A hepatic network of dendritic cells mediates CD4 T cell help outside lymphoid organs.

While CD4+ T cells are a prerequisite for CD8+ T cell-mediated protection against intracellular hepatotropic pathogens, the mechanisms facilitating the transfer of CD4-help to intrahepatic CD8+ T cells are unknown. Here, we developed an experimental system to investigate cognate CD4+ and CD8+ T cell responses to a model-antigen expressed de novo in hepatocytes and reveal that after initial priming, effector CD4+ and CD8+ T cells migrate into portal tracts and peri-central vein regions of the liver where they cluster with type-1 conventional dendritic cells. These dendritic cells are locally licensed by CD4+ T cells and expand the number of CD8+ T cells in situ, resulting in larger effector and memory CD8+ T cell pools. These findings reveal that CD4+ T cells promote intrahepatic immunity by amplifying the CD8+ T cell response via peripheral licensing of hepatic type-1 conventional dendritic cells and identify intrahepatic perivascular compartments specialized in facilitating effector T cell-dendritic cell interactions.

Chemokine receptor distribution on the surface of repolarizing T cells.

T cells migrate constitutively with a polarized morphology, underpinned by signaling compartmentalization and discrete cytoskeletal organizations, giving rise to a dynamic and expansive leading edge, distinct from the stable and constricted uropod at the rear. In vivo, the motion and function of T cells at various stages of differentiation is highly directed by chemokine gradients. When cognate ligands bind chemokine receptors on their surface, T cells respond by reorientating their polarity axis and migrating toward the source of the chemokine signal. Despite the significance of such chemotactic repolarization to the accurate navigation and function of T cells, the precise signaling mechanisms that underlie it remain elusive. Notably, it remained unclear whether the distribution of chemokine receptors on the T cell surface is altered during repolarization. Here, we developed parallel cell-secreted and microfluidics-based chemokine gradient delivery methods and employed both fixed imaging and live lattice light-sheet microscopy to investigate the dynamics of chemokine receptor CCR5 on the surface of primary murine CD8+ T cells. Our findings show that, during constitutive migration, chemokine receptor distribution is largely isotropic on the T cell surface. However, upon exposure to a CCL3 gradient, surface chemokine receptor distributions exhibit a transient bias toward the uropod. The chemokine receptors then progressively redistribute from the uropod to cover the T cell surface uniformly. This study sheds new light on the dynamics of surface chemokine receptor distribution during T cell repolarization, advancing our understanding of the signaling of immune cells in the complex chemokine landscapes they navigate.

Programming of Multicellular Patterning with Mechano-Chemically Microstructured Cell Niches.

Multicellular patterning of stem-cell-derived tissue models is commonly achieved via self-organizing activities triggered by exogenous morphogenetic stimuli. However, such tissue models are prone to stochastic behavior, limiting the reproducibility of cellular composition and forming non-physiological architectures. To enhance multicellular patterning in stem cell-derived tissues, a method for creating complex tissue microenvironments endowed with programmable multimodal mechano-chemical cues, including conjugated peptides, proteins, morphogens, and Young's moduli defined over a range of stiffnesses is developed. The ability of these cues to spatially guide tissue patterning processes, including mechanosensing and the biochemically driven differentiation of selected cell types, is demonstrated. By rationally designing niches, the authors engineered a bone-fat assembly from stromal mesenchyme cells and regionalized germ layer tissues from pluripotent stem cells. Through defined niche-material interactions, mechano-chemically microstructured niches enable the spatial programming of tissue patterning processes. Mechano-chemically microstructured cell niches thereby offer an entry point for enhancing the organization and composition of engineered tissues, potentiating structures that better recapitulate their native counterparts.

T cell cytoskeletal forces shape synapse topography for targeted lysis via membrane curvature bias of perforin.

Cytotoxic T lymphocytes (CTLs) lyse target cells by delivering lytic granules that contain the pore former perforin to the cytotoxic immunological synapse. Here, we establish that opposing cytoskeletal forces drive lytic granule polarization and simultaneously shape T cell synapse topography to enhance target perforation. At the cell rear, actomyosin contractility drives the anterograde movement of lytic granules toward the nucleus. At the synapse, dynein-derived forces induce negatively curved membrane pockets to which granules are transported around the nucleus. These highly concave degranulation pockets are located directly opposite positively curved bulges on the target cell membrane. We identify a curvature bias in the action of perforin, which preferentially perforates positively curved tumor cell membrane. Together, these findings demonstrate murine and human T cell-mediated cytotoxicity to be a highly tuned mechano-biochemical system, in which the forces that polarize lytic granules locally bend the synaptic membrane to favor the unidirectional perforation of the target cell.

T cell morphodynamics reveal periodic shape oscillations in three-dimensional migration.

T cells use sophisticated shape dynamics (morphodynamics) to migrate towards and neutralize infected and cancerous cells. However, there is limited quantitative understanding of the migration process in three-dimensional extracellular matrices (ECMs) and across timescales. Here, we leveraged recent advances in lattice light-sheet microscopy to quantitatively explore the three-dimensional morphodynamics of migrating T cells at high spatio-temporal resolution. We first developed a new shape descriptor based on spherical harmonics, incorporating key polarization information of the uropod. We found that the shape space of T cells is low-dimensional. At the behavioural level, run-and-stop migration modes emerge at approximately 150 s, and we mapped the morphodynamic composition of each mode using multiscale wavelet analysis, finding 'stereotyped' motifs. Focusing on the run mode, we found morphodynamics oscillating periodically (every approx. 100 s) that can be broken down into a biphasic process: front-widening with retraction of the uropod, followed by a rearward surface motion and forward extension, where intercalation with the ECM in both of these steps likely facilitates forward motion. Further application of these methods may enable the comparison of T cell migration across different conditions (e.g. differentiation, activation, tissues and drug treatments) and improve the precision of immunotherapeutic development.

SNX9-induced membrane tubulation regulates CD28 cluster stability and signalling.

T cell activation requires engagement of a cognate antigen by the T cell receptor (TCR) and the co-stimulatory signal of CD28. Both TCR and CD28 aggregate into clusters at the plasma membrane of activated T cells. While the role of TCR clustering in T cell activation has been extensively investigated, little is known about how CD28 clustering contributes to CD28 signalling. Here, we report that upon CD28 triggering, the BAR-domain protein sorting nexin 9 (SNX9) is recruited to CD28 clusters at the immunological synapse. Using three-dimensional correlative light and electron microscopy, we show that SNX9 generates membrane tubulation out of CD28 clusters. Our data further reveal that CD28 clusters are in fact dynamic structures and that SNX9 regulates their stability as well as CD28 phosphorylation and the resulting production of the cytokine IL-2. In summary, our work suggests a model in which SNX9-mediated tubulation generates a membrane environment that promotes CD28 triggering and downstream signalling events.

4D analysis of malaria parasite invasion offers insights into erythrocyte membrane remodeling and parasitophorous vacuole formation.

Host membrane remodeling is indispensable for viruses, bacteria, and parasites, to subvert the membrane barrier and obtain entry into cells. The malaria parasite Plasmodium spp. induces biophysical and molecular changes to the erythrocyte membrane through the ordered secretion of its apical organelles. To understand this process and address the debate regarding how the parasitophorous vacuole membrane (PVM) is formed, we developed an approach using lattice light-sheet microscopy, which enables the parasite interaction with the host cell membrane to be tracked and characterized during invasion. Our results show that the PVM is predominantly formed from the erythrocyte membrane, which undergoes biophysical changes as it is remodeled across all stages of invasion, from pre-invasion through to PVM sealing. This approach enables a functional interrogation of parasite-derived lipids and proteins in PVM biogenesis and echinocytosis during Plasmodium falciparum invasion and promises to yield mechanistic insights regarding how this is more generally orchestrated by other intracellular pathogens.

Cytotoxic T cells swarm by homotypic chemokine signalling.

Cytotoxic T lymphocytes (CTLs) are thought to arrive at target sites either via random search or following signals by other leukocytes. Here, we reveal independent emergent behaviour in CTL populations attacking tumour masses. Primary murine CTLs coordinate their migration in a process reminiscent of the swarming observed in neutrophils. CTLs engaging cognate targets accelerate the recruitment of distant T cells through long-range homotypic signalling, in part mediated via the diffusion of chemokines CCL3 and CCL4. Newly arriving CTLs augment the chemotactic signal, further accelerating mass recruitment in a positive feedback loop. Activated effector human T cells and chimeric antigen receptor (CAR) T cells similarly employ intra-population signalling to drive rapid convergence. Thus, CTLs recognising a cognate target can induce a localised mass response by amplifying the direct recruitment of additional T cells independently of other leukocytes.

Immune cells known as cytotoxic T lymphocytes, or CTLs for short, move around the body searching for infected or damaged cells that may cause harm. Once these specialised killer cells identify a target, they launch an attack, removing the harmful cell from the body. CTLs can also recognise and eliminate cancer cells, and can be infused into cancer patients as a form of treatment called adoptive cell transfer immunotherapy. Unfortunately, this kind of treatment does not yet work well on solid tumours because the immune cells often do not infiltrate them sufficiently. It is thought that CTLs arrive at their targets either by randomly searching or by following chemicals secreted by other immune cells. However, the methods used to map the movement of these killer cells have made it difficult to determine how populations of CTLs coordinate their behaviour independently of other cells in the immune system. To overcome this barrier, Galeano Niño, Pageon, Tay et al. employed a three-dimensional model known as a tumouroid embedded in a matrix of proteins, which mimics the tissue environment of a real tumour in the laboratory. These models were used to track the movement of CTLs extracted from mice and humans, as well as human T cells engineered to recognise cancer cells. The experiments showed that when a CTL identifies a tumour cell, it releases chemical signals known as chemokines, which attract other CTLs and recruit them to the target site. Further experiments and computer simulations revealed that as the number of CTLs arriving at the target site increases, this amplifies the chemokine signal being secreted, resulting in more and more CTLs being attracted to the tumour. Other human T cells that had been engineered to recognize cancer cells were also found to employ this method of mass recruitment, and collectively 'swarm' towards targeted tumours. These findings shed new light on how CTLs work together to attack a target. It is possible that exploiting the mechanism used by CTLs could help improve the efficiency of tumour-targeting immunotherapies. However, further studies are needed to determine whether these findings can be applied to solid tumours in cancer patients.

The Lifeact-EGFP mouse is a translationally controlled fluorescent reporter of T cell activation.

It has become increasingly evident that T cell functions are subject to translational control in addition to transcriptional regulation. Here, by using live imaging of CD8+ T cells isolated from the Lifeact-EGFP mouse, we show that T cells exhibit a gain in fluorescence intensity following engagement of cognate tumour target cells. The GFP signal increase is governed by Erk1/2-dependent distal T cell receptor (TCR) signalling and its magnitude correlates with IFN-γ and TNF-α production, which are hallmarks of T cell activation. Enhanced fluorescence was due to increased translation of Lifeact-EGFP protein, without an associated increase in its mRNA. Activation-induced gains in fluorescence were also observed in naïve and CD4+ T cells from the Lifeact-EGFP reporter, and were readily detected by both flow cytometry and live cell microscopy. This unique, translationally controlled reporter of effector T cell activation simultaneously enables tracking of cell morphology, F-actin dynamics and activation state in individual migrating T cells. It is a valuable addition to the limited number of reporters of T cell dynamics and activation, and opens the door to studies of translational activity and heterogeneities in functional T cell responses in situ.

Brightness-gated two-color coincidence detection unravels two distinct mechanisms in bacterial protein translation initiation.

Life on the molecular scale is based on a complex interplay of biomolecules under which the ability of binding is crucial. Fluorescence based two-color coincidence detection (TCCD) is commonly used to characterize molecular binding, but suffers from an underestimation of coincident events. Here, we introduce a brightness-gated TCCD which overcomes this limitation and benchmark our approach with two custom-made calibration samples. Applied to a cell-free protein synthesis assay, brightness-gated TCCD unraveled a previously disregarded mode of translation initiation in bacteria.

Flotillins promote T cell receptor sorting through a fast Rab5-Rab11 endocytic recycling axis.

The targeted endocytic recycling of the T cell receptor (TCR) to the immunological synapse is essential for T cell activation. Despite this, the mechanisms that underlie the sorting of internalised receptors into recycling endosomes remain poorly understood. To build a comprehensive picture of TCR recycling during T cell activation, we developed a suite of new imaging and quantification tools centred on photoactivation of fluorescent proteins. We show that the membrane-organising proteins, flotillin-1 and -2, are required for TCR to reach Rab5-positive endosomes immediately after endocytosis and for transfer from Rab5- to Rab11a-positive compartments. We further observe that after sorting into in Rab11a-positive vesicles, TCR recycles to the plasma membrane independent of flotillin expression. Our data suggest a mechanism whereby flotillins delineate a fast Rab5-Rab11a endocytic recycling axis and functionally contribute to regulate the spatial organisation of these endosomes.

Impact of Molecular Crowding on Translational Mobility and Conformational Properties of Biological Macromolecules.

Effects of molecular crowding on structural and dynamical properties of biological macromolecules do depend on the concentration of crowding agents but also on the molecular mass and the structural compactness of the crowder molecules. By employing fluorescence correlation spectroscopy (FCS), we investigated the translational mobility of several biological macromolecules ranging from 17 kDa to 2.7 MDa. Polyethylene glycol and Ficoll polymers of different molecular masses were used in buffer solutions to mimic a crowded environment. The reduction in translational mobility of the biological tracer molecules was analyzed as a function of crowder volume fractions and was generally more pronounced in PEG as compared to Ficoll solutions. For several crowding conditions, we observed a molecular sieving effect, in which the diffusion coefficient of larger tracer molecules is reduced to a larger extent than predicted by the Stokes-Einstein relation. By employing a FRET-based biosensor, we also showed that a multiprotein complex is significantly compacted in the presence of macromolecular crowders. Importantly, with respect to sensor in vivo applications, ligand concentration determining sensors would need a crowding specific calibration in order to deliver correct cytosolic ligand concentration.

Mechanoimmunology: molecular-scale forces govern immune cell functions.

Immune cell recognition of antigens is a pivotal process in initiating immune responses against injury, pathogens, and cancers. Breakthroughs over the past decade support a major role for mechanical forces in immune responses, laying the foundation for the emerging field of mechanoimmunology. In this Perspective, we discuss the mechanical forces acting at the level of ligand-receptor interactions and how they underpin receptor triggering, signal initiation, and immune cell activation. We also highlight the novel biophysical tools and advanced imaging techniques that have afforded us the recent progress in our understanding of the role of forces in immune cell functions.

Single-Molecule FRET Measurements in Additive-Enriched Aqueous Solutions.

The addition of high amounts of chemical denaturants, salts, viscosity enhancers or macro-molecular crowding agents has an impact on the physical properties of buffer solutions. Among others, the (microscopic) viscosity, the refractive index, the dielectric constant, and the ionic strength can be affected. Here, we systematically evaluate the importance of solvent characteristics with respect to single-molecule FRET (smFRET) data. First, we present a confocal based method for the determination of fluorescence quantum yields to facilitate a fast characterization of smFRET-samples at sub-nM-concentrations. As a case study, we analyze smFRET data of structurally rigid, double-stranded DNA-oligonucleotides in aqueous buffer and in buffers with specific amounts of glycerol, guanidine hydrochloride (GdnHCl), and sodium chloride (NaCl) added. We show that the calculation of interdye distances, without taking into account solvent-induced spectral and photophysical changes of the labels, leads to deviations of up to 4 Å from the real interdye distances. Additionally, we demonstrate that electrostatic dye-dye repulsions are negligible for the interdye distance regime considered here (>50 Å). Finally, we use our approach to validate the further compaction of the already unfolded state of phosphoglycerate kinase (PGK) with decreasing denaturant concentrations, a mechanism known as coil-globule transition.

Accurate fluorescence quantum yield determination by fluorescence correlation spectroscopy.

Here, we present a comparative method for the accurate determination of fluorescence quantum yields (QYs) by fluorescence correlation spectroscopy. By exploiting the high sensitivity of single-molecule spectroscopy, we obtain the QYs of samples in the microliter range and at (sub)nanomolar concentrations. Additionally, in combination with fluorescence lifetime measurements, our method allows the quantification of both static and collisional quenching constants. Thus, besides being simple and fast, our method opens up the possibility to photophysically characterize labeled biomolecules under application-relevant conditions and with low sample consumption, which is often important in single-molecule studies.

Conformational state distributions and catalytically relevant dynamics of a hinge-bending enzyme studied by single-molecule FRET and a coarse-grained simulation.

Over the last few decades, a view has emerged showing that multidomain enzymes are biological machines evolved to harness stochastic kicks of solvent particles into highly directional functional motions. These intrinsic motions are structurally encoded, and Nature makes use of them to catalyze chemical reactions by means of ligand-induced conformational changes and states redistribution. Such mechanisms align reactive groups for efficient chemistry and stabilize conformers most proficient for catalysis. By combining single-molecule Förster resonance energy transfer measurements with normal mode analysis and coarse-grained mesoscopic simulations, we obtained results for a hinge-bending enzyme, namely phosphoglycerate kinase (PGK), which support and extend these ideas. From single-molecule Förster resonance energy transfer, we obtained insight into the distribution of conformational states and the dynamical properties of the domains. The simulations allowed for the characterization of interdomain motions of a compact state of PGK. The data show that PGK is intrinsically a highly dynamic system sampling a wealth of conformations on timescales ranging from nanoseconds to milliseconds and above. Functional motions encoded in the fold are performed by the PGK domains already in its ligand-free form, and substrate binding is not required to enable them. Compared to other multidomain proteins, these motions are rather fast and presumably not rate-limiting in the enzymatic reaction. Ligand binding slightly readjusts the orientation of the domains and feasibly locks the protein motions along a preferential direction. In addition, the functionally relevant compact state is stabilized by the substrates, and acts as a prestate to reach active conformations by means of Brownian motions.

Inter-dye distance distributions studied by a combination of single-molecule FRET-filtered lifetime measurements and a weighted accessible volume (wAV) algorithm.

Förster resonance energy transfer (FRET) is an important tool for studying the structural and dynamical properties of biomolecules. The fact that both the internal dynamics of the biomolecule and the movements of the biomolecule-attached dyes can occur on similar timescales of nanoseconds is an inherent problem in FRET studies. By performing single-molecule FRET-filtered lifetime measurements, we are able to characterize the amplitude of the motions of fluorescent probes attached to double-stranded DNA standards by means of flexible linkers. With respect to previously proposed experimental approaches, we improved the precision and the accuracy of the inter-dye distance distribution parameters by filtering out the donor-only population with pulsed interleaved excitation. A coarse-grained model is employed to reproduce the experimentally determined inter-dye distance distributions. This approach can easily be extended to intrinsically flexible proteins allowing, under certain conditions, to decouple the macromolecule amplitude of motions from the contribution of the dye linkers.

Nanosecond dynamics of calmodulin and ribosome-bound nascent chains studied by time-resolved fluorescence anisotropy.

We report a time-resolved fluorescence anisotropy study of ribosome-bound nascent chains (RNCs) of calmodulin (CaM), a prototypical member of the EF-hand family of calcium-sensing proteins. As shown in numerous studies, in vitro protein refolding can differ substantially from biosynthetic protein folding, which takes place cotranslationally and depends on the rate of polypeptide chain elongation. A challenge in this respect is to characterize the adopted conformations of nascent chains before their release from the ribosome. CaM RNCs (full-length, half-length, and first EF-hand only) were synthesized in vitro. All constructs contained a tetracysteine motif site-specifically incorporated in the first N-terminal helix; this motif is known to react with FlAsH, a biarsenic fluorescein derivative. As the dye is rotationally locked to this helix, we characterized the structural properties and folding states of polypeptide chains tethered to ribosomes and compared these with released chains. Importantly, we observed decelerated tumbling motions of ribosome-tethered and partially folded nascent chains, compared to released chains. This indicates a pronounced interaction between nascent chains and the ribosome surface, and might reflect chaperone activity of the ribosome.