Abstract Rationale Understanding the motility dynamics of CD8+ cytotoxic T cells is of critical importance in the context of solid tumors, as greater T cell infiltration and killing activity is closely related to improved patient outcomes and therapeutic efficacy. Investigating T cell motility within the tumor microenvironment therefore provides insight into the endogenous immune response and may inform strategies to enhance T cell-based cytotoxic activity and therapies. However, conventional approaches to studying T cell movement and localization often depend on simplified two- or three-dimensional systems that fail to capture the structural complexity of living tissue, or on static histology that lacks temporal resolution. These shortcomings are particularly exacerbated in the context of the lung, a structurally unique and mechanically active environment. To overcome these limitations, we examine the behavior of native, migratory CD8+ T cells responding to pulmonary metastasis within the intact, functional murine lung via the crystal ribcage platform (Nature methods, 2023, Fig. 1A). By leveraging this platform, which enables high-resolution imaging of the lung while preserving physiological ventilation and perfusion, we characterize the effects of a range of physiological and immunological factors including tumor immunogenicity, tumor size, age, mechanical strain (i.e., tidal volume, TV), and therapeutic application on CD8+ T cell motility. Methods Models of pulmonary melanoma (Yummer1.7-H2B-GFP, B16F10-GFP) metastasis to the lung are induced in C57BL6/J mice (Fig. 1B). Native CD8+ T cells are labeled in situ using fluorophore-conjugated CD8-targeting nanobodies (Fig. 1C) before the lung is resected and imaged using the crystal ribcage while preserving active ventilation. Conditions probed include immunogenicity (strong vs weak immune response), tumor size, age (2-3 months vs 20-24 months), mechanical strain (high TV vs low TV), and therapeutic application (-PD1). Results Using the crystal ribcage, we imaged actively migrating, endogenous CD8+ T cells within the tumor microenvironment and surrounding noncancerous tissue. We find that immunogenicity increases T cell motility (Fig. 1D, 1E) while age (Fig. 1F, 1G) and high mechanical strain (Fig. 1H, 1I) notable decrease T cell speeds. Application of therapeutic doses of checkpoint blockade inhibition alter speeds of cells compared to no-treatment groups (Fig. 1J), with a pronounced reduction in speed in intratumoral populations. Conclusions Immune cell motility can be influenced by a variety of physiological and immunological factors. Using the crystal ribcage, we perturb the lung across a range of biological and mechanical axes and quantify changes in CD8+ T cell motility in the active, functional lung. This abstract is funded by: DP2HL168562, Beckman Young Investigator Award, NSF CAREER Award, DoD Idea Award
Regan et al. (Fri,) studied this question.