T cells expressing chimeric antigen receptors (CARs) are at the forefront of clinical treatment of cancers. Still, the nanoscale organization of CARs at the interface of CAR-Ts with target cells, which is essential for TCR-mediated T cell activation, remains poorly understood. Here, we studied the nanoscale organization of CARs targeting CD138 proteoglycans in such fixed and live interfaces, generated optimally for single-molecule localization microscopy. CARs showed significant self-association in nanoclusters that was enhanced in interfaces with on-target cells (SKOV-3, CAG, FaDu) relative to negative cells (OVCAR-3). CARs also segregated more efficiently from the abundant membrane phosphatase CD45 in CAR-T cells forming such interfaces. CAR clustering and segregation from CD45 correlated with the effector functions of Ca++ influx and target cell killing. Our results shed new light on the nanoscale organization of CARs on the surfaces of CAR-Ts engaging on- and off-target cells, and its potential significance for CAR-Ts' efficacy and safety.

Cell-cell interfaces convey mechanical and chemical information in multicellular systems. Microscopy has revealed intricate structure of such interfaces, yet typically with limited resolution due to diffraction and unfavourable orthogonal orientation of the interface to the coverslip. We present a simple and robust way to align cell-cell interfaces in parallel to the coverslip by adhering the interacting cells to two opposing coverslips. We demonstrate high-quality diffraction-limited and super-resolution imaging of interfaces (immune-synapses) between fixed and live CD8+ T-cells and either antigen presenting cells or melanoma cells. Imaging methods include bright-field, confocal, STED, dSTORM, SOFI, SRRF and large-scale tiled images. The low background, lack of aberrations and enhanced spatial stability of our method relative to existing cell-trapping techniques allow use of these methods. We expect that the simplicity and wide-compatibility of our approach will allow its wide dissemination for super-resolving the intricate structure and molecular organization in a variety of cell-cell interfaces.

Single-molecule-localization-microscopy (SMLM) enables superresolution imaging of biological samples down to ~ 10-20 nm and in single molecule detail. However, common SMLM reconstruction largely disregards information embedded in the entire intensity trajectories of individual emitters. Here, we develop and demonstrate an approach, termed time-correlated-SMLM (tcSMLM), that uses such information for enhancing SMLM reconstruction. Specifically, tcSMLM is shown to increase the spatial resolution and fidelity of SMLM reconstruction of both simulated and experimental data; esp. upon acquisition under stringent conditions of low SNR, high acquisition rate and high density of emitters. We further provide detailed guidelines and optimization procedures for effectively applying tcSMLM to data of choice. Importantly, our approach can be readily added in tandem to multiple SMLM and related superresolution reconstruction algorithms. Thus, we expect that our approach will become an effective and readily accessible tool for enhancing SMLM and superresolution imaging.

T cells engage antigen-presenting cells in search for cognate antigens via dynamic cell protrusions before forming a tight immune synapse. The spatiotemporal events that may lead to rapid TCR triggering and signal amplification in microvilli-driven isolated contacts, and in subsequent, more uniform contacts, remain poorly understood. Here, we combined interference-reflectance microscopy and single-molecule localization microscopy in live cells to resolve TCR-dependent signaling at tight cell contacts. We show that early contacts are sufficient for robust TCR triggering and ZAP-70 recruitment. With cell spreading, TCR activation and ZAP-70 recruitment increase and shift to the edges of the growing tight contacts. CD45 segregates from TCR at tight contacts and is enriched at high local curvature membrane. Surprisingly, cortical actin and LFA localized at contact regions of intermediate tightness. Our results show in molecular detail the roles of early and tight T cell contacts in T cell activation, as both sensing and decision-making entities.
Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.

Molecular interactions across intercellular interfaces serve to convey information between cells and to trigger appropriate cell functions. Examples include cell development and growth in tissues, neuronal and immune synapses (ISs). Here, we introduce an agent-based Monte-Carlo simulation of user-defined cellular interfaces. The simulation allows for membrane molecules, embedded at intercellular contacts, to diffuse and interact, while capturing the topography and energetics of the plasma membranes of the interface. We provide a detailed example related to pattern formation in the early IS. Using simulation predictions and three-color single molecule localization microscopy (SMLM), we detected the intricate mutual patterning of T cell antigen receptors (TCRs), integrins and glycoproteins in early T cell contacts with stimulating coverslips. The simulation further captures the dynamics of the patterning under the experimental conditions and at the IS with antigen presenting cells (APCs). Thus, we provide a generic tool for simulating realistic cell-cell interfaces, which can be used for critical hypothesis testing and experimental design in an iterative manner.