Direct Tumor Irradiation Potentiates Adoptive NK Cell Targeting Against Parental and Stemlike Cancer in Human Liver Cancer Models.

PURPOSE: Radiation therapy (RT) has been shown to effectively induce the expression of intercellular adhesion molecule-1 (ICAM-1), which is recognized by lymphocyte function-associated antigen 1 (LFA-1) expressed on natural killer (NK) cells. However, the potential synergistic antitumor immune response of tumor irradiation and administered NK cells has not been explored in intractable human liver cancers. Furthermore, NK cell targeting against both parental and cancer stemness has never been investigated. METHODS AND MATERIALS: Highly activated ex vivo NK cells were administered into the human liver tumor-bearing mice. Tumor direct RT was optimized according to tumor bearing site. HepG2 and Hep3B ICAM-1 knockout cells were generated using CRISPR/CAS9. Stemness tumor spheres were generated. NK cell cytolysis against parental and tumor sphere was evaluated using flow cytometry and real-time cytotoxicity assay. RESULTS: A combination of adoptive NK cell therapy with RT significantly improved therapeutic efficacy over monotherapies against subcutaneous, orthotopic, and metastatic human liver tumor models. Direct tumor irradiation potentiated NK cell recognition and conjugation against liver cancer through the LFA-1/ICAM-1 axis. Suppression of immune synapse formation on NK cells using high-affinity LFA-1 inhibitors or ICAM-1 knockout liver cancer induced "outside-in" signal blocking in NK cells, resulting in failure to eliminate liver tumor despite the combination therapy. NK cells effectively recognized and targeted triple-high epithelial cell adhesion molecule+CD133+CD24+ liver cancer expressing upregulated ICAM-1 in the irradiated tumor microenvironment, which led to prevention of the initiation of metastasis, improving survival in a metastatic model. In addition, the LFA-1/ICAM-1 axis interruption between NK cells and stemness liver tumor spheres significantly diminished NK cell cytolysis. Consistent with our preclinical data, the LFA-1/ICAM-1 axis correlated with survival outcomes in patients with metastatic cancer from the The Cancer Genome Atlas databases. CONCLUSIONS: NK cells in combination with tumor irradiation can provide synergistic therapeutic effects for NK cell recognition and elimination against both parental and stemlike liver cancer through LFA-1/ICAM-1.

GSK3ß Interacts With CRMP2 and Notch1 and Controls T-Cell Motility.

The trafficking of T-cells through peripheral tissues and into afferent lymphatic vessels is essential for immune surveillance and an adaptive immune response. Glycogen synthase kinase 3ß (GSK3ß) is a serine/threonine kinase and regulates numerous cell/tissue-specific functions, including cell survival, metabolism, and differentiation. Here, we report a crucial involvement of GSK3ß in T-cell motility. Inhibition of GSK3ß by CHIR-99021 or siRNA-mediated knockdown augmented the migratory behavior of human T-lymphocytes stimulated via an engagement of the T-cell integrin LFA-1 with its ligand ICAM-1. Proteomics and protein network analysis revealed ongoing interactions among GSK3ß, the surface receptor Notch1 and the cytoskeletal regulator CRMP2. LFA-1 stimulation in T-cells reduced Notch1-dependent GSK3ß activity by inducing phosphorylation at Ser9 and its nuclear translocation accompanied by the cleaved Notch1 intracellular domain and decreased GSK3ß-CRMP2 association. LFA-1-induced or pharmacologic inhibition of GSK3ß in T-cells diminished CRMP2 phosphorylation at Thr514. Although substantial amounts of CRMP2 were localized to the microtubule-organizing center in resting T-cells, this colocalization of CRMP2 was lost following LFA-1 stimulation. Moreover, the migratory advantage conferred by GSK3ß inhibition in T-cells by CHIR-99021 was lost when CRMP2 expression was knocked-down by siRNA-induced gene silencing. We therefore conclude that GSK3ß controls T-cell motility through interactions with CRMP2 and Notch1, which has important implications in adaptive immunity, T-cell mediated diseases and LFA-1-targeted therapies.

Receptor-Based Peptides for Inhibition of Leukotoxin Activity.

The Gram-negative bacterium Aggregatibacter actinomycetemcomitans, commonly associated with localized aggressive periodontitis (LAP), secretes an RTX (repeats-in-toxin) protein leukotoxin (LtxA) that targets human white blood cells, an interaction that is driven by its recognition of the lymphocyte function-associated antigen-1 (LFA-1) integrin. In this study, we report on the inhibition of LtxA-LFA-1 binding as an antivirulence strategy to inhibit LtxA-mediated cytotoxicity. Specifically, we designed and synthesized peptides corresponding to the reported LtxA binding domain on LFA-1 and characterized their capability to inhibit LtxA binding to LFA-1 and subsequent cytotoxic activity in human immune cells. We found that several of these peptides, corresponding to sequential ß-strands in the LtxA-binding domain of LFA-1, inhibit LtxA activity, demonstrating the effectiveness of this approach. Further investigations into the mechanism by which these peptides inhibit LtxA binding to LFA-1 reveal a correlation between toxin-peptide affinity and LtxA-mediated cytotoxicity, leading to a diminished association between LtxA and LFA-1 on the cell membrane. Our results demonstrate the possibility of using target-based peptides to inhibit LtxA activity, and we expect that a similar approach could be used to hinder the activity of other RTX toxins.

Mathematical Modeling of Synaptic Patterns.

During antigen recognition by T cells, a specific spatial structure is formed at the contact face to an antigen-presenting cell (APC), called an immunological synapse (IS). The IS supports bidirectional signaling and release of effector molecules and is widely studied both biologically and numerically, in order to understand the process of T cell activation and signaling. This specialized structure harbors a central area (central supramolecular activation cluster, cSMAC) populated by T cell receptor-peptide-major histocompatibility complex (TCR-pMHC ) interactions, hedged by a peripheral ring (peripheral supramolecular activation cluster, pSMAC) of integrin lymphocyte function associated-1 interactions with its immunoglobulin superfamily ligand intercellular adhesion molecule-1 (LFA-1-ICAM-1). These two regions form the "bull's eye" pattern characteristic of the mature IS.In theoretical studies, different modeling architectures, including partial differential equations (PDE) and agent-based models , have been developed with the purpose to answer mechanistic questions about the IS dynamics. In this chapter, we explain possible physiological mechanisms that lead to the formation of ISs and technical issues that may occur in the course of development of agent-based models.

LFA-1-dependent HuR nuclear export and cytokine mRNA stabilization in T cell activation.

Lymphokine gene expression is a precisely regulated process in T cell-mediated immune responses. In this study we demonstrate that engagement of the beta(2) integrin LFA-1 in human peripheral T cells markedly extends the half-life of TNF-alpha, GM-CSF, and IL-3 mRNA, as well as a chimeric beta-globin mRNA reporter construct containing a strongly destabilizing class II AU-rich element from the GM-CSF mRNA 3'-untranslated region. This integrin-enhanced mRNA stability leads to augmented protein production, as determined by TNF-alpha ELISPOT assays. Furthermore, T cell stimulation by LFA-1 promotes rapid nuclear-to-cytoplasmic translocation of the mRNA-stabilizing protein HuR, which in turn is capable of binding an AU-rich element sequence in vitro. Abrogation of HuR function by use of inhibitory peptides, or marked reduction of HuR levels by RNA interference, prevents LFA-1 engagement-mediated stabilization of T cell TNF-alpha or IFN-gamma transcripts, respectively. Thus, HuR-mediated mRNA stabilization, stimulated by integrin engagement and controlled at the level of HuR nuclear export, is critically involved in T cell activation.

The hepatitis C envelope 2 protein inhibits LFA-1-transduced protein kinase C signaling for T-lymphocyte migration.

BACKGROUND & AIMS: The ability of viruses to escape the host immune response represents a globally important problem related to a wide variety of pathogens. Hepatitis C is one of the major causes of liver disease worldwide. Clearance rates of this virus are low, and this condition normally involves a chronic inflammatory process. This raises a possibility that the virus may have developed mechanisms enabling it to evade T-cell-mediated immune surveillance. The aim of this study was to investigate the effect of the hepatitis C envelope protein E2 on LFA-1-stimulated T-cell migration and macrophage inflammatory protein (MIP-1alpha, MIP-1beta) secretion. METHODS: T cells were stimulated through the leukocyte function-associated molecule-1 (LFA-1) receptor by incubating with either intracellular adhesion molecule 1 (ICAM-1)-Fc fusion protein or anti-LFA-1 immobilized on 8-well chamber slides. Subcellular localization of protein kinase C (PKC)-beta, CD81, and LFA-1 was determined by immunofluorescence analysis. Lipid raft formation was assessed using the Cellomics Kineticscan reader. MIP-1alpha and MIP-1beta levels were detected by enzyme-linked immunosorbent assay. RESULTS: We report that the hepatitis C envelope protein E2 can dramatically inhibit T-lymphocyte motility and chemokine release induced via LFA-1 integrin ligation. We have demonstrated a novel T-lymphocyte-directed viral inhibitory mechanism involving the PKC-beta enzyme as a definitive intracellular target. E2-CD81 interaction stimulates translocation of PKC-beta to lipid rafts, thereby preventing its association with the centrosome and microtubule cytoskeleton, which is crucial to the process of T-cell migration. CONCLUSIONS: These studies identify a mechanism whereby the hepatitis C virus can evade the host immune response by inhibition of T-cell migration.

A primate model of renal ischemia-reperfusion injury for preclinical evaluation of the antileukocyte function associated antigen 1 monoclonal antibody odulimonab.

PURPOSE: We established a primate model to investigate the effects of the antileukocyte function associated antigen 1 (CD 11a) mAb odulimomab (Imtix-Sangstad, Lyon, France) for preventing renal ischemia-reperfusion injury. MATERIALS AND METHODS: We randomly divided 34 Macaca cynomolgus monkeys into groups 1 and 2, which received a renal autograft after 2 hours of cold ischemia, and groups 3 and 4, which received the autograft after 24 hours of cold ischemia. Before cold ischemia all harvested kidneys were subjected to 30 to 45 minutes of warm ischemia. Groups 1 and 3 monkeys were treated with an antileukocyte function associated antigen 1 mAb before cold ischemia and then for 3 days, while groups 2 and 4 monkeys received an IgG1 isotype control. In all groups renal function was investigated before warm ischemia and 72 hours after reperfusion. Serum creatinine and the leukocyte count were determined daily. Histological studies were done and lactoferrin was measured in the autotransplanted kidney 72 hours after reperfusion. RESULTS: A decrease in renal function was shown after 2 hours of cold ischemia with tubular necrosis and mild cell infiltration, while after 24 hours of cold ischemia there was severe renal failure with tubular and glomerular necrosis, and leukocyte infiltration. A significant improvement in renal function and decrease in kidney lactoferrin content was evident in group 1 compared to group 2 at 72 hours, while no significant difference was noted in groups 3 and 4. No difference in histological patterns was evident in treated and untreated animals. CONCLUSIONS: This study provides evidence for the validity of this ischemia-reperfusion injury model in primates. The protective effects of antileukocyte function associated antigen 1 mAb on renal injury was not as dramatic as in rodent models but a significant improvement in renal function was observed in treated animals after 2 hours of cold ischemia.

Effects of IL-8, Gro-alpha, and LTB(4) on the adhesive kinetics of LFA-1 and Mac-1 on human neutrophils.

Firm adhesion of rolling neutrophils on inflamed endothelium is dependent on beta(2) (CD18)-integrins and activating stimuli. LFA-1 (CD11a/CD18) appears to be more important than Mac-1 (CD11b/CD18) in neutrophil emigration at inflammatory sites, but little is known of the relative binding characteristics of these two integrins under conditions thought to regulate firm adhesion. The present study examined the effect of chemoattractants on the kinetics of LFA-1 and Mac-1 adhesion in human neutrophils. We found that subnanomolar concentrations of interleukin-8, Gro-alpha, and leukotriene B(4) (LTB(4)) induced rapid and optimal rates of LFA-1-dependent adhesion of neutrophils to intercellular adhesion molecule (ICAM)-1-coated beads. These optimal rates of LFA-1 adhesion were transient and decayed within 1 min after chemoattractant stimulation. Mac-1 adhesion was equally rapid initially but continued to rise for >/=6 min after stimulation. A fourfold higher density of ICAM-1 on beads markedly increased the rate of binding to LFA-1 but did not change the early and narrow time window for the optimal rate of adhesion. Using well-characterized monoclonal antibodies, we showed that activation of LFA-1 and Mac-1 by Gro-alpha was completely blocked by anti-CXC chemokine receptor R2, but activation of these integrins by interleukin-8 was most effectively blocked by anti-CXC chemokine receptor R1. The topographical distribution of beads also reflected significant differences between LFA-1 and Mac-1. Beads bound to Mac-1 translocated to the cell uropod within 4 min, but beads bound to LFA-1 remained bound to the lamellipodial regions at the same time. These kinetic and topographical differences may indicate distinct functional contributions of LFA-1 and Mac-1 on neutrophils.

Rat and rabbit plasma distribution of free and chylomicron-associated BIRT 377, a novel small molecule antagonist of LFA-1-mediated cell adhesion.

PURPOSE: The objectives of this study are to determine the plasma distribution of free and chylomicron-associated BIRT 377 within rats and rabbits. METHODS: For the rat studies free and chylomicron-associated BIRT 377 was incubated in plasma from CD 1 non-fasted rats for 60 minutes at 37 degrees C. Following incubation the plasma was separated into its lipoprotein and lipoprotein-deficient plasma (LPDP) fractions by three different methods and analyzed for BIRT 377 content by HPLC. For the rabbit studies New Zealand fasted white rabbits (3 kg; n=4) were administered an intravenous dose of free BIRT 377 (1 mg/kg). Following administration, serial blood samples were obtained and the plasma was analyzed for BIRT 377. The plasma conected at the 0.083-h time point was separated into each of its lipoprotein fractions and analyzed for BIRT 377. RESULTS: 37.8 +/- 1.2% of the original drug amount incubated in rat plasma was recovered within the lipoprotein-rich fraction. 41.5 +/- 0.4% of the original chylomicron-associated drug concentration incubated was recovered within the lipoprotein-rich fraction. The percentage of drug recovered within the TRL fraction was significantly greater following the incubation of chylomicron-associated BIRT 377 compared to free BIRT 377. In addition, BIRT 377 apparently follows a two-compartment pharmacokinetic model following single intravenous dose administration to rabbits. CONCLUSIONS: These findings suggest that plasma lipoprotein binding of BIRT 377 is evident and may be a factor in evaluating the pharmacological fate of this drug when administered to patients that exhibit changes in their plasma lipoprotein lipid.

Inhibition of the adherence of T-lymphocytes to epithelial cells by a cyclic peptide derived from inserted domain of lymphocyte function-associated antigen-1.

Tissue inflammation is characterized by aggravated leukocyte infiltration into the sites of inflammation. The mechanism requires the interactions of leukocyte adhesion-molecules and their ligands in the inflamed tissues. In this study, we demonstrate that a cyclic peptide cLAB.L [cyclol, 12-PenlTDGEATDSGC], derived from the "inserted" or I-domain of LFA-1 is able to inhibit the adherence of T-lymphocytes to the epithelial cell monolayers. This inhibition has been thought to involve the disruption of LFA-1/ICAM-1 interaction. The heterotypic adhesion of phorbol ester-activated Molt-3 cells and IFN-gamma-induced Caco-2 monolayers was inhibited upon treatment of the monolayers with monoclonal antibodies (MAbs) to adhesion molecules or with cLAB.L peptide. The adhesion can be inhibited by MAbs to ICAM-1, ICAM-2, and VCAM-1, and cLAB.L peptide in a concentration-dependent manner. However, none of the individual uses of these molecules led to a total inhibition. The inhibitory activity of cLAB.L is greatly reduced by low temperature and the absence of cell activation. Treatment of cLAB.L peptide may trigger an early event of apoptosis on activated but not on non-activated Molt-3 cells; no indication of peptide-induced apoptosis was found on Caco-2 cells. Taken together, data from this work suggest that cLAB.L may have applications to direct cell-targeted delivery during tissue inflammation.

Statins selectively inhibit leukocyte function antigen-1 by binding to a novel regulatory integrin site.

The beta2 integrin leukocyte function antigen-1 (LFA-1) has an important role in the pathophysiology of inflammatory and autoimmune diseases. Here we report that statin compounds commonly used for the treatment of hypercholesterolemia selectively blocked LFA-1-mediated adhesion and costimulation of lymphocytes. This effect was unrelated to the statins' inhibition of 3-hydroxy-3-methylglutaryl coenzyme-A reductase; instead it occurred via binding to a novel allosteric site within LFA-1. Subsequent optimization of the statins for LFA-1 binding resulted in potent, selective and orally active LFA-1 inhibitors that suppress the inflammatory response in a murine model of peritonitis. Targeting of the statin-binding site of LFA-1 could be used to treat diseases such as psoriasis, rheumatoid arthritis, ischemia/reperfusion injury and transplant rejection.

Chemokines stimulate human T lymphocyte transendothelial migration to utilize VLA-4 in addition to LFA-1.

Lymphocyte infiltration in inflammation is induced by the dual actions of chemokines and cell adhesion molecules. The role of LFA-1 and VLA-4 in chemokine-induced T cell transendothelial migration (TEM) across cytokine-activated endothelium has not been examined. LFA-1, but not VLA-4, mediated blood T cell TEM to RANTES, macrophage inflammatory protein-1alpha (MIP-1alpha), and stromal cell-derived factor-1 (SDF-1), and across tumor necrosis factor alpha (TNF-alpha) or interferon-gamma (IFN-gamma) -stimulated endothelial cells (EC). Chemokine stimulation in combination with TNF-alpha activation of EC induced TEM, which was partially mediated by VLA-4. SDF-1 increased a beta1-integrin activation epitope on T cells and enhanced VLA-4-mediated adhesion. Thus, LFA-1 mediates TEM under most conditions, but VLA-4 can also mediate TEM, although, in contrast to LFA-1, this requires exogenous chemokines and EC activation. In addition, an LFA-1- and VLA-4-independent pathway of lymphocyte TEM can also be induced by SDF-1.

LFA-1 expression on target cells promotes human immunodeficiency virus type 1 infection and transmission.

While CD4 and the chemokine receptors are the principal receptors for human immunodeficiency virus (HIV), other cellular proteins, such as LFA-1, are also involved in HIV infection. LFA-1 and its ligands, ICAM-1, ICAM-2, and ICAM-3, can be expressed on the cells infected by HIV, as well as on the HIV virions themselves. To examine the role of LFA-1 expressed on target cells in HIV infection, Jurkat-derived Jbeta2.7 T-cell lines that express either wild-type LFA-1, a constitutively active mutant LFA-1, or no LFA-1 were used. The presence of wild-type LFA-1 enhanced the initial processes of HIV infection, as well as the subsequent replication and transmission from cell to cell. In contrast, the constitutively active LFA-1 mutant failed to promote virus replication and spread, even though this mutant could help HIV enter cells and establish the initial infection. This study clearly demonstrates the contribution of LFA-1 in the different stages of HIV infection. Moreover, not only is LFA-1 expression important for initial HIV-cell interaction, subsequent replication, and transmission, but its activity must also be properly regulated.

Enhanced T-lineage acute lymphoblastic leukaemia cell survival on bone marrow stroma requires involvement of LFA-1 and ICAM-1.

The bone marrow (BM) microenvironment supports leukaemia cell survival and proliferation. The roles played by adhesive receptor interactions in the survival of T-lineage acute lymphoblastic leukaemia (T-ALL) cells on BM stromal cells are not well understood. Recently, we have developed an assay that partially recapitulates the BM microenvironment using HS-5 BM stromal cells. In this assay, the magnitude of ex vivo T-ALL lymphoblast survival predicts patient outcome. We examined the molecular basis for cell-cell adhesive events leading to T-ALL lymphoblast survival on HS-5 and on donor-derived BM stroma. Lympho cyte function-associated antigen-1 (LFA-1) on T-ALL cell lines bound intercellular adhesion molecule-1 (ICAM-1) on HS-5 monolayers, and survival was inhibited 85-98% with monoclonal antibodies directed against LFA-1 or ICAM-1. We compared these results with patient-derived T-ALL lymphoblasts co-cultured on either HS-5 BM or normal BM monolayers and found that LFA-1 and ICAM-1 were required, but not alone sufficient for ex vivo leukaemic cell survival. On normal BM stroma, but not HS-5 monolayers, two additional adhesion molecules, vascular cell adhesion molecule-1 (VCAM-1) and E-selectin, were highly expressed and contributed to T-ALL cell survival. This is the first report to demonstrate the importance of LFA-1/ICAM-1-mediated adhesion as a critical event in a cascade of cell surface receptor-ligand interactions that regulate T-ALL survival in the BM microenvironment.

Hydrogen peroxide induces LFA-1-dependent neutrophil adherence to cardiac myocytes.

Adult cardiac myocytes express intercellular adhesion molecule (ICAM)-1 in response to cytokine stimulation. This allows stable adhesion of chemotactically stimulated but not unstimulated neutrophils. In the current study, we demonstrated that brief exposure of ICAM-1-expressing cardiac myocytes to H(2)O(2) promoted transient adhesive interactions between myocytes and neutrophils without added chemotactic factors. This transient adhesion differed in two ways from the stable adhesion promoted by exogenous chemotactic factors. It occurred more rapidly, peaking within 15 min, and it was dependent on leukocyte function-associated antigen (LFA)-1 (CD11a/CD18) on the neutrophil interacting with ICAM-1 on the myocyte. In contrast, chemotactic factor-induced adhesion peaked at 60 min and was dependent on Mac-1 (CD11b/CD18). The transient adhesion could be completely inhibited by platelet-activating factor (PAF)-receptor antagonists WEB-2086 and SDZ-64-412. These results indicate that canine neutrophils may utilize both LFA-1 and Mac-1 to adhere to adult cardiac myocytes, with LFA-1 triggered by a PAF-like activity induced in myocytes by H(2)O(2).

Peptides derived from ICAM-1 and LFA-1 modulate T cell adhesion and immune function in a mixed lymphocyte culture.

BACKGROUND: The counter receptors intercellular adhesion molecule (ICAM)-1 and lymphocyte function-associated antigen (LFA)-1 are lymphocyte cell surface adhesion proteins the interaction of which can provide signals for T cell activation. This binding event is important in T cell function, migration, and general immune system regulation. The ability to inhibit this interaction with monoclonal antibodies has proved to be therapeutically useful for several allograft rejection and autoimmune disease models. METHODS: Short peptides representing counter-receptor contact domains of LFA-1 and ICAM-1 were examined for their ability to inhibit T cell adhesion and T cell function. RESULTS: Peptides encompassing amino acids Q1-C21 and D26-K50 of ICAM-1, I237-I261 and G441-G466 of the LFA-1 alpha-subunit, and D134-Q159 of the LFA-1 beta-subunit inhibited LFA-1/ICAM-1-dependent adhesion in a phorbol-12,13-dibutyrate-induced model of tonsil T cell homotypic adhesion. This inhibition was specific to the peptide sequence and occurred without stimulation of T cell proliferation. The peptides also were effective in preventing T cell function using a one-way mixed lymphocyte reaction model for bone marrow transplantation. CONCLUSIONS: Our data suggest that these peptides or their derivatives may be useful as therapeutic modulators of LFA-1/ICAM-1 interaction during organ transplants.

Ubiquitin-proteasome system is involved in induction of LFA-1/ICAM-1-dependent adhesion of HL-60 cells.

Membrane-permeable proteasome inhibitors, lactacystin (LC) and N-acetyl-Leu-Leu-norleucinal (ALLN), but not calpain inhibitor Z-Leu-leucinal (ZLL), prevented LFA-1/ICAM-1-dependent cellular adhesion of TPA-stimulated HL-60 cells. These proteasome inhibitors affected neither the induction of monocytic differentiation nor the accompanying protein-tyrosine phosphorylation. They suppressed the increase in the avidity of LFA-1 to ICAM-1 without changing the expression of these molecules. Immunoblotting using monoclonal antibody FK-1, which reacts specifically with polyubiquitinated proteins, demonstrated that the proteasome inhibitors caused the drastic accumulation of the polyubiquitinated proteins in the membrane fraction of TPA-treated HL-60 cells. This indicates that accompanying activation of LFA-1, TPA induces the polyubiquitination of the membrane proteins, which are rapidly degraded by proteasomes. These data taken together show that proteolysis mediated by the ubiquitin-proteasome system is a prerequisite for the induction of LFA-1-dependent adhesion of HL-60 cells.

A Ca2+ binding cyclic peptide derived from the alpha-subunit of LFA-1: inhibitor of ICAM-1/LFA-1-mediated T-cell adhesion.

The objective of this work is to study the conformation of cyclic peptide (1), cyclo (1, 12) Pen1-Gly2-Val3-Asp4-Val5-Asp6-Gln7-+ ++Asp8-Gly9-Glu10-Thr11-Cys12, in the presence and absence of calcium. Cyclic peptide 1 is derived from the divalent cation binding sequence of the alpha-subunit of LFA-1. This peptide has been shown to inhibit ICAM-1-LFA-1 mediated T-cell adhesion. In order to understand the structural requirements for this biologically active peptide, its solution structure was studied by nuclear magnetic resonance (NMR), circular dichroism (CD) and molecular dynamics simulations. This cyclic peptide exhibits two types of possible conformations in solution. Structure I is a loop-turn-loop type of structure, which is suitable to bind cations such as EF hand proteins. Structure II is a more extended structure with beta-hairpin bend at Asp4-Val5-Asp6-Gln7. There is evidence that alterations in the conformation of LFA-1 upon binding to divalent cations cause LFA-1 to bind to ICAM-1. To understand this mechanism, the cation-binding properties of the peptide were studied by CD and NMR. CD studies indicated that the peptide binds to calcium and forms a 1 : 1 (peptide: calcium) complex at low calcium concentrations and multiple types of complexes at higher cation concentrations. NMR studies indicated that the conformation of the peptide is not significantly altered upon binding to calcium. The peptide can inhibit T-cell adhesion by directly binding to ICAM-1 or by disrupting the interaction of the alpha and beta-subunits of LFA-1 protein. This study will help us to understand the mechanism(s) of action of this peptide and will improve our ability to design a better inhibitor of T-cell adhesion.

LFA-1- and ICAM-1-dependent homotypic aggregation of human thymocytes induced by JL1 engagement.

Cell-cell adhesion is essential for the appropriate immune response, differentiation, and migration of lymphocytes. This important physiological event is reflected in vitro by homotypic cell aggregation. We have previously reported that a 120 kDa cell surface glycoprotein, JL1, is a unique protein specifically expressed by immature double positive (DP) human thymocytes which are in the process of positive and negative selections through the interaction between thymocyte and antigen-presenting cells (APCs). The function of the JL1 molecule, however, is yet to be identified. We show here that anti-JL1 monoclonal antibody (mAb) induced the homotypic aggregation of human thymocytes in a temperature- and Mg2+-dependent manner. It required an intact cytoskeleton and the interaction between leucocyte function associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) since it was blocked by cytochalasin B and D, and mAb against LFA-1 and ICAM-1 which are known to be involved in the aggregation of thymocytes. Translocation of phosphatidylserine (PtdSer) through the cell membrane was not detected, implying that the molecular mechanism of JL-1-induced homotypic aggregation is different from that of CD99-induced homotypic aggregation. In summary, JL1 is a cell surface molecule that induces homotypic adhesion mediated by the LFA-1 and ICAM-1 interaction and cytoskeletal reorganization. These findings suggest that JL1 may be an important regulator of thymocyte development and thymocyte-APC interaction.

Acquisition of interleukin-5 secretion by human naive T-helper cells is regulated by distinct signals from both the T-cell receptor/CD3 complex and CD2.

We developed a human naive T-helper (Th) cell differentiation model with anti-CD3 monoclonal antibody (MoAb), using a B-cell line as source of costimulation. In this system, we examined the contribution of the T-cell receptor (TCR)/CD3-derived signals and that of lymphocyte function-associated antigen-1 (LFA-1) and CD2 in regulating Th-cell subset differentiation. We found that lowering the level of anti-CD3 MoAb decreased tumour necrosis factor-alpha (TNF-alpha) production, while increasing secretion of the Th2 cytokines, interleukin-5 (IL-5) and interleukin-13 (IL-13). Secretion of interferon-gamma (IFN-gamma) was not influenced by the strength of the anti-CD3 signal. Under conditions where Th0 cells are generated, co-culture with anti-CD2 F(ab')2 MoAb led to the generation of Th cells that secreted 30-35% less IL-5, while not affecting secretion of IFN-gamma or granulocyte-macrophage colony-stimulating factor (GM-CSF). By contrast, anti-CD18 MoAb F(ab')2 inhibited IFN-gamma and GM-CSF levels only in the primary stimulation, but did not affect cytokine levels after restimulation. Neither anti-CD2 nor anti-CD18 F(ab')2 MoAbs could alter cytokine secretion profiles of peripheral blood-derived memory/effector Th cells. Our results indicate that acquisition of IL-5 secretion capability by Th cells is regulated mainly by signals transduced by the TCR/CD3 complex and by the presence of interleukin-4 (IL-4), while the CD2/LFA-3 pathway plays an additional, but minor, role. These regulatory CD2-derived signals, however, are distinct from those generated by the TCR/CD3 complex.