Surface Ligand Density Research Articles

Controlling and Assessing the Surface Display of Cell-Binding Domains on Magnetite Conjugated Fluorescent Liposomes

Biological systems provide us with a diverse source of peptide-based ligands for cellular adhesion. Controlling and assessing the ligand surface density as well as tailoring the surface chemistry to have specific cellular adhesion properties are important in biomaterials design. In the following work, we provide a means for displaying peptide-based ligands on magnetic liposomes in which the surface density and chemistry may be controlled. Simultaneously, the conjugated vesicles provide a fluorescent signal for examining steric hindrance among surface ligands. In addition, the inherent magnetic and fluorescence features of this system revealed potential for magnet-based cell isolation and fluorescent labeling of adhered cells, respectively. Adhered cells were found to remain viable and proliferative, thereby allowing them to be used for subsequent evaluation. In a specific demonstration, we control the density of fibronectin-mimetic ligands on the polydiacetylene liposome surfaces. We find that steric limitation occurring at over 20% surface density result in decreased cell adhesion, in accord with related techniques. The magnetic-liposome system offers the means for not only separating cells adhered to the biomaterial, but also providing the ability to control and assess the biomaterial surface. This may prove particularly useful for examining combinations of peptide-based ligands or for evaluating the molecular-level ligand accessibility and its effect on cell attachment to a biomaterial surface.

Force Mapping during the Formation and Maturation of Cell Adhesion Sites with Multiple Optical Tweezers

Focal contacts act as mechanosensors allowing cells to respond to their biomechanical environment. Force transmission through newly formed contact sites is a highly dynamic process requiring a stable link between the intracellular cytoskeleton and the extracellular environment. To simultaneously investigate cellular traction forces in several individual maturing adhesion sites within the same cell, we established a custom-built multiple trap optical tweezers setup. Beads functionalized with fibronectin or RGD-peptides were placed onto the apical surface of a cell and trapped with a maximum force of 160 pN. Cells form adhesion contacts around the beads as demonstrated by vinculin accumulation and start to apply traction forces after 30 seconds. Force transmission was found to strongly depend on bead size, surface density of integrin ligands and bead location on the cell surface. Highest traction forces were measured for beads positioned on the leading edge. For mouse embryonic fibroblasts, traction forces acting on single beads are in the range of 80 pN after 5 minutes. If two beads were positioned parallel to the leading edge and with a center-to-center distance less than 10 µm, traction forces acting on single beads were reduced by 40%. This indicates a spatial and temporal coordination of force development in closely related adhesion sites. We also used our setup to compare traction forces, retrograde transport velocities, and migration velocities between two cell lines (mouse melanoma and fibroblasts) and primary chick fibroblasts. We find that maximal force development differs considerably between the three cell types with the primary cells being the strongest. In addition, we observe a linear relation between force and retrograde transport velocity: a high retrograde transport velocity is associated with strong cellular traction forces. In contrast, migration velocity is inversely related to traction forces and retrograde transport velocity.

Controlling Surface Ligand Density and Core Size of Alkanethiolate-Capped Pd Nanoparticles and Their Effects on Catalysis

This article presents systematic investigations on the relationship between the catalytic property and the surface ligand density/core size of thiolate ligand-capped Pd nanoparticles (PdNPs). The systematic variations in the two-phase synthesis of PdNPs generated from sodium S-dodecylthiosulfate were performed. The resulting PdNPs were characterized by transmission electron microscopy (TEM), thermogravimetric analysis (TGA), and (1)H NMR and UV-vis spectroscopy. The decrease in the molar equivalent of sodium S-dodecylthiosulfate (Bunte salts) resulted in the formation of nanoparticles with lower surface ligand density and larger particle core size. A decrease in the molar equivalent of tetra-n-octylammonium bromide or an increase in reaction temperature generated nanoparticles with higher surface ligand density and smaller particle core size. As the molar equivalent of NaBH(4) decreased, the particle core size increased. The catalysis studies on various PdNPs with different surface ligand density and average core size showed a strong correlation between the PdNP composition and the turnover frequency (TOF) of the isomerization of allyl alcohol. Optimized "good" PdNPs with lower surface ligand coverage and larger core size catalyzed the isomerization of various allyl alcohols to carbonyl analogues with high activity and selectivity.

Pd Nanoparticle-Catalyzed Isomerization vs Hydrogenation of Allyl Alcohol: Solvent-Dependent Regioselectivity

Our previous work has shown that alkanethiolate-capped Pd nanoparticles generated from sodium S-dodecylthiosulfate are excellent catalysts for selective isomerization of various allyl alcohols to the carbonyl analogues. The present work focuses on understanding the mechanism and the regioselectivity of Pd nanoparticles in different environments. First, the presence of H2 gas has turned out to be essential for the efficient catalytic isomerization reaction. This suggests that the mechanism likely involves the Pd-alkyl intermediate rather than the η3 π-allyl Pd hydride intermediate. Second, the Pd nanoparticles are found to convert allyl alcohol selectively to either propanal or 1-propanol depending on the type of solvent used for the catalytic reactions. The reaction pathway is most likely determined by steric hindrance, which is the result of the interaction between substrate and alkylthiolate ligands on Pd nanoparticles. Presumably, the conformation of alkylthiolate ligands changes upon the type of solvents, resulting in varying degree of available space close to the nanoparticle surface. In general, nonpolar or weakly polar solvents such as benzene and chloroform, respectively, promote the isomerization of allyl alcohol to propanal via the formation of the branched Pd-alkyl intermediate. On the other hand, polar protic solvents such as methanol and water foster the hydrogenation of allyl alcohol to 1-propanol involving the steric induced formation of a linear Pd-alkyl intermediate. Third, the use of sodium S-hexylthiosulfate instead of sodium S-dodecylthiosulfate for the synthesis of Pd nanoparticles results in nanoparticle catalysts with a lower regioselectivity toward isomerization over hydrogenation. This is due to the higher surface ligand density of hexanethiolate-capped Pd nanoparticles, which negatively impacts the formation of branched Pd-alkyl intermediate. The results clearly indicate that controlling the structure and surface density of alkanethiolate ligands around Pd nanoparticles can provide an opportunity to tune the activity and selectivity of nanoparticle catalysts. Lastly, the high stability of soluble nanocatalysts is demonstrated by recycling dodecanethiolate-capped Pd nanoparticles over 10 times for the isomerization reaction of allyl alcohol.

Fluorescence of Colloidal Gold Nanoparticles is Controlled by the Surface Adsorbate

AbstractFluorescent gold nanoparticles are important biological labels, in particular for combined optical and electron microscopy. It is reported that density and type of surface ligands have key influence on the dominant UV‐vis fluorescence band in positively and negatively charged gold nanoparticles capped with citrate, gold oxide, and cetyltrimethyl ammonium bromide (CTAB). The peak excitation and emission energies and fluorescence intensities vary with nanoparticle size, reflecting changes in surface charge and surface potential as well as a varying density of surface adsorbates. The fluorescence peak shifts, the evolution of zeta potentials, and fluorescence intensity trends are explained by a model of the principal fluorescence transitions that takes into account the nanoparticle surface conditions, such as the adhesion of ligands. Varying surface ligands is a simple strategy to optimize fluorescence intensity and to design spectral properties of gold nanoparticles.

Ligand density and clustering effects on endocytosis of folate modified nanoparticles

This study investigates the effects of surface ligand distribution pattern (ligand clustering and density) on the internalisation of nanoparticles by a bronchial epithelial in vitro model (Calu-3 cells cultured as polarised layers). Control of ligand clustering and its surface density was achieved through the use of ovalbumin as an intermediate species to anchor the ligand to the nanoparticle surface. The model particulate system consisted of polystyrene nanoparticles surface-decorated via the adsorption of ovalbumin with conjugated folate ligand. The density of the displayed ligand was manipulated by controlling the conjugation level of folate to ovalbumin, while ligand clustering was achieved by co-adsorption of varying mixtures of folate-ovalbumin conjugate (at different ligand density levels) and unconjugated ovalbumin. Increasing overall ligand density on the nanoparticle surface resulted in increased internalisation of modified nanoparticles by the cells, up to a saturation level. Surface ligand density also affected the cellular uptake pathway; from predominantly clathrin to predominantly caveolae-mediated as the ligand density is increased. We further demonstrate that surface clustering of the folate ligand enhances cellular internalisation of nanoparticles, relative to its dispersed surface distribution. Our work suggests a simple way to prepare a model system where surface manipulations of ligand density and its distribution are possible and which can be used to study nanoparticle–cell interaction processes.

Production of novel polymer monolithic columns, with stationary phase gradients, using cyclic olefin co-polymer (COC) optical filters

Polymer monolithic columns with controlled surface ligand density, providing stationary phase gradients within monolithic capillary columns, have been developed using photo-grafting through optical filters. Utilising commercially available cyclic olefin co-polymer (COC) films, the production of an optical filter capable of attenuating UV irradiation, in a tailored manner, was investigated. This novel optical filter was successfully applied to the surface modification of poly(BuMA-co-EDMA) monolithic columns in a multi-step grafting procedure. Fabricated columns were subjected to scanning capacitively coupled contactless conductivity (sC(4)D), to determine the distribution of the grafted functional groups, axially along the column. Further modification to produce a chelating stationary phase gradient of iminodiacetic acid (IDA) was demonstrated. To demonstrate the distribution of the IDA sites, a metal cation (Cu(2+)) was complexed to the IDA forming a chelate. Upon the formation of a complex of IDA with Cu(2+), an overall drop in conductive response was observed. The COC optical filter was also used in the fabrication of a grafted gradient of strong cation exchanger (SCX), sulphopropyl methacrylate (SPM) upon a polymer monolith, demonstrating the broader applicability of such a filter.

The role of poly(ethylene glycol) brush architecture in complement activation on targeted microbubble surfaces

Complement fixation to surface-conjugated ligands plays a critical role in determining the fate of targeted colloidal particles after intravenous injection. In the present study, we examined the immunogenicity of targeted microbubbles with various surface architectures and ligand surface densities using a flow cytometry technique. Targeted microbubbles were generated using a post-labeling technique with a physiological targeting ligand, cyclic arginine-glycine-asparagine (RGD), attached to the distal end of the poly(ethylene glycol) (PEG) moieties on the microbubble surface. Microbubbles were incubated in human serum, washed and then mixed with fluorescent antibodies specific for various serum components. We found that complement C3/C3b was the main human serum factor to bind in vitro to the microbubble surface, compared to IgG or albumin. We also investigated the effect of PEG brush architecture on C3/C3b fixation to the microbubble surface. RGD peptide was able to trigger a complement immune response, and complement C3/C3b fixation depended on microbubble size and RGD peptide surface density. When the targeting ligand was attached to shorter PEG chains that were shielded by a PEG overbrush layer (buried-ligand architecture), significantly less complement activation was observed when compared to the more traditional exposed-ligand motif. The extent of this protective role by the PEG chains depended on the overbrush length. Taken together, our results confirm that the buried-ligand architecture may significantly reduce ligand-mediated immunogenicity. More generally, this study illustrates the use of flow cytometry and microbubbles to analyze the surface interactions between complex biological media and surface-engineered biomaterials.

Using Microcontact Printing of Fibrinogen to Control Surface-Induced Platelet Adhesion and Activation

The ability to promote or inhibit specific platelet-surface interactions in well-controlled environments is crucial to studying fundamental adhesion and activation mechanisms. Here, microcontact printing was used to immobilize human fibrinogen covalently in the form of randomly placed, micrometer-sized islands at an overall surface coverage of 20, 50, or 85%. The nonprinted background region was blocked with covalently immobilized human albumin. Platelet adhesion and morphology on each substrate were assessed using combined differential interference and fluorescence microscopy. At 20% coverage, most of the fibrinogen surface features were small round islands, and platelet adhesion and spreading areas were limited by the position and the size of the islands. Platelet circularity, indicated the morphology was mostly rounded. At 50% coverage, some fibrinogen islands coalesced and platelet adhesion and spreading areas increased. Platelet morphology was controlled by the shape of underlying fibrinogen islands, leading to more irregular spreading. At 85% coverage, the fibrinogen pattern was completely interconnected and both platelet adhesion and the spreading area were significantly higher than at lower coverage. In addition, platelets also spread over the albumin regions, suggesting that after a critical surface density of fibrinogen ligands is reached, platelet spreading is no longer inhibited by albumin. Increasing the overall fibrinogen coverage resulted in higher activation levels defined by key morphological characteristics of the spreading platelet.

Fabrication of Multifaceted Micropatterned Surfaces with Laser Scanning Lithography.

The implementation of engineered surfaces presenting micrometer-sized patterns of cell adhesive ligands against a biologically inert background has led to numerous discoveries in fundamental cell biology. While existing surface patterning strategies allow for pattering of a single ligand it is still challenging to fabricate surfaces displaying multiple patterned ligands. To address this issue we implemented Laser Scanning Lithography (LSL), a laser-based thermal desorption technique, to fabricate multifaceted, micropatterned surfaces that display independent arrays of subcellular-sized patterns of multiple adhesive ligands with each ligand confined to its own array. We demonstrate that LSL is a highly versatile "maskless" surface patterning strategy that provides the ability to create patterns with features ranging from 450 nm to 100 μm, topography ranging from -1 to 17 nm, and to fabricate both stepwise and smooth ligand surface density gradients. As validation for their use in cell studies, surfaces presenting orthogonally interwoven arrays of 1×8 μm elliptical patterns of Gly-Arg-Gly-Asp-terminated alkanethiol self-assembled monolayers and human plasma fibronectin are produced. Human umbilical vein endothelial cells cultured on these multifaceted surfaces form adhesion sites to both ligands simultaneously and utilize both ligands for lamella formation during migration. The ability to create multifaceted, patterned surfaces with tight control over pattern size, spacing, and topography provides a platform to simultaneously investigate the complex interactions of extracellular matrix geometry, biochemistry, and topography on cell adhesion and downstream cell behavior.

Catalyst-Free Conjugation and In Situ Quantification of Nanoparticle Ligand Surface Density Using Fluorogenic Cu-Free Click Chemistry

A highly efficient method for functionalizing nanoparticles and directly quantifying conjugation efficiency and ligand surface density has been developed. Attachment of 3-azido-modifed RGD-peptides to PEGylated liposomes was achieved by using Cu-free click conditions. Upon coupling a fluorophore is formed, which could be utilized to monitor conjugation efficiency and the obtained ligand surface density in situ, without prior purification. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Cell receptor and surface ligand density effects on dynamic states of adhering circulating tumor cells

Dynamic states of cancer cells moving under shear flow in an antibody-functionalized microchannel are investigated experimentally and theoretically. The cell motion is analyzed with the aid of a simplified physical model featuring a receptor-coated rigid sphere moving above a solid surface with immobilized ligands. The motion of the sphere is described by the Langevin equation accounting for the hydrodynamic loadings, gravitational force, receptor-ligand bindings, and thermal fluctuations; the receptor-ligand bonds are modeled as linear springs. Depending on the applied shear flow rate, three dynamic states of cell motion have been identified: (i) free motion, (ii) rolling adhesion, and (iii) firm adhesion. Of particular interest is the fraction of captured circulating tumor cells, defined as the capture ratio, via specific receptor-ligand bonds. The cell capture ratio decreases with increasing shear flow rate with a characteristic rate. Based on both experimental and theoretical results, the characteristic flow rate increases monotonically with increasing either cell-receptor or surface-ligand density within certain ranges. Utilizing it as a scaling parameter, flow-rate dependent capture ratios for various cell-surface combinations collapse onto a single curve described by an exponential formula.

Retention and selectivity effects caused by bonding of a polar urea-type ligand to silica: A study on mixed-mode retention mechanisms and the pivotal role of solute–silanol interactions in the hydrophilic interaction chromatography elution mode

The separation properties of five silica packings bonded with 1-[3-(trimethoxysilyl)propyl]urea in the range of 0–3.67μmolm−2 were investigated in the hydrophilic interaction chromatography (HILIC) elution mode. An increase of the ligand surface density promoted retention of non-charged polar compounds and even more so for acids. An opposite trend was observed for bases, while the amphoteric compound tyrosine exhibited a U-shaped response profile. An overall partitioning retention mechanism was incompatible with these observations; rather, the substantial involvement of adsorptive interactions was implicated. Support for the latter was provided by column-specific changes in analyte retention and concomitant selectivity effects due to variations of salt concentration, type of salt, pH value, organic modifier content, and column temperature. Silica was more selective for separating compounds differing in charge state (e.g. tyramine vs. 4-hydroxybenzoic acid), while in cases where structural differences of solutes resided in non-charged polar groups (e.g. tyramine vs. 5-hydroxydopamine, nucleoside vs. nucleobase) more selective separations were obtained on bonded phases. Hierarchical cluster analysis of the home-made urea-type and three commercial amide-type bonded packings evinced considerable differences in separation properties. The present data emphasise that the role of the packing material under HILIC elution conditions is hardly just the polar support for a dynamic coating with a water-enriched layer. Three major retention mechanisms are claimed to be relevant on bare silica and the urea-type bonded packings: (i) HILIC-type partitioning, (ii) HILIC-type weak adsorption such as hydrogen bonding between solutes and ligands or solutes and silanols (potentially influenced by individual degrees of solvation, salt bridging, etc.), (iii) strong electrostatic (ionic) solute–silanol interactions (attractive/repulsive). Even when non-charged polar bonded phases are used, solute–silanol interactions should not be discounted, which makes them a prime parameter to be characterised by HILIC column tests. Multi/mixed-mode type separations seem to be common under HILIC elution conditions, associated with a great deal of selectivity increments. They are accessible and controllable by a careful choice of the type of packing, the mobile phase composition, and the temperature.

Numerical modeling of mass transport in microfluidic biomolecule-capturing devices equipped with reactive surfaces

This paper presents and compares three different designs including open-channel, circular-pillar and screen-plate microreactors for capturing and detection of biomolecules in a buffer liquid. In general, these capturing/detection devices consist of a flow cell containing one or several reactive surfaces loaded with ligand molecules. The critical issue in the design of an efficient device is the proximity of the biomolecules to the ligands in the capturing stage since the latter is immobilized on the reactive surface and the former is freely moving in the flow. The flow pattern and the geometry of the device are the key factors in this regard. The presented designs are numerically modeled and compared in terms of capture efficiency. Immersed biomolecules are assumed to behave like a continuum medium. The Navier-Stokes and convection–diffusion equations are solved in two dimensions and the concentration profile is obtained after a certain sampling period. The chemical reaction between the ligand and the biomolecule is included in the model through solving the reversible kinetic equation at the boundaries. Considering the level of performance, and ease of implementation, the screen plates are found to be the favourable option for the purpose of biomolecule removal. The effects of the change in the geometric parameters (i.e., the number of plates and reactive side preference) and physicochemical parameters (i.e., the diffusion constant, ligand surface density, and forward and backward reaction rates all combined in non-dimensional numbers) on the capture efficiency of the screen plates are thoroughly inspected and the corresponding results are plotted.

Role of surface charge density in nanoparticle-templated assembly of bromovirus protein cages.

Self-assembling icosahedral protein cages have potentially useful physical and chemical characteristics for a variety of nanotechnology applications, ranging from therapeutic or diagnostic vectors to building blocks for hierarchical materials. For application-specific functional control of protein cage assemblies, a deeper understanding of the interaction between the protein cage and its payload is necessary. Protein-cage encapsulated nanoparticles, with their well-defined surface chemistry, allow for systematic control over key parameters of encapsulation such as the surface charge, hydrophobicity, and size. Independent control over these variables allows experimental testing of different assembly mechanism models. Previous studies done with Brome mosaic virus capsids and negatively charged gold nanoparticles indicated that the result of the self-assembly process depends on the diameter of the particle. However, in these experiments, the surface-ligand density was maintained at saturation levels, while the total charge and the radius of curvature remained coupled variables, making the interpretation of the observed dependence on the core size difficult. The current work furnishes evidence of a critical surface charge density for assembly through an analysis aimed at decoupling the surface charge and the core size.

Glyconanomaterials: synthesis, characterization, and ligand presentation.

Glyconanomaterials, nanomaterials carrying surface-tethered carbohydrate ligands, have emerged and demonstrated increasing potential in biomedical imaging, therapeutics, and diagnostics. These materials combine the unique properties of nanometer-scale objects with the ability to present multiple copies of carbohydrate ligands, greatly enhancing the weak affinity of individual ligands to their binding partners. Critical to the performance of glyconanomaterials is the proper display of carbohydrate ligands, taking into consideration of the coupling chemistry, the type and length of the spacer linkage, and the ligand density. This article provides an overview of the coupling chemistry for attaching carbohydrate ligands to nanomaterials, and discusses the need for thorough characterization of glyconanomaterials, especially quantitative analyses of the ligand density and binding affinities. Using glyconanoparticles synthesized by a versatile photocoupling chemistry, methods for determining the ligand density by colorimetry and the binding affinity with lectins by a fluorescence competition assay are determined. The results show that the multivalent presentation of carbohydrate ligands significantly enhances the binding affinity by several orders of magnitude in comparison to the free ligands in solution. The effect is sizeable even at low surface ligand density. The type and length of the spacer linkage also affect the binding affinity, with the longer linkage promoting the association of bound ligands with the corresponding lectins.

Effects of surface ligand density on lipid-monolayer-mediated 2D assembly of proteins

The two-dimensional (2D) assembly of the protein streptavidin on a biotin-bearing lipid monolayer was studied as a function of the surface density of biotin, a protein-binding ligand, by means of in situ X-ray scattering and optical Brewster angle microscopy measurements at the liquid–vapor interface. Although this model system has been studied extensively, the relationship between the surface biotin density and the adsorption, 2D phase behavior, and binding state of streptavidin has yet to be determined quantitatively. The observed equilibrium phase behavior provides direct structural evidence that the 2D crystallization of the lipid-bound streptavidin occurs as a density-driven first-order phase transition. The minimum biotin density required for the 2D crystallization of streptavidin is found to be remarkably close to the density of the ligand-binding sites in the protein crystal. Moreover, both above and below this transition, the observed biotin-density dependence of protein adsorption is well described by the binding of biotin-bearing lipids at both of the two available sites per streptavidin molecule. These results imply that even in the low-density noncrystalline phase, the bound proteins share a common, fixed orientation relative to the surface normal, and that the 2D crystallization occurs when the lateral protein density reaches 50–70% of the 2D crystal density. This study demonstrates that in addition to a well-defined molecular orientation, high lateral packing density is essential to the 2D crystallization of proteins.

Identification of different donor-acceptor structures via Förster Resonance Energy Transfer (FRET) in quantum-dot-perylene bisimide assemblies.

Nanoassemblies are formed via self-assembly of ZnS capped CdSe quantum dots (QD) and perylene bisimide (PBI) dyes. Upon assembly formation the QD photoluminescence is quenched, as can be detected both via single particle detection and ensemble experiments in solution. Quenching has been assigned to FRET and NON-FRET processes. Analysis of FRET allows for a distinction between different geometries of the QD dye assemblies. Time-resolved single molecule spectroscopy reveals intrinsic fluctuations of the PBI fluorescence lifetime and spectrum, caused by rearrangement of the phenoxy side groups. The distribution of such molecular conformations and their changed dynamics upon assembly formation are discussed in the scope of FRET efficiency and surface ligand density.

Protein-Directed Spatial Rearrangement of Glycolipids at the Air−Water Interface for Bivalent Protein Binding: In Situ Infrared Reflection Absorption Spectroscopy

Lateral rearrangement of lipids on the surfaces of cell membranes plays an important role in multivalent interactions because a ligand for the second and subsequent binding events can be delivered through the lateral rearrangement. The binary monolayers containing glycolipids with mannose moieties at the air-water interface before and after binding of concanavalin A (Con A) have been investigated in detail using infrared reflection absorption spectroscopy (IRRAS). The spatial rearrangement of glycolipids in the binary monolayers directed by Con A in the subphase facilitated to match with protein binding pockets and minimize steric hindrance of neighboring carbohydrate ligands for bivalent protein binding. The amounts of specifically bound proteins were almost independent of surface glycolipid density at the air-water interface, different from the dependence of the amounts on surface ligand density at the solid-water interface with limited glycolipid rearrangement. Besides, hydrocarbon chains of the glycolipids in the monolayers were even reoriented favorable to the access of the ligands to the proteins for enhanced binding.

Cytokine Induced Killer Cells Can Kill Target through Antigen/TCR Dependent and Independent Mechanisms: New Clinical Perspectives of Utilisation.

Abstract 3024Poster Board II-1000Cytokine induced killer (CIK) cells cultures can easily be obtained by stimulating PBMCs with monoclonal antibody anti-CD3 OKT3, IFNgamma and IL2. After 3-4 weeks at least 3 separate populations are present in the culture: CD3+/CD56-/CD8+ precursors (40.5 ± 19.9%), CD3-CD56+ NK cells (2.5 ± 1.5%) and CD3+/CD56+/CD8+ CIK cells (56.9 ± 21%) which show a T EMRA phenotype (Franceschetti et al., Exp Hematol. 37, 616-628, 2009). CIK cultures are currently used in allogeneic or autologous settings as potential anti-neoplastic effectors for adoptive transfer clinical approaches. We have further characterised the mechanism of target cell recognition and role of activating receptors and of lytic mediators in the cytotoxicity of purified CD3+/CD56+/CD8+ CIK cells. We have observed that CIK cells can kill targets through at least two distinct mechanisms: the first TCR-dependent and antigen-specific and the second non TCR-dependent. Indeed, upon TCR/CD3 crosslinking in CIK cells we observe ERK-1/2 phosphorylation, IFNgamma production (mean 32.6% of positive cells by intracellular staining) and TNFalpha production (mean 19.6%). CD3 ligation by OKT3 results in a significant increase over time in the percentage of CIK cells undergoing degranulation evaluated as CD107a positive cells (respectively 15.5 ± 2.2% at 60', 24.4 ± 1% at 120', 32,9 ± 8.7% at 180' and 34.2 ± 11.1% at 240'). CD3 ligation on CIK cells can induce cytotoxicity in a reverse Ab-dependent killing assay. Addition of OKT3 enhances also the cytotoxicity of CIK cells against K562 leukaemic target (from 16 ± 5% to 50 ± 4 %, E:T 10:1; p<0.001). The TCR/CD3 mediated activation is blocked by pre-treatment with cyclosporine A, confirming the role of calcium-regulated phosphatase calcineurin in the CD3-linked degranulation pathway. Interestingly, CIK cells retain functional activity as antigen-specific memory T cells. Indeed in case of CIK cultures obtained from CMV-seropositive donors, CMV-specific CD3+/CD56+ CIK cells can be generated. CMV-specific CIK cells immunopurified by HLA-peptide tetramers are able to specifically recognise and kill autologous but not allogeneic PHA-blasts pulsed with pp65495-503 but not with irrelevant peptide (average lysis 63 ± 8%, E:T 10:1) and to produce IFNgamma following antigenic stimulation (26.4 ± 7%). Similar data are obtained with EBV-specific T cells. Moreover, CIK cells also show non-MHC-restricted cytotoxicity against numerous leukemic target cell lines. The same CMV-specific purified CIK population shows to posses non MHC-restricted cytotoxic properties against several leukemic targets (average lysis 44 ± 8%, E:T 10:1). NKG2D, described as receptor that can trigger TCR-independent cytotoxicity in activated T cells, is expressed on all CIK cells (mean 99%, MFI 251). Although we can show that NKG2D is functional in these cells since cross-linking with anti-NKG2D mAb leads to ERK phophorylation, blocking of this receptor with mAb does not affect the cytolysis of leukaemic targets. NKG2D crosslinking in CIK cells is not sufficient to induce granule esocytosis and INFgamma or TNFalpha production. No correlation is found between expression and surface density of NKG2D ligands on leukaemic cell targets and their susceptibility to CIK-mediated lysis. Unrestricted CIK-mediated cytotoxicity occurs in the absence of measurable degranulation, is not affected by cyclosporin A, but is inhibited by 70% in presence of brefeldin A, a surface upregulation of glycopolypeptide molecules inhibitor, suggesting a role for Fas-ligand family molecules. CIK cells indeed express TNFalpha (mean 16%, MFI 56), FasL (mean 34%, MFI 34) and TRAIL (mean 37%, MFI 49). The role of this death ligands in leukaemic cell killing is currently under investigation. These data clearly show that CD3+/CD56+/CD8+ CIK cells are activated T EMRA cells which have retained their TCR/CD3 complex usage and their antigen specificity but have acquired unrestricted anti-leukaemic activity. The identification of the molecules involved in this killing is still under investigation. These data open up the possibility of multiple clinical use of CIK cultures, including anti-leukaemic and anti-infective usage particularly in immunodeficient patients. DisclosuresNo relevant conflicts of interest to declare.