Quantitative Imaging Approaches to Study the CAR Immunological Synapse

Please cite this article in press as: Mukherjee et al., Quantitative Imaging Approaches to Study the CAR Immunological Synapse, Molecular Therapy (2017), http://dx.doi.org/10.1016/j.ymthe.2017.06.003

Review

Quantitative Imaging Approaches to Study the CAR Immunological Synapse Malini Mukherjee,1,2,3 Emily M. Mace,1,3 Alexandre F. Carisey,1,3 Nabil Ahmed,1,2 and Jordan S. Orange1,2,3 1Department

of Pediatrics, Baylor College of Medicine, Houston, TX 77025, USA; 2Cell and Gene Therapy, Baylor College of Medicine, Houston, TX 77025, USA; 3Center

for Human Immunobiology, Texas Children’s Hospital, Houston, TX 77030, USA

The lytic immunological synapse (IS) is a discrete structural entity formed after the ligation of specific activating receptors that leads to the destruction of a cancerous cell. The formation of an effector cell IS in cytotoxic T lymphocytes or natural killer cells is a hierarchical and stepwise rearrangement of structural and signaling components and targeted release of the contents of lytic granules. While recent advances in the generation and testing of cytotoxic lymphocytes expressing chimeric antigen receptors (CARs) has demonstrated their efficacy in the targeted lysis of tumor targets, the contribution and dynamics of IS components have not yet been extensively investigated in the context of engineered CAR cells. Understanding the biology of the CAR IS will be a powerful approach to efficiently guide the engineering of new CARs and help identify mechanistic problems in existing CARs. Here, we review the formation of the lytic IS and describe quantitative imaging-based measurements using multiple microscopy techniques at a single cell level that can be used in conjunction with established population-based assays to provide insight into the important cytotoxic function of CAR cells. The inclusion of this approach in the pipeline of CAR product design could be a novel and valuable innovation for the field. Chimeric antigen receptors (CARs) are engineered chimeric proteins that combine the extracellular portion of a highly specific antibody single-chain variable fragment (scFv) targeted to a cancer antigen with an intracellular signaling chain of the T cell receptor (TCR) (Figure 1). The predecessors of currently applied CARs were formed by linking the variable domains of antibodies (VH + VL, Figure 1) to the CD3z unit of the TCR, hence using the conventional TCR signaling machinery with antibody type-specificity as activation stimulus.1 These early studies demonstrated that the TCR z cytoplasmic domain alone linked to a transmembrane receptor could successfully reconstitute TCR signaling2 and paved the way to a first-generation single-chain CAR in which a scFv was linked to the CD3z cytoplasmic domain.3 The second- and third-generation CARs were developed by inserting domains from co-stimulatory receptors into the singlechain CAR4 (Figure 1) or on a second chain to allow trans co-stimulation via a chimeric co-stimulatory receptor (CCR).5 Second-generation CARs targeting CD19 on B cell cancers and with a CD28 co-stimulatory domain6 or 4-1BB signaling capacity provoked potent cytotoxicity against acute lymphoblastic leukemia (ALL). These

were among the first CAR molecules successfully applied in clinical trials.7 The success of CD19 CAR T cells in treating and inducing complete remission of B cell cancers underscores their powerful impact on cancer immunotherapy. Ongoing research is being conducted to identify new CAR targets in cancers and further advance CAR cell design using T cells, NK (natural killer) cells, and NKT (natural killer T) cells as effectors. Co-stimulatory domains from other T cell surface receptors are being included (such as OX408), with the hope of harnessing their cell survival and proliferation abilities and hence improve efficacy. As this field progresses and incorporates greater sophistication in the design and testing of promising CARs, understanding the mechanism of their relative success or failure will rely increasingly on our understanding of the underlying cell biology of CAR effector cells. Basic comparative observations of the cytotoxic activities of CAR engineered T cells and T/NK cells hint at a generally conserved overall mechanism.9 To mediate the destruction of a targeted cell, conventional T cells form a highly organized and regulated immunological synapse (IS) with target cells.10,11 The role of this structure is to generate activation signaling and promote directed secretion of lytic granules onto the targeted cell until its death by apoptosis. Extraordinary advances have been made in the understanding of the cytolytic immune cell synapse by the application of quantitative imaging studies using diffraction-limited and super-resolution microscopy.12–16 However, little was known about whether the IS formed by CAR-expressing cells is similar in structure and behavior to the one observed in the various types of CTLs (cytotoxic lymphocytes). The missing link was demonstrated only recently9 and the impact of this is 2-fold. The study established that some of the spatio-temporal organization of specific individual components of a CAR molecule impacts the effectiveness of CAR-mediated killing; additionally, the outcome of the cytotoxic activity of the CAR could be accurately described by the quantitative analysis of known features of IS formation in T and NK cells. This work suggests that the landmark attributes of the IS of T/NK cells can be identified in the CAR-mediated IS and can be used to evaluate CAR T cell cytotoxicity, http://dx.doi.org/10.1016/j.ymthe.2017.06.003. Correspondence: Malini Mukherjee, PhD, Department of Pediatrics, Baylor College of Medicine, 1102 Bates Street, Suite 330, Houston, TX 77025, USA. E-mail: [email protected]

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Review

Figure 1. Evolution of CAR Designs Schematic model of typical CAR designs each representing a different generation (main panel). The elementary blocks used for their design are put back into their original context in the insets: the top panel shows the domains of a monoclonal antibody used to create the target-specific scFv domain of the CAR, while the bottom inset depicts the signaling receptors (CD3z, CD28, 4-1BB, OX40) that provide the CAR intracellular signaling domain. The first-generation CAR has a single activation signal, while the secondgeneration CAR integrates activation and a single co-stimulatory signal in cis. The third generation of CARs includes more than one co-stimulatory signal that can be presented in cis or in trans as a co-stimulatory receptor (CCR) when paired with a first-generation CAR. mAb, monoclonal antibody.

providing the required proof of principle to justify the use of microscopy-based assays described in the present work. An extension of the already established quantitative imaging approaches to visualize and understand the CAR cell synapse is thus relevant and timely. In this review, we first recapitulate the key features and the timeline of the IS from its formation until the death of the sensitive target cell, drawing from the knowledge established using T and NK cells. Next, we use the description of the general steps of the current CAR design pipeline to emphasize the challenges that can benefit from microscopy-based assays. Then, we describe in greater details four quantitative methods, some of which have been instrumental to elucidate complex diseases and immunodeficiencies, and how they can potentially predict functionality of CAR cytolytic synapse. Besides the existing set of methods derived from NK/T cell biology, we suggest future directions for the development of innovative quantitative imaging studies to characterize CAR cell behavior. Finally, we focus on how these approaches can be practically applied to ongoing research by outlining the improved roadmap for the development and mechanistic validation of a novel type of CAR. Immune Synapse Formation: A Multi-step Process

The IS, first visualized by high-resolution microscopy over 25 years ago, represents a specialized interface formed between an immune effector cell and a target or co-stimulatory cell.10,11 Originally described in the context of T cell-antigen-presenting cell (APC) interactions, the definition has been expanded to many different types of

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immune cell-cell interactions, including the NK cell lytic and inhibitory synapses,17 T cell lytic synapses,18,19 and NK to dendritic cell (DC) synapses.20 The original criteria for an IS still hold true, however, and include direct contact with a target cell, polarization of the immune cell, and productive signaling across the synapse.21,22 The formation of a cytotoxic cell immune synapse has been extensively reviewed by others and is a multi-step process that requires elaborate and coordinated molecular and structural rearrangements within the target and effector cells.23 This process is dependent not only on the affinity and avidity of receptors and ligands but also on their density, conformation, and localization within the immune synapse. In utilizing the interaction between receptors and ligands to initiate contactdependent cellular functions, there are a series of steps with which the immune cell can progress to access immune cell function. These can be divided into three broad stages of immune synapse formation: the initiation, effector, and termination stages (Figure 2). The initiation stage includes the interaction with a potential target cell by the effector cell through tethering and adhesion receptors. This leads to engagement of activation receptors such as the TCR with cognate antigen-presenting MHC (major histocompatibility complex) molecules. In a lytic synapse, the effector stage represents the molecular and cell biological rearrangements that enable the effector cell to kill its target cell. These include critical and mostly sequential mechanical processes such as signaling via dynamic microclusters (MCs) of activating receptors, filamentous (F)-actin polymerization at the IS,

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Review

Figure 2. Schematic Representation of the Critical Steps of the Killing of a Sensitive Target by a CAR-Expressing Effector Cell The “initiation” stage includes the recognition and the ligation of the CAR receptor with its ligand at the surface of the target cell. Following intracellular signaling, the receptors at the surface of the effector converge and aggregate toward the interface between the two cells where the IS is forming. The following stage is referred to as the “effector stage” and encompasses multiple discrete steps: the accumulation of filamentous actin at the IS, the convergence of the lytic granules around the microtubule organizing center (MTOC), the polarization of the granule-loaded MTOC against the plasma membrane at the IS and, ultimately, the release of the cytolytic granules in the IS cleft leading to the killing of the target cell. The final stage closing the cycle is the “termination” stage: the target cell carries on its apoptotic process and then separates from the effector, allowing the latter to proceed with the killing of another target cell, a mechanism called serial killing. If the effector cannot regenerate its granule content, it remains depleted and enters a stage of exhaustion. Note that the receptor and ligands at the cell surface of the cells are only depicted in one panel for clarity purposes but are present at all stages.

granule convergence, and microtubule organizing center (MTOC) polarization, all of which are precisely timed events that orchestrate the final lytic kill. The termination stage includes detachment of the effector from its killed target cell, a step that is required for immune homeostasis, as failure in detachment can lead to prolonged immune reaction and hyperinflammation.24 Cytolytic cells both in vivo and in vitro have rapid and efficient sequential killing ability, although both T and NK cells have significant heterogeneity in this capacity.25–28 When applied to the direct comparison of CAR- and TCR-mediated killing, this approach shows similar initial kinetics of serial killing, with individual CAR T cells mediating multiple lytic hits. However, attenuation of CAR-mediated killing occurred faster than for TCR-mediated killing, typically as a result of the rapid CAR downregulation.29 While CAR T and CAR NK cells are derived from their parent cytotoxic cell and therefore share many cell intrinsic components, they are

artificially engineered cells that use an antibody-antigen-based recognition of targets and might have altered dynamics for IS formation. It is thus insightful to analyze in great detail the timeline of CAR cell IS formation and lytic killing of its target cell using key intrinsic parameters that can be reproducibly interrogated and would be generalizable to most lytic CAR cells. Besides providing additional measures to interrogate CAR functionality, these approaches can serve as important checkpoints in the existing CAR design pipeline to further offset unanticipated problems as discussed below. CAR Design Pipeline

To identify the parameters of CARs that can be tuned following the evaluation of their killing potential, we must first briefly outline the critical steps in their design that present opportunities for alteration. The CAR design pipeline begins with the identification of a suitable antigen target overexpressed by a cancer cell, and then the scFv allows

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Review the selection of the most relevant and variable domain for the selected antigen, followed by optimization of all other components of the CAR molecule (spacer, transmembrane, and endodomains). The final CAR molecule is then expressed in PBMCs (peripheral blood mononuclear cells) after viral infection of the construct (Figure 3). Iterative testing of the final CAR product, both in vitro and in vivo, is then performed to achieve a better CAR, and suboptimal functioning can be a result of a design problem in any one of the CAR modules. For example, CAR scFv internalization upon antigen binding can result in inefficient CAR targeting and elimination of cancer cells. Inefficient CAR-mediated killing could be due to a less than optimal tumor antigen-to-CAR interaction caused by a suboptimal ligation of the scFv domain. Recent studies have reported that proximity of a target epitope to the CAR is crucial for efficient lytic synapse formation30 and imply this as an important design consideration for new CAR molecules. Antigen escape or low-affinity/avidity binding between CAR exodomains and target antigens can also result in reduced synapse strength or duration, both leading to ineffective killing. Similarly, the design of the transmembrane domain can dramatically affect the mobility of the receptor within the plasma membrane. It can be hypothesized that any limitation in CAR mobility would prevent sufficient aggregation of the receptor and might even impair activation altogether. The selection of the CAR endodomains is a critical step as well, as they can trigger tonic signaling within the CAR-expressing cell, resulting in effector suicide (self-killing) or fratricide (killing each other). These and other problems reported by us31–33 in the CAR validation process suggest that currently established evaluations fall short and that an innovative and more detailed approach should be considered. Alongside traditional assays currently used to validate CAR functionality, we propose the addition of a quantitative imaging evaluation of the CAR lytic synapse to identify and improve upon CAR design. Specifically, our published and unpublished work has identified four generalizable measures of the CAR lytic synapse and successfully used them to interrogate CAR functionality and efficacy early in the CAR development pipeline. The four measures described here are (1) CAR and antigen aggregation at the IS, (2) F-actin polymerization, (3) granule convergence to the MTOC, and (4) MTOC polarization to the IS. Readily Quantifiable Parameters of CAR Lytic Synapse Efficiency and Their Rationale CAR and Antigen Aggregation at the CAR IS

CAR T cells depend on the high-affinity binding of the engineered CAR and target antigen expressed on the tumor cell surface to mediate killing. The affinity and avidity between the CAR extracellular domain (scFv) and the antigen as well as the length and flexibility of the hinge region can significantly affect CAR binding. Studies of the extracellular CAR domain have revealed that modifications in the affinity of CARs to their target antigens can significantly affect their cytotoxicity.34 Also, designing CAR T cells to uniquely

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target membrane-bound tumor antigens and not soluble forms of the same protein can improve CAR-mediated killing.35,36 In some instances, antigen-independent aggregation of CARs can cause tonic signaling leading to the premature exhaustion of CAR T cells.37 CAR hinge/spacer domain designs have also been reported to play an important role in the final cytolytic outcome by improving accessibility of target antigen by the CAR.38 Furthermore, in the context of targeting solid tumors that have a highly heterogeneous surface antigen profile, and antigen escape after CAR treatment can result in treatment failure, designing optimal CARs that can co-target more than one antigen is of essence.39 These studies point toward the utility of technologies that can be applied to a direct visual examination of the CAR T cell IS, with special emphasis on the CAR-antigen interaction and its correlation with downstream signaling and cytotoxicity. The recent report of a tandem CAR T molecule that co-targets Her2 and interleukin (IL)-13Ra2 expressed on glioblastoma illustrates the application of these methods. Super-resolution fluorescence imaging using STED (stimulated emission depletion) microscopy revealed that tandem CARs form a bivalent synapse, with distinct dimers of the individual target antigens aggregating at the IS. This is correlated with increased synapse strength with greater F-actin accumulation and MTOC polarization at the tandem CAR synapse. We thus propose the measurement of CAR and antigen aggregation at the IS as an initial parameter to evaluate CAR cytotoxicity. Practically, CAR and antigen aggregation at the IS can be readily measured by immunofluorescence microscopy (Figure 4A). The fractions of antigen and CAR at the IS are measured by drawing a region of interest (ROI) to identify only the target cell (for antigen) and effector cell (for CAR) and then using an intensity threshold to measure accumulation of each at the IS, as described previously by Banerjee and Orange40 for measurements of F-actin accumulation. Additionally, the use of the most recently developed super-resolution imaging generally grouped under the term of SMLM (single-molecule localization microscopy) can provide additional measurement down to the stoichiometry of the receptor-ligand couple or the multi-chain CAR at the cell membrane. For live cell imaging studies, the fusion of a CAR with a fluorescent protein allows quantification of recruitment and accumulation at the IS. These may be combined with live/dead cell reporter dyes to provide a direct feedback on the cytotoxicity of the current CAR, all of which could be used as iterative feedback for improvement on specific CAR designs. F-Actin Polymerization and Rearrangement

As illustrated in Figure 4, the next measurable step after the ligation of the CAR with its ligand on the sensitive target cell is the reorganization of the cytoskeleton resulting in the formation of an IS by rearrangement and de novo polymerization of actin monomers at the IS.41,42 Filamentous actin is arguably the most important structural component of the immune synapse and has been the focus of imaging-based studies of lytic synapse function for more than a decade.42 F-actin-mediated cytoskeletal rearrangements allow

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Review

Figure 3. Typical Workflow of CAR Design and Proposed Imaging-Based Interrogations The middle pipeline describes the successive steps leading to the production of an efficient CAR that can be evaluated in clinical trials. On the left side are the most common bulk assays currently in use to optimize and test CAR products. On the right side of the central stem is the list of microscopy-based experiments that we propose to gain a better insight into the mechanism of action of the current CAR. The quantitative results of these assays can be used iteratively to optimize the various stages of the CAR design pipeline.

migrating T cells to migrate and pause upon cognate peptide-loaded MHC (pMHC) recognition. This holds true for the migration of NK cells and during their surveillance of the neighboring cells as well, albeit there the search is for ligands for NK cell-activating receptors.43,44 Typically, the crucial roles for F-actin polymerization and rearrangement at the different stages of T cell synapse formation have been visualized by fluorescence microscopy using multiple T cell activation systems including antigen-loaded supported lipid bilayer (SLB)45 or coated glass coverslips,10 reviewed by Kumari et al.46 Pharmacological agents that block actin polymerization, including latrunculin A and cytochalasin D, have confirmed the critical role of F-actin polymerization specifically for maintaining T cell signaling downstream of TCR and LFA-1 engagement.47 Routinely, F-actin is detected for fluorescence microscopy using fluorophoreconjugated phalloidin. But the development of F-actin reporters compatible with live cell studies such as LifeAct48 and F-Tractin49 have emphasized the complex dynamics of F-actin that occur during synapse formation and function,50,51 challenging the originally simplistic view drawn from the study of fixed samples. Beyond the scaffolding role of the filamentous network of F-actin, myosin IImediated contractility of actin fibers in T cells regulates the assembly and movement of the newly generated TCR-MC (T cell receptor microcluster), as well as MTOC relocation.52–54 In NK cells, the actin cytoskeleton undergoes reorganization throughout the synapse to form a pervasive filamentous mesh. This specialized structure plays a critical role in the regulation of lytic granule secretion through the formation of minimally sized clearances.55–57 Because of its crucial role in IS formation, F-actin accumulation at the synapse has been a hallmark of a stable and functional cytolytic IS in T and NK cells. Impairment of the polymerization of actin at the IS

could be a consequence of defects in actin nucleation promoting factors such as Wiskott-Aldrich syndrome protein (WASp) and could lead to profound immunodeficiencies with aberrant or dysfunctional cytotoxic cells.58 Additionally, mutations in other F-actin regulators including Coronin1A,59,60 Vav1,61,62 and Dedicator of cytokinesis protein 8 (DOCK8)63–65 lead to immunodeficiency presumably as a direct result of defects in T cell homeostasis and trafficking, migration, or compromised IS formation. Quantitative imaging-based methods have been used to highlight the role of aberrant actin nucleation in deficient lytic function in the context of primary immunodeficiency diseases, and this has been used to monitor the rescue of function following lentiviral gene therapy in the case of WAS.66–68 When applied to CAR T cells, measurement of actin accumulation at the IS mirrors quantifiable differences in CAR cytotoxic potential as measured by better killing of primary glioblastoma (GBM) target cells in standard 15Cr-release assays and in in vivo intratumoral injection of CAR T cells in established GBM xenografts.9 This underscores its utility as a prognostic measure of CAR functional outcome. Given the dynamic range of this measure, we believe that this is an opportunity to gauge synapse strength as well. While conventional cytotoxicity assays indicate bulk target killing potential of a CAR product, measurement of F-actin directly assesses a much more limited step of the cytotoxic process and can hint at which stage of the design pipeline must be optimized. Measurement of F-actin at the IS may be done on fixed or live cell conjugates (Figure 4B). After acquisition of a three-dimensional volume encompassing the conjugate, a segmentation mask is used to isolate the volume of the effector from the target. Using an adjustable threshold to only include the signal from the F-actin staining present

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Review

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Review at the synapse, the fluorescence intensity of F-actin at the synapse relative to unconjugated regions of the cortex of the CAR T cell is calculated. Other methods to measure F-actin accumulation include ROI selection at the IS and calculation of F-actin present thereof as a product of mean fluorescence intensity (MFI) and volume of the ROI selected.40 This last method does not eliminate contribution of the target cell F-actin; therefore, a background subtraction of target cell surface un-polarized F-actin is necessary.

One variation of this method requires the measurement of the distance between each individual granule and the MTOC for all the granules in an effector (Figure 4C). These individual distances are then averaged to provide an averaged distance per cell. The principal benefit of this alternative method is the possibility to plot the standard deviation of the distribution of the granules to MTOC distances in each cell to facilitate identification of outliers (stranded granules, cell membrane-docked granules, or false detections).

Granule Convergence

The last alternative method for the measurement of granule convergence only relies on the detection of lytic granules. The distance between the center of each granule and the centroid of the region defined by all the granules (area-weighted distance calculation) is recorded and averaged per cell. This last method, albeit valid, shows a smaller dynamic range than the previously mentioned ones and should be only used when the localization of the MTOC is not practically possible.

Granule convergence is an early step in immune synapse formation and can occur independently of F-actin accumulation and commitment to cytotoxicity.69 First described in NK cells and more recently in CTLs,51 this rapid convergence of lytic granules from their cytoplasmic distribution toward the MTOC occurs prior to MTOC polarization to the synapse and thus poises the lytic effector cell to deliver targeted release of granules (Figure 4C). Lytic granule convergence is a coordinated process controlled by cytokine signaling or adhesion/activation receptor ligation.69–71 Quantitative measurements of both fixed cell and live cell images have been used to determine the distance of lytic granules to the MTOC in cytotoxic T cells and NK cells.51,69–72 Lytic granule convergence can be measured using at least two different approaches. One way is to use an area-weighted distance calculation to find the centroid of the lytic granules in the focal plane of the MTOC and measure the average distance between the granule centroid and the MTOC centroid as described in Mentlik et al.69 and Hsu et al.73 As convergence increases, the measured distance is reduced. The MTOC can be identified by immunofluorescence using antibodies directed against pericentrin, beta-tubulin, or gammatubulin or using silicon rhodamine (SiR) tubulin compound for live imaging. With the more general tubulin detection techniques that are less specific for MTOC, the MTOC needs to be defined as the accumulated foci of tubulin fluorescence as demonstrated by James et al.70 Lytic granules can be detected by an immunostaining of any of their major components74 (i.e., perforin or granzyme B, or by addition of fluorescent acidotropic probes such as LysoTrackers). As the distance of the lytic granules to the MTOC changes over time, granule convergence measurements in time-lapse imaging can be plotted and compared between various effector cells.

MTOC Polarization

A separate and independent measurement of the distance between the MTOC and the IS reflects MTOC polarization. This cytoskeletal reorientation is considered a later step toward cytotoxicity.23 Measurements of granule convergence have been used in combination with MTOC polarization to dissect the mechanism of cytotoxic dysfunction in primary immunodeficiencies. For example, mutations in RASGRP1,75 DOCK8,76,77 WASp,58 Rab27a,78 and MYH979,80 result in T and NK cells lacking the ability to polarize and/or converge their lytic machinery to the IS. Recent quantitative imaging-based studies have highlighted the importance of coordinated granule convergence and polarization to the IS in cytolytic T cells, a pathway that is frequently deregulated in some cancers, allowing target cell escape and survival.81 The lack of polarization of MTOC in chronic lymphocytic leukemia (CLL) patient-derived T cells in the presence autologous B cells81 provides a direct mechanistic insight into the cytotoxic failure of these cells and highlights the importance of our imaging-based approach to monitor localization of lytic granules and MTOC in activated effectors. Quantitative live cell imaging studies have contributed to understanding the spatio-temporal kinetics of lytic granule movement following delivery to the IS in response to TCR signals of varying affinity.82,83 These studies show

Figure 4. Simplified Schematics alongside Representative Volume Projections of Microscopy Data of CAR NK Cell/Target Cell Conjugates Demonstrating Four Quantifiable Parameters of IS Formation (A) CAR-antigen aggregation, (B) F-actin accumulation, (C) granule convergence, and (D) MTOC polarization. (A) The first panel series describes the accumulation of CAR at the IS formed with the target cell as one of the first measurable steps in CAR-mediated cytotoxicity. The representative fluorescence microscopy image shows the region overlapping between the CD19 antigen of the target cell (blue) and the mEmeraldGFP-CD19-CAR of the CAR T cell (green). A quantifiable parameter can be extracted by measuring the fluorescence intensity of the CD19-CAR at the IS versus the total fluorescence intensity in the effector and expressed as a ratio. (B) The second panel series shows accumulation of F-actin at the IS (green) as the second measurable step in CAR-mediated cytotoxicity. In the microscopy image, a region of interest (ROI) is drawn to indicate the accumulation of filamentous actin at the interface between effector and target cells. Once again, this can be quantified with a ratio between the fluorescence intensity in this region compared to the total intensity in the entire effector cell. (C) The third panel series illustrates the convergence of the lytic granules around the MTOC. The distance from each individual granule (here stained for perforin, red) to the MTOC (here stained for pericentrin, blue) is materialized by a red line in the schematic and the example dataset. The mean distance between all the granules and the MTOC in each cell is an accurate descriptor of their state of convergence. (D) The bottom panel series shows polarization of the MTOC to the IS, which marks the fourth readily measurable stage. The length of the shortest line drawn from the MTOC (here stained for pericentrin, blue) to the IS (here materialized by a staining for filamentous actin, green) measures the progression of polarization. The example dataset in the rightmost panel shows a MTOC together with converged granules approaching the surface of the IS. Scale bars represent 1 mm.

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Review a more direct accumulation of granules to the central IS in response to higher-affinity antigen, although live cell studies of CTL in culture with target cells bearing antigenic peptides of different strengths show similar kinetics of lytic granule accumulation in each.28 MTOC polarization can be measured from three-dimensional microscopy datasets. In CAR T cells engaged in a conjugate with a sensitive target cell, this involves the measurement of the distance between the centrosome and the geometric center of the IS. The centrosome again is the core component of the MTOC and can be highlighted by immunofluorescent staining of pericentrin. Alternative markers of the MTOC, such as gamma- or beta-tubulin, can be used. In the latter, the MTOC will be located as the brightest point of the microtubule network (as discussed above) from which the microtubules radiate. For live cell imaging, SiR tubulin, a derivative of the microtubule-binding drug docetaxel, can be used. The distance between the centrosome and the IS, defined by immunofluorescence for synaptic components, is drawn as a straight line measuring the shortest distance between the two (Figure 4D). In an NK cell conjugated to a sensitive target, this distance can be shorter than a micrometer. Alternatively, the degree of MTOC polarization can also be evaluated by dividing the area of the effector into four sectors, with the first sector being the one closest to the target cell, and recording the quadrant containing the MTOC.84,85 The level of polarization can then be scored as the proportion of cells displaying their MTOC in the front quadrant compared to non-polarized control cells, which would display no preferential localization. As demonstrated in recent applications of quantitative imaging tools, MTOC polarization can be used as an accurate CAR T cell pre-clinical benchmark in evaluating novel CAR designs.9 A second observation of cytotoxic defects in CAR NK cells resulting from failure to polarize MTOC to IS has recently been made in CLL patient-derived NK cells transduced with CD19 CAR (E. Liu, Y. Tong, G. Dotti, H. Shaim, B. Savoldo, M.M., J.O., X. Wan, X. Lu, A. R., M. Gagea, P. Banerjee, R. Cai, M.H. Bdaiwi, R. Basar, M. Muftuoglu, L. Li, D. Marin, W. Wierda, M. Keating, R. Champlin, E. Shpall, and K. Rezvani, unpublished data), providing a proof of principle for this parameter to be used as a quantifiable measurement of cytotoxic success or failure of CAR therapy cells. Like F-actin, these parameters can be used iteratively to further dissect the results obtained from standard in vitro assays used to evaluate CAR cytotoxic potential at the development stages of rational CAR design. Future Development of Microscopy-Based Approaches for FineTuning CARs

The four measurements reviewed above have all demonstrated their discriminatory power, which can be used advantageously during the manufacturing of CAR products. Many more tools can be applied to solve the challenges faced during the modeling of innovative CARs and are presented in the box on the right in Figure 3. Although this remains to be tested for this category of receptors, FRAP86 and

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FLIP87 methodologies (fluorescence recovery after photobleaching or fluorescence loss in photobleaching) are a practical way to measure the lateral mobility of transmembrane receptors. They can also be used on conjugates between CAR effectors and target cells to monitor the internalization and the binding of the chimeric receptor. Another parameter that is relatively difficult to assess using the traditional bulk assays is the efficiency of formation of the IS by the effector cells. Using multiparameter live cell imaging, it is easily conceivable to follow the CAR-expressing cells using a fluorescent marker and to measure the duration of their contact with target cells. Moreover, the addition of a viability dye in the medium during the experiment would allow measurement of target death, effector suicide and/or fratricide, CAR persistence, and serial killing capacity. One possible limitation of this assay would be the limited throughput of conventional fluorescence microscopy but the next generation of fully automated high-throughput systems is already widely available. Additionally, exciting new technologies are being developed to allow the acquisition of multiplexed experiments88,89 using spheroids, representing a dramatic experimental shift that is bringing us closer to the in vivo context. Our proposed panel of methods for CAR IS evaluation does not consider alternate ways by which cytotoxic cells can kill target cells and the potential role of these pathways in CAR-mediated killing. For example, studies in T cells have shown that granule transport to the T cell synapse can take place independent of the MTOC.90,91 Recent studies in T cells using a planar lipid bilayer system have indicated the dynamic exocytosis of TCR-containing microvesicles at the T cell immune synapse as a way to maintain TCR signal strength in an antigen avidity-dependent manner.92 It is unknown whether these mechanisms are also in place in CAR T cells and could contribute to CAR-mediated cytotoxicity. However, both of these complex and subtle situations can be addressed by microscopy using fluorescently tagged CAR T cells and markers for degranulation. Our ongoing and future imaging-based studies add insight with additional imaging components that can be helpful during the evaluation of important CAR design parameters. The transfer of the CAR engineered cell into in vivo animal models will still be required as the ultimate step before clinical trials to address safety and tolerance issues. Nevertheless, we believe that the use of microscopy-based approaches could significantly reduce their failure rate as well as make the process more time- and cost-effective. The development of entirely new CAR products combining multiple intracellular signaling chains in the CAR construct along with cytokines in armored CARs and TRUCKs93 (T cell redirected for universal cytokine killing) is an ideal opportunity to put in action the approaches reviewed here and to test the predictability of these methods in CAR function. Roadmap for the Optimization of the Design of Novel CARs Using Microscopy-Based Validation Methods

The first CARs described by Eshhar and colleagues used a single CD3z chain of the native TCR complex to drive downstream

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Review signaling (Figure 1).3 Several groups reported that the limited cytotoxic and proliferative potential of these CARs could be overcome by the inclusion of co-stimulatory domains in the CAR itself. Those second-generation CARs that had either a CD28 or 4-1BB co-stimulatory domain had significantly superior proliferative and survival advantage to CAR cells in vivo. Subsequent to this success, the list of co-stimulatory domains used for CAR T and CAR NK cells was expanded and multiple second-generation CARs now exist. The success and limitations of these second-generation CARs have been extensively reviewed by others.94,95 In addition to incorporating the co-stimulatory domains in CARs in cis, several studies demonstrated the feasibility of using co-stimulation or co-inhibition in trans (i.e., expressed by a separate transgene, CCR, Figure 1). This arrangement could either provide natural co-stimulatory signals to a T cell or be used as a “split CAR” system allowing to recognize a pattern of antigens to increase the specificity of CAR T cells.5 It has not been clear how these auxiliary receptors (CCRs) interact with CAR molecules at the IS and whether the co-stimulatory/co-inhibitory potential of these receptors is modulated by the extent of their recruitment to the IS. These novel CARs provide opportunities for further development and mechanistic understanding by application of the quantitative imaging approach described here. Once more, the use of super-resolution STED microscopy9 can directly evaluate the localization and co-engagement of multiple CARs and CCRs on the same cell using different fluorescently tagged receptors. Evaluation of the mechanics of the cytolytic process via quantitative imaging will help in contributing to the prioritization of CAR cell designs and strategies to allow for early identification of the most effective. As solid tumors continue to present the most challenging and immunosuppressive atmosphere for CAR T cell therapy by secreting immunosuppressive cytokines like transforming growth factor (TGF)-b or IL-10, intense efforts are now ongoing to enhance and repolarize the tumor microenvironment using cytokine-armored CARs called TRUCKs. These TRUCKs have been designed to directly secrete pro-inflammatory cytokines like IL-12, allowing them to function and survive better in a suppressive solid tumor microenvironment,96 as summarized in a recent review.93 Live cell imaging microscopy using fluorescently tagged CAR effectors would allow us to monitor improved TRUCK survival and proliferation over several days. Unlike a bulk proliferation assay, imaging approaches could allow for assessment of any positive correlation between the expression level of the transcript and the survival of the CAR-expressing effector. The capacity to record data at a single cell level allows this level of investigation that is not otherwise accessible in the study of non-clonal populations. Modifications to the regulators of the expression level can then be implemented to maximize survival and function. A way to measure the direct functional outcome in the formation of an improved cytolytic synapse for these novel CARs (TRUCKs) has not yet been proposed. An ongoing study by Rezvani and colleagues (E. Liu, Y. Tong, G. Dotti, H. Shaim, B. Savoldo, M.M., J.O., X. Wan, X. Lu, A. R., M. Gagea, P. Banerjee, R. Cai, M.H. Bdaiwi, R. Basar, M. Muftuoglu, L. Li, D. Marin, W. Wierda, M. Keating, R. Champlin,

E. Shpall, and K. Rezvani, unpublished data) using cord blood-derived NK cell CARs to target CD19 expressed by B cell CLL cells is exploiting this novel approach and shows enhanced MTOC polarization and F-actin accumulation in the IS of IL-15 “armored” CD19-28z-CAR over and above the CD19-28z-CAR alone. These findings are validated by in vivo studies that showed superior cytolytic performance of these CARs, confirming the predictability value of this approach. We propose that the application of our methodology will allow for their more accelerated development, improvement, and refinement as well as provide a mechanistic understanding of the failure of some existing novel second- and third-generation CARs. CAR Immune Synapse: A Paradigm Shift?

It is not known whether the engineered CAR immune synapse presents a paradigm shift from the traditional understanding of an effector cell (T or NK cell) synapse: while the latter is entirely a genome-driven entity responding to natural cues within the body’s microenvironment, the former (CAR synapse) has been manipulated by specific alterations in the CAR modules to tune CAR-mediated cytotoxicity. To gain this level of sophistication and control over the CAR synapse, a better understanding of the existing biology of CAR cells must be established. As signal strength differences in T cells have been ascribed to many variables including TCR antigen recognition strength,97,98 there is likely to be a wide array of synapse biology in CAR T cells. Future studies in our group are focused on exploring these yet-unknown aspects of CAR-presenting cells using our methods described above and those derivative from them. Sample Preparation and Acquisition for SIM and Confocal Microscopy

CAR T or CAR NK cell and target cell conjugates were seeded on no. 1.5 coverslips (Corning) pre-coated with 0.01% poly L lysine (PLL) (Sigma Aldrich) and were left to attach for 30 min in a humidified incubator at 37 C and 5% CO2 in pre-warmed media. Conjugates were fixed using 4% PFA (Electron Microscopy Science) with 0.1% Triton X-100 (Sigma Aldrich) at room temperature and were then carefully washed with PBS and blocked for 1 hr with 3% BSA (w/v) in PBS. Staining using phalloidin-Alexa Fluor 568 (Thermo Fisher Scientific), rabbit anti-pericentrin (Abcam), goat anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific), anti-CD19 Alexa Fluor 594 human antibody (Biolegend), and anti-perforin-Alexa Fluor 647 (Biolegend) was performed for 1 hr at room temperature in the blocking buffer. Coverslips were mounted using Vectashield H-1000 (Vector Labs) and slides were kept at 4 C prior to imaging. Images for the receptor aggregation panel were acquired using a Leica TCS SP8 laser scanning confocal microscope (Leica Microsystems) with an HCX Pl Apo 100/1.4 numerical aperture (NA) oil objective. A tunable white light laser provided the excitation illumination for mEmerald-CD19-CAR and the fluorescent dyes mentioned above. Emission was detected using time-gated HyD detectors operating in standard mode, and images were collected as a z stack to cover the entire volume of the conjugate. Data was acquired with LAS AF

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Review software (Leica Microsystems) and then exported to Volocity software (PerkinElmer) for analysis. Images for all the other panels were imaged using a GE OMX (version 3) microscope in structured illumination microscopy (SIM) illumination mode. Fluorescence was collected through a 60/1.4 NA oil objective and captured on a sCMOS camera at 286 MHz across a 1024  1024-pixel area with no binning and a pixel size of 80 nm controlled by SoftWorx. Each z plane was reconstructed using three orientations and five phase shifts and a Wiener filter constant of 0.005 before applying a Gaussian filter with a sigma value of 80 nm (1 pixel). Raw data were assembled into panels using Fiji99 and subjected to signal rescaling using linear transformation for display in the figures. Final figures with the cartoons were made in Illustrator (version 2017.1; Adobe).

ACKNOWLEDGMENTS

12. Huppa, J.B., Axmann, M., Mörtelmaier, M.A., Lillemeier, B.F., Newell, E.W., Brameshuber, M., Klein, L.O., Schütz, G.J., and Davis, M.M. (2010). TCR-peptideMHC interactions in situ show accelerated kinetics and increased affinity. Nature 463, 963–967. 13. Lillemeier, B.F., Mörtelmaier, M.A., Forstner, M.B., Huppa, J.B., Groves, J.T., and Davis, M.M. (2010). TCR and Lat are expressed on separate protein islands on T cell membranes and concatenate during activation. Nat. Immunol. 11, 90–96. 14. Mace, E.M., and Orange, J.S. (2014). Visualization of the immunological synapse by dual color time-gated stimulated emission depletion (STED) nanoscopy. J. Vis. Exp. 85, 51100. 15. Purbhoo, M.A., Liu, H., Oddos, S., Owen, D.M., Neil, M.A., Pageon, S.V., French, P.M., Rudd, C.E., and Davis, D.M. (2010). Dynamics of subsynaptic vesicles and surface microclusters at the immunological synapse. Sci. Signal. 3, ra36. 16. Williamson, D.J., Owen, D.M., Rossy, J., Magenau, A., Wehrmann, M., Gooding, J.J., and Gaus, K. (2011). Pre-existing clusters of the adaptor Lat do not participate in early T cell signaling events. Nat. Immunol. 12, 655–662. 17. Davis, D.M., Chiu, I., Fassett, M., Cohen, G.B., Mandelboim, O., and Strominger, J.L. (1999). The human natural killer cell immune synapse. Proc. Natl. Acad. Sci. USA 96, 15062–15067. 18. McGavern, D.B., Christen, U., and Oldstone, M.B. (2002). Molecular anatomy of antigen-specific CD8(+) T cell engagement and synapse formation in vivo. Nat. Immunol. 3, 918–925.

The authors thank Dr. Malcolm K. Brenner and Dr. Maksim Mamonkin for critical reading of the manuscript.

19. Stinchcombe, J.C., Bossi, G., Booth, S., and Griffiths, G.M. (2001). The immunological synapse of CTL contains a secretory domain and membrane bridges. Immunity 15, 751–761.

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