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.

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.

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.

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.

Active site formation, not bond kinetics, limits adhesion rate between human neutrophils and immobilized vascular cell adhesion molecule 1.

The formation of receptor ligand bonds at the interface between different cells and between cells and substrates is a widespread phenomenon in biological systems. Physical measurements of bond formation rates between cells and substrates have been exploited to increase our understanding of the biophysical mechanisms that regulate bond formation at interfaces. Heretofore, these measurements have been interpreted in terms of simple bimolecular reaction kinetics. Discrepancies between this simple framework and the behavior of neutrophils adhering to surfaces expressing vascular cell adhesion molecule 1 (VCAM-1) motivated the development of a new kinetic framework in which the explicit formation of active bond formation sites (reaction zones) are a prerequisite for bond formation to occur. Measurements of cells interacting with surfaces having a wide range of VCAM-1 concentrations, and for different durations of contact, enabled the determination of novel kinetic rate constants for the formation of reaction zones and for the intrinsic bond kinetics. Comparison of these rates with rates determined previously for other receptor-ligand pairs points to a predominant role of extrinsic factors such as surface topography and accessibility of active molecules to regions of close contact in determining forward rates of bond formation at cell interfaces.

Adhesion between human neutrophils and immobilized endothelial ligand vascular cell adhesion molecule 1: divalent ion effects.

Integrin-mediated adhesion of circulating neutrophils to endothelium during inflammation involves multiple adhesion molecules on both neutrophils and endothelium. Most studies of neutrophil adhesion have focused on adhesion to ICAM-1 (mediated by beta(2) integrins), but interaction with the endothelial ligand vascular cell adhesion molecule 1 (VCAM-1) may also play a role in neutrophil adhesion to activated endothelium. In this study we demonstrate significant adhesion between neutrophils and VCAM-1 mediated by beta(1) integrins, principally via alpha(4)beta(1) (VLA-4). We characterize the dynamics of adhesion in terms of rate constants for a two-step bond formation process, the first involving juxtaposition of active molecules with substrate and the second involving bond formation. The results indicate that the first step is rate limiting for VLA-4-VCAM-1 interactions. Changing divalent cation composition affects these coefficients, implicating molecular conformational changes as a key step in the process.

Activation of human neutrophil Mac-1 by anion substitution.

Substituting the medium chloride with glucuronate or glutamate causes a rapid, 10 to 30-fold, increase in the binding of the monoclonal antibody, CBRM1/5, which recognizes the high-affinity conformation of the Mac-1 integrin. This change is reflected in functional adhesion assays that show increased adhesion to ICAM-1 coated beads. Blocking antibodies indicate that the increased adhesion is almost entirely due to Mac-1. The inhibitor NPPB (100 microM) reduces Cl(-) efflux into low Cl(-) medium by 75%, and blocks increased CBRM1/5 binding after stimulation with fMLP or TNF-alpha, but has no effect on the anion substitution induced increase in CBRM1/5 binding or adhesion to immobilized ICAM-1. Thus, changes in external anion composition, not internal chloride or increases in Cl(-) efflux, are responsible for Mac-1 activation. This effect is substantial. The percentage of Mac-1 in the high affinity state approaches 100% in glutamate and 50% in glucuronate, a far greater response than what is observed after stimulation with fMLP.

Immobilized IL-8 Triggers Phagocytosis and Dynamic Changes in Membrane Microtopology in Human Neutrophils.

The interaction of leukocytes with surface bound ligands can be limited by the location of the molecules relative to the surface topology of the cell. In this report, we examine the dynamic response of neutrophils to IL-8-fractalkine chimera immobilized on bead surfaces, taking into account changes in receptor occupancy resulting from changes in surface topography. As a readout for receptor signaling, we observe the dynamics of calcium release in neutrophils following contact with the IL-8 coated surface. After a delay that depended on the initial area of contact and the surface density of IL-8, the cell began to phagocytose the IL-8 coated bead. This appeared to be a pre-requisite for release of calcium, which typically followed shortly after the initiation of phagocytosis. In separate experiments, effective kinetic coefficients for the formation of bonds between immobilized IL-8 and receptors on the cell surface were determined. Using these coefficients, we were able to estimate the number of bound receptors in the nascent contact zone. Kinetic modeling of the signaling response predicted that cell spreading and a concomitant increase in the density of occupied receptors would be required for the experimentally observed calcium dynamics. Postulating that there is an increase in receptor occupancy resulting from smoothing of the cell surface as it is stretched over the bead enabled us to obtain model predictions consistent with experimental observations. This study reveals the likely importance of membrane microtopology as a rate-limiting property and potential means of regulation of cell responses stimulated by two-dimensional surface interactions.

Uropod elongation is a common final step in leukocyte extravasation through inflamed vessels.

The efficient trafficking of immune cells into peripheral nonlymphoid tissues is key to enact their protective functions. Despite considerable advances in our understanding of cell migration in secondary lymphoid organs, real-time leukocyte recruitment into inflamed tissues is not well characterized. The conventional multistep paradigm of leukocyte extravasation depends on CD18 integrin-mediated events such as rapid arrest and crawling on the surface of the endothelium and transmigration through the endothelial layer. Using enhanced three-dimensional detection of fluorescent CD18 fusion proteins in a newly developed knockin mouse, we report that extravasating leukocytes (neutrophils, monocytes, and T cells) show delayed uropod detachment and become extremely elongated before complete transmigration across the endothelium. Additionally, these cells deposit CD18(+) microparticles at the subendothelial layer before retracting the stretched uropod. Experiments with knockout mice and blocking antibodies reveal that the uropod elongation and microparticle formation are the result of LFA-1-mediated adhesion and VLA-3-mediated cell migration through the vascular basement membrane. These findings suggest that uropod elongation is a final step in the leukocyte extravasation cascade, which may be important for precise regulation of leukocyte recruitment into inflamed tissues.

Signaling and Dynamics of Activation of LFA-1 and Mac-1 by Immobilized IL-8.

The dynamic response of neutrophils to interleukin-8 (IL-8) is of central interest in inflammation. Chemokine -induced ß(2) integrin dependent adhesion can take several minutes after initial contact with IL-8 as evidenced by increased cell adhesion to intracellular adhesion molecule 1 (ICAM-1). The goal of this study is to identify signaling events that are critical for this response. We demonstrate that neither the PI3K inhibitor wortmannin, nor the PKC inhibitor bisindolymaleimide had any effect on IL-8 induced adhesion to ICAM-1. However, inhibition of PLC with U73122 or stopping the release of intracellular calcium by its downstream effector IP3 with caffeine or 2-aminoethoxydiphenyl borate completely blocked the adhesive response. Chelation of intracellular calcium with BAPTA or extracellular calcium with EGTA completely abrogated neutrophil adhesion to ICAM-1. This adhesion is mediated by LFA-1 (α(L)ß(2)) within first 300 seconds after chemokine stimulation, followed by Mac-1 (α(M)ß(2)) mediated adhesion, beginning 350 seconds after stimulus. Inhibition of p38MAP kinase results in a time course similar to Mac-1 inhibition, consistent with published evidence that Mac-1 mediated adhesion is p38MAP kinase dependent. These findings confirm a PLC dependent, PKC independent pathway from chemokine stimulus to integrin activation previously identified in other cell types, and demonstrate distinct dynamics and different requirements for LFA-1 vs. Mac-1 activation in primary human neutrophils.

Leukocyte-endothelial cell interactions are linked to vascular permeability via ICAM-1-mediated signaling.

Two key characteristics of the inflammatory response are the recruitment of leukocytes to inflamed tissue as well as changes in vessel permeability. We explored the relationship between these two processes using intravital confocal microscopy in cremasters of anesthetized (65 mg/kg Nembutal ip) mice. We provide direct evidence that intercellular adhesion molecule-1 (ICAM-1) links leukocyte-endothelial cell interactions and changes in solute permeability (Ps). Importantly, we show that arterioles, not just venules, respond to proinflammatory stimuli, thus contributing to microvascular exchange. We identified two independent, ICAM-1-mediated pathways regulating Ps. Under control conditions in wild-type (WT) mice, there is a constitutive PKC-dependent pathway (Ps = 1.0 +/- 0.10 and 2.2 +/- 0.46 x 10(-6) cm/s in arterioles and venules, respectively), which was significantly reduced in ICAM-1 knockout (KO) mice (Ps = 0.54 +/- 0.07 and 0.77 +/- 0.11 x 10(-6) cm/s). The PKC inhibitor bisindolylmaleimid l (1 micromol/l in 0.01% DMSO) decreased P(s) in WT mice to levels similar to those in ICAM-1 KO mice. Likewise, a PKC activator (phorbol-12-myristate-acetate; 1 micromol/l in 0.01% DMSO) successfully restored Ps in ICAM-1 KO vessels to be not different from that of the WT controls. On the other hand, during TNF-alpha-induced inflammation, Ps in WT mice was significantly increased (2-fold in venules and 2.5-fold in arterioles) in a Src-dependent and PKC-independent manner. The blockade of Src (PP2; 2 micromol/l in 0.01% DMSO) but not PKC significantly reduced the TNF-alpha-dependent increase in Ps. We conclude that ICAM-1 plays an essential role in the regulation of Ps in microvessels and that there are two separate (constitutive and inducible) signaling pathways that regulate permeability under normal and inflamed conditions.

Dynamics of increased neutrophil adhesion to ICAM-1 after contacting immobilized IL-8.

Changing affinity of beta(2)-integrins on neutrophils for their ligands on endothelium is a critical, regulated step in the inflammatory response. In this report, the dynamics of the neutrophil response to the inflammatory chemokine interleukin-8 (IL-8) is examined. Human IL-8 was immobilized on beads and brought into contact with neutrophils selected from whole blood samples. Resulting changes in cellular adhesion were assessed by measuring the adhesion frequency between a human neutrophil and a bead coated with the endothelial ligand ICAM-1 (intercellular adhesion molecule-1). Cells engulfed the IL-8 coated beads within a few tens of seconds, and most of the cells exhibited an increase in adhesion to ICAM-1 after approximately 5 to 10 min of contact with IL-8 at room temperature (3 to 5 min at 37 degrees C). Neither monocyte chemotactic protein-1 (MCP-1) nor anti-CD45-coated beads caused any changes in adhesion to ICAM-1. IL-8 induced adhesion was blocked by antibody against CD18. At lower surface density of chemokine, approximately 20% of IL-8 coated beads adhered but were not engulfed by the cells, although the increase in adhesion for ICAM-1 was still effected. Heterogeneity in the cellular response and variability between donors was also noted.

Micromechanical tests of adhesion dynamics between neutrophils and immobilized ICAM-1.

Strong, integrin-mediated adhesion of neutrophils to endothelium during inflammation is a dynamic process, requiring a conformational change in the integrin molecule to increase its affinity for its endothelial counterreceptors. To avoid general activation of the cell, Mg(2+) was used to induce the high-affinity integrin conformation, and micromechanical methods were used to determine adhesion probability to beads coated with the endothelial ligand ICAM-1. Neutrophils in Mg(2+) bind to the beads with much greater frequency and strength than in the presence of Ca(2+). An increase in adhesion strength and frequency was observed with both increasing temperature and contact duration (from 2 s to 1 min, 21 or 37 degrees C). The dependence of adhesion probability on contact time or receptor density yielded estimates of the effective reverse rate constant, k(r), and the equilibrium association constant, K(a), for binding of neutrophils to ICAM-1 coated surfaces in Mg(2+): k(r) approximately 0.7 s(-1) and the product K(a)rho(c) approximately 2.4 x 10(-4), where rho(c) is the density of integrin on the cell surface.