ST3GAL1 and ßII-spectrin pathways control CAR T cell migration to target tumors.

Adoptive transfer of genetically engineered chimeric antigen receptor (CAR) T cells is becoming a promising treatment option for hematological malignancies. However, T cell immunotherapies have mostly failed in individuals with solid tumors. Here, with a CRISPR-Cas9 pooled library, we performed an in vivo targeted loss-of-function screen and identified ST3 ß-galactoside α-2,3-sialyltransferase 1 (ST3GAL1) as a negative regulator of the cancer-specific migration of CAR T cells. Analysis of glycosylated proteins revealed that CD18 is a major effector of ST3GAL1 in activated CD8+ T cells. ST3GAL1-mediated glycosylation induces the spontaneous nonspecific tissue sequestration of T cells by altering lymphocyte function-associated antigen-1 (LFA-1) endocytic recycling. Engineered CAR T cells with enhanced expression of ßII-spectrin, a central LFA-1-associated cytoskeleton molecule, reversed ST3GAL1-mediated nonspecific T cell migration and reduced tumor growth in mice by improving tumor-specific homing of CAR T cells. These findings identify the ST3GAL1-ßII-spectrin axis as a major cell-intrinsic program for cancer-targeting CAR T cell migration and as a promising strategy for effective T cell immunotherapy.

Changes in endothelial glycocalyx layer protective ability after inflammatory stimulus.

Leukocyte adhesion to the endothelium is an important early step in the initiation and progression of sepsis. The endothelial glycocalyx layer (EGL) has been implicated in neutrophil adhesion and barrier dysfunction, but studies in this area are few. In this report, we examine the hypothesis that damage to the structure of the EGL caused by inflammation leads to increased leukocyte adhesion and endothelial barrier dysfunction. We used human umbilical vein endothelial cells enzymatically treated to remove the EGL components hyaluronic acid (HA) and heparan sulfate (HS) as a model for EGL damage. Using atomic force microscopy, we show reductions in EGL thickness after removal of either HA or HS individually, but the largest decrease, comparable with TNF-α treatment, was observed when both HA and HS were removed. Interestingly, removal of HS or HA individually did not affect neutrophil adhesion significantly, but removal of both constituents resulted in increased neutrophil adhesion. To test EGL contributions to endothelial barrier properties, we measured transendothelial electrical resistance (TEER) and diffusion of fluorescently labeled dextran (10 kDa molecular weight) across the monolayer. Removal of EGL components decreased TEER but had an insignificant effect on dextran diffusion rates. The reduction in TEER suggests that disruption of the EGL may predispose endothelial cells to increased rates of fluid leakage. These data support the view that damage to the EGL during inflammation has significant effects on the accessibility of adhesion molecules, likely facilitates leukocyte adhesion, and may also contribute to increased rates of fluid transport into tissues.

Constitutive Model of Erythrocyte Membranes with Distributions of Spectrin Orientations and Lengths.

We present an analytical hyperelastic constitutive model of the red blood cell (erythrocyte) membrane based on recently improved characterizations of density and microscopic structure of its spectrin network from proteomics and cryo-electron tomography. The model includes distributions of both orientations and natural lengths of spectrin and updated copy numbers of proteins. By applying finite deformation to the spectrin network, we obtain the total free energy and stresses in terms of invariants of shear and area deformation. We generalize an expression of the initial shear modulus, which is independent of the number of molecular orientations within the network and also derive a simplified version of the model. We apply the model and its simplified version to analyze micropipette aspiration computationally and analytically and explore the effect of local cytoskeletal density change. We also explore the discrepancies among shear modulus values measured using different experimental techniques reported in the literature. We find that the model exhibits hardening behavior and can explain many of these discrepancies. Moreover, we find that the distribution of natural lengths plays a crucial role in the hardening behavior when the correct copy numbers of proteins are used. The initial shear modulus values we obtain using our current model (5.9-15.6 pN/µm) are close to the early estimates (6-9 pN/µm). This new, to our knowledge, constitutive model establishes a direct connection between the molecular structure of spectrin networks and constitutive laws and also defines a new picture of a much denser spectrin network than assumed in prior studies.

Endothelial Glycocalyx Layer Properties and Its Ability to Limit Leukocyte Adhesion.

The endothelial glycocalyx layer (EGL), which consists of long proteoglycans protruding from the endothelium, acts as a regulator of inflammation by preventing leukocyte engagement with adhesion molecules on the endothelial surface. The amount of resistance to adhesive events the EGL provides is the result of two properties: EGL thickness and stiffness. To determine these, we used an atomic force microscope to indent the surfaces of cultured endothelial cells with a glass bead and evaluated two different approaches for interpreting the resulting force-indentation curves. In one, we treat the EGL as a molecular brush, and in the other, we treat it as a thin elastic layer on an elastic half-space. The latter approach proved more robust in our hands and yielded a thickness of 110 nm and a modulus of 0.025 kPa. Neither value showed significant dependence on indentation rate. The brush model indicated a larger layer thickness (∼350 nm) but tended to result in larger uncertainties in the fitted parameters. The modulus of the endothelial cell was determined to be 3.0-6.5 kPa (1.5-2.5 kPa for the brush model), with a significant increase in modulus with increasing indentation rates. For forces and leukocyte properties in the physiological range, a model of a leukocyte interacting with the endothelium predicts that the number of molecules within bonding range should decrease by an order of magnitude because of the presence of a 110-nm-thick layer and even further for a glycocalyx with larger thickness. Consistent with these predictions, neutrophil adhesion increased for endothelial cells with reduced EGL thickness because they were grown in the absence of fluid shear stress. These studies establish a framework for understanding how glycocalyx layers with different thickness and stiffness limit adhesive events under homeostatic conditions and how glycocalyx damage or removal will increase leukocyte adhesion potential during inflammation.

Ultrathin Dual-Scale Nano- and Microporous Membranes for Vascular Transmigration Models.

Selective cellular transmigration across the microvascular endothelium regulates innate and adaptive immune responses, stem cell localization, and cancer cell metastasis. Integration of traditional microporous membranes into microfluidic vascular models permits the rapid assay of transmigration events but suffers from poor reproduction of the cell permeable basement membrane. Current microporous membranes in these systems have large nonporous regions between micropores that inhibit cell communication and nutrient exchange on the basolateral surface reducing their physiological relevance. Here, the use of 100 nm thick continuously nanoporous silicon nitride membranes as a base substrate for lithographic fabrication of 3 µm pores is presented, resulting in a highly porous (≈30%), dual-scale nano- and microporous membrane for use in an improved vascular transmigration model. Ultrathin membranes are patterned using a precision laser writer for cost-effective, rapid micropore design iterations. The optically transparent dual-scale membranes enable complete observation of leukocyte egress across a variety of pore densities. A maximal density of ≈14 micropores per cell is discovered beyond which cell-substrate interactions are compromised giving rise to endothelial cell losses under flow. Addition of a subluminal extracellular matrix rescues cell adhesion, allowing for the creation of shear-primed endothelial barrier models on nearly 30% continuously porous substrates.

Finite element modeling to analyze TEER values across silicon nanomembranes.

Silicon nanomembranes are ultrathin, highly permeable, optically transparent and biocompatible substrates for the construction of barrier tissue models. Trans-epithelial/endothelial electrical resistance (TEER) is often used as a non-invasive, sensitive and quantitative technique to assess barrier function. The current study characterizes the electrical behavior of devices featuring silicon nanomembranes to facilitate their application in TEER studies. In conventional practice with commercial systems, raw resistance values are multiplied by the area of the membrane supporting cell growth to normalize TEER measurements. We demonstrate that under most circumstances, this multiplication does not 'normalize' TEER values as is assumed, and that the assumption is worse if applied to nanomembrane chips with a limited active area. To compare the TEER values from nanomembrane devices to those obtained from conventional polymer track-etched (TE) membranes, we develop finite element models (FEM) of the electrical behavior of the two membrane systems. Using FEM and parallel cell-culture experiments on both types of membranes, we successfully model the evolution of resistance values during the growth of endothelial monolayers. Further, by exploring the relationship between the models we develop a 'correction' function, which when applied to nanomembrane TEER, maps to experiments on conventional TE membranes. In summary, our work advances the the utility of silicon nanomembranes as substrates for barrier tissue models by developing an interpretation of TEER values compatible with conventional systems.

Piezo1 regulates mechanotransductive release of ATP from human RBCs.

Piezo proteins (Piezo1 and Piezo2) are recently identified mechanically activated cation channels in eukaryotic cells and associated with physiological responses to touch, pressure, and stretch. In particular, human RBCs express Piezo1 on their membranes, and mutations of Piezo1 have been linked to hereditary xerocytosis. To date, however, physiological functions of Piezo1 on normal RBCs remain poorly understood. Here, we show that Piezo1 regulates mechanotransductive release of ATP from human RBCs by controlling the shear-induced calcium (Ca(2+)) influx. We find that, in human RBCs treated with Piezo1 inhibitors or having mutant Piezo1 channels, the amounts of shear-induced ATP release and Ca(2+) influx decrease significantly. Remarkably, a critical extracellular Ca(2+) concentration is required to trigger significant ATP release, but membrane-associated ATP pools in RBCs also contribute to the release of ATP. Our results show how Piezo1 channels are likely to function in normal RBCs and suggest a previously unidentified mechanotransductive pathway in ATP release. Thus, we anticipate that the study will impact broadly on the research of red cells, cellular mechanosensing, and clinical studies related to red cell disorders and vascular disease.

A simple approach for bioactive surface calibration using evanescent waves.

When investigating the interaction of cells with surfaces, it is becoming increasingly important to perform quantitative measurements of surface protein density to understand reaction kinetics. Previously, to calibrate a surface for an experiment one would have to use a radiometric assay or strip the surface with acid and perform a mass quantification. Although both of these methodologies have been proven to be effective measurement techniques for surface quantification, they can be time consuming and require substantial amounts of material. The latter is particularly problematic when working with specialized molecules or constructs that may be expensive to produce and/or only available in small quantities. Here we present a simple method to measure the intensity and penetration depth of an evanescent wave, and use this information to quantify the density of surface molecules in a microscopic region of a transparent surface.

Halloysite Nanotube Coatings Suppress Leukocyte Spreading.

The nanoscale topography of adhesive surfaces is known to be an important factor governing cellular behavior. Previous work has shown that surface coatings composed of halloysite nanotubes enhance the adhesion, and therefore capture of, rare target cells such as circulating tumor cells. Here we demonstrate a unique feature of these coatings in their ability to reduce the adhesion of leukocytes and prevent leukocyte spreading. Surfaces were prepared with coatings of halloysite nanotubes and functionalized for leukocyte adhesion with E-selectin, and the dilution of nanotube concentration revealed a threshold concentration below which cell spreading became comparable to smooth surfaces. Evaluation of surface roughness characteristics determined that the average distance between discrete surface features correlated with adhesion metrics, with a separation distance of ∼2 µm identified as the critical threshold. Computational modeling of the interaction of leukocytes with halloysite nanotube-coated surfaces of varying concentrations demonstrates that the geometry of the cell surface and adhesive counter-surface produces a significantly diminished effective contact area compared to a leukocyte interacting with a smooth surface.

Opposing roles for RhoH GTPase during T-cell migration and activation.

T cells spend the majority of their time perusing lymphoid organs in search of cognate antigen presented by antigen presenting cells (APCs) and then quickly recirculate through the bloodstream to another lymph node. Therefore, regulation of a T-cell response is dependent upon the ability of cells to arrive in the correct location following chemokine gradients ("go" signal) as well as to receive appropriate T-cell receptor (TCR) activation signals upon cognate antigen recognition ("stop" signal). However, the mechanisms by which T cells regulate these go and stop signals remain unclear. We found that overexpression of the hematopoietic-specific RhoH protein in the presence of chemokine signals resulted in decreased Rap1-GTP and LFA-1 adhesiveness to ICAM-1, thus impairing T-cell chemotaxis; while in the presence of TCR signals, there were enhanced and sustained Rap1-GTP and LFA-1 activation as well as prolonged T:APC conjugates. RT-PCR analyses of activated CD4(+) T cells and live images of T-cell migration and immunological synapse (IS) formation revealed that functions of RhoH took place primarily at the levels of transcription and intracellular distribution. Thus, we conclude that RhoH expression provides a key molecular determinant that allows T cells to switch between sensing chemokine-mediated go signals and TCR-dependent stop signals.

Cell surface topography is a regulator of molecular interactions during chemokine-induced neutrophil spreading.

Adhesive interactions between neutrophils and endothelium involve chemokine-induced neutrophil spreading and subsequent crawling on the endothelium to sites of transmigration. We investigated the importance of cell topography in this process using immunofluorescence, scanning electron microscopy, and live-cell imaging using total internal reflectance microscopy to observe redistribution of key membrane proteins, both laterally and relative to surface topography, during neutrophil spreading onto glass coated with interleukin 8. During formation of the lamellipod, L-selectin is distributed on microvilli tips along the top of the lamellipodium, whereas the interleukin 8 receptors CXCR1 and CXCR2 and the integrin LFA-1 (αLß2) were present at the interface between the lamellipodium and the substrate. Total internal reflection fluorescence imaging indicated that LFA-1 and both chemokine receptors redistributed into closer contact with the substrate as the cells spread onto the surface and remodeled their topography. A geometric model of the surface remodeling with nonuniform distribution of molecules and a realistic distribution of microvilli heights was matched to the data, and the fits indicated a 1000-fold increase in the concentration of chemokine receptors and integrins available for bond formation at the interface. These observations imply that topographical remodeling is a key mechanism for regulating cell adhesion and surface-induced activation of cells.

Sided Stimulation of Endothelial Cells Modulates Neutrophil Trafficking in an In Vitro Sepsis Model.

While the role of dysregulated polymorphonuclear leukocyte (PMN) transmigration in septic mediated tissue damage is well documented, strategies to mitigate aberrant transmigration across endothelium have yet to yield viable therapeutics. Recently, microphysiological systems (MPS) have emerged as novel in vitro mimetics that facilitate the development of human models of disease. With this advancement, aspects of endothelial physiology that are difficult to assess with other models can be directly probed. In this study, the role of endothelial cell (EC) apicobasal polarity on leukocyte trafficking response is evaluated with the µSiM-MVM (microphysiological system enabled by a silicon membrane - microvascular mimetic). Here, ECs are stimulated either apically or basally with a cytokine cocktail to model a septic-like challenge before introducing healthy donor PMNs into the device. Basally oriented stimulation generated a stronger PMN transmigratory response versus apical stimulation. Importantly, healthy PMNs are unable to migrate towards a bacterial peptide chemoattractant when ECs are apically stimulated, which mimics the attenuated PMN chemotaxis seen in sepsis. Escalating the apical inflammatory stimulus by a factor of five is necessary to elicit high PMN transmigration levels across endothelium. These results demonstrate that EC apicobasal polarity modulates PMN transmigratory behavior and provides insight into the mechanisms underlying sepsis.

Dynamics of adhesion molecule domains on neutrophil membranes: surfing the dynamic cell topography.

Lateral organization and mobility of adhesion molecules play a significant role in determining the avidity with which cells can bind to target cells or surfaces. Recently, we have shown that the lateral mobility of the principal adhesion molecules on neutrophils is lower for rolling associated adhesion molecules (RAAMs: L-selectin and PSGL-1) than for ß2 integrins (LFA-1 and Mac-1). Here we report that all four adhesion molecules exhibit distinct punctate distributions that are mobile on the cell surface. Using uniform illumination image correlation microscopy, we measure the lateral mobility of these topologically distinct domains. For all four molecules, we find that diffusion coefficients calculated from domain mobility agree with measurements we made previously using fluorescence recovery after photobleaching. This agreement indicates that the transport of receptors on the surface of the resting neutrophil is dominated by the lateral movement of domains rather than individual molecules. The diffusion of pre-assembled integrin domains to zones of neutrophil/endothelial contact may provide a mechanism to facilitate high avidity adhesion during the earliest stages of firm arrest.

Activation effects on the physical characteristics of T lymphocytes.

The deformability of leukocytes is relevant to a wide array of physiological and pathophysiological behaviors. The goal of this study is to provide a detailed, quantitative characterization of the mechanical properties of T cells and how those properties change with activation. We tested T cells and CD8+ cells isolated from peripheral blood samples of healthy donors either immediately (naïve population) or after 7 days of activation in vitro. Single-cell micropipette aspiration was used to test the mechanical properties. T cells exhibit the general characteristics of a highly viscous liquid drop with a cortical "surface" tension, T cort. The time course of each cell entry into the micropipette was measured at two different aspiration pressures to test for shear thinning behavior. The data were analyzed in the framework of an approximate mechanical model of the cell deformation to determine the cortical tension, the cell volume, the magnitude of the initial cell entry, the characteristic viscosity µ o, and the shear thinning coefficient, b. Activation generally caused increases in cellular resistance to deformation and a broadening of the distribution of cell properties. The cell volume increased substantially upon cell activation from ∼200 µm3 to ∼650 µm3. Naive and activated T cells had similar mean cortical tension (∼150 pN/µm). However, compared to naïve CD8+ cells, the cortical tension of activated CD8+ cells increased significantly to ∼250 pN/µm. Dynamic resistance of naive CD8+ T cells, as reflected in their characteristic viscosity, was ∼870 Pa and significantly increased to 1,180 Pa after in vitro activation. The magnitude of the instantaneous projection length as the cell enters the pipette (L init) was more than doubled for activated vs. naive cells. All cell types exhibited shear thinning behavior with coefficients b in the range 0.5-0.65. Increased cell size, cortical tension, and characteristic viscosity all point to increased resistance of activated T cells to passage through the microvasculature, likely contributing to cell trapping. The increased initial elastic response of cells after activation was unexpected and could point to instability in the cell that might contribute to spontaneous cell motility.

A computer vision approach for analyzing label free leukocyte trafficking dynamics on a microvascular mimetic.

High-content imaging techniques in conjunction with in vitro microphysiological systems (MPS) allow for novel explorations of physiological phenomena with a high degree of translational relevance due to the usage of human cell lines. MPS featuring ultrathin and nanoporous silicon nitride membranes (µSiM) have been utilized in the past to facilitate high magnification phase contrast microscopy recordings of leukocyte trafficking events in a living mimetic of the human vascular microenvironment. Notably, the imaging plane can be set directly at the endothelial interface in a µSiM device, resulting in a high-resolution capture of an endothelial cell (EC) and leukocyte coculture reacting to different stimulatory conditions. The abundance of data generated from recording observations at this interface can be used to elucidate disease mechanisms related to vascular barrier dysfunction, such as sepsis. The appearance of leukocytes in these recordings is dynamic, changing in character, location and time. Consequently, conventional image processing techniques are incapable of extracting the spatiotemporal profiles and bulk statistics of numerous leukocytes responding to a disease state, necessitating labor-intensive manual processing, a significant limitation of this approach. Here we describe a machine learning pipeline that uses a semantic segmentation algorithm and classification script that, in combination, is capable of automated and label-free leukocyte trafficking analysis in a coculture mimetic. The developed computational toolset has demonstrable parity with manually tabulated datasets when characterizing leukocyte spatiotemporal behavior, is computationally efficient and capable of managing large imaging datasets in a semi-automated manner.

LFA-1 and Mac-1 define characteristically different intralumenal crawling and emigration patterns for monocytes and neutrophils in situ.

To exit blood vessels, most (∼80%) of the lumenally adhered monocytes and neutrophils crawl toward locations that support transmigration. Using intravital confocal microscopy of anesthetized mouse cremaster muscle, we separately examined the crawling and emigration patterns of monocytes and neutrophils in blood-perfused unstimulated or TNF-α-activated venules. Most of the interacting cells in microvessels are neutrophils; however, in unstimulated venules, a greater percentage of the total monocyte population is adherent compared with neutrophils (58.2 ± 6.1% versus 13.6 ± 0.9%, adhered/total interacting), and they crawl for significantly longer distances (147.3 ± 13.4 versus 61.8 ± 5.4 µm). Intriguingly, after TNF-α activation, monocytes crawled for significantly shorter distances (67.4 ± 9.6 µm), resembling neutrophil crawling. Using function-blocking Abs, we show that these different crawling patterns were due to CD11a/CD18 (LFA-1)- versus CD11b/CD18 (Mac-1)-mediated crawling. Blockade of either Mac-1 or LFA-1 revealed that both LFA-1 and Mac-1 contribute to monocyte crawling; however, the LFA-1-dependent crawling in unstimulated venules becomes Mac-1 dependent upon inflammation, likely due to increased expression of Mac-1. Mac-1 alone was responsible for neutrophil crawling in both unstimulated and TNF-α-activated venules. Consistent with the role of Mac-1 in crawling, Mac-1 block (compared with LFA-1) was also significantly more efficient in blocking TNF-α-induced extravasation of both monocytes and neutrophils in cremaster tissue and the peritoneal cavity. Thus, mechanisms underlying leukocyte crawling are important in regulating the inflammatory responses by regulating the numbers of leukocytes that transmigrate.

Molecular mechanisms underlying the heterogeneous barrier responses of two primary endothelial cell types to sphingosine-1-phosphate.

Sphingosine-1-phosphate (S1P) signals to enhance or destabilize the vascular endothelial barrier depending on the receptor engaged. Here, we investigated the differential barrier effects of S1P on two influential primary endothelial cell (EC) types, human umbilical vein endothelial cells (HUVECs) and human pulmonary microvascular endothelial cells (HPMECs). S1PR1 (barrier protective) and S1PR3 (barrier disruptive) surface and gene expression were quantified by flow cytometry and immunofluorescence, and RT-qPCR, respectively. Functional evaluation of EC monolayer permeability in response to S1P was quantified with transendothelial electrical resistance (TEER) and small molecule permeability. S1P significantly enhanced HUVEC barrier function, while promoting HPMEC barrier breakdown. Immunofluorescence and flow cytometry analysis showed select, S1PR3-high HPMECs, suggesting susceptibility to barrier destabilization following S1P exposure. Reevaluation of HPMEC barrier following S1P exposure under inflamed conditions demonstrated synergistic barrier disruptive effects of pro-inflammatory cytokine and S1P. The role of the Rho-ROCK signaling pathway under these conditions was confirmed through ROCK1/2 inhibition (Y-27632). Thus, the heterogeneous responses of ECs to S1P signaling are mediated through Rho-ROCK signaling, and potentially driven by differences in the surface expression of S1PR3.

The Modular µSiM Reconfigured: Integration of Microfluidic Capabilities to Study In Vitro Barrier Tissue Models under Flow.

Microfluidic tissue barrier models have emerged to address the lack of physiological fluid flow in conventional "open-well" Transwell-like devices. However, microfluidic techniques have not achieved widespread usage in bioscience laboratories because they are not fully compatible with traditional experimental protocols. To advance barrier tissue research, there is a need for a platform that combines the key advantages of both conventional open-well and microfluidic systems. Here, a plug-and-play flow module is developed to introduce on-demand microfluidic flow capabilities to an open-well device that features a nanoporous membrane and live-cell imaging capabilities. The magnetic latching assembly of this design enables bi-directional reconfiguration and allows users to conduct an experiment in an open-well format with established protocols and then add or remove microfluidic capabilities as desired. This work also provides an experimentally-validated flow model to select flow conditions based on the experimental needs. As a proof-of-concept, flow-induced alignment of endothelial cells and the expression of shear-sensitive gene targets are demonstrated, and the different phases of neutrophil transmigration across a chemically stimulated endothelial monolayer under flow conditions are visualized. With these experimental capabilities, it is anticipated that both engineering and bioscience laboratories will adopt this reconfigurable design due to the compatibility with standard open-well protocols.

The Modular µSiM: A Mass Produced, Rapidly Assembled, and Reconfigurable Platform for the Study of Barrier Tissue Models In Vitro.

Advanced in vitro tissue chip models can reduce and replace animal experimentation and may eventually support "on-chip" clinical trials. To realize this potential, however, tissue chip platforms must be both mass-produced and reconfigurable to allow for customized design. To address these unmet needs, an extension of the µSiM (microdevice featuring a silicon-nitride membrane) platform is introduced. The modular µSiM (m-µSiM) uses mass-produced components to enable rapid assembly and reconfiguration by laboratories without knowledge of microfabrication. The utility of the m-µSiM is demonstrated by establishing an hiPSC-derived blood-brain barrier (BBB) in bioengineering and nonengineering, brain barriers focused laboratories. In situ and sampling-based assays of small molecule diffusion are developed and validated as a measure of barrier function. BBB properties show excellent interlaboratory agreement and match expectations from literature, validating the m-µSiM as a platform for barrier models and demonstrating successful dissemination of components and protocols. The ability to quickly reconfigure the m-µSiM for coculture and immune cell transmigration studies through addition of accessories and/or quick exchange of components is then demonstrated. Because the development of modified components and accessories is easily achieved, custom designs of the m-µSiM shall be accessible to any laboratory desiring a barrier-style tissue chip platform.

Activated integrin VLA-4 localizes to the lamellipodia and mediates T cell migration on VCAM-1.

Lymphocyte migration from blood into lymphoid tissues or to sites of inflammation occurs through interactions between cell surface integrins and their ligands expressed on the vascular endothelium and the extracellular matrix. VLA-4 (alpha(4)beta(1)) is a key integrin in the effective trafficking of lymphocytes. Although it has been well established that integrins undergo functionally significant conformational changes to mediate cell adhesion, there is no mechanistic information that explains how these are dynamically and spatially regulated during lymphocyte polarization and migration. Using dynamic fluorescence resonance energy transfer analysis of a novel VLA-4 FRET sensor under total internal reflection fluorescence microscopy, we show that VLA-4 activation localizes to the lamellipodium in living cells. During T cell migration on VCAM-1, VLA-4 activation concurs with spatial redistribution of chemokine receptor and active Rap1 at the leading edge. Selective inhibition of the activated VLA-4 at the leading edge with a small molecule inhibitor is sufficient to block T cell migration. These data suggest that a subpopulation of activated VLA-4 is mainly localized to the leading edge of polarized human T cells and is critical for T cell migration on VCAM-1.