Mayhem under the microscope: T cell cytotoxicity and serial killers captured in situ.

Abstract

2540 Background: Developing a deeper understanding of the dynamics of immune cell-mediated cytotoxicity is critical to advancing immunotherapy and cell therapy. The results from the multidisciplinary effort reported here include numerous measurements and movies of immune cell-mediated cytotoxicity with striking examples of serial killing, foraging, path-tracking, triple killing events, measurements of cytokine gradients at tumor margins, and other dynamics. Some cytotoxic events revealed peak apoptotic signatures just minutes after T Cell engagement. Methods: In vitro studies of immune cell killing are traditionally performed using time-lapse imaging and biochemical assays, but these methods are often limited by spatial and temporal resolution, throughput, and the ability to extract the dynamics of cellular interactions. This study integrates high-resolution and high-speed laser scanning confocal microscopy with artificial intelligence (AI), and machine learning (ML) approaches to provide a high-resolution data-driven analysis of immune cell killing dynamics in vitro. In these studies, we use human CAR T cells with an anti-cd19.28z and Burkitt Lymphoma. Results: We have engineered a perfusion-enabled 3D culture system integrated microscopy to assess cellular dynamics for extended periods of time. Perfusion culture maintains the interstitial flow of liquid culture media, clearing the microenvironment of toxic metabolites and reactive oxygen species. This platform uses a Liquid-Like Solids (LLS) to mimic the transport dynamics of a capillary bed. Integrated microscopy allows in situ quantification of spatiotemporal cytokine concentrations, immune cell tracking, immune cell killing dynamics, and invasion dynamics. Cytokine on and off-rates were referenced alongside measured bead fluorescence intensities and positions to fit spatiotemporal reaction-diffusion models out to a 1,600 um radius. Fast-scanning confocal microscopy facilitated in-situ observation of the evolutionary dynamics of tumor progression. In-situ cytokine measurements revealed local IL-8 concentrations reached a maximum value of 2 ng ml-1 after 10 hours. A cellular production rate was estimated at 2 molecules cell-1 s-1. Conclusions: T Cell cytotoxicity is shown to be incredibly heterogeneous spanning from minutes to hours. The platform developed in this study demonstrates a powerful method for real-time, high-resolution imaging of cancer-immune interactions within a controlled 3D environment. By leveraging in situ fast-scanning fluorescence microscopy, the platform enables precise quantification of spatiotemporal cytokine concentrations, T cell motility, proliferation, cytotoxic activity, and tumor invasion patterns.