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[Formula: see text] 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 a significant increase in the lytic hit-delivery rate, [Formula: see text], as the main kinetic change associated with the enhanced cytotoxicity of SIN-treated T cells. The target encounter rate, [Formula: see text], could not be reliably compared between conditions because it was practically non-identifiable in the untreated dataset. Global sensitivity analysis further showed that increasing lytic hit delivery has a stronger effect on tumour control than 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.