Immune synapse molecules knockout in induced pluripotent stem cells enables broad NK cell resistance in T cell progeny.

INTRODUCTION: Allogeneic iPSCs provide a potential source for regenerative T cell therapies, yet their clinical application remains limited by immune rejection driven by human leukocyte antigen (HLA) incompatibility. Knockout of β2-microglobulin (B2M) eliminates HLA class I expression and protects against CD8(+) T cell-mediated killing, but paradoxically provokes natural killer (NK) cell activation via the "missing-self" response. Existing approaches seek to enhance inhibitory signaling or dampen activating signals, yet these approaches achieve only partial NK evasion due to the extraordinary heterogeneity of NK receptor repertoires. METHODS: We developed a broader and more universal immune evasion strategy by targeting immune synapse (IS) adhesion molecules that critical for NK-target cell engagement. Building on our previously reported hypoimmunogenic iPSC-derived T cell (iT cell) platform (B2M(KO)CIITA(KO)PVR(KO) with HLA-E overexpression), we engineered double-knockout (dKO) iT cells lacking CD54 (ICAM-1) and CD58 (LFA-3) using CRISPR/Cas9-mediated gene editing. These two ligands play key roles in stabilizing NK-target immunological synapses. RESULTS: Functionally, dKO iT cells exhibited marked resistance to NK cell-mediated cytotoxicity both in vitro and in vivo in human IL-15 transgenic mouse model, while maintaining iT cell cytotoxic effector functions. By disrupting the physical interface required for NK engagement, this approach provides broad protection against diverse NK cell subsets and complements existing HLA-focused immune evasion strategies. CONCLUSIONS: Our findings establish a potentially versatile platform for generating universal, NK-resistant iT cells and advance the translational potential of iPSC-based immunotherapies.