High-throughput time-lapse imaging of B16 target cell killing by ovalbumin-specific CD8⁺ T cells

Cytotoxic T Lymphocytes (CTLs) mediate tumor clearance and anti-tumoral responses through the elimination of target cancerous cells upon antigen recognition and TCR engagement. The killing of cancerous cells mediated by CTLs can be modulated by the strength of the TCR engagement, notably through the antigen affinity to the TCR, the quality of the immune synapse, the time of contact between CTLs and targets, among other parameters. Unveiling the mechanisms, kinetics and factors influencing cancer cell elimination by CTLs is essential for the amelioration of lymphocyte-based immunotherapies, such as CAR-T cells, and the understanding of tumoral clearance in vivo. Here, we provide a method to precisely monitor the cytotoxic capacity of murine CTLs in an antigen-specific manner, using ovalbumin (OVA) specific CTLs expressing the modified OT1 TCR and four different tumoral B16-F10 cell lines expressing OVA peptides with variable affinity for the OT1 TCR. We present step-by-step the implementation of a high throughput image-based pipeline allowing, on one hand, the time-lapse monitoring of CTL-mediated cytotoxicity and, on the other hand, the measurement of morphological parameters of CTLs and target cells based on fluorescent labeling. This method is suitable for ex-vivo exploration of lymphocyte cytotoxicity and related parameters, and can be adapted for mechanistic studies or screening approaches.