cells Independent of Nck Recruitment to the γδ T Cell Antigen Receptor

T lymphocytes expressing the gamma delta T cell receptor (gamma delta TCR) can recognize antigens expressed by tumor cells and subsequently kill these cells. gamma delta T cells are indeed used in cancer immunotherapy clinical trials. The anti-CD3 epsilon antibody UCHT1 enhanced the in vitro tumor killing activity of human gamma delta T cells by an unknown molecular mechanism. Here, we demonstrate that Fab fragments of UCHT1, which only bind monovalently to the.d TCR, also enhanced tumor killing by expanded human V gamma 9V delta 2 gamma delta T cells or pan-gamma delta T cells of the peripheral blood. The Fab fragments induced Nck recruitment to the gamma delta TCR, suggesting that they stabilized the.d TCR in an active CD3 epsilon conformation. However, blocking the Nck-CD3 epsilon interaction in gamma delta T cells using the small molecule inhibitor AX-024 neither reduced the gamma delta T cells' natural nor the Fab-enhanced tumor killing activity. Likewise, Nck recruitment to CD3 epsilon was not required for intracellular signaling, CD69 and CD25 up-regulation, or cytokine secretion by gamma delta T cells. Thus, the Nck-CD3 epsilon interaction seems to be dispensable in gamma delta T cells.