Poster P19 - Enhancement of T Cell Transmigration and Infiltration Into the Solid Tissues by Engineering of the Lymphocyte’s Mechanobiological Phenotype.

Introduction: Engineering the invasive cytotoxic T-cells to infiltrate solid tumors is paramount to successful immunotherapies of solid tumor cancers. Resolving this problem has been riddled with numerous challenges rooted in the need for an adequate mechanobiological model of T cell motility within confined microenvironments of solid tissues. We have deciphered the invasive behaviors of the primary human CD8+ cytotoxic T-cells and determined that dynein motor protein-generated forces that emerge in the microtubular (MT) network are the principal driving forces that enable T-cells’ transmigration, infiltration, and effective movement of their nuclei within the tightly confined environments (nucleokinesis). Surprisingly, myosin-driven cell contractile forces play no principal role during lymphocytes’ migration, transmigration, and invasive dynamics. We also determined that septin GTPase activity is a molecular switch that activates dynein-driven T-cell locomotion. Specifically, SEP9 and SEP7 regulate the amoeboid-mesenchymal T-cell transition via the MT post-translational modifications and activation of the dynein motricity. By targeting septin’s GTPase activity with the novel inhibitor (UR214-9), we have significantly enhanced the T- and NK-cell transmigration efficiency and responsiveness to the guidance cues.

Materials and

Methods: We isolated primary human CD8+ T-cells from whole human blood for our experiments. These cells were then activated using anti-CD3 and anti-CD28 antibodies in the presence of IL-2 interleukin. To test T-cell adhesiveness and guidance along their principal adhesion ligand (ICAM-1), we used (1) 1-µm-wide micro-patterned lanes and grids on polyacrylamide surfaces as 2D system and (2) reticular collagen matrices and transmigration (transwell) membranes as 3D systems. This setup allowed us to observe and measure the movement and behavior of the T- and NK-cells under spatially and mechanically controlled conditions. The live confocal microscopy enabled the direct real-time observation of the T-cell dynamics, structural changes, responsiveness to their microenvironments, and reorganization of their organelles' spatiotemporal positioning. AI deep-learning and automated algorithms enabled the quantification of the lymphocytes’ structural and migratory dynamics. Pharmacological targeting of septin’s GTPase activity with UR214-9 enabled studying of the T-cell amoeboid-mesenchymal transitioning. Dynarrestin, Dynapyrazole-A, and Ciliobrevin-D targeted dynein’s motricity and binding to the MT network to dissect and decipher the mechanobiological role of the dynein-generated forces during lymphocyte migration within the mechano-structurally various 2D and 3D biomimetic environments. Super-resolution microscopy (iSIM, STED, AiryScan-2) revealed the lymphocyte's organization and organelles’ positioning during the amoeboid-mesenchymal transition.

Results, Conclusions, and Discussions: Targeted inhibition of septin GTPase activity causes the immediate transition of T and NK cells from the amoeboid phenotype towards mesenchymal-like cell dynamics, exhibiting extreme cell structural conformity and elongation along the narrow guidance cues. This phenomenon is accompanied by the substantially enhanced T and NK cell transmigration efficiency. The observed cell morphological transformation and enhanced conformity also reflect the cell’s ability to fit into the tight spatial confinements of the solid tissues, supported by enhanced transmigration. These observations and conclusions were orthogonally tested by measuring lymphocyte transmigration/migration in the transmigration chambers (transwells), 3D granular scaffolds, 3D collagen matrices, and live tissues. Pharmacological inhibition or genetic suppression of the dynein’s motricity in the mesenchymal-like lymphocytes abrogates nucleokinesis, decreases cell conformity and adhesion to the guidance cues and confinements, and arrests cell transmigration. On the other hand, myosin-generated forces were non-essential for all listed above behaviors.
Septin inhibition-induced mesenchymal-like lymphocyte phenotype displays an enhanced oscillatory nucleus translocation driven by the dynein motors along the microtubules (nucleokinesis). We demonstrated that this phenomenon is essential for effective T and NK cell migration and transmigration through spatially tight environments. Similarly, we show that metastatic cancer cells share the identical dynein-driven migratory mechanism as the T and NK cells. Analysis of the septin expression profile indicates that immune and mesenchymal cancers display the upregulation of the SEP9 isoform-1 (SEP9_i1) that acts as both dynein-dynactin complex activator and as MAP-like protein with MT-stabilizing function. We demonstrated that these SEP9_i1 functions require inhibiting its GTPase activity to get released from the septin heteropolymers. Thus, we conclude that SEP9_i1 is the key to engineering immune cell infiltrating migration phenotype.

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