Integrin LFA-1 Research Articles

CD47 plays a critical role in T-cell recruitment by regulation of LFA-1 and VLA-4 integrin adhesive functions

CD47 plays an important but incompletely understood role in the innate and adaptive immune responses. CD47, also called integrin-associated protein, has been demonstrated to associate in cis with β1 and β3 integrins. Here we test the hypothesis that CD47 regulates adhesive functions of T-cell α4β1 (VLA-4) and αLβ2 (LFA-1) in in vivo and in vitro models of inflammation. Intravital microscopy studies reveal that CD47(-/-) Th1 cells exhibit reduced interactions with wild-type (WT) inflamed cremaster muscle microvessels. Similarly, murine CD47(-/-) Th1 cells, as compared with WT, showed defects in adhesion and transmigration across tumor necrosis factor-α (TNF-α)-activated murine endothelium and in adhesion to immobilized intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion protein 1 (VCAM-1) under flow conditions. Human Jurkat T-cells lacking CD47 also showed reduced adhesion to TNF-α-activated endothelium and ICAM-1 and VCAM-1. In cis interactions between Jurkat T-cell β2 integrins and CD47 were detected by fluorescence lifetime imaging microscopy. Unexpectedly, Jurkat CD47 null cells exhibited a striking defect in β1 and β2 integrin activation in response to Mn(2+) or Mg(2+)/ethylene glycol tetraacetic acid treatment. Our results demonstrate that CD47 associates with β2 integrins and is necessary to induce high-affinity conformations of LFA-1 and VLA-4 that recognize their endothelial cell ligands and support leukocyte adhesion and transendothelial migration.

Novel Automated Tracking Analysis of Particles Subjected to Shear Flow: Kindlin-3 Role in B Cells

Shear flow assays are used to mimic the influence of physiological shear force in diverse situations such as leukocyte rolling and arrest on the vasculature, capture of nanoparticles, and bacterial adhesion. Analysis of such assays usually involves manual counting, is labor-intensive, and is subject to bias. We have developed the Leukotrack program that incorporates a novel (to our knowledge) segmentation routine capable of reliable detection of cells in phase contrast images. The program also automatically tracks rolling cells in addition to those that are more firmly attached and migrating in random directions. We demonstrate its use in the analysis of lymphocyte arrest mediated by one or more active conformations of the integrin LFA-1. Activation of LFA-1 is a multistep process that depends on several proteins including kindlin-3, the protein that is mutated in leukocyte adhesion deficiency-III patients. We find that the very first stage of LFA-1-mediated attaching is unable to proceed in the absence of kindlin-3. Our evidence indicates that kindlin-3-mediated high-affinity LFA-1 controls both the early transient integrin-dependent adhesions in addition to the final stable adhesions made under flow conditions.

OP0195 Genetic and Pharmacologic Inhibition of MST1 Blocks Lymphocyte Function and Protects Against Inflammation and Autoimmunity

BackgroundThe mammalian sterile 20-like kinase 1 (MST1) is a MAP4 kinase which acts upstream of a wide range of signal transduction pathways. It can modify signaling events mediated by Jnk,...

NK cell licensing modulates NK cell conjugation to target cells via altered activation receptor function (P1063)

Abstract MHC class I recognition by inhibitory receptors on natural killer (NK) cells promotes a state of increased NK cell responsiveness through a process known as licensing/education. In the absence of either MHC class I expression or inhibitory receptors, NK cells exhibit a decreased responsive state. Thus NK cell licensing is a critical aspect of NK cell biology. However, understanding how licensing modulates natural cytotoxicity is limited. Using pure populations of unlicensed human NK cells, we were able to evaluate the effect of licensing on several NK cell functions. Our findings reveal that NK cell licensing does not affect lytic granule polarization but significantly enhances NK cell conjugation to target cells. Furthermore, our data show that activation receptors in unlicensed NK cells fail to provide strong ‘inside-out’ signaling for the beta2 integrin LFA-1. NK cell adhesion to target cells is a crucial initial step in NK cell-mediated cytotoxicity. The reduced conjugation of unlicensed NK cells may contribute to their hyporesponsive state.

Regulated cytoskeletal control of ICAM-1 mobility on the dendritic cell plasma membrane enhances T cell activation (P1205)

Abstract T cell activation involves actin-dependent assembly and centripetal movement of signaling microclusters at the Immune Synapse (IS). Model stimulatory surfaces that limit microcluster dynamics enhance TCR signaling, but the possibility that antigen presenting cells can modulate T cell signaling by regulating ligand distribution is largely unexplored. Dendritic cells (DCs) form a multi-focal IS rather than the concentric pattern seen with freely mobile ligands, suggesting that DCs can control receptor-ligand localization. We have measured the lateral mobility of T cell ligands on the DC membrane, and find that ICAM-1, the key ligand for the integrin LFA-1, is selectively constrained via interactions with the actin cytoskeleton. ICAM-1 mobility decreases further upon LPS-induced DC maturation, concomitant with upregulation moesin and α-actinin 1, two actin-binding proteins that bind the cytoplasmic tail of ICAM-1. ICAM-1 co-localizes with moesin at punctate sites on the DC surface. Mutation of the ICAM-1 cytoplasmic tail or suppression of moesin and/or α-actinin liberates ICAM-1 lateral mobility. Importantly, liberating ICAM-1 from cytoskeletal constraints decreases the ability of DCs to induce the proliferation of naïve CD4 T cells. Taken together, our data show that the DC actin cytoskeleton actively controls ligand mobility and thus enhances T cell activation, and raise the possibility that DCs modulate T cell signaling by actively regulating molecular dynamics at the IS.

Tuning tumor immunity through integrin transregulation (P5077)

Abstract Recent trials using adoptive cellular immunotherapy for cancer have shown the exciting potential of this approach. However, ex vivo-modified tumor-specific T cells are limited by poor homing to solid tumors. Surprisingly this problem has received relatively little attention. α4 integrins are crucial in governing the migration of T cells to sites of infection and inflammation. Blocking phosphorylation of α4 integrin by mutating Ser988 to Ala prevents release of the adaptor protein paxillin, and boosts LFA-1-dependent migration in trans. We thus generated a mouse with this mutation to test effects on leukocyte migration in vivo. Here we report that the α4(S988A) mutation, which boosts VLA-4/LFA-1 integrin transregulation, selectively increases migration of lymphocytes to both inflammatory and tumor sites; myeloid cell homing was unaffected. Melanoma tumor growth was greatly reduced, associated with increased migration of α4(S988A) T cells to tumors, suggesting that boosting integrin trans-regulation is a promising approach to improve adoptive cellular immunotherapy for cancer.

Recombinant production of human ICAM-1 chimeras by single step on column refolding and purification

The interaction of the adhesion molecule of the immunoglobulin family intercellular adhesion molecule 1 (ICAM-1) with its ligands such that the integrins LFA-1 and Mac-1 is crucial for the regulation of several physiological and pathophysiological processes like cell mediated-elimination of tumor or virus infected cells, cancer metastasis or inflammatory autoimmune processes. Thus, production of milligrams of protein is required to perform structural and functional studies as well as design novel approaches to find out new inhibitors of ICAM-1/LFA-1 interaction. Here we report on the production of a recombinant human ICAM-1 chimera comprising the first two extracellular domains of ICAM-1 linked to the Fc fraction of a human IgG1. To this aim we have used a cost-effective method based on the expression of a His-tagged protein in Escherichia coli followed by a single step of refolding and purification on immobilized metal affinity columns. This method is able to produce 3mg/l of bacterial culture in just 72h with purity greater than 95%. The identity and the native structure of refolded human ICAM-1 chimera were confirmed by biochemical and biophysical studies including SDS-electrophoresis, immunoblot, circular dichroism, isothermal titration calorimetry and fluorescence spectroscopy. Native folding and functional activity of the chimera were further confirmed by different cell biology studies, including B cell adhesion, T cell binding and inhibition of NK cell function. These studies indicate a high biological activity of the protein since it induces a 200-fold increase/mg of protein in B cell adhesion and the inhibitory dose 50 to block cell-mediated cytotoxicity is 10pg/effector cell. These analyses show that our protocol is able to produce a recombinant human ICAM-1 chimera fully active and useful to analyze the biological processes in which ICAM-1/LFA-1 interaction is critically involved.

Monocyte signal transduction receptors in active and latent tuberculosis.

The mechanisms that promote either resistance or susceptibility to TB disease remain insufficiently understood. Our aim was to compare the expression of cell signaling transduction receptors, CD14, TLR2, CD206, and β2 integrin LFA-1 on monocytes from patients with active TB or nonmycobacterial lung disease and healthy individuals with M.tb latency and uninfected controls to explain the background of the differences between clinical and subclinical forms of M.tb infection. A simultaneous increase in the expression of the membrane bound mCD14 receptor and LFA-1 integrin in patients with active TB may be considered a prodrome of breaking immune control by M.tb bacilli in subjects with the latent TB and absence of clinical symptoms.

Expression and Function of the Homeostatic Molecule Del-1 in Endothelial Cells and the Periodontal Tissue

Developmental endothelial locus-1 (Del-1) is an endothelial cell-secreted protein that limits the recruitment of neutrophils by antagonizing the interaction between the LFA-1 integrin on neutrophils and the intercellular adhesion molecule (ICAM)-1 on endothelial cells. Mice with genetic or age-associated Del-1 deficiency exhibit increased neutrophil infiltration in the periodontium resulting in inflammatory bone loss. Here we investigated additional novel mechanisms whereby Del-1 could interfere with neutrophil recruitment and inflammation. Treatment of human endothelial cells with Del-1 did not affect the expression of endothelial molecules involved in the leukocyte adhesion cascade (ICAM-1, VCAM-1, and E-selectin). Moreover, genetic or age-associated Del-1 deficiency did not significantly alter the expression of these adhesion molecules in the murine periodontium, further ruling out altered adhesion molecule expression as a mechanism whereby Del-1 regulates leukocyte recruitment. Strikingly, Del-1 inhibited ICAM-1-dependent chemokine release (CXCL2, CCL3) by neutrophils. Therefore, Del-1 could potentially suppress the amplification of inflammatory cell recruitment mediated through chemokine release by infiltrating neutrophils. Interestingly, Del-1 was itself regulated by inflammatory stimuli, which generally exerted opposite effects on adhesion molecule expression. The reciprocal regulation between Del-1 and inflammation may contribute to optimally balance the protective and the potentially harmful effects of inflammatory cell recruitment.

Arrest Functions of the MIF Ligand/Receptor Axes in Atherogenesis

Macrophage migration inhibitory factor (MIF) has been defined as an important chemokine-like function (CLF) chemokine with an essential role in monocyte recruitment and arrest. Adhesion of monocytes to the vessel wall and their transendothelial migration are critical in atherogenesis and many other inflammatory diseases. Chemokines carefully control all steps of the monocyte recruitment process. Those chemokines specialized in controlling arrest are typically immobilized on the endothelial surface, mediating the arrest of rolling monocytes by chemokine receptor-triggered pathways. The chemokine receptor CXCR2 functions as an important arrest receptor on monocytes. An arrest function has been revealed for the bona fide CXCR2 ligands CXCL1 and CXCL8, but genetic studies also suggested that additional arrest chemokines are likely to be involved in atherogenic leukocyte recruitment. While CXCR2 is known to interact with numerous CXC chemokine ligands, the CLF chemokine MIF, which structurally does not belong to the CXC chemokine sub-family, was surprisingly identified as a non-cognate ligand of CXCR2, responsible for critical arrest functions during the atherogenic process. MIF was originally identified as macrophage migration inhibitory factor (this function being eponymous), but is now known as a potent inflammatory cytokine with CLFs including chemotaxis and leukocyte arrest. This review will cover the mechanisms underlying these functions, including MIF’s effects on LFA1 integrin activity and signal transduction, and will discuss the structural similarities between MIF and the bona fide CXCR2 ligand CXCL8 while emphasizing the structural differences. As MIF also interacts with CXCR4, a chemokine receptor implicated in CXCL12-elicited lymphocyte arrest, the arrest potential of the MIF/CXCR4 axis will also be scrutinized as well as the recently identified role of pericyte MIF in attracting leukocytes exiting through venules as part of the pericyte “motility instruction program.”

Abstract 18596: Conjugated Linoleic Acid Targets β2 Integrin Expression to Suppress Monocyte Adhesion

Recruitment of monocytes to the damaged endothelium characterizes the early stages of atherosclerosis. Conjugated linoleic acids (CLA) are a group of naturally occurring fatty acids which inhibit the progression and induce regression of pre-established atherosclerosis. We have previously shown that CLA inhibits human peripheral blood monocyte (HPBMC) migration. Here we demonstrate that CLA suppresses adhesion of monocytes to endothelial cells, via inhibition of β2 integrins. We further show that CLA suppresses the CXCR4 signalling pathway thus inhibiting crawling of monocytes towards CXCL12 chemokine. HPBMCs were treated with 10uM of CLA isomers (c9,t11-CLA and t10,c12-CLA), a CLA blend (80:20, c9t,11:t10,c12), control lipids and 5uM of PPARγ agonist (troglitazone) prior to adhesion to HAECs. c9,t11-CLA and CLA blend treatment inhibited monocyte adhesion to HAECs (63±3%, p<0.01 and 68±3%, p<0.01, respectively). Furthermore, c9,t11-CLA and CLA blend inhibited CD18, the β chain of β2 integrin, mRNA (48±14%, p<0.05 and 35±3%, p<0.001) and protein expression (44±13%, p<0.05, and 35±3%, p<0.001) via a PPARγ[[Unable to Display Character: ]]dependent mechanism. Flow cytometry analysis showed that c9,t11-CLA and CLA blend decreased external surface expression of both β chain (10±4%, p<0.05 and 20±5%, p<0.01), and α subunits (CD11a, 13±6%, p<0.05 and 30±5%, p<0.01; CD11b, 8±3%, p<0.05 and 27±7%, p<0.01), suggesting that CLA’s effect on CD18 transcription results in dysfunctional αβ complex formation, altering expression of LFA-1 and Mac-1 integrins. Furthermore, c9,t11-CLA and CLA blend almost completely abolished (by 82% and 89%, p<0.001) adhesion of monocytes to ICAM-1. Immunocytochemistry of adherent cells further confirmed the inhibition of cell spreading. Importantly, CLA suppresses monocyte response to chemoattraction, preventing crawling of cells through a reduction, of CXCR4 expression (c9,t11-CLA, 47±10%, p<0.05; CLA blend, 33±5%, p<0.01). Our findings suggest that CLA inhibits monocyte recruitment by suppressing their sensitivity to chemokines and by inhibiting their ability to firmly attach to ICAM-1.

Pan-Selectin Antagonist GMI-1070 Affects Biomarkers of Adhesion, Activation and the Coagulation Cascade in Sickle Cell Adults At Steady State

AbstractAbstract 87 Introduction:Engagement of selectins by their ligands leads to cellular activation and adhesion and plays a role in thrombus formation. In sickle cell disease (SCD), the selectins underlie vaso-occlusion, which results in vaso-occlusive crisis (VOC). In SCD patients high levels of soluble E-selectin (sEsel) are associated with increased mortality (Kato, BJH 2005). In addition, SCD patients exhibit chronic activation of the coagulation cascade and of leukocytes. Previously, we showed in animal models of SCD VOC that pan-selectin antagonist GMI-1070 reduced arrested RBC/WBC aggregates and improved blood flow and survival. In a Phase 1 study of SCD adults at steady state (not in VOC), GMI-1070 inhibited neutrophil activation and platelet/neutrophil aggregate formation and increased circulating neutrophils. Herein we report on the effect of GMI-1070 on biomarkers of monocyte, endothelial cell, and coagulation cascade activation; and on the effect of hydroxycarbamide (hydroxyurea or HU) on these biomarkers for patients on this trial. Methods:SCD adults at steady state (n=15) received an IV loading dose of GMI-1070 (20 mg/kg) and a second dose ten hours later (10 mg/kg). Safety and PK were reported elsewhere. HU use was noted. Biomarker blood samples were drawn prior to treatment, and at 4, 8, 24 and 48 hrs after the loading dose. Analytes measured included: soluble adhesion molecules sEsel, soluble P-selectin (sPsel) and intracellular adhesion molecule-1 (ICAM-1) by multiplex ELISA; tissue factor and thrombin-antithrombin complexes (TF and TAT) by ELISA; and, surface expression of monocyte b2 integrins MAC-1 & LFA-1; and platelet-monocyte aggregates (PMA) by flow cytometry. Expression levels were compared against pre-treatment, and stratified by HU use. Analysis was by ANOVA F-test from mixed effects model. Data are reported for 11 subjects (biomarkers) and 15 subjects (neutrophils). Results:Soluble adhesion markers were reduced after 8 hrs (sEsel), or 4 and 8 hrs (sPsel; ICAM-1). Tissue factor (TF) was reduced at 4 and 8 hrs. Thrombin-antithrombin complex (TAT) levels and expression of MAC-1 and LFA-1 were reduced at all time points. The percentage of PMA was reduced at 8 hrs. (Table)BiomarkerBaseline Level (SD)4 hrs8 hrs24 hrs48 hrssE-sel (ng/mL)117.2 (48.6)86.8 (14.0)101.5 (40.9)**109.5 (56.9)112.2 (42.8)sP-sel (ng/mL)180.4 (154.0)60.7 (17.2)*140.2 (97.1)*127.8 (112.7)143.4 (106.2)ICAM-1 (ng/mL)239.6 (177.8)126.6 (58.2)*209.5 (130.6)**208.1 (156.1)214.6 (139.6)TF (pg/mL)466.7 (313.4)272.5 (65.9)**365.7 (124.0)*349.3 (70.1)356.4 (97.2)TAT (ng/mL)145.4 (94.4)42.2 (24.2)***83.6 (64.7)***66.8 (50.0)***98.4 (64.0)**MAC-1 (MFI)3278.5 (1302.4)2724.7 (347.1)**2337.5 (896.8)**2821.1 (876.7)**2543.2 (1148.4)**LFA-1 (MFI)815.3 (271.8)758.8 (4.8)**661.4 (222.7)**702.7 (242.7)*677.2 (238.4)**PMA (%)82 (20)87 (6)61 (30)*69 (23)73 (22)*p<0.05;**p<0.01;***p<0.001.When HU use was considered (HU–No; HU–Yes), the levels of sEsel, sPsel, ICAM-1, TF, TAT, MAC-1, and neutrophil counts were higher and more variable at baseline in the HU-No group, significantly so for ICAM-1 (p=0.048) and MAC-1 (p=0.001). After GMI-1070, significant reduction from baseline was seen in both groups: in the HU-No group for ICAM-1, TF, PMA, LFA-1, and in the HU-Yes group for sEsel, MAC-1, TF, TAT. Neutrophil counts increased at 24 hours in the HU-No group only (p=0.001). Conclusion:In this small sample of adults with SCD at steady state, selectin inhibition by treatment with GMI-1070 resulted in reduction in soluble adhesion markers, leukocyte activation, PMA, and markers of coagulation activation. This suggests that selectin inhibition affects downstream processes in vivo, continuing after plasma clearance of >97% of the drug. This may represent interference in the processes that lead to VOC in SCD. A phase 2 study is underway to further explore the effect of this experimental drug on these markers and in the treatment of VOC. Disclosures:Magnani:GlycoMimetics: Employment, Equity Ownership. Kuypers:GlycoMimetics: Research Funding. Patton:GlycoMimetics: Employment. Larkin:GlycoMimetics: Research Funding. Styles:GlycoMimetics: Research Funding. DeCastro:GlycoMimetics: Research Funding. Telen:GlycoMimetics: Research Funding. Wun:GlycoMimetics: Research Funding. Thackray:GlycoMimetics: Employment, Equity Ownership.

A role for Rap2 in recycling the extended conformation of LFA-1 during T cell migration

SummaryT lymphocytes make use of their major integrin LFA-1 to migrate on surfaces that express ICAM-1 such as blood vessels and inflamed tissue sites. How the adhesions are turned over in order to supply traction for this migration has not been extensively investigated. By following the fate of biotinylated membrane LFA-1 on T lymphocytes, we show in this study that LFA-1 internalization and re-exposure on the plasma membrane are linked to migration. Previously we demonstrated the GTPase Rap2 to be a regulator of LFA-1-mediated migration. SiRNA knockdown of this GTPase inhibits both LFA-1 internalization and also its ability to be re-exposed, indicating that Rap2 participates in recycling of LFA-1 and influences its complete endocytosis–exocytosis cycle. Confocal microscopy images reveal that the intracellular distribution of Rap2 overlaps with endosomal recycling vesicles. Although the homologous GTPase Rap1 is also found on intracellular vesicles and associated with LFA-1 activation, these two homologous GTPases do not co-localize. Little is known about the conformation of the LFA-1 that is recycled. We show that the extended form of LFA-1 is internalized and in Rap2 siRNA-treated T lymphocytes the trafficking of this LFA-1 conformation is disrupted resulting in its intracellular accumulation. Thus LFA-1-mediated migration of T lymphocytes requires Rap2-expressing vesicles to recycle the extended form of LFA-1 that we have previously found to control migration at the leading edge.

Complex structure of engineered modular domains defining molecular interaction between ICAM-1 and integrin LFA-1.

Intermolecular contacts between integrin LFA-1 (αLβ2) and ICAM-1 derive solely from the integrin αL I domain and the first domain (D1) of ICAM-1. This study presents a crystal structure of the engineered complex of the αL I domain and ICAM-1 D1. Previously, we engineered the I domain for high affinity by point mutations that were identified by a directed evolution approach. In order to examine αL I domain allostery between the C-terminal α7-helix (allosteric site) and the metal-ion dependent adhesion site (active site), we have chosen a high affinity variant without mutations directly influencing either the position of the α7-helix or the active sites. In our crystal, the αL I domain was found to have a high affinity conformation to D1 with its α7-helix displaced downward away from the binding interface, recapitulating a current understanding of the allostery in the I domain and its linkage to neighboring domains of integrins in signaling. To enable soluble D1 of ICAM-1 to fold on its own, we also engineered D1 to be functional by mutations, which were found to be those that would convert hydrogen bond networks in the solvent-excluded core into vdW contacts. The backbone structure of the β-sandwich fold and the epitope for I domain binding of the engineered D1 were essentially identical to those of wild-type D1. Most deviations in engineered D1 were found in the loops at the N-terminal region that interacts with human rhinovirus (HRV). Structural deviation found in engineered D1 was overall in agreement with the function of engineered D1 observed previously, i.e., full capacity binding to αL I domain but reduced interaction with HRV.

Anti-LFA-1 antibodies enhance metastasis of ocular lymphoma to the brain and contralateral eye

Previously we demonstrated that intraperitoneal (IP) inoculation of Rev-2-T-6 mouse lymphoma into syngeneic Balb/c hosts resulted in brain metastasis, migration along the optic nerve sheath, and ocular infiltration. In a second model: intravitreal inoculation of Rev-2-T-6 cells, the developing lymphoma was largely confined within the eye, seldom breaching the retinal pigment epithelium to reside in the choroid and sclera. There was no retrograde infiltration into the brain. Here, we describe a third, complementary model, whereby intravitreal inoculation of Rev-2-T-6 cells into Balb/c mice, followed by repeated IP inoculations of anti-LFA-1/CD11a monoclonal antibodies, results in extensive infiltration of the choroid, sclera, conjunctiva, eyelids and orbit. Furthermore, the lymphoma cells metastasize along the optic nerve sheath into the brain, and through the contralateral optic nerve tract into the contralateral eye. There is no systemic involvement of the lymphoma. Furthermore, anti-LFA-1 treatment results in elevated levels of serum anti-Rev-2-T-6 antibodies. Inoculation of Rev-2-T-6 cells into the vitreous of severe combined immune deficient mice demonstrates a course of clinical signs and histopathological findings similar to those in immune-competent mice treated with anti-LFA-1 antibodies, including invasion of the contralateral eye. Taken together, these findings suggest that confinement of Rev-2-T-6 lymphoma cells to the eye depends on active immune surveillance using a population of effector cells expressing the cell surface integrin LFA-1. Impairing this protection enhances tumor aggressiveness within the eye, and the likelihood of early retrograde lymphoma metastasis into the brain and the contralateral eye.

L-Plastin Regulates Polarization and Migration in Chemokine-Stimulated Human T Lymphocytes

Chemokines such as SDF-1α play a crucial role in orchestrating T lymphocyte polarity and migration via polymerization and reorganization of the F-actin cytoskeleton, but the role of actin-associated proteins in this process is not well characterized. In this study, we have investigated a role for L-plastin, a leukocyte-specific F-actin-bundling protein, in SDF-1α-stimulated human T lymphocyte polarization and migration. We found that L-plastin colocalized with F-actin at the leading edge of SDF-1α-stimulated T lymphocytes and was also phosphorylated at Ser(5), a site that when phosphorylated regulates the ability of L-plastin to bundle F-actin. L-plastin phosphorylation was sensitive to pharmacological inhibitors of protein kinase C (PKC), and several PKC isoforms colocalized with L-plastin at the leading edge of SDF-1α-stimulated lymphocytes. However, PKC ζ, an established regulator of cell polarity, was the only isoform that regulated L-plastin phosphorylation. Knockdown of L-plastin expression with small interfering RNAs demonstrated that this protein regulated the localization of F-actin at the leading edge of chemokine-stimulated cells and was also required for polarization, lamellipodia formation, and chemotaxis. Knockdown of L-plastin expression also impaired the Rac1 activation cycle and Akt phosphorylation in response to SDF-1α stimulation. Furthermore, L-plastin also regulated SDF-1α-mediated lymphocyte migration on the integrin ligand ICAM-1 by influencing velocity and persistence, but in a manner that was independent of LFA-1 integrin activation or adhesion. This study, therefore, demonstrates an important role for L-plastin and the signaling pathways that regulate its phosphorylation in response to chemokines and adds L-plastin to a growing list of proteins implicated in T lymphocyte polarity and migration.

The Gαq/11 Proteins Contribute to T Lymphocyte Migration by Promoting Turnover of Integrin LFA-1 through Recycling

The role of Gαi proteins coupled to chemokine receptors in directed migration of immune cells is well understood. In this study we show that the separate class of Gαq/11 proteins is required for the underlying ability of T cells to migrate both randomly and in a directed chemokine-dependent manner. Interfering with Gαq or Gα11 using dominant negative cDNA constructs or siRNA for Gαq causes accumulation of LFA-1 adhesions and stalled migration. Gαq/11 has an impact on LFA-1 expression at plasma membrane level and also on its internalization. Additionally Gαq co-localizes with LFA-1- and EEA1-expressing intracellular vesicles and partially with Rap1- but not Rab11-expressing vesicles. However the influence of Gαq is not confined to the vesicles that express it, as its reduction alters intracellular trafficking of other vesicles involved in recycling. In summary vesicle-associated Gαq/11 is required for the turnover of LFA-1 adhesion that is necessary for migration. These G proteins participate directly in the initial phase of recycling and this has an impact on later stages of the endo-exocytic pathway.

Th17 cell LFA-1 engagement-mediated IL-17A and GM-CSF mRNA stabilization is HuR-dependent (54.14)

Abstract Recent studies demonstrate that the β2 integrin LFA-1 is required for entry of encephalitogenic Th17 cells into the brain parenchyma during murine experimental autoimmune encephalomyelitis (EAE). We previously showed that LFA-1 engagement by its ligand, intracellular adhesion molecule-1 (ICAM-1), in human T cells results in translocation of the RNA-binding protein HuR and stabilization of TNFα and IFNγ mRNAs. The role of HuR in the regulation of Th17 cell-mediated inflammation is unknown. We hypothesize that LFA-1 engagement in Th17 cells leads to increased neuropathology-relevant cytokine expression at inflammatory sites through HuR-mediated mRNA stabilization. LFA-1 engagement led to HuR nuclear-to-cytoplasmic translocation and stabilization of both IL-17A and GM-CSF transcripts in mouse EL4 T cells, in vitro differentiated mouse Th17 cells and human primary T cells. EL4 and human primary T cell adhesion to ICAM-1 led to increased levels of IL-17A and GM-CSF mRNA in HuR immunoprecipitates. LFA-1 engagement-stimulated IL-17A and GM-CSF mRNA stabilization was lost in HuR-deficient Th17 cells generated from RORcγt-Cre/HuRflox/flox mice. We conclude that LFA-1 engagement induces HuR-dependent stabilization of the otherwise labile GM-CSF and IL-17A transcripts in Th17 cells, and that this may be highly relevant in Th17 cell-induced CNS pathology. Future experiments will focus on the role of Th17 cell HuR in the EAE model.

Antigen-induced release and retroviral subversion of TCR-enriched microvesicles at the CD4+ T cell immunological synapse (58.5)

Abstract The immunological synapse (IS) is a specialized junction that forms between T cells and antigen presenting cells, which is critical for effective T cell antigen recognition and effector functions. Its spatiotemporal evolution results in laterally segregated supramolecular domains composed of a peripheral adhesive ring of the β2 integrin LFA-1 and its ligand ICAM-1, and centrally accumulated T cell receptors (TCR). The molecular and cell biological basis for supramolecular domain organization at the IS remains unclear. Here we show, using novel correlative optical-electron microscopy methods in conjunction with electron tomography, that centrally accumulated TCR is located on the surface of microvesicles that are shed at the IS center. The ESCRT I member TSG101 sorts TCR into microvesicles. TCR-enriched microvesicles (TEMs) are transferred from T cell to B cell during cognate antigen recognition, and B cells initiate intracellular signals in response to TEMs, suggesting their involvement in intercellular communication between immune cells. Expression of the HIV GAG structural polyprotein in human CD4+ T cells, displaces TCR from microvesicles at the IS, resulting in the release of GAG-containing virus-like particles. These findings provide an ultrastructural basis for supramolecular domain organization at the IS, and identify an ESCRT-dependent mechanism for production of stimulatory TEMs, which may be co-opted during infection for the release of enveloped viruses.

Glia maturation factor gamma regulates the migration and adherence of human T lymphocytes

BackgroundLymphocyte migration and chemotaxis are essential for effective immune surveillance. A critical aspect of migration is cell polarization and the extension of pseudopodia in the direction of movement. However, our knowledge of the underlying molecular mechanisms responsible for these events is incomplete. Proteomic analysis of the isolated leading edges of CXCL12 stimulated human T cell lines was used to identify glia maturation factor gamma (GMFG) as a component of the pseudopodia. This protein is predominantly expressed in hematopoietic cells and it has been shown to regulate cytoskeletal branching. The present studies were undertaken to examine the role of GMFG in lymphocyte migration.ResultsMicroscopic analysis of migrating T-cells demonstrated that GMFG was distributed along the axis of movement with enrichment in the leading edge and behind the nucleus of these cells. Inhibition of GMFG expression in T cell lines and IL-2 dependent human peripheral blood T cells with shRNAmir reduced cellular basal and chemokine induced migration responses. The failure of the cells with reduced GMFG to migrate was associated with an apparent inability to detach from the substrates that they were moving on. It was also noted that these cells had an increased adherence to extracellular matrix proteins such as fibronectin. These changes in adherence were associated with altered patterns of β1 integrin expression and increased levels of activated integrins as detected with the activation specific antibody HUTS4. GMFG loss was also shown to increase the expression of the β2 integrin LFA-1 and to increase the adhesion of these cells to ICAM-1.ConclusionsThe present studies demonstrate that GMFG is a component of human T cell pseudopodia required for migration. The reduction in migration and increased adherence properties associated with inhibition of GMFG expression suggest that GMFG activity influences the regulation of integrin mediated adhesion.