Microvilli are actin-based microscopic membrane protrusions that are present in a wide variety of immune cells. Scanning electron microscopy (SEM) revealed that the T cell surface is covered by microvilli. Growing evidence shows that microvilli play important roles in T cell antigen detection and signal transduction. T cell microvilli are highly dynamic and constantly scan and palpate the opposing antigen-presenting cell (APC) surface in search of antigens. Visualizing the rapid movement of microvilli that are only hundreds of nanometers in size requires imaging technologies with high spatial and temporal resolution. Lattice light-sheet microscopy can achieve diffraction-limited resolution in all three dimensions with a temporal resolution of seconds, making it the perfect tool for studying dynamic events of microvilli during T cell antigen detection and activation. In this chapter, we describe a protocol for imaging localization and movement of T cell microvilli and surface receptors using lattice light-sheet microscopy.

T cells typically recognize their ligands using a defined cell biology – the scanning of their membrane microvilli to palpate their environment – while that same membrane scaffolds T cell receptors (TCRs) that can signal upon ligand binding. Chimeric antigen receptors (CARs) present both a therapeutic promise as well as a tractable means to study the interplay between receptor affinity, microvillar dynamics and T cell function. CARs are often built using single-chain variable fragments (scFvs) with far greater affinity than that of natural TCRs. We used high resolution lattice lightsheet (LLS) and total internal reflection fluorescence (TIRF) imaging to visualize microvillar scanning in the context of variations in CAR design. This demonstrated that conventional CARs hyper-stabilized microvillar contacts relative to TCRs. Reducing the affinity and/or avidity of binding brought synapse microvillar dynamics into natural ranges, normalized synapse resolution and improved downstream effector function. This work highlights the importance of understanding the underlying cell biology when designing receptors for optimal antigen engagement.

T cell exhaustion is a major impediment to antitumor immunity. However, it remains elusive how other immune cells in the tumor microenvironment (TME) contribute to this dysfunctional state. Here, we show that the biology of tumor-associated macrophages (TAMs) and exhausted T cells (T-ex) in the TME is extensively linked. We demonstrate that in vivo depletion of TAMs reduces exhaustion programs in tumor-infiltrating CD8(+) T cells and reinvigorates their effector potential. Reciprocally, transcriptional and epigenetic profiling reveals that T-ex express factors that actively recruit monocytes to the TME and shape their differentiation. Using lattice light sheet microscopy, we show that TAM and CD8(+) T cells engage in unique, long-lasting, antigen-specific synaptic interactions that fail to activate T cells but prime them for exhaustion, which is then accelerated in hypoxic conditions. Spatially resolved sequencing supports a spatiotemporal self-enforcing positive feedback circuit that is aligned to protect rather than destroy a tumor.

T cells typically recognize their ligands using a defined cell biology—the scanning of their membrane microvilli (MV) to palpate their environment—while that same membrane scaffolds T cell receptors (TCRs) that can signal upon ligand binding. Chimeric antigen receptors (CARs) present both a therapeutic promise and a tractable means to study the interplay between receptor affinity, MV dynamics and T cell function. CARs are often built using single-chain variable fragments (scFvs) with far greater affinity than that of natural TCRs. We used high-resolution lattice lightsheet (LLS) and total internal reflection fluorescence (TIRF) imaging to visualize MV scanning in the context of variations in CAR design. This demonstrated that conventional CARs hyper-stabilized microvillar contacts relative to TCRs. Reducing receptor affinity, antigen density, and/or multiplicity of receptor binding sites normalized microvillar dynamics and synapse resolution, and effector functions improved with reduced affinity and/or antigen density, highlighting the importance of understanding the underlying cell biology when designing receptors for optimal antigen engagement.

During immune surveillance, CD8 T cells scan the surface of antigen-presenting cells using dynamic microvillar palpation and movements as well as by having their receptors preconcentrated into patches. Here, we use real-time lattice light-sheet microscopy to demonstrate the independence of microvillar and membrane receptor patch scanning. While T cell receptor (TCR) patches can distribute to microvilli, they do so stochastically and not preferentially as for other receptors such as CD62L. The distinctness of TCR patch movement from microvillar movement extends to many other receptors that form patches that also scan independent of the TCR. An exception to this is the CD8 coreceptor which largely comigrates in patches that overlap with or are closely adjacent to those containing TCRs. Microvilli that assemble into a synapse contain various arrays of the engaged patches, notably of TCRs and the inhibitory receptor PD-1, creating a pastiche of occupancies that vary from microvillar contact to contact. In summary, this work demonstrates that localization of receptor patches within the membrane and on microvillar projections is random prior to antigen detection and that such random variation may play into the generation of many individually composed receptor patch compositions at a single synapse.

T cells typically recognize their ligands using microvilli to scan for cognate antigen and scaffold T cell receptors (TCRs) during antigen recognition. Conventional chimeric antigen receptors (CARs) are often built using single-chain variable fragments (scFvs) with far greater affinity than that of natural TCRs. The implications of this for T cell function are not well understood. Using high-resolution microscopy, we studied the membrane dynamics in cells bearing anti-human epidermal growth factor receptor 2 (HER2) CARs and found these hyper-stabilized microvillar contacts relative to TCRs. While these CARs also impaired synapse resolution, a monomerized CAR with a lower affinity scFv rescued synapse dynamics and improved antigen-dose discrimination. The dimeric low-affinity CAR improved synapse dynamics and minimized early indicators of exhaustion, while maintaining or improving effector functions. This work highlights the importance of designing CAR binding dynamics that more closely resemble natural TCR antigen sensing to optimize T cell quality and function.

In order to drive productive tumor-infiltrating lymphocyte (TIL) function, myeloid populations must direct antigens to the lymph node, including to resident antigen-presenting cells (APCs) that have never touched the tumor. It has long been supposed that APCs trade antigens with one another, but the dominant cell biology underlying that remains unknown. We used and assays together with lattice light sheet and multiphoton imaging to show that myeloid cells carry tumor antigen-laden vesicles that they ‘trade’ with one another as they reach distant sites. This accounts for the majority of antigen displayed to T cells and provides tumors with a mechanism to access APCs that differentially direct T cell activation away from memory phenotypes. This work defines efficient cell biology that drives the first steps of TIL generation and represents a new frontier for engineering tumoral immunity.

Generation of tumor-infiltrating lymphocytes begins when tumor antigens reach the lymph node (LN) to stimulate T cells, yet we know little of how tumor material is disseminated among the large variety of antigen-presenting dendritic cell (DC) subsets in the LN. Here, we demonstrate that tumor proteins are carried to the LN within discrete vesicles inside DCs and are then transferred among DC subsets. A synapse is formed between interacting DCs and vesicle transfer takes place in the absence of free exosomes. DCs -containing vesicles can uniquely activate T cells, whereas DCs lacking them do not. Understanding this restricted sharing of tumor identity provides substantial room for engineering better anti-tumor immunity.

During antigen detection, T-cells survey the surface of antigen-presenting cells (APCs), which may display mainly nonstimulatory peptide-loaded major histocompatibility complexes (pMHCs) and only rare cognate antigen in a process involving close (nanometer-scale) membrane apposition. Thus, T-cell must solve a classic trade-off between speed and sensitivity. It has long been supposed that microvilli on T-cells act as sensory organs to enable search, but their strategy has been unknown. We used lattice light-sheet microscopy and quantum dot-enabled synaptic contact mapping microscopy to show how microvilli on the surface of T-cells search opposing cells and surfaces before and during antigen recognition. We uncovered that microvilli on T-cell surfaces dynamically survey the majority of opposing surfaces within one minute through anomalous diffusion. T-cell receptor (TCR) recognition resulted in selective stabilization of receptor-occupied protrusions, which was independent of tyrosine kinase signaling and the actin cytoskeleton. We revealed that TCRs on activated T-cells were nonuniformly distributed on cell membrane: some TCRs were concentrated on microvilli, while other TCRs formed high-density patches on flatter membrane regions. Many microvilli were TCR-occupied, but a small population of microvilli were found not occupied by TCRs. TCR high-density patches moved relative to microvilli. During T-cell-APC interaction, we observed T-cell microvilli projected deep into 3D pockets formed by veil structures on the surface of dendritic cells (DC), and DC membrane also conformed to accommodate T-cell microvilli, which increased the effective close-contact area between T-cell and APC. Such scanning pattern enabled T-cells to efficiently scan more APC surface in given time. This work defines the efficienT-cellular search process against which ligand detection takes place in T-cells. Citation Format: En Cai, Kyle Marchuk, Casey Beppler, Matthew Krummel. Microvilli enable efficient T-cell antigen search and ligand detection [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B148.

During immune surveillance, T cells survey the surface of antigen-presenting cells. In searching for peptide-loaded major histocompatibility complexes (pMHCs), they must solve a classic trade-off between speed and sensitivity. It has long been supposed that microvilli on T cells act as sensory organs to enable search, but their strategy has been unknown. We used lattice light-sheet and quantum dot-enabled synaptic contact mapping microscopy to show that anomalous diffusion and fractal organization of microvilli survey the majority of opposing surfaces within 1 minute. Individual dwell times were long enough to discriminate pMHC half-lives and T cell receptor (TCR) accumulation selectively stabilized microvilli. Stabilization was independent of tyrosine kinase signaling and the actin cytoskeleton, suggesting selection for avid TCR microclusters. This work defines the efficient cellular search process against which ligand detection takes place.

Previous studies tracking AMPA receptor (AMPAR) diffusion at synapses observed a large mobile extrasynaptic AMPAR pool. Using super-resolution microscopy, we examined how fluorophore size and photostability affected AMPAR trafficking outside of, and within, post-synaptic densities (PSDs) from rats. Organic fluorescent dyes (≈4 nm), quantum dots, either small (≈10 nm diameter; sQDs) or big (>20 nm; bQDs), were coupled to AMPARs via different-sized linkers. We find that >90% of AMPARs labeled with fluorescent dyes or sQDs were diffusing in confined nanodomains in PSDs, which were stable for 15 min or longer. Less than 10% of sQD-AMPARs were extrasynaptic and highly mobile. In contrast, 5–10% of bQD-AMPARs were in PSDs and 90–95% were extrasynaptic as previously observed. Contrary to the hypothesis that AMPAR entry is limited by the occupancy of open PSD ‘slots’, our findings suggest that AMPARs rapidly enter stable ‘nanodomains’ in PSDs with lifetime >15 min, and do not accumulate in extrasynaptic membranes.

We imaged transiently transfected live glutamate receptors (iGluRs), namely AMPAR and NMDAR, with super-resolution in three-dimension, labeled with differently sized fluorophores. We used small organic fluorescent dyes (∼ 4 nm), small quantum dots (sQD, ∼10 nm in diameter), or big (commercial) quantum dots (bQD, ∼ 20 nm in diameter). The iGluRs were imaged along with a synaptic protein, Homer1c, using fiducial markers that eliminate stage drift, and under conditions where the receptor cross-linking is monitored or eliminated. With small probes under basal conditions, we find that both AMPAR and NMDAR are predominantly within the synapse (∼84-95%), in contrast to bQDs, which show only 5-10% within the synapse. The results can likely be explained by cross-linking and inhibited mobility of the iGluRs labeled with bQDs, but not with sQDs or organic fluorophores. Hence, this data indicates that there is not a highly diffusible pool of extrasynaptic iGluRs in the plasma membrane, as has been widely reported. In addition, within a synapse, the distribution is non-homogeneous, perhaps due to the non-homogeneous distribution of glutamate.

VMD as a Software for Visualization and Quantitative Analysis of Super Resolution Imaging and Single Particle Tracking Yanxin Liu1,2, John E. Stone2, En Cai1, Jingyi Fei1, Sang Hak Lee1, Seongjin Park1, Taekjip Ha1, Paul R. Selvin1, Klaus Schulten1,2. Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, IL, USA, Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, IL, USA. Super resolution imaging and high-precision single particle tracking are promising techniques to study biomolecular trafficking and localization as well as intracellular structures. The tremendous amount of data acquired from the experiments pose a great visualization and analysis challenge. VMD, a software for visualizing and analyzing trajectories from molecular dynamics simulations, has the ability to handle hundreds of millions of particles and hundreds of thousands of frames in a time-dependent manner. We extended VMD with new functions for handling data from super-resolution imaging and highprecision single particle tracking experiments and performed visualization and data analysis. The analysis can take advantage of VMD’s existing capability to display particles in various representations and to compute system properties employing GPUs. The Tcl/Tk interface in VMD adds a layer of flexibility to implement project-specific functionality specified by users. The use of VMD is demonstrated for two examples: (1) employing STORM to reveal the organization of long non-coding RNAs in nuclear bodies; (2) combining single particle tracking and PALM to study AMPA receptor trafficking at live synapses.

Understanding various dynamic phenomena in synapses is important for explaining how the brain works. One important phenomenon is the dynamic behavior of glutamate receptors, namely AMPAR and NMDAR. In this study, we used 3D super-accurate microscopy (FIONA) and super-resolution microscopy (PALM/STORM), with approximately 10-40 nm accuracy/resolution, to determine the diffusion constants and spatial location of NMDAR. We also used small quantum dots which have the brightness and photostability of the more commonly used commercial quantum dots, but have a reduced size (∼8-10 nm diameter, compared to ∼20 nm for the commercial ones). We found that NMDAR exists primarily in the synapses, but does have a presence just outside the synapse, and in the spine, and in the dendrites. We found that NMDAR, when in the synapse, is primarily in nanodomains, i.e. sub-synaptic regions where the NMDAR diffuses around approximately 10-times slower then when in the non-synaptic cell membrane. Looking over the course of 15 minutes, we find that the nanodomain itself, tend to move around, with ∼10x slower than NMDAR in the cell membrane. We also found that diffusion of NMDARs changed after co-agonists treatment.

Synapses are the fundamental structures for signal transmission in neurons. A synapse is a cell-cell junction composed of pre-synapse and post-synapse between neurons. The structure of synapses is dynamically changing upon external stimuli. In this work, we used super-resolution imaging technique (PALM) to study the synaptic changes after inducing long-term potentiation (LTP). In particular, after LTP induction, we investigated the changes in distances between 1. A presynaptic protein (synapsin and postsynaptic scaffold proteins (Homer1 and PSD95) and 2. Two different postsynaptic scaffold proteins, namely Homer1 and PSD95. We labeled these proteins using photo-activable proteins such as mEOS2, Dronpa, and mGeos, and obtained two-color PALM imaging for each pair of synaptic proteins. Chromatic aberration was corrected for a more precise measurement of the distances. We found out that, after LTP induction, the distance between Homer1 and PSD95 decreased significantly from 330 nm to 90 nm; the distance between synapsin and PSD95 also decreased from 243 nm to 125 nm. Moreover, we carried out fast PALM imaging, which allows us to take super-resolution images every minute, in order to observe the volume change of synapses during LTP. Observation of the time-dependent distribution of PSD95 and Homer1 has shown that the average volume of synapses is increasing after LTP induction from 0.05 μm3 to 0.076 μm3.

Quantum dots are promising candidates for single molecule imaging due to their exceptional photophysical properties, including their intense brightness and resistance to photobleaching. They are also notorious for their blinking. Here we report a novel way to take advantage of quantum dot blinking to develop an imaging technique in three-dimensions with nanometric resolution. We first applied this method to simulated images of quantum dots, and then to quantum dots immobilized on microspheres. We achieved imaging resolutions (FWHM) of 8-17 nm in the x-y plane and 58 nm (on coverslip) or 81 nm (deep in solution) in the z-direction, approximately 3-7 times better than what has been achieved previously with quantum dots. This approach was applied to resolve the 3D distribution of epidermal growth factor receptor (EGFR) molecules at, and inside of, the plasma membrane of resting basal breast cancer cells.

We developed a coating method to produce functionalized small quantum dots (sQDs), about 9 nm in diameter, that were stable for over a month. We made sQDs in four emission wavelengths, from 527 to 655 nm and with different functional groups. AMPA receptors on live neurons were labeled with sQDs and postsynaptic density proteins were visualized with super-resolution microscopy. Their diffusion behavior indicates that sQDs access the synaptic clefts significantly more often than commercial QDs.

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (>200 µm), localization precision of 2-3 nm can be achieved.

Super resolution imaging and high-precision single particle tracking are promising techniques to study biomolecular trafficking and localization as well as intracellular structures. The tremendous amount of data acquired from the experiments pose a great visualization and analysis challenge. VMD, a software for visualizing and analyzing trajectories from molecular dynamics simulations, has the ability to handle hundreds of millions of particles and hundreds of thousands of frames in a time-dependent manner. We extended VMD with new functions for handling data from super-resolution imaging and high-precision single particle tracking experiments and performed visualization and data analysis. The analysis can take advantage of VMD's existing capability to display particles in various representations and to compute system properties employing GPUs. The Tcl/Tk interface in VMD adds a layer of flexibility to implement project-specific functionality specified by users. The use of VMD is demonstrated for two examples: (1) employing STORM to reveal the organization of long non-coding RNAs in nuclear bodies; (2) combining single particle tracking and PALM to study AMPA receptor trafficking at live synapses.