Decoupling T Cell Cytotoxicity: A CCL21+ICAM1-Based Synthetic Immune Niche Enhances Tumor Elimination by Accelerating Lytic Hit Delivery

Abstract

Preserving T cell cytotoxic function during ex vivo expansion remains a major challenge for adoptive cancer immunotherapy. A synthetic immune niche (SIN) composed of immobilized CCL21 and ICAM1 was shown to improve T cell expansion while preserving cytotoxicity; however, it remains unclear which specific step of the T cell killing process is enhanced by the SIN stimulation. Here, we combined advanced imaging based on time-lapse microscopy with a mean-field model to analyze the distinct stages in killing of B16 melanoma cells by CD8+ T cells. This framework enabled us to resolve the tumor cell killing process into discrete steps throughout target cell engagement with SIN-treated T cells and lytic hit delivery. We found that tumor cell death is best explained by a multi-hit process, requiring approximately four discrete hits to trigger cell death. Model-based analysis identified an increase in the lytic hit delivery as the parameter that best accounts for the enhanced cytotoxicity of SIN-treated T cells, a difference not explained by changes in target encounter frequency or conjugate stability. Global sensitivity analysis further showed that tumor control is more strongly improved by enhancing lytic hit delivery than by increasing target encounter rates. These findings fundamentally reorient our understanding of optimized T cell manufacturing, suggesting that the lytic execution step may be the primary rate-limiting bottleneck in this in-vitro system, and that engineering strategies targeting granule polarization or discharge warrant prioritization alongside affinity-enhancement approaches.