LFA-1 Research Articles

Abstract 408: The Role of the Chemokine Receptor CXCR3 - Integrin Lymphocyte Function Associated Antigen 1 (LFA1) - Intercellular Adhesion Molecule 1 (ICAM1) Axis in T Lymphocyte Mediated Cardiac Inflammation in Pressure Overload Induced Heart Failure

Background: Left ventricular (LV) dysfunction and Heart Failure (HF) are associated in humans and mice with increased circulating chemokines CXCL9, CXCL10 and CXCL11. Expression of their receptor, CXCR3, in circulating T cells is associated with disease progression in HF patients. We recently published that ICAM1 is significantly upregulated in the LV endothelium and contributes to the progression of HF by regulating T cell recruitment to the LV in a mouse model of pressure overload (PO) induced HF. As CXCR3 can signal to integrins upon chemokine recognition and induce T cell adhesion to endothelial cells under flow conditions in vitro , we hypothesize that chemokine signaling through CXCR3 contributes to LV T cell recruitment and regulates integrin dependent adhesion to ICAM1 in PO induced HF. Methods and results: We used the mouse model of Thoracic Aortic Constriction (TAC) to induce LV remodeling and HF in WT and ICAM1 -/- mice, cardiac fibroblasts (CFB) isolated from adult C57/BL6 mice, flow cytometry and qPCR in mouse tissues and in cells cultured in vitro . The LV expression of CXCL9, 10 and 11 was upregulated in WT mice in response to 2 and 4 weeks TAC as compared to Sham, correlating with ICAM1, IL-6 and IL-1β upregulation and increased CXCR3+ T cell LV infiltration. In vitro , CFB cultured with CXCR3+ T cells or with their culture supernatants, induced CXCL9, 10 and 11, as compared to control media cultured CFB, suggesting CFB are a source of these chemokines under stress. 4 weeks post TAC in WT mice, LV recruited CXCR3+ T cells, as well as mediastinal lymph node and circulating CXCR3+ lymphocytes expressed higher levels of the integrin LFA1, the main ligand of ICAM1, than CXCR3- T cells. The same pattern was observed in systemic CXCR3+ T cells in ICAM1 -/- mice, which, in contrast to WT mice, had reduced LV infiltrated T cells. Conclusion: Our data supports a CXCR3-LFA1-ICAM1 axis being involved in non- ischemic HF. Further studies will determine the CXCR3 ligands triggering T cell LFA1 activation through CXCR3 and whether this mechanism regulates the pathology of non- ischemic HF via ICAM-1 mediated adhesion.

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

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. 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. 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. Unlike the mechanism reported in other malignancies, NKG2D-mediated cytotoxicity does not constitute the major killing mechanism of CIK cells against NB.

Development of lifitegrast: a novel T-cell inhibitor for the treatment of dry eye disease.

Dry eye disease (DED) is a multifactorial disorder of the ocular surface characterized by symptoms of discomfort, decreased tear quality, and chronic inflammation that affects an estimated 20 million patients in the US alone. DED is associated with localized inflammation of the ocular surface and periocular tissues leading to homing and activation of T cells, cytokine release, and development of hyperosmolar tears. This inflammatory milieu results in symptoms of eye dryness and discomfort. Homing of T cells to the ocular surface is influenced by the binding of lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18; αLβ2), a cell surface adhesion protein, to its cognate ligand, intercellular adhesion molecule-1 (ICAM-1; CD54), which is expressed on inflamed ocular/periocular epithelium and vascular endothelium. LFA-1/ICAM-1 binding within the immunologic synapse enables both T-cell activation and cytokine release. Lifitegrast is a novel T-cell integrin antagonist that is designed to mimic the binding epitope of ICAM-1. It serves as a molecular decoy to block the binding of LFA-1/ICAM-1 and inhibits the downstream inflammatory process. In vitro studies have demonstrated that lifitegrast inhibits T-cell adhesion to ICAM-1-expressing cells and inhibits secretion of pro-inflammatory cytokines including interferon gamma, tumor necrosis factor alpha, macrophage inflammatory protein 1 alpha, interleukin (IL)-1α, IL-1β, IL-2, IL-4, and IL-6, all of which are known to be associated with DED. Lifitegrast has the potential to be the first pharmaceutical product approved in the US indicated for the treatment of both symptoms and signs of DED. Clinical trials involving over 2,500 adult DED patients have demonstrated that topically administered lifitegrast 5.0% ophthalmic solution can rapidly reduce the symptoms of eye dryness and decrease ocular surface staining with an acceptable long-term safety profile. The purpose of this review is to highlight the developmental story – from bench top to bedside – behind the scientific rationale, engineering, and clinical experience of lifitegrast for the treatment of DED.

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.

Artificial antigen-presenting cells expressing HLA class II molecules as an effective tool for amplifying human specific memory CD4(+) T cells.

Owing to their multiple immune functions, CD4(+) T cells are of major interest for immunotherapy in chronic viral infections and cancer, as well as for severe autoimmune diseases and transplantation. Therefore, standardized methods allowing rapid generation of a large number of CD4(+) T cells for adoptive immunotherapy are still awaited. We constructed stable artificial antigen-presenting cells (AAPCs) derived from mouse fibroblasts. They were genetically modified to express human leukocyte antigen (HLA)-DR molecules and the human accessory molecules B7.1, Intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-3 (LFA-3). AAPCs expressing HLA-DR1, HLA-DR15 or HLA-DR51 molecules and loaded with peptides derived from influenza hemagglutinin (HA), myelin basic protein (MBP) or factor VIII, respectively, activated specific CD4(+) T-cell clones more effectively than Epstein-Barr virus (EBV)-transformed B cells. We also showed that AAPCs were able to take up and process whole Ag proteins, and present epitopes to specific T cells. In primary cultures, AAPCs loaded with HA peptide allowed generation of specific Th1 lymphocytes from healthy donors as demonstrated by tetramer and intracellular cytokine staining. Although AAPCs were less effective than autologous peripheral blood mononuclear cells (PBMCs) to stimulate CD4(+) T cells in primary culture, AAPCs were more potent to reactivate and expand memory Th1 cells in a strictly Ag-dependent manner. As the availability of autologous APCs is limited, the AAPC system represents a stable and reliable tool to achieve clinically relevant numbers of CD4(+) T cells for adoptive immunotherapy. For fundamental research in immunology, AAPCs are also useful to decipher mechanisms involved in the development of human CD4 T-cell responses.

IL-1β limits the extent of human 6-sulfo LacNAc dendritic cell (slanDC)-mediated NK cell activation and regulates CD95-induced apoptosis.

To function optimally, human blood natural killer (NK) cells need to communicate with other immune cells. Previously, it has been shown that NK cells communicate with 6-sulfo LacNAc dendritic cells (slanDCs), which are able to stimulate NK cells in vitro. In this study, we investigated how slanDCs regulate the level of NK cell activation. The secretion of interleukin (IL)-1β by slanDCs during coculture with NK cells increased as a result of signaling via intercellular adhesion molecule-1 on slanDCs following its interaction with lymphocyte function-associated antigen-1 on NK cells. IL-1β induced the expression of Fas receptor (CD95) on NK cells. The binding of Fas ligand (CD178) to CD95 induced the apoptosis of activated NK cells. Moreover, IL-1β also induced increased cyclooxygenase-2 expression in slanDCs, which in turn enabled the cells to secrete prostaglandin (PG)-E2. Consequently, PGE2 acted as a suppressing agent, tuning down the activation level of NK cells. In summary, IL-1β limits the level of NK cell activation by inducing apoptosis and suppression as a homeostatic regulatory function.

Inhibition of Bacterial Toxin Activity by the Nuclear Stain, DRAQ5™.

The repeats-in-toxin family of toxins includes proteins produced by Gram negative bacteria such as Escherichia coli (α-hemolysin), Bordetella pertussis (adenylate cyclase toxin), and Aggregatibacter actinomycetemcomitans (LtxA), which contribute to the pathogenesis of these organisms by killing host cells. In the case of LtxA produced by A. actinomycetemcomitans, white blood cells are targeted, allowing the bacteria to avoid clearance by the host immune system. In its association with target cells, LtxA binds to a receptor, lymphocyte function-associated antigen-1, as well as membrane lipids and cholesterol, before being internalized via a lysosomal-mediated pathway. The motivation for this project comes from our discovery that DRAQ5™, a membrane-permeable nuclear stain, prevents the internalization of LtxA in a Jurkat T cell line. We hypothesized that DRAQ5™, in crossing the plasma membrane, alters the properties of the membrane to inhibit LtxA internalization. To investigate how DRAQ5™ interacts with the lipid membrane to prevent LtxA internalization, we used studied DRAQ5™-mediated membrane changes in model membranes using a variety of techniques, including differential scanning calorimetry and fluorescence spectroscopy. Our results suggest that DRAQ5™ inhibits the activity of LtxA by decreasing the fluidity of the cellular lipid membrane, which decreases LtxA binding. These results present an interesting possible anti-virulence strategy; by altering bacterial toxin activity by modifying membrane fluidity, it may be possible to inhibit the pathogenicity of A. actinomycetemcomitans.

The positive influence of NKT cells on liver regeneration in a subseptic setting depends on lymphocyte function-associated antigen 1

The positive influence of NKT cells on liver regeneration in a subseptic setting depends on lymphocyte function-associated antigen 1

Leukocyte integrin αLβ2 headpiece structures: The αI domain, the pocket for the internal ligand, and concerted movements of its loops

High-resolution crystal structures of the headpiece of lymphocyte function-associated antigen-1 (integrin αLβ2) reveal how the αI domain interacts with its platform formed by the α-subunit β-propeller and β-subunit βI domains. The αLβ2 structures compared with αXβ2 structures show that the αI domain, tethered through its N-linker and a disulfide to a stable β-ribbon pillar near the center of the platform, can undergo remarkable pivoting and tilting motions that appear buffered by N-glycan decorations that differ between αL and αX subunits. Rerefined β2 integrin structures reveal details including pyroglutamic acid at the β2 N terminus and bending within the EGF1 domain. Allostery is relayed to the αI domain by an internal ligand that binds to a pocket at the interface between the β-propeller and βI domains. Marked differences between the αL and αX subunit β-propeller domains concentrate near the binding pocket and αI domain interfaces. Remarkably, movement in allostery in the βI domain of specificity determining loop 1 (SDL1) causes concerted movement of SDL2 and thereby tightens the binding pocket for the internal ligand.

Proteome Based Construction of the Lymphocyte Function-Associated Antigen 1 (LFA-1) Interactome in Human Dendritic Cells.

The β2-integrin lymphocyte function-associated antigen 1 (LFA-1) plays an important role in the migration, adhesion and intercellular communication of dendritic cells (DCs). During the differentiation of human DCs from monocyte precursors, LFA-1 ligand binding capacity is completely lost, even though its expression levels were remained constant. Yet LFA-1-mediated adhesive capacity on DCs can be regained by exposing DCs to the chemokine CCL21, suggesting a high degree of regulation of LFA-1 activity during the course of DC differentiation. The molecular mechanisms underlying this regulation of LFA-1 function in DCs, however, remain elusive. To get more insight we attempted to identify specific LFA-1 binding partners that may play a role in regulating LFA-1 activity in DCs. We used highly sensitive label free quantitative mass-spectrometry to identify proteins co-immunoprecipitated (co-IP) with LFA-1 from ex vivo generated DCs. Among the potential binding partners we identified not only established components of integrin signalling pathways and cytoskeletal proteins, but also several novel LFA-1 binding partners including CD13, galectin-3, thrombospondin-1 and CD44. Further comparison to the LFA-1 interaction partners in monocytes indicated that DC differentiation was accompanied by an overall increase in LFA-1 associated proteins, in particular cytoskeletal, signalling and plasma membrane (PM) proteins. The here presented LFA-1 interactome composed of 78 proteins thus represents a valuable resource of potential regulators of LFA-1 function during the DC lifecycle.

PI3Kδ promotes CD4(+) T-cell interactions with antigen-presenting cells by increasing LFA-1 binding to ICAM-1.

Activation of T lymphocytes by peptide/major histocompatibility complex on antigen-presenting cells (APCs) involves dynamic contacts between the two cells, during which T cells undergo marked morphological changes. These interactions are facilitated by integrins. Activation of the T cells increases the binding of the integrin lymphocyte function-associated antigen 1 (LFA-1) expressed by T cells to intercellular adhesion molecule (ICAM)-1 and ICAM-2 expressed by APCs. The signalling pathways that control integrin affinities are incompletely defined. The phosphoinositide 3-kinases (PI3Ks) generate second-messenger signalling molecules that control cell growth, proliferation, differentiation and trafficking. Here we show that in T cells, PI3Kδ attenuates the activation of Rac1, but sustains the activation of Rap1. Consequently, PI3Kδ increases LFA-1-dependent adhesion to form stable conjugates with APCs. Increased Rap1 activity and LFA-1 adhesion were only in part mediated by the downstream kinase Akt, suggesting the involvement of additional phosphatidylinositol(3,4,5)P3-binding proteins. These results establish a link between PI3K activity, cytoskeletal changes and integrin binding and help explain the impaired T-cell-dependent immune responses in PI3Kδ-deficient mice.

Lung ICAM-1 and ICAM-2 support spontaneous intravascular effector lymphocyte entrapment but are not required for neutrophil entrapment or emigration inside endotoxin-inflamed lungs.

The pulmonary vasculature constitutively expresses the integrin lymphocyte function-associated antigen-1 ligands intercellular adhesion molecule (ICAM)-1 and -2. In this study, effector T cells were temporarily entrapped by the lung vasculature on their way to inflamed lymph nodes, and this entrapment was strongly reduced in ICAM-1 and -2 double-deficient mice (79 and 86% reduction for CD8(+) and CD4(+) effectors, respectively, compared with wild-type mice). Although the pulmonary vasculature has been suggested to be masked by the heparan sulfate-containing glycocalyx, which is susceptible to heparanase-mediated shedding, lung and lymphocyte heparanase have been found to be unnecessary for this entrapment. Systemic LPS induced rapid neutrophil entrapment in the lung vasculature, but in contrast to T-cell entrapment, this sequestration was ICAM-1, ICAM-2, and heparanase independent. Furthermore, neutrophil migration into the bronchoalveolar space induced by LPS inhalation and LPS-induced leakage of red blood cells into this space were not dependent on lung ICAMs or heparanase activity. Nevertheless, heparanase was critical for neutrophil accumulation in smoke-exposed lungs. Our results indicate that, whereas T cells use ICAM-1 and -2 for temporary pulmonary entrapment, neutrophils get sequestered and extravasate into inflamed lungs independent of ICAMs. This is the first demonstration that the pulmonary vasculature is differentially recognized by T cells and neutrophils.-Petrovich, E., Feigelson, S. W., Stoler-Barak, L., Hatzav, M., Solomon, A., Bar-Shai, A., Ilan, N., Li, J.-P., Engelhardt, B., Vlodavsky, I., Alon, R. Lung ICAM-1 and ICAM-2 support spontaneous intravascular effector lymphocyte entrapment but are not required for neutrophil entrapment or emigration inside endotoxin-inflamed lungs.

Gnb isoforms control a signaling pathway comprising Rac1, Plcβ2, and Plcβ3 leading to LFA-1 activation and neutrophil arrest in vivo

AbstractChemokines are required for leukocyte recruitment and appropriate host defense and act through G protein-coupled receptors (GPCRs), which induce downstream signaling leading to integrin activation. Although the α and β subunits of the GPCRs are the first intracellular molecules that transduce signals after ligand binding and are therefore indispensable for downstream signaling, relatively little is known about their contribution to lymphocyte function-associated antigen 1 (LFA-1) activation and leukocyte recruitment. We used knockout mice and short hairpin RNA to knock down guanine nucleotide binding protein (GNB) isoforms (GNB1, GNB2, GNB4, and GNB5) in HL60 cells and primary murine hematopoietic cells. Neutrophil function was assessed by using intravital microscopy, flow chamber assays, and chemotaxis and biochemistry studies. We unexpectedly discovered that all expressed GNB isoforms are required for LFA-1 activation. Their downregulation led to a significant impairment of LFA-1 activation, which was demonstrated in vitro and in vivo. Furthermore, we showed that GPCR activation leads to Ras-related C3 botulinum toxin substrate 1 (Rac1)-dependent activation of both phospholipase C β2 (Plcβ2) and Plcβ3. They act nonredundantly to produce inositol triphosphate–mediated intracellular Ca2+ flux and LFA-1 activation that support chemokine-induced arrest in vivo. In a complex inflammatory disease model, Plcβ2-, Plcβ3-, or Rac1-deficient mice were protected from lipopolysaccharide-induced lung injury. Taken together, we demonstrated that all Gnb isoforms are required for chemokine-induced downstream signaling, and Rac1, Plcβ2, and Plcβ3 are critically involved in integrin activation and leukocyte arrest.

Substrate stiffness of endothelial cells directs LFA-1/ICAM-1 interaction: A physical trigger of immune-related diseases?

In blood vessels, substrate stiffness of the endothelium varies between different body locations and increases during the progression of multiple sclerosis. As a crucial step of the immune response, lymphocyte function associated antigen-1 (LFA-1)/ intercellular adhesion molecule-1 (ICAM-1) interaction occurs in various tissues and plays a pivotal role in atherosclerosis. However, the contribution of the physical property of endothelium substrate, such as the stiffness, to LFA-1/ICAM-1 interaction and immune-related diseases progression remains largely unknown. In this study, we investigated the influence of substrate stiffness on the adhesion force of LFA-1/ICAM-1 bond and ICAM-1 expression on the endothelial cell apical surface with an improved in vitro model. A silica microsphere-functionalized atomic force microscopy (AFM) tip was linked to LFA-1 via a polyethylene glycol (PEG) chain, and then approached toward human aortic endothelial cells (HAECs) on polyacrylamide gels of different stiffnesses. The results showed that the adhesion force was elevated on stiff substrates, while the expression of ICAM-1 on the HAECs surface was not influenced by substrate stiffness. A low-dose blebbistatin treatment (5μmol/L) reduced the adhesion force on both substrates while a high dose blebbistatin treatment (50μmol/L)) eliminated the adhesion between LFA-1 and ICAM-1, indicating that endothelium substrate stiffness directs the LFA-1/ICAM-1 interaction in a myosin II-dependent manner. These results help to describe the relationship between substrate stiffness and myosin II-dependent LFA-1/ICAM-1 interaction, and may increase the understanding of the pathogenesis and treatment of immune-related diseases.

Lifitegrast: First LFA-1/ICAM-1 antagonist for treatment of dry eye disease.

Dry eye disease is an extremely common condition affecting millions worldwide. The underlying pathophysiological mechanism is thought to be localized inflammation of the ocular surface resulting in the localization of T cells at this surface followed by their activation and subsequent liberation of cytokines. This effect on T cells results from the binding of lymphocyte function-associated antigen-1 (LFA-1) located on T cells to intercellular adhesion molecule 1 (ICAM-1) expressed on inflamed epithelium and endothelium, and on T cells. Lifitegrast is a T-cell integrin antagonist designed to mimic ICAM-1, thus blocking the interaction of LFA-1 and ICAM-1. Lifitegrast enters the systemic circulation to a limited extent thus reducing the likelihood of unwanted systemic reactions. Clinical trials in over 2,500 subjects with dry eye disease have shown that 5.0% lifitegrast given by ocular instillation causes a significant reduction in objective and subjective signs and symptoms of the disease. These beneficial effects are associated with a relatively low incidence of unwanted effects, almost all local in nature. In light of these findings, lifitegrast was approved by the Food and Drug Administration (FDA) in 2016 for the treatment of dry eye disease, the first drug with this mechanism of action to be so approved.

Rap1-GTP Augments TGF-b-Mediated Signaling in T Lymphocytes Via a Mechanism Dependent on the b Chain of LFA-1 Integrin

Integrin-mediated adhesion of lymphocytes to antigen presenting cells (APCs) is a critical event linking innate and adaptive immunity. Integrins function as bidirectional receptors and can transmit signals from both sides of the plasma membrane, a property referred to as inside-out and outside-in signaling. Lymphocyte adhesion to APC is mainly accomplished through the principle adhesion molecule on the lymphocyte surface, the lymphocyte functional antigen 1 (LFA-1), which binds to intercellular adhesion molecule 1 (ICAM-1) on the surface of APCs. In order to mediate its function, LFA-1 must be activated via a process, which results in conformation changes of the receptor that extends the ectodomains of the α and β chains leading to a high affinity state. Among the few signaling molecules that have been implicated in integrin activation in hematopoietic cells are the small GTPase Rap1A (thereafter named Rap1) and its downstream effectors RapL and RIAM. In response to Rap1-GTP, RapL regulates LFA-1 activation by interacting with the integrin α chain, whereas RIAM mediates recruitment of talin to the cytoplasmic tail of the β chain leading to its conformational change to the high affinity state. To understand the role of Rap1 in T cell responses we generated transgenic (Tg) mice that selectively express the active, GTP-bound Rap1 mutant Rap1E63 in mature T cells. Rap1E63-Tg and littermate control mice had no statistically significant difference in absolute thymocyte numbers and differentiation profiles. In contrast, in peripheral lymphoid organs, Rap1E63-Tg mice had a significant reduction in total T cells but a 4-fold increase in the CD4+ CD103+ T cell fraction. CD103 (also known as αEβ7 integrin) defines a subset of peripherally generated Treg with potent suppressive function. TGF-β is the strongest stimulus for induction of CD103 (αEβ7). To examine whether Rap1-GTP can affect TGF-β-mediated signaling in T cells, we used stable Jurkat T cell lines expressing GTP-bound Rap1 mutant, Rap1E63, or Jurkat T cell lines in which the endogenous Rap1 was depleted by shRNA (Rap1-KD), and also primary T cells from Rap1E63-Tg mice and Rap1-KO mice. After interaction with two membrane-bound receptors, TGF-βRI and II, TGF-β propagates downstream signaling via the Smad family transcription factors. Incubation of Rap1E63 Jurkat T cells with TGF-β resulted in enhanced and sustained phosphorylation of Smad2 and Smad3, which was observed with very low concentrations of TGF-β that were incapable of inducing detectable phosphorylation of Smads in control Jurkat T cells. In contrast, diminished level and duration of Smad2 and Smad3 phosphorylation was observed in Rap1-KD Jurkat T cells. Similar patterns of responses to those observed in Rap1E63 Jurkat T cells and in Rap1-KD Jurkat T cells were observed in primary mouse T cells isolated from Rap1E63-Tg mice and Rap1-KO mice, respectively. To investigate whether the LFA-1 integrin α and/or β chain had an active role in the enhanced TGF-β-mediated signaling in the presence of Rap1-GTP, we used Rap1E63 Jurkat T cells in which RapL or RIAM were depleted by shRNA (Rap1E63/RapL-KD and Rap1E63/RIAM-KD) because these adaptors selectively regulate the LFA-1 α and the LFA-1 β chain, respectively, downstream of Rap1-GTP. Although in Rap1E63/RapL-KD cells the enhanced TGF-β-induced Smad3 phosphorylation remained unaffected, in Rap1E63/RIAM-KD cells the enhanced TGF-β-induced Smad3 phosphorylation was abrogated. To investigate the biological relevance of these observations, we used T cells from Rap1E63-Tg mice crossed with mice deficient for the LFA-1 α chain. TGF-β resulted in enhanced Smad3 phosphorylation in T cells from Rap1E63-Tg/LFA1-a KO mice similarly to T cells from Rap1E63-Tg mice, indicating that this effect was not dependent on the activation of LFA-1 α chain. In contrast, T cells from RIAMflox/flox -Lck-Cre mice, in which activation of the LFA-1 β chain is impaired, displayed abrogated activation of Smad3 in response to TGF-β. Our data reveal a novel mechanism by which Rap1 regulates T cell responses via outside-in integrin signaling and may have important implications on TGF-β-mediated T cell homeostasis, differentiation and immune quiescence. DisclosuresNo relevant conflicts of interest to declare.

Mechanisms of LtxA (Leukotoxin), a Potent New Anti-Inflammatory Agent for the Treatment of Alopecia Areata.

Alopecia areata is an autoimmune condition where activated, pro-inflammatory white blood cells (WBCs) attack the hair follicles, resulting in hair loss. Migration of these activated WBCs from the blood stream and into the follicle tissue requires interaction between the integrin, lymphocyte function-associated antigen-1 (LFA-1) on WBCs, and ICAM-1 on vascular endothelial cells. High levels of active LFA-1 are uniquely expressed on WBCs that are involved in autoimmune and inflammatory conditions. The natural biologic agent LtxA (Leukothera) preferentially targets and depletes disease activated and malignant WBCs by binding to active LFA-1. The experimental drug has demonstrated significant therapeutic efficacy against autoimmune/inflammatory conditions such as psoriasis and allergic asthma in mouse models for these diseases. In addition, when injected into rodents, rhesus macaques, and dogs, LtxA was demonstrated to be physiologically active, biologically specific, and extremely well-tolerated. LFA-1 is an attractive target for therapy because it is only normally present on WBCs and has been shown to be activated and overexpressed on WBCs that are responsible for autoimmune/inflammatory conditions.

Celecoxib increases lung cancer cell lysis by lymphokine-activated killer cells via upregulation of ICAM-1.

The antitumorigenic mechanism of the selective cyclooxygenase-2 (COX-2) inhibitor celecoxib is still a matter of debate. Using lung cancer cell lines (A549, H460) and metastatic cells derived from a lung cancer patient, the present study investigates the impact of celecoxib on the expression of intercellular adhesion molecule 1 (ICAM-1) and cancer cell lysis by lymphokine-activated killer (LAK) cells. Celecoxib, but not other structurally related selective COX-2 inhibitors (i.e., etoricoxib, rofecoxib, valdecoxib), was found to cause a substantial upregulation of ICAM-1 protein levels. Likewise, ICAM-1 mRNA expression was increased by celecoxib. Celecoxib enhanced the susceptibility of cancer cells to be lysed by LAK cells with the respective effect being reversed by a neutralizing ICAM-1 antibody. In addition, enhanced killing of celecoxib-treated cancer cells was reversed by preincubation of LAK cells with an antibody to lymphocyte function associated antigen 1 (LFA-1), suggesting intercellular ICAM-1/LFA-1 crosslink as crucial event within this process. Finally, celecoxib elicited no significant increase of LAK cell-mediated lysis of non-tumor bronchial epithelial cells, BEAS-2B, associated with a far less ICAM-1 induction as compared to cancer cells. Altogether, our data demonstrate celecoxib-induced upregulation of ICAM-1 on lung cancer cells to be responsible for intercellular ICAM-1/LFA-1 crosslink that confers increased cancer cell lysis by LAK cells. These findings provide proof for a novel antitumorigenic mechanism of celecoxib.

Detection of Diffusion Heterogeneity in Single Particle Tracking Trajectories Using a Hidden Markov Model with Measurement Noise Propagation.

We develop a Bayesian analysis framework to detect heterogeneity in the diffusive behaviour of single particle trajectories on cells, implementing model selection to classify trajectories as either consistent with Brownian motion or with a two-state (diffusion coefficient) switching model. The incorporation of localisation accuracy is essential, as otherwise false detection of switching within a trajectory was observed and diffusion coefficient estimates were inflated. Since our analysis is on a single trajectory basis, we are able to examine heterogeneity between trajectories in a quantitative manner. Applying our method to the lymphocyte function-associated antigen 1 (LFA-1) receptor tagged with latex beads (4 s trajectories at 1000 frames s−1), both intra- and inter-trajectory heterogeneity were detected; 12–26% of trajectories display clear switching between diffusive states dependent on condition, whilst the inter-trajectory variability is highly structured with the diffusion coefficients being related by D 1 = 0.68D 0 − 1.5 × 104 nm2 s−1, suggestive that on these time scales we are detecting switching due to a single process. Further, the inter-trajectory variability of the diffusion coefficient estimates (1.6 × 102 − 2.6 × 105 nm2 s−1) is very much larger than the measurement uncertainty within trajectories, suggesting that LFA-1 aggregation and cytoskeletal interactions are significantly affecting mobility, whilst the timescales of these processes are distinctly different giving rise to inter- and intra-trajectory variability. There is also an ‘immobile’ state (defined as D < 3.0 × 103 nm2 s−1) that is rarely involved in switching, immobility occurring with the highest frequency (47%) under T cell activation (phorbol-12-myristate-13-acetate (PMA) treatment) with enhanced cytoskeletal attachment (calpain inhibition). Such ‘immobile’ states frequently display slow linear drift, potentially reflecting binding to a dynamic actin cortex. Our methods allow significantly more information to be extracted from individual trajectories (ultimately limited by time resolution and time-series length), and allow statistical comparisons between trajectories thereby quantifying inter-trajectory heterogeneity. Such methods will be highly informative for the construction and fitting of molecule mobility models within membranes incorporating aggregation, binding to the cytoskeleton, or traversing membrane microdomains.

Aggregatibacter actinomycetemcomitans leukotoxin induces cytosol acidification in LFA-1 expressing immune cells.

Studies have suggested that Aggregatibacter actinomycetemcomitans leukotoxin (LtxA) kills human lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18)-bearing immune cells through a lysosomal-mediated mechanism. Lysosomes are membrane-bound cellular organelles that contain an array of acid hydrolases that are capable of breaking down biomolecules. The lysosomal membrane bilayer confines the pH-sensitive enzymes within an optimal acidic (pH 4.8) environment thereby protecting the slightly basic cytosol (pH 6.8-7.5). In the current study, we have probed the effect of LtxA-induced cytolysis on lysosomal integrity in two different K562 erythroleukemia cell lines. K562-puro/LFA-1 cells were stably transfected with CD11a and CD18 cDNA to express LFA-1 on the cell surface while K562-puro, which does not express LFA-1, served as a control. Following treatment with 100 ng ml(-1) LtxA cells were analyzed by live cell imaging in conjunction with time-lapse confocal microscopy and by flow cytometry. Using a pH-sensitive indicator (pHrodo(®)) we demonstrated that the toxin causes a decrease in the intracellular pH in K562-puro/LFA-1 cells that is noticeable within the first 15 min of treatment. This process correlated with the disappearance of lysosomes in the cytosol as determined by both acridine orange and LysoTracker(®) Red DND-99 staining. These changes were not observed in K562-puro cells or when heat inactivated toxin was added to K562-puro/LFA-1. Our results suggest that LtxA induces lysosomal damage, cytosol acidification, which is followed by cell death in K562-puro/LFA-1 cells.