Enhancement of the killing capacity of CD8+ T-cells following stimulation with substrate-bound CCL21 and ICAM 1

Adoptive cancer immunotherapies use our immune T-cells for an effective arrest of cancer development. The main challenge of these immunotherapies is the necessity to effectively expand the cancer-specific T-cell population while maintaining or even enhancing their functionality. In an attempt to meet this challenge, a novel approach was recently developed in our laboratory, based on ex-vivo culturing of CD8+ T-cells on a synthetic surface that mimics the natural immune environment in the lymph node, termed “synthetic immune niche” (SIN). Previous studies have shown that substrate-attached CCL21 and ICAM1 enhance the proliferation rate as well as the cytotoxicity of CD8+ T cells. To accurately monitor the changes in CD8+ T-cells, caused by the SIN stimulation, we developed an image-based approach for quantifying the cells’ killing capacity and the morphological changes induced in the T-cells by the SIN. The assay uses an advanced light microscopy and deep learning-based segmentation software that identifies the T cells and target cells during the killing process and analyzes various parameters related to the activation state of T-cells and the killing dynamics. We found that the increased killing capacity of T-cells pre-treated by the SIN, corresponds to specific changes in T-cells area and circularity. The results show that the SIN induces an increase in cell area and decrease in cell circularity, which are, apparently highly active cells with high killing capacity. Our results suggest that this SIN may be used for generating T-cells with augmented cytotoxic functions, for use in cancer immunotherapy.