ICAM-1 and ICAM-2 Are Differentially Expressed and Up-Regulated on Inflamed Pulmonary Epithelium, but Neither ICAM-2 nor LFA-1: ICAM-1 Are Required for Neutrophil Migration Into the Airways In Vivo.

Neutrophil migration into the airways is an important process to fight infection and is mediated by cell adhesion molecules. The intercellular adhesion molecules, ICAM-1 (CD54) and ICAM-2 (CD102) are known ligands for the neutrophil integrins, lymphocyte function associated antigen (LFA)-1 (αLß2; CD11a/CD18), and macrophage-1 antigen (Mac-1;αMß2;CD11b/CD18) and are implicated in leukocyte migration into the lung. However, it is ill-defined how neutrophils exit the lung and the role for ICAMs in trans-epithelial migration (TEpM) across the bronchial or alveolar epithelium. We found that human and murine alveolar epithelium expressed ICAM-1, whilst the bronchial epithelium expressed ICAM-2, and both were up-regulated during inflammatory stimulation in vitro and in inflammatory lung diseases such as cystic fibrosis. Although ß2 integrins interacting with ICAM-1 and -2 mediated neutrophil migration across human bronchial epithelium in vitro, neither ICAM-2 nor LFA-1 binding of ICAM-1 mediated murine neutrophil migration into the lung or broncho-alveolar space during LPS-induced inflammation in vivo. Furthermore, TEpM of neutrophils themselves resulted in increased epithelial junctional permeability and reduced barrier function in vitro. This suggests that although ß2 integrins interacting with ICAMs may regulate low levels of neutrophil traffic in healthy lung or early in inflammation when the epithelial barrier is intact; these interactions may be redundant later in inflammation when epithelial junctions are disrupted and no longer limit TEpM.

Tensile force transmitted through LFA-1 bonds mechanoregulate neutrophil inflammatory response.

Recruitment of leukocytes to sites of acute inflammation is guided by spatial and temporal cues that ensure appropriate cell numbers infiltrate the tissue at precise locations to protect it from infection and initiate repair. On inflamed endothelium, neutrophil rolling via selectins elicits cytosolic calcium release from endoplasmic reticulum (ER)-stores that are synergistic with chemokine signaling to activate formation of high affinity (HA) LFA-1 bonds to ICAM-1, which is necessary to anchor cells against the drag force of blood flow. Bond tension on LFA-1 within the area of adhesive contact with endothelium elicits calcium entry through calcium release-activated calcium channel protein 1 (Orai-1) membrane channels that in turn activate neutrophil shape change and migration. We hypothesized that mechanotransduction via LFA-1 is mediated by assembly of a cytosolic molecular complex consisting of Kindlin-3, receptor for activated C kinase 1 (RACK1), and Orai1. Initiation of Ca2+ flux at sites of adhesive contact required a threshold level of shear stress and increased with the magnitude of bond tension transduced across as few as 200 HA LFA-1. A sequential mechanism triggered by force acting on LFA-1/Kindlin-3 precipitated dissociation of RACK1, which formed a concentration gradient above LFA-1 bond clusters. This directed translocation of ER proximal to Orai1, where binding of inositol 1,4,5-triphosphate receptor type 1 and activation via stromal interaction molecule 1 elicited Ca flux and subsequent neutrophil shape change and motility. We conclude that neutrophils sense adhesive traction on LFA-1 bonds on a submicron scale to direct calcium influx, thereby ensuring sufficient shear stress of blood flow is present to trigger cell arrest and initiate transmigration at precise regions of vascular inflammation.

Frontline Science: Kindlin-3 is essential for patrolling and phagocytosis functions of nonclassical monocytes during metastatic cancer surveillance.

Nonclassical monocytes maintain vascular homeostasis by patrolling the vascular endothelium, responding to inflammatory signals, and scavenging cellular debris. Nonclassical monocytes also prevent metastatic tumor cells from seeding new tissues, but whether the patrolling function of nonclassical monocytes is required for this process is unknown. To answer this question, we utilized an inducible-knockout mouse that exhibits loss of the integrin-adaptor protein Kindlin-3 specifically in nonclassical monocytes. We show that Kindlin-3-deficient nonclassical monocytes are unable to patrol the vascular endothelium in either the lungs or periphery. We also find that Kindlin-3-deficient nonclassical monocytes cannot firmly adhere to, and instead "slip" along, the vascular endothelium. Loss of patrolling activity by nonclassical monocytes was phenocopied by ablation of LFA-1, an integrin-binding partner of Kindlin-3. When B16F10 murine melanoma tumor cells were introduced into Kindlin-3-deficient mice, nonclassical monocytes showed defective patrolling towards tumor cells and failure to ingest tumor particles in vivo. Consequently, we observed a significant, 4-fold increase in lung tumor metastases in mice possessing Kindlin-3-deficient nonclassical monocytes. Thus, we conclude that the patrolling function of nonclassical monocytes is mediated by Kindlin-3 and essential for these cells to maintain vascular endothelial homeostasis and prevent tumor metastasis to the lung.

Expression of TSLC1 in patients with HAM/TSP.

Human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is chronic myelopathy characterized by slowly progressive spastic paraparesis and urinary dysfunction. A few biomarkers in the cerebrospinal fluid are known to be related to disease activity, but no biomarker has been reported in peripheral blood. This study aims to explore the expression level of the adhesion molecule during the expression level of the adhesion molecule among HAM/TSP disease activity. In lymphocyte function-associated antigen 1 and DNAX accessory molecule 1, no variation in expression levels specific to HTLV-1 infection was observed in CD4-positive T cells; however, TSLC1 expression was higher in HAM patients than in asymptomatic carriers and non-infected persons. TSLC1 tended to be higher in patients whose symptoms were worsening. On the contrary, the expression level of TSLC1 in CD8-positive T cells was lower in HAM patients than in asymptomatic carriers, and this tendency was stronger in patients whose symptoms had deteriorated. No significant correlation was found between TSLC1 and either of the transcription factors Tax or HBZ in any T cell group. Therefore, TSLC1 expression in CD4-positive T cells might be a useful biomarker of HAM/TSP disease activity.

A Multivalent ICAM-1 Binding Nanoparticle which Inhibits ICAM-1 and LFA-1 Interaction Represents a New Tool for the Investigation of Autoimmune-Mediated Dry Eye.

The autoimmune disorder, Sjögren's syndrome (SS), is characterized by lymphocytic infiltration and loss of function of exocrine glands such as the lacrimal gland (LG) and salivary gland. SS-associated changes in the LG are associated with the development of autoimmune-mediated dry eye disease. We have previously reported the accumulation of intercellular adhesion molecule 1 (ICAM-1) in the LG of Non-Obese Diabetic (NOD) mice, a murine model of autoimmune-mediated dry eye in SS, in both LG acinar cells and infiltrating lymphocytes. ICAM-1 initiates T-cell activation and can trigger T-cell migration through binding to lymphocyte function-associated 1 antigen (LFA). To modulate this interaction, this study introduces a new tool, a multivalent biopolymeric nanoparticle assembled from a diblock elastin-like polypeptide (ELP) using the S48I48 (SI) ELP scaffold fused with a mouse ICAM-1 targeting peptide to form IBP-SI. IBP-SI forms a multivalent, monodisperse nanoparticle with a radius of 21.9 nm. Unlike the parent SI, IBP-SI binds mouse ICAM-1 and is internalized by endocytosis into transfected HeLa cells before it accumulates in lysosomes. In vitro assays measuring lymphocyte adhesion to Tumor Necrosis Factor TNF-α-treated bEnd.3 cells, which express high levels of ICAM-1, show that adhesion is inhibited by IBP-SI but not by SI, with IC50 values of 62.7 µM and 81.2 µM, respectively, in two different assay formats. IBP-SI, but not SI, also blocked T-cell proliferation in a mixed lymphocyte reaction by 74% relative to proliferation in an untreated mixed cell reaction. These data suggest that a biopolymeric nanoparticle with affinity for ICAM-1 can disrupt ICAM-1 and LFA interactions in vitro and may have further utility as an in vivo tool or potential therapeutic.

MAP4K4 negatively regulates CD8 T cell-mediated antitumor and antiviral immunity.

During cytotoxic T cell activation, lymphocyte function-associated antigen-1 (LFA-1) engages its ligands on antigen-presenting cells (APCs) or target cells to enhance T cell priming or lytic activity. Inhibiting LFA-1 dampens T cell-dependent symptoms in inflammation, autoimmune diseases, and graft-versus-host disease. However, the therapeutic potential of augmenting LFA-1 function is less explored. Here, we show that genetic deletion or inhibition of mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) enhances LFA-1 activation on CD8 T cells and improves their adherence to APCs or LFA-1 ligand. In addition, loss of Map4k4 increases CD8 T cell priming, which culminates in enhanced antigen-dependent activation, proliferation, cytokine production, and cytotoxic activity, resulting in impaired tumor growth and improved response to viral infection. LFA-1 inhibition reverses these phenotypes. The ERM (ezrin, radixin, and moesin) proteins reportedly regulate T cell-APC conjugation, but the molecular regulator and effector of ERM proteins in T cells have not been defined. In this study, we demonstrate that the ERM proteins serve as mediators between MAP4K4 and LFA-1. Last, systematic analyses of many organs revealed that inducible whole-body deletion of Map4k4 in adult animals is tolerated under homeostatic conditions. Our results uncover MAP4K4 as a potential target to augment antitumor and antiviral immunity.

ß2 Integrins-Multi-Functional Leukocyte Receptors in Health and Disease.

ß2 integrins are heterodimeric surface receptors composed of a variable α (CD11a-CD11d) and a constant ß (CD18) subunit and are specifically expressed by leukocytes. The α subunit defines the individual functional properties of the corresponding ß2 integrin, but all ß2 integrins show functional overlap. They mediate adhesion to other cells and to components of the extracellular matrix (ECM), orchestrate uptake of extracellular material like complement-opsonized pathogens, control cytoskeletal organization, and modulate cell signaling. This review aims to delineate the tremendous role of ß2 integrins for immune functions as exemplified by the phenotype of LAD-I (leukocyte adhesion deficiency 1) patients that suffer from strong recurrent infections. These immune defects have been largely attributed to impaired migratory and phagocytic properties of polymorphonuclear granulocytes. The molecular base for this inherited disease is a functional impairment of ß2 integrins due to mutations within the CD18 gene. LAD-I patients are also predisposed for autoimmune diseases. In agreement, polymorphisms within the CD11b gene have been associated with autoimmunity. Consequently, ß2 integrins have received growing interest as targets in the treatment of autoimmune diseases. Moreover, ß2 integrin activity on leukocytes has been implicated in tumor development.

Human Neutrophils Will Crawl Upstream on ICAM-1 If Mac-1 Is Blocked.

The recruitment of neutrophils to sites of inflammatory insult is a hallmark of the innate immune response. Neutrophil recruitment is regulated by a multistep process that includes cell rolling, activation, adhesion, and transmigration through the endothelium commonly referred to as the leukocyte adhesion cascade. After selectin-mediated braking, neutrophils migrate along the activated vascular endothelium on which ligands, including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), are expressed. Previous studies have shown that two cells that commonly home from blood vessel to tissue-T cells and hematopoietic stem and progenitor cells-use the integrin lymphocyte functional antigen-1 (LFA-1) to migrate against the direction of shear flow once adherent on ICAM-1 surfaces. Like T cells and hematopoietic stem and progenitor cells, neutrophils express LFA-1, but they also express macrophage-1 antigen (Mac-1), which binds to ICAM-1. Previous reports have shown that neutrophils will not migrate against the direction of flow on ICAM-1, but we hypothesized this was due to the influence of Mac-1. Here, we report that both the HL-60 neutrophil-like cell line and primary human neutrophils can migrate against the direction of fluid flow on ICAM-1 surfaces via LFA-1 if Mac-1 is blocked; otherwise, they migrate downstream. We demonstrate this both on ICAM-1 surfaces and on activated endothelium. In sum, both LFA-1 and Mac-1 binding ICAM-1 play a critical role in determining the direction of neutrophil migration along the endothelium, and their interaction may play an important role in controlling neutrophil trafficking during inflammation.

Filamin A Phosphorylation at Serine 2152 by the Serine/Threonine Kinase Ndr2 Controls TCR-Induced LFA-1 Activation in T Cells.

The integrin LFA-1 (CD11a/CD18) plays a critical role in the interaction of T cells with antigen presenting cells (APCs) to promote lymphocyte differentiation and proliferation. This integrin can be present either in a closed or in an open active conformation and its activation upon T-cell receptor (TCR) stimulation is a critical step to allow interaction with APCs. In this study we demonstrate that the serine/threonine kinase Ndr2 is critically involved in the initiation of TCR-mediated LFA-1 activation (open conformation) in T cells. Ndr2 itself becomes activated upon TCR stimulation and phosphorylates the intracellular integrin binding partner Filamin A (FLNa) at serine 2152. This phosphorylation promotes the dissociation of FLNa from LFA-1, allowing for a subsequent association of Talin and Kindlin-3 which both stabilize the open conformation of LFA-1. Our data suggest that Ndr2 activation is a crucial step to initiate TCR-mediated LFA-1 activation in T cells.

ICAM-1-LFA-1 Dependent CD8+ T-Lymphocyte Aggregation in Tumor Tissue Prevents Recirculation to Draining Lymph Nodes.

The quantity of T-lymphocytes reaching the draining lymph nodes from tumors is likely important to mount effective distant responses and for the establishment of long term systemic memory. Looking into mechanisms behind lymphocyte egress, we directed our attention to leukocyte adhesion mechanisms inside tumors. Here we demonstrate that activated T-cells form intra-tumor aggregates in a LFA-1-ICAM-1-dependent fashion in mouse models of melanoma and breast cancer. We also provide evidence of the presence of T-cell clusters in primary human melanoma. Disruption of LFA-1-ICAM-1 interactions, and thereby T-cell clustering, enhances the arrival of activated CD8+ T-cells to tumor draining lymph nodes in both transplanted and spontaneous cancer models. Interestingly, upon ICAM-1 blockade, the expression of the chemotactic receptor CCR7 augments in tumor infiltrating lymphocytes and in in-vitro de-clustered T cells, as well as their ability to transmigrate across lymphatic endothelial cells. We propose that ICAM-1-mediated homotypic T-lymphocyte aggregation may serve as a tumor-mediated immune retention mechanism entrapping activated CD8+ T cells in the tumor microenvironment. Modulation of T-cell adhesion may be of use to improve the transit of activated lymphocytes toward the lymph nodes and their subsequent recirculation.

Protein tyrosine phosphatase PTPN22 regulates LFA-1 dependent Th1 responses.

A missense C1858T single nucleotide polymorphism within PTPN22 is a strong genetic risk factor for the development of multiple autoimmune diseases. PTPN22 encodes a protein tyrosine phosphatase that negatively regulates immuno-receptor proximal Src and Syk family kinases. Notably, PTPN22 negatively regulates kinases downstream of T-cell receptor (TCR) and LFA-1, thereby setting thresholds for T-cell activation. Alterations to the quality of TCR and LFA-1 engagement at the immune synapse and the regulation of downstream signals can have profound effects on the type of effector T-cell response induced. Here we describe how IFNγ+ Th1 responses are potentiated in Ptpn22-/- T-cells and in T-cells from mice expressing Ptpn22R619W (the mouse orthologue of the human genetic variant) as they age, or following repeated immune challenge, and explore the mechanisms contributing to the expansion of Th1 cells. Specifically, we uncover two LFA-1-ICAM dependent mechanisms; one T-cell intrinsic, and one T-cell extrinsic. Firstly, we found that in vitro anti-CD3/LFA-1 induced Th1 responses were enhanced in Ptpn22-/- T-cells compared to WT, whereas anti-CD3/anti-CD28 induced IFNy responses were similar. These data were associated with an enhanced ability of Ptpn22-/- T-cells to engage ICAM-1 at the immune synapse when incubated on planar lipid bilayers, and to form conjugates with dendritic cells. Secondly, we observed a T-cell extrinsic mechanism whereby repeated stimulation of WT OT-II T-cells with LPS and OVA323-339 pulsed Ptpn22-/- bone marrow derived dendritic cells (BMDCs) was sufficient to enhance Th1 cell development compared to WT BMDCs. Furthermore, this response could be reversed by LFA-1 blockade. Our data point to two related but distinct mechanisms by which PTPN22 regulates LFA-1 dependent signals to enhance Th1 development, highlighting how perturbations to PTPN22 function over time to regulate the balance of the immune response.

Aggregatibacter (Actinobacillus) actimycetemcomitans leukotoxin and human periodontitis - A historic review with emphasis on JP2.

Aggregatibacter (Actinobacillus) actimycetemcomitans (Aa) is a gram-negative bacterium that colonizes the human oral cavity and is causative agent for localized aggressive (juvenile) periodontitis (AgP). In the middle of 1990s, a specific JP2 clone of belonging to the cluster of serotype b strains of Aa with highly leukotoxicity (leukotoxin, LtxA) able to kill human immune cells was isolated. JP2 clone of Aa was strongly associated with in particularly in rapidly progressing forms of aggressive periodontitis. The JP2 clone of Aa is transmitted through close contacts. Therefore, AgP patients need intense monitoring of their periodontal status as the risk for developing severely progressing periodontitis lesions are relatively high. Furthermore, timely periodontal treatment, including periodontal surgery supplemented by the use of antibiotics, is warranted. More importantly, periodontal attachment loss should be prevented by early detection of the JP2 clone of Aa by microbial diagnosis testing and/or preventive means.

Chimeric antigen receptor T cells form nonclassical and potent immune synapses driving rapid cytotoxicity.

Chimeric antigen receptor T (CAR-T) cells are effective serial killers with a faster off-rate from dying tumor cells than CAR-T cells binding target cells through their T cell receptor (TCR). Here we explored the functional consequences of CAR-mediated signaling using a dual-specific CAR-T cell, where the same cell was triggered via TCR (tcrCTL) or CAR (carCTL). The carCTL immune synapse lacked distinct LFA-1 adhesion rings and was less reliant on LFA to form stable conjugates with target cells. carCTL receptors associated with the synapse were found to be disrupted and formed a convoluted multifocal pattern of Lck microclusters. Both proximal and distal receptor signaling pathways were induced more rapidly and subsequently decreased more rapidly in carCTL than in tcrCTL. The functional consequence of this rapid signaling in carCTL cells included faster lytic granule recruitment to the immune synapse, correlating with faster detachment of the CTL from the target cell. This study provides a mechanism for how CAR-T cells can debulk large tumor burden quickly and may contribute to further refinement of CAR design for enhancing the quality of signaling and programming of the T cell.

Talin Plays a Critical Role in the Maintenance of the Regulatory T Cell Pool.

Talin, a cytoskeletal protein essential in mediating integrin activation, has been previously shown to be involved in the regulation of T cell proliferation and function. In this study, we describe a role for talin in maintaining the homeostasis and survival of the regulatory T (Treg) cell pool. T cell-specific deletion of talin in Tln1fl/flCd4Cre mice resulted in spontaneous lymphocyte activation, primarily due to numerical and functional deficiencies of Treg cells in the periphery. Peripheral talin-deficient Treg cells were unable to maintain high expression of IL-2Rα, resulting in impaired IL-2 signaling and ultimately leading to increased apoptosis through downregulation of prosurvival proteins Bcl-2 and Mcl-1. The requirement for talin in maintaining high IL-2Rα expression by Treg cells was due, in part, to integrin LFA-1-mediated interactions between Treg cells and dendritic cells. Collectively, our data suggest a critical role for talin in Treg cell-mediated maintenance of immune homeostasis.

Endogenous developmental endothelial locus-1 limits ischaemia-related angiogenesis by blocking inflammation.

We have recently identified endothelial cell-secreted developmental endothelial locus-1 (Del-1) as an endogenous inhibitor of ß2-integrin-dependent leukocyte infiltration. Del-1 was previously also implicated in angiogenesis. Here, we addressed the role of endogenously produced Del-1 in ischaemia-related angiogenesis. Intriguingly, Del-1-deficient mice displayed increased neovascularisation in two independent ischaemic models (retinopathy of prematurity and hind-limb ischaemia), as compared to Del-1-proficient mice. On the contrary, angiogenic sprouting in vitro or ex vivo (aortic ring assay) and physiological developmental retina angiogenesis were not affected by Del-1 deficiency. Mechanistically, the enhanced ischaemic neovascularisation in Del-1-deficiency was linked to higher infiltration of the ischaemic tissue by CD45+ haematopoietic and immune cells. Moreover, Del-1-deficiency promoted ß2-integrin-dependent adhesion of haematopoietic cells to endothelial cells in vitro, and the homing of hematopoietic progenitor cells and of immune cell populations to ischaemic muscles in vivo. Consistently, the increased hind limb ischaemia-related angiogenesis in Del-1 deficiency was completely reversed in mice lacking both Del-1 and the ß2-integrin LFA-1. Additionally, enhanced retinopathy-associated neovascularisation in Del-1-deficient mice was reversed by LFA-1 blockade. Our data reveal a hitherto unrecognised function of endogenous Del-1 as a local inhibitor of ischaemia-induced angiogenesis by restraining LFA-1-dependent homing of pro-angiogenic haematopoietic cells to ischaemic tissues. Our findings are relevant for the optimisation of therapeutic approaches in the context of ischaemic diseases.

Association of Ribonuclease T2 Gene Polymorphisms With Decreased Expression and Clinical Characteristics of Severity in Crohn's Disease.

BACKGROUND & AIMS: Variants in the tumor necrosis factor superfamily member 15 gene (TNFSF15, also called TL1A) have been associated with risk for inflammatory bowel disease (IBD). TL1A affects expression of multiple cytokines to promote mucosal inflammation. Little is known about the TL1A-response pathways that regulate cytokine expression. We investigated T-cell gene expression patterns to determine the mechanisms by which TL1A regulates cytokine production, and whether these associate with outcomes of patients with Crohn's disease (CD). METHODS: Peripheral T cells isolated from normal donors were cultured with TL1A. We performed gene expression profile analysis by RNA sequencing of subsets of interferon gamma (IFNG)-producing and non-producing cells purified by flow cytometry. Unsupervised hierarchical clustering analysis was used to identify gene expression differences between these subsets. Ribonuclease T2 gene (RNASET2) expression and methylation were assessed by quantitative trait loci analyses. Clinical characteristics of patients (complications, resistance to therapy, and recurrence time) were associated with single nucleotide polymorphisms in RNASET2. We performed motif screening to identify polymorphisms that disrupt transcription factor binding sites. Levels of RNASET2 were knocked down with small interfering RNA in CD4+ T cells and the effect on protein expression was determined by proteomic analysis and cytokine production. Cell aggregation was measured by flow cytometry. RESULTS: We identified 764 genes with at least a 2-fold difference in TL1A-mediated expression between IFNG-secreting and non-secreting T cells (P < 1 × 10-5). Many of these genes were located near IBD susceptibility variants. RNASET2 was the only IBD risk-associated gene with >5-fold down-regulation in the IFNG-secreting subset. RNASET2 disease risk variants were associated with decreased expression in peripheral and mucosal tissues and DNA hypermethylation in CD patients requiring surgical intervention. RNASET2 disease risk variants were associated in CD patients with more complicated disease or resistance to therapy, defined in part by failed response to treatment, increased length of intestinal resection, shorter time to repeat surgery, and high Rutgeerts score (>2) in postoperative endoscopy. The RNASET2 variant rs2149092 was predicted to disrupt a consensus binding site for the transcription factor ETS within an enhancer region. Expression of RNASET2 correlated with expression of ETS. RNASET2 knockdown in T cells increased expression of IFNG and intercellular adhesion molecule 1 (ICAM1) and induced T-cell aggregation. A blocking antibody against (ILFA1), disrupting the lymphocyte function-associated antigen 1-intercellular adhesion molecule 1 interaction, reduced T-cell production of IFNG. CONCLUSIONS: We identified decreased expression of RNASET2 as a component of TL1A-mediated increase in production of IFNG and as a potential biomarker for patients with severe CD. Further study of the role of RNASET2 in regulating mucosal inflammation may lead to development of novel therapeutic targets.

HS1 deficiency impairs neutrophil recruitment in vivo and activation of the small GTPases Rac1 and Rap1.

Neutrophil extravasation is a critical step of the innate immune system's response to inflammation. This multistep process is tightly regulated by adhesion and signaling molecules in the endothelium and neutrophils. Activation of the ß2 integrin LFA-1 is critical for adhesion of leukocytes to postcapillary venules. This step requires coordinated activation of signaling pathways in chemokine-stimulated neutrophils, including GTPase activation and cytoskeletal remodeling, leading to conformational changes in LFA-1. Hematopoietic cell-specific lyn substrate 1 (HS1) is a cortactin-related and leukocyte-specific actin-binding protein (ABP) that regulates several processes in various immune cells. It has been shown in vitro that HS1 is important for neutrophil chemotaxis and transendothelial migration of NK cells, but its role in neutrophil extravasation in vivo has not been investigated yet. Intravital microscopy of CXCL1-stimulated cremaster venules revealed an increased rolling velocity and reduced neutrophil adhesion and transmigration in HS1 knockout (KO) mice. CXCL1-induced rapid neutrophil arrest in vivo and adhesion under flow conditions in vitro were also reduced significantly. Whereas random motility of neutrophils was unaffected, chemotaxis toward a CXCL1 gradient was reduced in the absence of HS1. Further analysis of the underlying mechanisms demonstrated that HS1 controls CXCL1-induced activation of the small GTPases Ras-related C3 botulinum toxin substrate 1 (Rac1) and Ras-related protein 1 (Rap1), thus supporting LFA-1-mediated neutrophil adhesion. Importantly, with the use of Rac1 KO neutrophils, we could show that Rac1 acts upstream of Rap1. Our results establish HS1 as an important regulator of proper Rac1 and Rap1 activation and neutrophil extravasation.

LFA-1 Activation in NK Cells and Their Subsets: Influence of Receptors, Maturation, and Cytokine Stimulation.

The integrin LFA-1 is essential for efficient activation and for cytotoxicity of NK cells because it initiates the assembly of the immunological synapse and mediates firm adhesion to the target. LFA-1 is also needed to polarize the cytotoxic machinery of the NK cell toward the target cell. The binding affinity and avidity of integrins can be regulated via inside-out signals from other receptors. In this article, we investigate the signals necessary to activate LFA-1 in human NK cells. Our data show that LFA-1 has a low ligand-binding activity in resting human NK cells, but it can be stimulated by triggering activating receptors, such as 2B4 or CD16, or by coactivation of different receptor combinations. Short-term stimulation of freshly isolated NK cells with cytokines, such as IL-15, IL-12, or IL-18, does not activate LFA-1 but increases the responsiveness of the cells to subsequent receptor stimulation. Different NK cell subsets vary in their ability to induce LFA-1 binding activity after activating receptor stimulation. Interestingly, the NK cell subsets that are more mature and possess higher cytotoxic potential also show the highest activation of LFA-1, which correlated with the expression of the small calcium-binding protein S100A4. Our data suggest that regulation of LFA-1 is one reason for the different activity of NK cells during differentiation.

L-plastin regulates the stability of the immune synapse of naive and effector T-cells.

T-cells need to be tightly regulated during their activation and effector phase to assure an appropriate defence against cancer or pathogens and - vice versa - to avoid autoimmune reactions. Regulatory signals are provided via the immune synapse between T-cells and antigen-presenting cells (APCs) or target cells. The stability and kinetics of immune synapse formation is critical for proper T-cell functions. It requires dynamic rearrangements of the actin cytoskeleton necessary for organized spatio-temporal redistribution of receptors and adhesion molecules. We identified glucocorticoid-sensitive phosphorylation of serine 5 on the actin-bundling protein L-plastin as one important signalling event for this regulation. Using imaging flow cytometry as well as confocal and super-resolution microscopy we showed that L-plastin relocalizes to the immune synapse upon antigen encounter, where it associates with the ß2-subunit of LFA-1 (CD11a/CD18). Interfering with L-plastin expression or activation leads to a defective LFA-1 recruitment and unstable T-cell/APC contacts. Consequently, the lack of L-plastin diminishes T-cell activation, proliferation and proximal effector responses such as cytokine production. On the other hand, a pro-oxidative milieu leads to prolonged activation of L-plastin resulting in a stronger enrichment of LFA-1 in the cytolytic immune synapse. Concomitant stabilization of conjugates formed by cytotoxic T-cells (CTLs) and their target cells impairs the ability of CTLs to kill more than one target cells (serial killing), which de facto leads to a downregulation of T-cell cytotoxicity. Together, we demonstrate that activation and spacial distribution of L-plastin regulates the maturation and stability of activating and cytolytic immune synapses important for T-cell activation and effector functions.

The Integrin LFA-1 Controls T Follicular Helper Cell Generation and Maintenance.

T follicular helper (Tfh) cells are a CD4+ T cell subset critical for long-lived humoral immunity. We hypothesized that integrins play a decisive role in Tfh cell biology. Here we show that Tfh cells expressed a highly active form of leukocyte function-associated antigen-1 (LFA-1) that was required for their survival within the germinal center niche. In addition, LFA-1 promoted expression of Bcl-6, a transcriptional repressor critical for Tfh cell differentiation, and inhibition of LFA-1 abolished Tfh cell generation and prevented protective humoral immunity to intestinal helminth infection. Furthermore, we demonstrated that expression of Talin-1, an adaptor protein that regulates LFA-1 affinity, dictated Tfh versus Th2 effector cell differentiation. Collectively, our results define unique functions for LFA-1 in the Tfh cell effector program and suggest that integrin activity is important in lineage decision-making events in the adaptive immune system.