Light-inducible T cell engagers trigger, tune, and shape the activation of primary T cells.

To mount appropriate responses, T cells integrate complex sequences of receptor stimuli perceived during transient interactions with antigen-presenting cells. Although it has been hypothesized that the dynamics of these interactions influence the outcome of T cell activation, methodological limitations have hindered its formal demonstration. Here, we have engineered the Light-inducible T cell engager (LiTE) system, a recombinant optogenetics-based molecular tool targeting the T cell receptor (TCR). The LiTE system constitutes a reversible molecular switch displaying exquisite reactivity. As proof of concept, we dissect how specific temporal patterns of TCR stimulation shape T cell activation. We established that CD4+ T cells respond to intermittent TCR stimulation more efficiently than their CD8+ T cells counterparts and provide evidence that distinct sequences of TCR stimulation encode different cytokine programs. Finally, we show that the LiTE system could be exploited to create light-activated bispecific T cell engagers and manipulate tumor cell killing. Overall, the LiTE system provides opportunities to understand how T cells integrate TCR stimulations and to trigger T cell cytotoxicity with high spatiotemporal control.

Prominent in vivo influence of single interneurons in the developing barrel cortex.

Spontaneous synchronous activity is a hallmark of developing brain circuits and promotes their formation. Ex vivo, synchronous activity was shown to be orchestrated by a sparse population of highly connected GABAergic 'hub' neurons. The recent development of all-optical methods to record and manipulate neuronal activity in vivo now offers the unprecedented opportunity to probe the existence and function of hub cells in vivo. Using calcium imaging, connectivity analysis and holographic optical stimulation, we show that single GABAergic, but not glutamatergic, neurons influence population dynamics in the barrel cortex of non-anaesthetized mouse pups. Single GABAergic cells mainly exert an inhibitory influence on both spontaneous and sensory-evoked population bursts. Their network influence scales with their functional connectivity, with highly connected hub neurons displaying the strongest impact. We propose that hub neurons function in tailoring intrinsic cortical dynamics to external sensory inputs.

Functional Mapping of Adhesiveness on Live Cells Reveals How Guidance Phenotypes Can Emerge From Complex Spatiotemporal Integrin Regulation.

Immune cells have the ubiquitous capability to migrate disregarding the adhesion properties of the environment, which requires a versatile adaptation of their adhesiveness mediated by integrins, a family of specialized adhesion proteins. Each subtype of integrins has several ligands and several affinity states controlled by internal and external stimuli. However, probing cell adhesion properties on live cells without perturbing cell motility is highly challenging, especially in vivo. Here, we developed a novel in vitro method using micron-size beads pulled by flow to functionally probe the local surface adhesiveness of live and motile cells. This method allowed a functional mapping of the adhesiveness mediated by VLA-4 and LFA-1 integrins on the trailing and leading edges of live human T lymphocytes. We show that cell polarization processes enhance integrin-mediated adhesiveness toward cell rear for VLA-4 and cell front for LFA-1. Furthermore, an inhibiting crosstalk of LFA-1 toward VLA-4 and an activating crosstalk of VLA-4 toward LFA-1 were found to modulate cell adhesiveness with a long-distance effect across the cell. These combined signaling processes directly support the bistable model that explains the emergence of the versatile guidance of lymphocyte under flow. Molecularly, Sharpin, an LFA-1 inhibitor in lymphocyte uropod, was found involved in the LFA-1 deadhesion of lymphocytes; however, both Sharpin and Myosin inhibition had a rather modest impact on adhesiveness. Quantitative 3D immunostaining identified high-affinity LFA-1 and VLA-4 densities at around 50 and 100 molecules/µm2 in basal adherent zones, respectively. Interestingly, a latent adhesiveness of dorsal zones was not grasped by immunostaining but assessed by direct functional assays with beads. The combination of live functional assays, molecular imaging, and genome editing is instrumental to characterizing the spatiotemporal regulation of integrin-mediated adhesiveness at molecular and cell scales, which opens a new perspective to decipher sophisticated phenotypes of motility and guidance.

Leukocyte transmigration and longitudinal forward-thrusting force in a microfluidic Transwell device.

Transmigration of leukocytes across blood vessels walls is a critical step of the immune response. Transwell assays examine transmigration properties in vitro by counting cells passages through a membrane; however, the difficulty of in situ imaging hampers a clear disentanglement of the roles of adhesion, chemokinesis, and chemotaxis. We used here microfluidic Transwells to image the cells' transition from 2D migration on a surface to 3D migration in a confining microchannel and measure cells longitudinal forward-thrusting force in microchannels. Primary human effector T lymphocytes adhering with integrins LFA-1 (αLß2) had a marked propensity to transmigrate in Transwells without chemotactic cue. Both adhesion and contractility were important to overcome the critical step of nucleus penetration but were remarkably dispensable for 3D migration in smooth microchannels deprived of topographic features. Transmigration in smooth channels was qualitatively consistent with a propulsion by treadmilling of cell envelope and squeezing of cell trailing edge. Stalling conditions of 3D migration were then assessed by imposing pressure drops across microchannels. Without specific adhesion, the cells slid backward with subnanonewton forces, showing that 3D migration under stress is strongly limited by a lack of adhesion and friction with channels. With specific LFA-1 mediated adhesion, stalling occurred at around 3 and 6 nN in 2 × 4 and 4 × 4 µm2 channels, respectively, supporting that stalling of adherent cells was under pressure control rather than force control. The stall pressure of 4 mbar is consistent with the pressure of actin filament polymerization that mediates lamellipod growth. The arrest of adherent cells under stress therefore seems controlled by the compression of the cell leading edge, which perturbs cells front-rear polarization and triggers adhesion failure or polarization reversal. Although stalling assays in microfluidic Transwells do not mimic in vivo transmigration, they provide a powerful tool to scrutinize 2D and 3D migration, barotaxis, and chemotaxis.

Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells.

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ranging from nanometers to micrometers and microseconds to minutes, respectively. Such a broad range of biological processes challenges conventional microscopy approaches. Here, we detail the procedure for implementing spot variation Fluorescence Correlation Spectroscopy (svFCS) measurements using a classical fluorescence microscope that has been customized. The protocol includes a specific performance check of the svFCS setup and the guidelines for molecular diffusion measurements by svFCS on the plasma membrane of living cells under physiological conditions. Additionally, we provide a procedure for disrupting plasma membrane raft nanodomains by cholesterol oxidase treatment and demonstrate how these changes in the lateral organization of the plasma membrane might be revealed by svFCS analysis. In conclusion, this fluorescence-based method can provide unprecedented details on the lateral organization of the plasma membrane with the appropriate spatial and temporal resolution.

Longitudinal Intravital Microscopy Reveals Axon Degeneration Concomitant With Inflammatory Cell Infiltration in an LPC Model of Demyelination.

Demyelination and axon degeneration are major events in all neurodegenerative diseases, including multiple sclerosis. Intoxication of oligodendrocytes with lysophosphatidylcholine (LPC) is often used as a selective model of focal and reversible demyelination thought to have no incidence for neurons. To characterize the cascade of cellular events involved in LPC-induced demyelination, we have combined intravital coherent antistoke Raman scattering microscopy with intravital two-photon fluorescence microscopy in multicolor transgenic reporter mice. Moreover, taking advantage of a unique technique of spinal glass window implantation, we here provide the first longitudinal description of cell dynamics in the same volume of interest over weeks after insults. We have detected several patterns of axon-myelin interactions and classified them in early and advanced events. Unexpectedly, we have found that oligodendrocyte damages are followed by axon degeneration within 2 days after LPC incubation, and this degeneration is amplified after the recruitment of the peripheral proinflammatory cells at day 4. Beyond day 7, the recovery of axon number and myelin takes 3 more weeks postlesion and involves a new wave of anti-inflammatory innate immune cells at day 14. Therefore, recurrent imaging over several weeks suggests an important role of peripheral immune cells in regulating both the axonal and oligodendroglial fates and thereby the remyelination status. Better understanding the recruitment of peripheral immune cells during demyelinating events should help to improve diagnosis and therapy.

Author Correction: Phosphoinositides regulate the TCR/CD3 complex membrane dynamics and activation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Effects of VEGF blockade on the dynamics of the inflammatory landscape in glioblastoma-bearing mice.

BACKGROUND: Targeting angiogenesis has been and continues to be an attractive therapeutic modality in glioblastoma (GBM) patients. However, GBM rapidly becomes refractory to anti-VEGF therapies. Myeloid cell infiltration is an important determinant of tumor progression. Given that VEGF is a modulator of the innate immune response we sought to analyze the dynamics of this response in a mouse model of GBM undergoing anti-VEGF therapy. METHODS: We grafted GL261-DsRed cells in transgenic Thy1-CFP//LysM-EGFP//CD11c-EYFP reporter mice. We combined recurrent spectral two-photon imaging with multiparametric cytometry, immunostaining, and brain clearing to characterize at two critical stages of tumor development (day 21 and day 28 after tumor grafting) the nature and spatial distribution of the innate response in control and bevacizumab-treated mice. RESULTS: We report that at an early stage (21 day), VEGF blockade has a detectable effect on the number of microglial cells but only a mild effect on the number of infiltrating myeloid cells. At a later stage (day 28), the treatment resulted in a specific adjustment of dendritic cell subsets. In treated mice, the number of monocytes and their monocyte-derived dendritic cells (moDC) progeny was increased by approximately twofold compared to untreated mice. In agreement, by in vivo quantitative imaging, we observed that treatment increased the number of LysM-EGFP cells traveling in tumor blood vessels and doubled the densities of both infiltrated LysM-EGFP monocytes and double-labeled EGFP/EYFP moDC. The treatment also led to an increased density of conventional cDCs2 subset together with a decrease of cDCs1 subset, necessary for the development of anti-tumor immunity. Finally, we describe differential spatial cell distributions and two immune cell-traveling routes into the brain. LysM-EGFP cells distributed as a gradient from the meninges towards the tumor whereas CD11c-EYFP/MHCII+ cells were located in the basal area of the tumor. Brain clearing also revealed a flow of CD11c-EYFP cells following the corpus callosum. CONCLUSION: We uncovered new features in the dynamics of innate immune cells in GBM-bearing mice and deciphered precisely the key populations, i.e., DC subsets controlling immune responses, that are affected by VEGF blockade. Since despite differences, human pathogenesis presents similarities with our mouse model, the data provide new insights into the effect of bevacizumab at the cellular level.

A Theoretical High-Density Nanoscopy Study Leads to the Design of UNLOC, a Parameter-free Algorithm.

Single-molecule localization microscopy (SMLM) enables the production of high-resolution images by imaging spatially isolated fluorescent particles. Although challenging, the result of SMLM analysis lists the position of individual molecules, leading to a valuable quantification of the stoichiometry and spatial organization of molecular actors. Both the signal/noise ratio and the density (Dframe), i.e., the number of fluorescent particles per µm2 per frame, have previously been identified as determining factors for reaching a given SMLM precision. Establishing a comprehensive theoretical study relying on these two parameters is therefore of central interest to delineate the achievable limits for accurate SMLM observations. Our study reports that in absence of prior knowledge of the signal intensity α, the density effect on particle localization is more prominent than that anticipated from theoretical studies performed at known α. A first limit appears when, under a low-density hypothesis (i.e., one-Gaussian fitting hypothesis), any fluorescent particle distant by less than ∼600 nm from the particle of interest biases its localization. In fact, all particles should be accounted for, even those dimly fluorescent, to ascertain unbiased localization of any surrounding particles. Moreover, even under a high-density hypothesis (i.e., multi-Gaussian fitting hypothesis), a second limit arises because of the impossible distinction of particles located too closely. An increase in Dframe is thus likely to deteriorate the localization precision, the image reconstruction, and more generally the quantification accuracy. Our study firstly provides a density-signal/noise ratio space diagram for use as a guide in data recording toward reaching an achievable SMLM resolution. Additionally, it leads to the identification of the essential requirements for implementing UNLOC, a parameter-free and fast computing algorithm approaching the Cramér-Rao bound for particles at high-density per frame and without any prior knowledge of their intensity. UNLOC is available as an ImageJ plugin.

Phosphoinositides regulate the TCR/CD3 complex membrane dynamics and activation.

Phosphoinositides (PIs) play important roles in numerous membrane-based cellular activities. However, their involvement in the mechanism of T cell receptor (TCR) signal transduction across the plasma membrane (PM) is poorly defined. Here, we investigate their role, and in particular that of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] in TCR PM dynamics and activity in a mouse T-cell hybridoma upon ectopic expression of a PM-localized inositol polyphosphate-5-phosphatase (Inp54p). We observed that dephosphorylation of PI(4,5)P2 by the phosphatase increased the TCR/CD3 complex PM lateral mobility prior stimulation. The constitutive and antigen-elicited CD3 phosphorylation as well as the antigen-stimulated early signaling pathways were all found to be significantly augmented in cells expressing the phosphatase. Using state-of-the-art biophotonic approaches, we further showed that PI(4,5)P2 dephosphorylation strongly promoted the CD3ε cytoplasmic domain unbinding from the PM inner leaflet in living cells, thus resulting in an increased CD3 availability for interactions with Lck kinase. This could significantly account for the observed effects of PI(4,5)P2 dephosphorylation on the CD3 phosphorylation. Our data thus suggest that PIs play a key role in the regulation of the TCR/CD3 complex dynamics and activation at the PM.

A straightforward STED-background corrected fitting model for unbiased STED-FCS analyses.

Combining stimulated emission depletion and fluorescence correlation spectroscopy (STED-FCS) provides a powerful and sensitive tool for studying the molecular dynamics in live cells with high spatio-temporal resolution. STED-FCS gives access to molecular diffusion characteristic at the nanoscale occurring within short period of times. However due to the incomplete suppression of fluorescence in the STED process, the STED-FCS point spread function (PSF) deviates from a Gaussian shape and challenges the analysis of the auto-correlation curves obtained by FCS. Here, we model the effect of the incomplete fluorescence suppression in STED-FCS experiments and propose a new fitting model improving the accuracy of the diffusion times and average molecule numbers measurements. The implementation of a STED module with pulsed laser source on a commercial confocal/FCS microscope allowed us to apply the STED-background corrected model to fit the STED-FCS measurements. The experimental results are in good accordance with the theoretical analysis both for the number of molecules and the diffusion time which decrease accordingly with the STED power.

Transverse chemical interface detection with coherent anti-Stokes Raman scattering microscopy.

Transverse "chemical" interfaces are revealed with a conventional two beam narrowband coherent anti-Stokes Raman scattering microscopy setup in a collinear configuration. The exciting "pump" and "Stokes" beams are focused on the sample in two opposite directions. The subtraction of the two generated anti-Stokes signals gives rise to a signal that is directly proportional to the pure Raman spectrum of the resonant medium. This property is used to highlight an interface between glass and N,N-dimethylformamide (DMF) and recover the pure Raman spectrum of DMF around its 1408 cm(-1) vibrational band.

Double-clad hollow core photonic crystal fiber for coherent Raman endoscope.

Performing label free coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) in endoscope imaging is a challenge, with huge potential clinical benefit. To date, this goal has remained inaccessible because of the inherent coherent Raman noise that is generated in the fiber itself. By developing double-clad hollow core photonic crystal fiber, we demonstrate coherent anti-Stokes Raman scattering and stimulated Raman scattering in an 'endoscope-like' scheme. Both the excitation beams and the collected CARS and SRS signals travel through the same fiber. No CARS and SRS signals are generated within the hollow core fiber even for temporally overlapping pump and Stokes beams, leading to excellent image quality. The CARS and SRS signals generated in the sample are coupled back into a high numerical aperture multimode cladding surrounding the central photonic crystal cladding. We demonstrate this scheme by imaging molecular vibrational bonds of organic crystal deposited on a glass surface.

Optical parametric oscillator-based light source for coherent Raman scattering microscopy: practical overview.

We present the assets and constraints of using optical parametric oscillators (OPOs) to perform point scanning nonlinear microscopy and spectroscopy with special emphasis on coherent Raman spectroscopy. The different possible configurations starting with one OPO and two OPOs are described in detail and with comments that are intended to be practically useful for the user. Explicit examples on test samples such as nonlinear organic crystal, polystyrene beads, and fresh mouse tissues are given. Special emphasis is given to background-free coherent Raman anti-Stokes scattering (CARS) imaging, including CARS hyperspectral imaging in a fully automated mode with commercial OPOs.

Revisiting the Young's double slit experiment for background-free nonlinear Raman spectroscopy and microscopy.

In the Young's double slit experiment, the spatial shift of the interference pattern projected onto a screen is directly related to the phase difference between the fields diffracted by the two slits. We apply this property to fields emitted by nonlinear processes and thus demonstrate background-free coherent anti-Stokes Raman scattering microscopy near an axial interface between a resonant and a nonresonant medium. This method is relevant to remove the nonresonant background in other coherent resonant processes.

Influence of birefringence on polarization resolved nonlinear microscopy and collagen SHG structural imaging.

We analyze the influence of the anisotropy of molecular and biological samples on polarization resolved nonlinear microscopy imaging. We show in particular the detrimental influence of birefringence on Second Harmonic Generation (SHG) and Two-Photon Excited Fluorescence (TPEF) polarization resolved microscopy imaging, which, if not accounted for, can lead to an erroneous determination of the sample properties and thus to a misinterpretation of the read-out information. We propose a method to measure this birefringence and account for this effect in nonlinear polarization resolved experiments.

Polarization distortion effects in polarimetric two-photon microscopy.

We present a global analysis of experimental factors affecting polarization responses in two-photon inverted microscopy. The role of reflection optics and high numerical aperture focusing is investigated in two-photon fluorescence, which can be extended to other nonlinear processes. We show that both effects strongly distort polarization responses and can lead to misleading extraction of molecular order information from polarimetric measurements. We describe a model accounting for these effects and develop a calibration technique for the determination of polarization parameters in the sample plane using two-photon fluorescence polarimetry in liquids.

Absolute radiance imaging using parametric image amplification.

We show that parametric image amplification can be used to achieve a 2D radiance map directly expressed in photons per spatiotemporal mode. Radiance images of incoherent signals with less than one photon per mode (typically 10(-2)) are resolved.