PI3K in T Cell Adhesion and Trafficking.

PI3K signalling is required for activation, differentiation, and trafficking of T cells. PI3Kδ, the dominant PI3K isoform in T cells, has been extensively characterised using PI3Kδ mutant mouse models and PI3K inhibitors. Furthermore, characterisation of patients with Activated PI3K Delta Syndrome (APDS) and mouse models with hyperactive PI3Kδ have shed light on how increased PI3Kδ activity affects T cell functions. An important function of PI3Kδ is that it acts downstream of TCR stimulation to activate the major T cell integrin, LFA-1, which controls transendothelial migration of T cells as well as their interaction with antigen-presenting cells. PI3Kδ also suppresses the cell surface expression of CD62L and CCR7 which controls the migration of T cells across high endothelial venules in the lymph nodes and S1PR1 which controls lymph node egress. Therefore, PI3Kδ can control both entry and exit of T cells from lymph nodes as well as the recruitment to and retention of T cells within inflamed tissues. This review will focus on the regulation of adhesion receptors by PI3Kδ and how this contributes to T cell trafficking and localisation. These findings are relevant for our understanding of how PI3Kδ inhibitors may affect T cell redistribution and function.

Stromal cell-derived DEL-1 inhibits Tfh cell activation and inflammatory arthritis.

The secreted protein developmental endothelial locus 1 (DEL-1) regulates inflammatory cell recruitment and protects against inflammatory pathologies in animal models. Here, we investigated DEL-1 in inflammatory arthritis using collagen-induced arthritis (CIA) and collagen Ab-induced arthritis (CAIA) models. In both models, mice with endothelium-specific overexpression of DEL-1 were protected from arthritis relative to WT controls, whereas arthritis was exacerbated in DEL-1-deficient mice. Compared with WT controls, mice with collagen VI promoter-driven overexpression of DEL-1 in mesenchymal cells were protected against CIA but not CAIA, suggesting a role for DEL-1 in the induction of the arthritogenic Ab response. Indeed, DEL-1 was expressed in perivascular stromal cells of the lymph nodes and inhibited Tfh and germinal center B cell responses. Mechanistically, DEL-1 inhibited DC-dependent induction of Tfh cells by targeting the LFA-1 integrin on T cells. Overall, DEL-1 restrained arthritis through a dual mechanism, one acting locally in the joints and associated with the anti-recruitment function of endothelial cell-derived DEL-1; the other mechanism acting systemically in the lymph nodes and associated with the ability of stromal cell-derived DEL-1 to restrain Tfh responses. DEL-1 may therefore be a promising therapeutic for the treatment of inflammatory arthritis.

Aggregatibacter actinomycetemcomitans Leukotoxin (LtxA) Requires Death Receptor Fas, in Addition to LFA-1, To Trigger Cell Death in T Lymphocytes.

Leukotoxin (LtxA) (trade name, Leukothera) is a protein secreted by the oral bacterium Aggregatibacter actinomycetemcomitansA. actinomycetemcomitans is an oral pathogen strongly associated with development of localized aggressive periodontitis. LtxA acts as a virulence factor for A. actinomycetemcomitans by binding to the ß2 integrin lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) on white blood cells (WBCs) and causing cell death. In addition, because of its specificity for malignant and activated WBCs, LtxA is being investigated as a therapeutic agent for treatment of hematological malignancies and autoimmune diseases. Here, we report the successful generation and characterization of Jurkat T lymphocytes with deletions in CD18, CD11a, and Fas that were engineered using CRISPR/Cas9 gene editing. Using these clones, we demonstrate the specificity of LtxA for cells expressing LFA-1. We also demonstrate the requirement of the cell death receptor Fas for LtxA-mediated cell death in T lymphocytes. We show that LFA-1 and Fas are early events in the LtxA-mediated cell death cascade as caspase activation and mitochondrial perturbation do not occur in the absence of either receptor. To our knowledge, LtxA is the first molecule, other than FasL, known to require the Fas death receptor to initiate cell death. Knowledge of the mechanism of cell death induced by LtxA will facilitate the understanding of LtxA as a bacterial virulence factor and development of it as a potential therapeutic agent.

Microvascular Mechanisms of Polyphosphate-Induced Neutrophil-Endothelial Cell Interactions in vivo.

BACKGROUND: Polyphosphates (PolyPs) have been reported to exert pro-inflammatory effects. However, the molecular mechanisms regulating PolyP-provoked tissue accumulation of leukocytes are not known. The aim of the present investigation was to determine the role of specific adhesion molecules in PolyP-mediated leukocyte recruitment. METHODS: PolyPs and TNF-α were intrascrotally administered, and anti-P-selectin, anti-E-selectin, anti-P-selectin glycoprotein ligand-1 (PSGL-1), anti-membrane-activated complex-1 (Mac-1), anti-lymphocyte function antigen-1 (LFA-1), and neutrophil depletion antibodies were injected intravenously or intraperitoneally. Intravital microscopy of the mouse cremaster microcirculation was used to examine leukocyte-endothelium interactions and recruitment in vivo. RESULTS: Intrascrotal injection of PolyPs increased leukocyte accumulation. Depletion of neutrophils abolished PolyP-induced leukocyte-endothelium interactions, indicating that neutrophils were the main leukocyte subtype responding to PolyP challenge. Immunoneutralization of P-selectin and PSGL-1 abolished PolyP-provoked neutrophil rolling, adhesion, and emigration. Moreover, immunoneutralization of Mac-1 and LFA-1 had no impact on neutrophil rolling but markedly reduced neutrophil adhesion and emigration evoked by PolyPs. CONCLUSION: These results suggest that P-selectin and PSGL-1 exert important roles in PolyP-induced inflammatory cell recruitment by mediating neutrophil rolling. In addition, our data show that Mac-1 and LFA-1 are necessary for supporting PolyP-triggered firm adhesion of neutrophils to microvascular endothelium. These novel findings define specific molecules as potential targets for pharmacological intervention in PolyP-dependent inflammatory diseases.

Intramembrane ionic protein-lipid interaction regulates integrin structure and function.

Protein transmembrane domains (TMDs) are generally hydrophobic, but our bioinformatics analysis shows that many TMDs contain basic residues at terminal regions. Physiological functions of these membrane-snorkeling basic residues are largely unclear. Here, we show that a membrane-snorkeling Lys residue in integrin αLß2 (also known as lymphocyte function-associated antigen 1 [LFA-1]) regulates transmembrane heterodimer formation and integrin adhesion through ionic interplay with acidic phospholipids and calcium ions (Ca2+) in T cells. The amino group of the conserved Lys ionically interacts with the phosphate group of acidic phospholipids to stabilize αLß2 transmembrane association, thus keeping the integrin at low-affinity conformation. Intracellular Ca2+ uses its charge to directly disrupt this ionic interaction, leading to the transmembrane separation and the subsequent extracellular domain extension to increase adhesion activity. This Ca2+-mediated regulation is independent on the canonical Ca2+ signaling or integrin inside-out signaling. Our work therefore showcases the importance of intramembrane ionic protein-lipid interaction and provides a new mechanism of integrin activation.

Blockade of adhesion molecule lymphocyte function-associated antigen-1 improves long-term heart allograft survival in mixed chimeras.

BACKGROUND: The mixed chimerism approach for intentional induction of donor-specific tolerance was shown to be successful in various models from mice to humans. For transplant patients, the approach would obviate the need for long-term immunosuppression and associated side effects; moreover, it would preclude the risk of late graft loss due to chronic rejection. Widespread clinical application is hindered by toxicities related to recipient pre-conditioning. Herein we aimed to investigate a clinically relevant protocol for tolerance induction to cardiac allografts, sparing CD40 blockade or T-cell depletion. METHODS: B6 mice were conditioned with non-myeloablative total body irradiation, fully mismatched BALB/c bone marrow cells, and short-term therapy, based on either anti- lymphocyte function-associated antigen-1 (anti-LFA-1) or anti-CD40L. Multilineage chimerism was followed by flow-cytometric analysis, tolerance was assessed with skin and heart allografts from fully or major histocompatibility complex-mismatched donors. Mechanisms of tolerance were investigated by analysis of donor-specific antibodies (DSAs), mixed lymphocyte reaction (MLR) assays, and deletion of donor-reactive T cells. RESULTS: We found that the combination of cytotoxic T-lymphocyte antigen 4 immunoglobulin (CTLA4Ig) and rapamycin with LFA-1 blockade enhanced bone marrow engraftment and led to more efficient T-cell engraftment and subsequent tolerization. Although fully mismatched skin grafts were chronically rejected, primarily vascularized heart allografts survived indefinitely and without signs of chronic rejection, independent of minor antigen mismatches. CONCLUSIONS: We have demonstarted a robust protocol for the induction of tolerance for cardiac allografts in the absence of CD40 blockade. Our findings demonstrate the potential of a clinically relevant minimal conditioning protocol designed to induce lifelong immunologic tolerance toward cardiac allografts.

Forced-rupture of cell-adhesion complexes reveals abrupt switch between two brittle states.

Cell adhesion complexes (CACs), which are activated by ligand binding, play key roles in many cellular functions ranging from cell cycle regulation to mediation of cell extracellular matrix adhesion. Inspired by single molecule pulling experiments using atomic force spectroscopy on leukocyte function-associated antigen-1 (LFA-1), expressed in T-cells, bound to intercellular adhesion molecules (ICAM), we performed constant loading rate (rf) and constant force (F) simulations using the self-organized polymer model to describe the mechanism of ligand rupture from CACs. The simulations reproduce the major experimental finding on the kinetics of the rupture process, namely, the dependence of the most probable rupture forces (f*s) on ln rf (rf is the loading rate) exhibits two distinct linear regimes. The first, at low rf, has a shallow slope, whereas the slope at high rf is much larger, especially for a LFA-1/ICAM-1 complex with the transition between the two occurring over a narrow rf range. Locations of the two transition states (TSs) extracted from the simulations show an abrupt change from a high value at low rf or constant force, F, to a low value at high rf or F. This unusual behavior in which the CACs switch from one brittle (TS position is a constant over a range of forces) state to another brittle state is not found in forced-rupture in other protein complexes. We explain this novel behavior by constructing the free energy profiles, F(Λ)s, as a function of a collective reaction coordinate (Λ), involving many key charged residues and a critical metal ion (Mg2+). The TS positions in F(Λ), which quantitatively agree with the parameters extracted using the Bell-Evans model, change abruptly at a critical force, demonstrating that it, rather than the molecular extension, is a good reaction coordinate. Our combined analyses using simulations performed in both the pulling modes (constant rf and F) reveal a new mechanism for the two loading regimes observed in the rupture kinetics in CACs.

The Role of Mst1 in Lymphocyte Homeostasis and Function.

The Hippo pathway is an evolutionarily conserved pathway crucial for regulating tissue size and for limiting cancer development. However, recent work has also uncovered key roles for the mammalian Hippo kinases, Mst1/2, in driving appropriate immune responses by directing T cell migration, morphology, survival, differentiation, and activation. In this review, we discuss the classical signaling pathways orchestrated by the Hippo signaling pathway, and describe how Mst1/2 direct T cell function by mechanisms not seeming to involve the classical Hippo pathway. We also discuss why Mst1/2 might have different functions within organ systems and the immune system. Overall, understanding how Mst1/2 transmit signals to discrete biological processes in different cell types might allow for the development of better drug therapies for the treatments of cancers and immune-related diseases.

Ubc9 Binds to ADAP and Is Required for Rap1 Membrane Recruitment, Rac1 Activation, and Integrin-Mediated T Cell Adhesion.

Although the immune adaptor adhesion and degranulation-promoting adaptor protein (ADAP) acts as a key mediator of integrin inside-out signaling leading to T cell adhesion, the regulation of this adaptor during integrin activation and clustering remains unclear. We now identify Ubc9, the sole small ubiquitin-related modifier E2 conjugase, as an essential regulator of ADAP where it is required for TCR-induced membrane recruitment of the small GTPase Rap1 and its effector protein RapL and for activation of the small GTPase Rac1 in T cell adhesion. We show that Ubc9 interacted directly with ADAP in vitro and in vivo, and the association was increased in response to anti-CD3 stimulation. The Ubc9-binding domain on ADAP was mapped to a nuclear localization sequence (aa 674-700) within ADAP. Knockdown of Ubc9 by short hairpin RNA or expression of the Ubc9-binding-deficient ADAP mutant significantly decreased TCR-induced integrin adhesion to ICAM-1 and fibronectin, as well as LFA-1 clustering, although it had little effect on the TCR proximal signaling responses and TCR-induced IL-2 transcription. Furthermore, downregulation of Ubc9 impaired TCR-mediated Rac1 activation and attenuated the membrane targeting of Rap1 and RapL, but not Rap1-interacting adaptor molecule. Taken together, our data demonstrate for the first time, to our knowledge, that Ubc9 acts as a functional binding partner of ADAP and plays a selective role in integrin-mediated T cell adhesion via modulation of Rap1-RapL membrane recruitment and Rac1 activation.

Strong adhesion by regulatory T cells induces dendritic cell cytoskeletal polarization and contact-dependent lethargy.

Dendritic cells are targeted by regulatory T (T reg) cells, in a manner that operates as an indirect mode of T cell suppression. In this study, using a combination of single-cell force spectroscopy and structured illumination microscopy, we analyze individual T reg cell-DC interaction events and show that T reg cells exhibit strong intrinsic adhesiveness to DCs. This increased DC adhesion reduces the ability of contacted DCs to engage other antigen-specific cells. We show that this unusually strong LFA-1-dependent adhesiveness of T reg cells is caused in part by their low calpain activities, which normally release integrin-cytoskeleton linkage, and thereby reduce adhesion. Super resolution imaging reveals that such T reg cell adhesion causes sequestration of Fascin-1, an actin-bundling protein essential for immunological synapse formation, and skews Fascin-1-dependent actin polarization in DCs toward the T reg cell adhesion zone. Although it is reversible upon T reg cell disengagement, this sequestration of essential cytoskeletal components causes a lethargic state of DCs, leading to reduced T cell priming. Our results reveal a dynamic cytoskeletal component underlying T reg cell-mediated DC suppression in a contact-dependent manner.

Cytotoxic potential of IL-15-activated cytokine-induced killer cells against human neuroblastoma cells.

BACKGROUND: Neuroblastoma (NB) is the most common solid extracranial tumor in childhood. Despite advances in therapy, the prognosis is poor and optimized therapies are urgently needed. Therefore, we investigated the antitumor potential of interleukin-15 (IL-15)-activated cytokine-induced killer (CIK) cells against different NB cell lines. PROCEDURE: CIK cells were generated from peripheral blood mononuclear cells by the stimulation with interferon-γ (IFN-γ), IL-2, OKT-3 and IL-15 over a period of 10-12 days. The cytotoxic activity against NB cells was analyzed by nonradioactive Europium release assay before and after blocking of different receptor-ligand interactions relevant in CIK cell-mediated cytotoxicity. RESULTS: The final CIK cell products consisted in median of 83% (range: 75.9-91.9%) CD3+ CD56- T cells, 14% (range: 5.2-20.7%) CD3+ CD56+ NK-like T cells and 2% (range: 0.9-4.8%) CD3- CD56+ NK cells. CIK cells expanded significantly upon ex vivo stimulation with median rates of 22.3-fold for T cells, 58.3-fold for NK-like T cells and 2.5-fold for NK cells. Interestingly, CD25 surface expression increased from less than equal to 1% up to median 79.7%. Cytotoxic activity of CIK cells against NB cells was in median 34.7, 25.9 and 34.8% against the cell lines UKF-NB-3, UKF-NB-4 and SK-N-SH, respectively. In comparison with IL-2-stimulated NK cells, CIK cells showed a significantly higher cytotoxicity. Antibody-mediated blocking of the receptors NKG2D, TRAIL, FasL, DNAM-1, NKp30 and lymphocyte function-associated antigen-1 (LFA-1) significantly reduced lytic activity, indicating that diverse cytotoxic mechanisms might be involved in CIK cell-mediated NB killing. CONCLUSIONS: Unlike the mechanism reported in other malignancies, NKG2D-mediated cytotoxicity does not constitute the major killing mechanism of CIK cells against NB.

DNA-based nanoparticle tension sensors reveal that T-cell receptors transmit defined pN forces to their antigens for enhanced fidelity.

T cells are triggered when the T-cell receptor (TCR) encounters its antigenic ligand, the peptide-major histocompatibility complex (pMHC), on the surface of antigen presenting cells (APCs). Because T cells are highly migratory and antigen recognition occurs at an intermembrane junction where the T cell physically contacts the APC, there are long-standing questions of whether T cells transmit defined forces to their TCR complex and whether chemomechanical coupling influences immune function. Here we develop DNA-based gold nanoparticle tension sensors to provide, to our knowledge, the first pN tension maps of individual TCR-pMHC complexes during T-cell activation. We show that naïve T cells harness cytoskeletal coupling to transmit 12-19 pN of force to their TCRs within seconds of ligand binding and preceding initial calcium signaling. CD8 coreceptor binding and lymphocyte-specific kinase signaling are required for antigen-mediated cell spreading and force generation. Lymphocyte function-associated antigen 1 (LFA-1) mediated adhesion modulates TCR-pMHC tension by intensifying its magnitude to values >19 pN and spatially reorganizes the location of TCR forces to the kinapse, the zone located at the trailing edge of migrating T cells, thus demonstrating chemomechanical crosstalk between TCR and LFA-1 receptor signaling. Finally, T cells display a dampened and poorly specific response to antigen agonists when TCR forces are chemically abolished or physically "filtered" to a level below ∼12 pN using mechanically labile DNA tethers. Therefore, we conclude that T cells tune TCR mechanics with pN resolution to create a checkpoint of agonist quality necessary for specific immune response.

Adhesive Mechanisms of Histone-Induced Neutrophil-Endothelium Interactions in the Muscle Microcirculation.

BACKGROUND: Extracellular histones released during cell damage have the capacity to cause tissue injury associated with increased leukocyte accumulation. However, the molecular mechanisms regulating histone-induced leukocyte recruitment remain elusive. The objective of this study was to examine the role of adhesion molecules in histone-dependent leukocyte accumulation by use of intravital microscopy of the mouse cremaster microcirculation. METHODS: Histone 3 and TNF-α were intrascrotally administered, and anti-P-selectin, anti-P-selectin glycoprotein ligand-1 (PSGL-1), anti-membrane-activated complex-1 (Mac-1), anti-lymphocyte function antigen-1 (LFA-1) antibody and neutrophil depletion antibody were injected intravenously or intraperitoneally. RESULTS: Intrascrotal injection of histone 3 dose-dependently increased leukocyte recruitment. Neutrophil depletion abolished intravascular and extravascular leukocytes after histone 3 challenge, suggesting that neutrophils were the dominating leukocyte subtype responding to histone stimulation. Pretreatment with an anti-P-selectin and an anti-PSGL-1 antibody abolished histone-stimulated neutrophil rolling, adhesion and emigration. When the anti-P-selectin or the anti-PSGL-1 antibody was administrated after histone 3 stimulation, neutrophil rolling was reduced, whereas the number of firmly adherent and emigrated neutrophils were unchanged, suggesting that the inhibitory effect of blocking P-selectin and PSGL-1 on neutrophil adhesion and recruitment was due to the reduction in neutrophil rolling. Moreover, pretreatment with antibodies against Mac-1 and LFA-1 had no effect of neutrophil rolling but abolished adhesion and emigration evoked by histone 3. Thus, our data demonstrate that P-selectin and PSGL-1 play an important role in histone-induced inflammatory cell recruitment by mediating neutrophil rolling as a precondition for histone-provoked firm adhesion and emigration in vivo. Moreover, we conclude that both Mac-1 and LFA-1 are critical in supporting histone-provoked firm adhesion of neutrophils to endothelial cells. CONCLUSION: These novel findings define specific selectins and integrins as potential targets for pharmacological intervention in histone-dependent inflammatory diseases.

Neutrophils promote Alzheimer's disease-like pathology and cognitive decline via LFA-1 integrin.

Inflammation is a pathological hallmark of Alzheimer's disease, and innate immune cells have been shown to contribute to disease pathogenesis. In two transgenic models of Alzheimer's disease (5xFAD and 3xTg-AD mice), neutrophils extravasated and were present in areas with amyloid-ß (Aß) deposits, where they released neutrophil extracellular traps (NETs) and IL-17. Aß42 peptide triggered the LFA-1 integrin high-affinity state and rapid neutrophil adhesion to integrin ligands. In vivo, LFA-1 integrin controlled neutrophil extravasation into the CNS and intraparenchymal motility. In transgenic Alzheimer's disease models, neutrophil depletion or inhibition of neutrophil trafficking via LFA-1 blockade reduced Alzheimer's disease-like neuropathology and improved memory in mice already showing cognitive dysfunction. Temporary depletion of neutrophils for 1 month at early stages of disease led to sustained improvements in memory. Transgenic Alzheimer's disease model mice lacking LFA-1 were protected from cognitive decline and had reduced gliosis. In humans with Alzheimer's disease, neutrophils adhered to and spread inside brain venules and were present in the parenchyma, along with NETs. Our results demonstrate that neutrophils contribute to Alzheimer's disease pathogenesis and cognitive impairment and suggest that the inhibition of neutrophil trafficking may be beneficial in Alzheimer's disease.

Integrin LFA-1 regulates cell adhesion via transient clutch formation.

Integrin LFA-1 regulates immune cell adhesion and trafficking by binding to ICAM-1 upon chemokine stimulation. Integrin-mediated clutch formation between extracellular ICAM-1 and the intracellular actin cytoskeleton is important for cell adhesion. We applied single-molecule tracking analysis to LFA-1 and ICAM-1 in living cells to examine the ligand-binding kinetics and mobility of the molecular clutch under chemokine-induced physiological adhesion and Mn(2+)-induced tight adhesion. Our results show a transient LFA-1-mediated clutch formation that lasts a few seconds and leads to a transient lower-mobility is sufficient to promote cell adhesion. Stable clutch formation was observed for Mn(2+)-induced high affinity LFA-1, but was not required for physiological adhesion. We propose that fast cycling of the clutch formation by intermediate-affinity integrin enables dynamic cell adhesion and migration.

JAK tyrosine kinases promote hierarchical activation of Rho and Rap modules of integrin activation.

Lymphocyte recruitment is regulated by signaling modules based on the activity of Rho and Rap small guanosine triphosphatases that control integrin activation by chemokines. We show that Janus kinase (JAK) protein tyrosine kinases control chemokine-induced LFA-1- and VLA-4-mediated adhesion as well as human T lymphocyte homing to secondary lymphoid organs. JAK2 and JAK3 isoforms, but not JAK1, mediate CXCL12-induced LFA-1 triggering to a high affinity state. Signal transduction analysis showed that chemokine-induced activation of the Rho module of LFA-1 affinity triggering is dependent on JAK activity, with VAV1 mediating Rho activation by JAKs in a Gαi-independent manner. Furthermore, activation of Rap1A by chemokines is also dependent on JAK2 and JAK3 activity. Importantly, activation of Rap1A by JAKs is mediated by RhoA and PLD1, thus establishing Rap1A as a downstream effector of the Rho module. Thus, JAK tyrosine kinases control integrin activation and dependent lymphocyte trafficking by bridging chemokine receptors to the concurrent and hierarchical activation of the Rho and Rap modules of integrin activation.

The role of ß2-integrins and CD44 in intrahepatic leukocyte sequestration.

BACKGROUND: Intrahepatic leukocyte sequestration is a component of the systemic inflammatory response, and can be triggered by systemic immune dysfunction during sepsis. METHODS: To examine leukocyte sequestration over time during endotoxemia, its influence on liver function, and the role of specific cell adhesion molecules, endotoxemia was induced in mice by intraperitoneal application of lipopolysaccharides. Leukocyte sequestration was measured at different times after induction using fluorescence microscopy. Liver injury was evaluated by measuring liver enzymes and tissue histology. RESULTS: Endotoxin induces a strong leukocyte sequestration in the liver microvasculature. This was associated with an induction of liver injury, as reflected by an increase in enzyme levels and histomorphologic changes. Intrahepatic leukocyte sequestration was reduced in CD44(-/-), but not in intercellular adhesion molecule-1 (ICAM-1)(-/-), lymphocyte function-associated antigen-1(-/-), and macrophage-1(-/-) antigen mice. Leukocyte sequestration dropped in ICAM-1(-/-), lymphocyte function-associated antigen-1(-/-), and macrophage-1(-/-) mice in later stages, but remained stable in wild-type and CD44(-/-) animals. Reduced leukocyte sequestration in CD44(-/-) mice was accompanied by a significant decrease in transferase levels. CONCLUSIONS: Endotoxemia induces stable intra-sinusoidal leukocyte sequestration, which contributes to liver injury. At the initial stage of the endotoxemia, leukocyte sequestration depends on CD44 but is independent of ICAM-1 and ß2-integrins. Intercellular adhesion molecule-1 and ß2-integrins, but not CD44, stabilize leukocyte sequestration during the later stage of endotoxemia. The molecular modulation of intrahepatic leukocyte sequestration may have important therapeutic implications in sepsis, reducing liver injury, and improving immune defense capabilities.

Gimme a brake: HPK1 regulates LFA-1 and neutrophil traction.

In this issue of Blood, Jakob et al report that hematopoietic progenitor kinase 1 (HPK1) participates during signaling of neutrophil recruitment by acting as a regulator of the adhesiveness of the b2-integrin lymphocyte function-associated antigen 1 (LFA-1) during acute inflammation.

Naive B-cell trafficking is shaped by local chemokine availability and LFA-1-independent stromal interactions.

It is not known how naive B cells compute divergent chemoattractant signals of the T-cell area and B-cell follicles during in vivo migration. Here, we used two-photon microscopy of peripheral lymph nodes (PLNs) to analyze the prototype G-protein-coupled receptors (GPCRs) CXCR4, CXCR5, and CCR7 during B-cell migration, as well as the integrin LFA-1 for stromal guidance. CXCR4 and CCR7 did not influence parenchymal B-cell motility and distribution, despite their role during B-cell arrest in venules. In contrast, CXCR5 played a nonredundant role in B-cell motility in follicles and in the T-cell area. B-cell migration in the T-cell area followed a random guided walk model, arguing against directed migration in vivo. LFA-1, but not α4 integrins, contributed to B-cell motility in PLNs. However, stromal network guidance was LFA-1 independent, uncoupling integrin-dependent migration from stromal attachment. Finally, we observed that despite a 20-fold reduction of chemokine expression in virus-challenged PLNs, CXCR5 remained essential for B-cell screening of antigen-presenting cells. Our data provide an overview of the contribution of prototype GPCRs and integrins during naive B-cell migration and shed light on the local chemokine availability that these cells compute.