Human T-cell receptor triggering requires inactivation of Lim kinase-1 by Slingshot-1 phosphatase.

Actin dynamics control early T-cell receptor (TCR) signalling during T-cell activation. However, the precise regulation of initial actin rearrangements is not completely understood. Here, we have investigated the regulatory role of the phosphatase Slingshot-1 (SSH1) in this process. Our data show that SSH1 rapidly polarises to nascent cognate synaptic contacts and later relocalises to peripheral F-actin networks organised at the mature immunological synapse. Knockdown of SSH1 expression by CRISPR/Cas9-mediated genome editing or small interfering RNA reveal a regulatory role for SSH1 in CD3ε conformational change, allowing Nck binding and proper downstream signalling and immunological synapse organisation. TCR triggering induces SSH1-mediated activation of actin dynamics through a mechanism mediated by Limk-1 inactivation. These data suggest that during early TCR activation, SSH1 is required for rapid F-actin rearrangements that mediate initial conformational changes of the TCR, integrin organisation and proximal signalling events for proper synapse organisation. Therefore, the SSH1 and Limk-1 axis is a key regulatory element for full T cell activation.

CRISPR/Cas9-mediated genome editing assists protein dynamics studies in live cells.

Spatial and temporal regulation of molecular reactions dictates cell fate. Thus, studying molecular dynamics is essential to understand how cells decide what to do and the fundamental perturbations causing disease. Classically, molecular dynamics has been studied by protocols based in the overexpression of fluorescent fusion proteins. However, overexpression is associated to altered stoichiometry, molecular dynamics and subcellular distribution. We here discuss the necessity to study molecular dynamics of fluorescent fusion proteins expressed under physiological mechanisms in the cell, pointing to CRISPR/Cas9-mediated genome editing as the ideal means to do so. Current genome editing protocols enable us to study molecular dynamics while avoiding drawbacks associated to overexpression.

Bispecific Antibody Format and the Organization of Immunological Synapses in T Cell-Redirecting Strategies for Cancer Immunotherapy.

T cell-redirecting strategies have emerged as effective cancer immunotherapy approaches. Bispecific antibodies (bsAbs) are designed to specifically recruit T cells to the tumor microenvironment and induce the assembly of the immunological synapse (IS) between T cells and cancer cells or antigen-presenting cells. The way that the quality of the IS might predict the effectiveness of T cell-redirecting strategies, including those mediated by bsAbs or by chimeric antigen receptors (CAR)-T cells, is currently under discussion. Here we review the organization of the canonical IS assembled during natural antigenic stimulation through the T cell receptor (TCR) and to what extent different bsAbs induce T cell activation, canonical IS organization, and effector function. Then, we discuss how the biochemical parameters of different formats of bsAbs affect the effectivity of generating an antigen-induced canonical IS. Finally, the quality of the IS assembled by bsAbs and monoclonal antibodies or CAR-T cells are compared, and strategies to improve bsAb-mediated T cell-redirecting strategies are discussed.