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• W2167079782 abstract "Alefacept is a chimeric protein combining CD58 immunoglobulin-like domain 1 with human IgG1 Fc. Alefacept mediates adhesion by bridging CD2 on T cells to activating Fc receptors on effector cells, but the equilibrium binding parameters have not been determined. Alefacept mediated T cell killing by NK cells and adhesion between CD2- and CD16-expressing cells at an optimum concentration of 100 nm. We introduce novel measurements with supported planer bilayers, from which key two-dimensional and three-dimensional parameters can be determined by data fitting. Alefacept competitively inhibited cell bilayer adhesion mediated by the CD2–CD58 interaction. Alefacept mediated maximal adhesion of CD2+ T cells to CD16B, an Fc receptor, in planar bilayers at 500 nm. A mechanistic model for alefacept-mediated cell-bilayer adhesion allowed fitting of the data and determination of two-dimensional binding parameters. These included the density of bonds in the adhesion area, which grew to maintain a consistent average bond density of 200 molecules/μm2 and two-dimensional association constants of 3.1 and 630 μm2 for bivalently and monovalently bound forms of alefacept, respectively. The maximum number of CD16 bound and the fit value of 4,350 CD2 per cell are much lower than the 40,000 CD2 per cell measured with anti-CD2 Fab. These results suggest that additional information is needed to correctly predict Alefacept-mediated bridge formation. Alefacept is a chimeric protein combining CD58 immunoglobulin-like domain 1 with human IgG1 Fc. Alefacept mediates adhesion by bridging CD2 on T cells to activating Fc receptors on effector cells, but the equilibrium binding parameters have not been determined. Alefacept mediated T cell killing by NK cells and adhesion between CD2- and CD16-expressing cells at an optimum concentration of 100 nm. We introduce novel measurements with supported planer bilayers, from which key two-dimensional and three-dimensional parameters can be determined by data fitting. Alefacept competitively inhibited cell bilayer adhesion mediated by the CD2–CD58 interaction. Alefacept mediated maximal adhesion of CD2+ T cells to CD16B, an Fc receptor, in planar bilayers at 500 nm. A mechanistic model for alefacept-mediated cell-bilayer adhesion allowed fitting of the data and determination of two-dimensional binding parameters. These included the density of bonds in the adhesion area, which grew to maintain a consistent average bond density of 200 molecules/μm2 and two-dimensional association constants of 3.1 and 630 μm2 for bivalently and monovalently bound forms of alefacept, respectively. The maximum number of CD16 bound and the fit value of 4,350 CD2 per cell are much lower than the 40,000 CD2 per cell measured with anti-CD2 Fab. These results suggest that additional information is needed to correctly predict Alefacept-mediated bridge formation. Immunoadhesins are biopharmaceuticals that take the basic framework of antibodies and replace the antigen-binding domain with the ectodomain of adhesion molecules (1Zettlmeissl G. Gregersen J.P. Duport J.M. Mehdi S. Reiner G. Seed B. DNA Cell Biol. 1990; 9: 347-353Crossref PubMed Scopus (62) Google Scholar, 2Byrn R.A. Mordenti J. Lucas C. Smith D. Marsters S.A. Johnson J.S. Cossum P. Chamow S.M. Wurm F.M. Gregory T. Groopman J.E. Capon D.J. Nature. 1990; 344: 667-670Crossref PubMed Scopus (139) Google Scholar). The common fragment of IgG (Fc) portion is thought to link to immune effector mechanisms to destroy cancer cells and or over-reactive immune cells. One example of this approach is the drug alefacept, which combines the CD2 binding domain of the adhesion molecule CD58 (LFA-3) with human IgG1 Fc in a single polypeptide chain. Alefacept is approved for treatment of psoriasis. Alefacept mediates reduction of circulating memory T cells in patients and mediates Fc receptor (FcR) 2The abbreviations used are: FcR, Fc receptor; PBL, human peripheral blood lymphocytes; FITC, fluorescein isothiocyanate; GPI, glycophosphatidylinositol; MR, maximal release; SR, spontaneous release; GFP, green fluorescent protein; BF, bright field; IRM, interference reflection microscopy; D, diffusion coefficient. 2The abbreviations used are: FcR, Fc receptor; PBL, human peripheral blood lymphocytes; FITC, fluorescein isothiocyanate; GPI, glycophosphatidylinositol; MR, maximal release; SR, spontaneous release; GFP, green fluorescent protein; BF, bright field; IRM, interference reflection microscopy; D, diffusion coefficient.-dependent cell-mediated killing of T cells in vitro (3Majeau G.R. Meier W. Jimmo B. Kioussis D. Hochman P.S. J. Immunol. 1994; 152: 2753-2767PubMed Google Scholar, 4Ellis C.N. Krueger G.G. N. Engl. J. Med. 2001; 345: 248-255Crossref PubMed Scopus (567) Google Scholar). It is hypothesized that alefacept both reduces deleterious effector functions of activated T cells by blocking interaction of CD2 with CD58 and deletes autoaggressive T cells through FcR-dependent killing. When CD16A is the activating FcR each of the individual interactions of alefacept is low affinity with a Kd of 1.5 μm for the CD2–CD58 interaction and Kd of 0.91 μm for the Fc-CD16 interaction (5van der Merwe P.A. Barclay A.N. Mason D.W. Davies E.A. Morgan B.P. Tone M. Krishnam A.K.C. Ianelli C. Davis S.J. Biochemistry. 1994; 33: 10149-10160Crossref PubMed Scopus (190) Google Scholar, 6Dustin M.L. Golan D.E. Zhu D.M. Miller J.M. Meier W. Davies E.A. van der Merwe P.A. J. Biol. Chem. 1997; 272: 30889-30898Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 7Majeau G.R. Whitty A. Yim K. Meier W. Hochman P.S. Cell Adhes Commun. 1999; 7: 267-279Crossref PubMed Scopus (33) Google Scholar, 8Galon J. Robertson M.W. Galinha A. Mazieres N. Spagnoli R. Fridman W.H. Sautes C. Eur. J. Immunol. 1997; 27: 1928-1932Crossref PubMed Scopus (45) Google Scholar). How these solution affinities relate to interactions in an adhesion area is not clear. These interactions are proposed to take place in the two-dimensional interface between cells, but there are only limited data on such interactions and no quantitative data on formation of the trimolecular bridges as proposed for alefacept. Determining such parameters in a model system would be a first step to development of a system of two-dimensional pharmacology to better predict in vivo behavior in cell-cell contact areas based on in vitro measured interaction parameters. Information like affinity and maximum binding has been mainstays of modern pharmacology for over 50 years. The availability of such tools for adhesion mediating drugs would aid in development of effective biological and small molecule drugs based on bridging surface receptor to induce adhesion or cell-cell signaling. An equilibrium model to predict the ability of immunoadhesins, and related compounds including antibodies, to mediate adhesion would be useful to evaluate current and future pharmaceuticals. The chemistry of these interactions at cell interfaces has been poorly understood due to the technical difficulty of quantifying molecular interactions in cell-cell interfaces. One approach to this problem has been to study the molecular interactions in the hybrid cell-supported planar bilayer system (6Dustin M.L. Golan D.E. Zhu D.M. Miller J.M. Meier W. Davies E.A. van der Merwe P.A. J. Biol. Chem. 1997; 272: 30889-30898Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 9Dustin M.L. Ferguson L.M. Chan P.Y. Springer T.A. Golan D.E. J. Cell Biol. 1996; 132: 465-474Crossref PubMed Scopus (199) Google Scholar, 10Bromley S.K. Iaboni A. Davis S.J. Whitty A. Green J.M. Shaw A.S. Weiss A. Dustin M.L. Nat. Immunol. 2001; 2: 1159-1166Crossref PubMed Scopus (245) Google Scholar). Glass-supported planar bilayers consist of a phospholipid bilayer supported on a layer of water molecules on a glass, quartz, or SiO2 surface (11Weis R.M. Balakrishnan K. Smith B.A. McConnell H.M. J. Biol. Chem. 1982; 257: 6440-6445Abstract Full Text PDF PubMed Google Scholar). The bilayer can contain fluorescently labeled receptors and control molecules that are freely mobile in the bilayer. Interaction between these receptors and cell surface molecules in hybrid cell-supported planar bilayer junctions, referred to subsequently as an adhesion area, induces accumulation of the fluorescent receptors and partial exclusion of non-interacting control molecules in the bilayer, which can be detected by fluorescence microscopy. Varying the density of receptors in the substrate enables analysis of adhesion area growth and two-dimensional affinity measurements using the Golan-Zhu method, which makes the assumption that receptor interactions are confined to the adhesion site, while free receptors diffuse over the entire cell surface (6Dustin M.L. Golan D.E. Zhu D.M. Miller J.M. Meier W. Davies E.A. van der Merwe P.A. J. Biol. Chem. 1997; 272: 30889-30898Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 12Zhu D.M. Dustin M.L. Cairo C.W. Golan D.E. Biophys. J. 2007; 92: 1022-1034Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The interactions of CD2 with CD58 has been studied in detail using supported planar bilayers and the Golan-Zhu analysis revealing a two-dimensional Kd in the range of 1.1–7.6 molecules/μm2 and a maximal binding (Bmax) close to the total number of cell surface CD2 accessible by antibodies (6Dustin M.L. Golan D.E. Zhu D.M. Miller J.M. Meier W. Davies E.A. van der Merwe P.A. J. Biol. Chem. 1997; 272: 30889-30898Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 12Zhu D.M. Dustin M.L. Cairo C.W. Golan D.E. Biophys. J. 2007; 92: 1022-1034Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). These results are consistent with self-assembly of an ordered interface into which CD2 diffuses freely. Our hypothesis is that immunoadhesin-mediated adhesion works by similar self-assembly of ordered junctions between cell membranes containing target epitopes and Fc receptors. In this report we describe a detailed equilibrium model for alefacept mediated adhesion. We test the model by fitting experimental data and derive estimates for key parameters. The model incorporates a growth rule based on constant density of bonds that is well fit by the data. A key prediction of the model is that the concentration of alefacept needed to initiate an adhesion area is inversely proportion to the square of the CD2 density. This prediction is important because it implies that a 5-fold higher expression of a receptor on a target compared with a bystander cell will result in initiation of interaction with the target at 25-fold lower concentration of alefacept than needed to initiate adhesion to the bystander. We experimentally verify this prediction. The data indicate that rules governing access of alefacept-CD2 complexes to the adhesion area are not fully understood and identify areas for further study. Human Peripheral Blood Lymphocytes (PBL)—In Fig. 1, lymphocytes were separated from freshly drawn peripheral blood (from healthy volunteers employed at Biogen Idec) using Ficoll-hypaque gradient centrifugation, as previously described (3Majeau G.R. Meier W. Jimmo B. Kioussis D. Hochman P.S. J. Immunol. 1994; 152: 2753-2767PubMed Google Scholar). In Fig. 5 human peripheral blood T cells were obtained from the New York Blood Center (New York, NY). Naive and memory cells were isolated by negative selection with magnetic beads (Miltenyi Biotech, Auburn, CA).FIGURE 5CD2 expression and alefacept-mediated adhesion. A, peripheral blood T cells were separated into naive and memory populations using appropriate MACS kits to obtain >90% pure populations. Cells were stained with saturating concentrations of FITC-TS2/18 (solid lines) or control antibody FITC-YN1/1 (dashed lines), washed, and analyzed. Naive (1× thickness line), memory (2× thickness line), and effector (3× thickness line) were analyzed and calibrated to IgG bound using FITC calibration beads and antibody F:P ratios. Data are representative of three experiments. B, percent adhesion was analyzed with naive T cells (open triangles), memory T cells (open squares) or in vitro activated T cell blasts (filled circles) on planar bilayers containing 500 molecules/μm2 CD16B and the indicated concentration of Alefacept at 24 °C for 60 min. In separate experiments plotted on the same graph, adhesion of Jurkat cells (filled triangles) or Jurkat cells with 80% of CD2 blocked by TS2/18 Fab (filled squares) to bilayers with 500 molecules/μm2 CD16B was measured at two concentrations of alefacept spanning a 25-fold range. Adhesion was scored by interference reflection microscopy and bright field microscopy and confirmed by evaluation of CD16 fluorescence in the adhesion area. At least 10 fields were analyzed with greater than 200 input cells. Symbols are listed in legend. Data are representative of three experiments for each point. C, forward scatter analysis for cell populations analyzed in A. Data are representative of three experiments. D, titration of TS2/18 Fab (x and + symbols are two Fab preps) to determine the concentration needed to block 80% of CD2 molecules (dashed lines). “Saturation” binding of TS2/18 IgG (filled circles) is shown for comparison. Data are representative of three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cells Lines—The CD16A+ CD2+ Jurkat cells used in Fig. 1 were obtained from P. Anderson (Dana Farber Cancer Institute, Boston, MA). The CD2+ CHO transfectants used in Fig. 1 were provided by Cathy Hession (Biogen Idec, Inc. Cambridge, MA) (3Majeau G.R. Meier W. Jimmo B. Kioussis D. Hochman P.S. J. Immunol. 1994; 152: 2753-2767PubMed Google Scholar). CD16B+ Jurkat cells were obtained from J. Green and E. R. Brown (University of California, San Francisco, CA) (13Green J.M. Schreiber A.D. Brown E.J. J. Cell Biol. 1997; 139: 1209-1217Crossref PubMed Scopus (41) Google Scholar). Jurkat cells used in Figs. 2, 3, 4, 5, 6 were originally obtained from A. Chan (Genentech, S. San Francisco, CA). The gene encoding enhanced green fluorescence protein (eGFP) was fused to the C terminus of a human CD2 cDNA using standard molecular biology techniques, and the resulting fusion construct was verified by sequencing (14Douglass A.D. Vale R.D. Cell. 2005; 121: 937-950Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar). Jurkat cells were transiently transfected with CD2-GFP by electroporation and allowed to recover for 72–96 h prior to imaging.FIGURE 3Alefacept effect on CD2 lateral mobility and adhesion formation. A, single molecule analysis of short range diffusion of CD2-GFP on untreated Jurkat cells. n = 716 particles, D = 0.18 μm2/s. The black area includes all events that may be “immobile” in that their movement is less than or equal to that of GFP attached to glass, whereas the gray area includes particles with greater diffusion that GFP attached to glass. By this criterion 87% of the particles are unambiguously mobile. B, short-range diffusion analysis on cells treated with 0.5 μm alefacept. n = 827 particles. D = 0.08 μm2/s. By the criteria applied in C, 62% of the particles are mobile. Data are representative of two experiments. C, images of alefacept-mediated bridging of CD2-expressing Jurkat T cells to a planar bilayer containing GPI-linked Cy3-CD16B. 600 molecules/μm2 CD16B; 250 molecules/μm2 CD48. The soluble ligand is buffer control or 0.5 μm alefacept. Scale bar = 10 μm. D, titration of alefacept with CD16B bilayers (1200 molecule/μm2). Amount of bound CD16B in the interface (open squares) and percent adhesion (filled circles) at indicated concentrations of soluble alefacept. Each point represents the mean of greater than 100 adhesion areas. E, titration of CD16B density in the bilayer at the optimal concentration of alefacept (0.5 μm). Each point is a mean of six experiments in which each data point is determined by greater than 100 Jurkat cell adhesion areas.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4Fitting of data to a model for immunoadhesin-mediated bridge formation. A, mechanistic steps in immunoadhesin-mediated adhesion. The 7 reaction steps considered in the bivalent model and the relationships that must be satisfied based on the law of detailed balance (equations shown). B and C, CD16B binding under conditions where the initial CD16B density equals 1200 molecules/μm2 (B) and 625 molecules/μm2 (C). Each data point is determined by greater than 100 Jurkat T cell adhesion areas in which the area (filled symbols) and bound CD16 number (open symbols) was measured. In the model, the density of bridges in the adhesion area is constant (β = B/A). The solid lines in B and C are the simultaneous fit of the model to the area and bound CD16 data at the two CD16B densities using nonlinear least squares regression based on a finite difference Lenvenberg-Marquardt algorithm. The model predicts that bivalent alefacept-CD2 interaction dominates a low alefacept concentration (dot-dash-dash line), while monovalent binding dominates at higher alefacept concentration (dashed line). In the fit the following parameters were held constant: Acell = 800 μm2, K = 6.7 × 105 m–1, KR = 1.3 × 106 m–1, and Kx = 0.033 μm2. The best fit values of the four parameters that were varied were: β = 200 molecules/μm2, Kb1 = 3.1 μm2, Kb2 = 630 μm2, and NT = 4,350 CD2 per cell. A reasonable fit could not be obtained if we fixed NT = 40,000 CD2 per cell. D, straight line fit of bound CD16B versus adhesion area. Data for bilayers containing initial CD16B density of 1200 molecules/μm2 (squares) or 625 molecules/μm2 (circles) corresponding to data in panels B and C. Simple linear regression sets β = 190 molecules/μm2, close to the fit of 200 molecules/μm2 that takes into account all model parameters. E, predictions of Lmin and Lmax. Symbols are calculated values. The solid line has a slope of 1, and the dashed line a slope of –2 indicating that Lmax is directly proportional to the CD2 density and that Lmin is inversely proportional to the square of the CD2 density. Note that the model predicts that below 320 CD2 per cell there is no alefacept concentration at which adhesion occurs.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6CD2 distribution and inhibition by soluble IgG. A, Jurkat T cells were stained with FITC-CD2.1 Fab and allowed to interact with planar bilayers containing 200 molecules/μm2 CD58 or 500 molecules/μm2 CD16 in the presence of 1 μm alefacept. Confocal microscopy was used to obtain a BF image, an IRM image, and a 488-nm laser excitation, 530/30-nm band pass confocal scan through the middle of the cell (FITC-CD2.1). The faint signal from the cell on CD58 indicates extensive redistribution of CD2 to the interface. Scale bar = 10 μm. B, percent CD2 loss from the cells was determined for 100 cells in each condition relative to FITC-CD2.1 Fab-labeled Jurkat cells adhering to CD80, which does not induce any reorganization of CD2, to determine, which was used to obtain the 100% value. The “non-contact” value was directly measured and the “contact” value was calculated based on the assumption that all CD2 lost from the non-contact surface is located in the interface. C, Jurkat T cell adhesion to supported planar bilayers containing 450 molecules/μm2 CD16B at 500 nm alefacept and the indicated concentration of protein A-purified human IgG. Adhesion was scored by interference reflection microscopy and bright field microscopy and confirmed by evaluation of CD16 fluorescence in the adhesion area. At least 10 fields were analyzed with greater than 200 input cells. Data are representative of three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Alefacept—The fusion protein designated herein as alefacept is composed of the first extracellular domain of CD58 fused to the hinge, CH2 and CH3 domains of human IgG1 (15Miller G.T. Hochman P.S. Meier W. Tizard R. Bixler S.A. Rosa M.D. Wallner B.P. J. Exp. Med. 1993; 178: 211-222Crossref PubMed Scopus (171) Google Scholar). For a subset of experiments, to eliminate the possibility that the results might be influenced by aggregated protein that might be present at very low concentrations, the material was further purified by size exclusion chromatography on Superose-6 (Amersham Biosciences). Briefly, 2 ml of alefacept (15 mg/ml) were loaded onto a 1.6 × 100 cm Superose-6 column equilibrated in phosphate-buffered saline at a flow rate of 40 ml/h. Individual fractions were analyzed for aggregate content by analytical size exclusion chromatography using a TSK-3000 column (Toso-Haas, Montgomeryville, PA). Fractions with an aggregate content of <0.5% were pooled, concentrated by ultracentrifugation using YM-30 Centriprep filters (Millipore, Billerica, MA), aliquoted and stored at –70 °C. The final aggregate content was determined to be to be <0.5% after the concentrated pool (12.4 mg/ml) had been put through one freeze/thaw cycle. Molar concentrations of alefacept were calculated with a molecular weight of 72,000. Alefacept-mediated Adhesion Assay—CHO cells transfected with human CD2 were grown to confluence in wells of flat bottom 96-well plates. Wells were washed twice, and 50 μlof varying concentrations of alefacept were added. Plates were then washed or unwashed as indicated. 105 CD16+ Jurkat cells, which were labeled with BCECF-AM according to the manufacturer's directions (Invitrogen-Molecular Probes Inc., Eugene, OR), were then added in 50 μl to wells. Plates were incubated for 0.5 h at 37 °C or 24 °C. Wells were then washed four times. Background signal was that of wells without cells, incubated and washed as were the experimental groups. Total input was the signal released from 105 BCECF-AM labeled cells. The percent binding was determined as the (experimental minus background counts) divided by (total input minus background counts) × 100. Cytotoxicity Assay—Targets were human PBL blasts. Briefly, 106 PBL were cultured with 10 μg/ml final concentration of PHA for 3 days. PHA blasts were then collected, washed, 100 μCi of 51Cr was added to 107 cells, which were then incubated at 37 °C for 1 h, washed, counted, and resuspended in complete medium. 5 × 103 target cells in 20 μl were added to wells of round bottom 96 well microtiter plates. PBLs freshly prepared from normal donors on the day of the assay were added to the target cells to make a total volume of 200 μl and an effector to target ratio of 50:1. Alefacept was added to wells at the indicated concentrations. Microtiter plates were incubated in a 37 °C, 5% CO2 incubator for 4 h, centrifuged, and 150 μl of supernatant was removed to measure released 51Cr in a Liquid Scintillation Counter (Wallac Inc., Gaithersburg, MD). Spontaneous release (SR) was counts released from target cells in the presence of medium alone, and maximum release (MR) was counts released from target cells incubated in the presence of 0.5% Triton. Percent lysis was calculated as (experimental minus SR cpm) divided by (MR cpm minus SR cpm). Previously it was shown that fusion protein mediated cytotoxicity was inhibited by the addition of blocking mAbs specific for CD16 to the 4-h assay cultures. Lysis is dependent on the expression of CD2 by the target cells (3Majeau G.R. Meier W. Jimmo B. Kioussis D. Hochman P.S. J. Immunol. 1994; 152: 2753-2767PubMed Google Scholar). Quantitative Flow Cytometry—Flow cytometry experiments were performed on a Becton Dickinson FacsCalibur. FITC labeling of antibodies and determination of the fluoresceine: protein ratio was determined using absorption spectroscopy (16Wells A.F. Miller C.E. Nadel M.K. Appl. Microbiol. 1966; 14: 271-275Crossref PubMed Google Scholar). Calibration was performed using FITC standard beads obtained from Bangs Laboratories (Fishers, IN). Antibody-stained cells were washed twice prior to analysis. Alefacept binding modified from Majeau et al. (7Majeau G.R. Whitty A. Yim K. Meier W. Hochman P.S. Cell Adhes Commun. 1999; 7: 267-279Crossref PubMed Scopus (33) Google Scholar). Alefacept was labeled with 0.8 FITC/alefacept. Binding was analyzed by flow cytometry without washing away free alefacept. Because the sample stream does not mix with the sheath fluid prior to reaching the interrogation point there was no alefacept dilution prior to analysis. When the free FITC-alefacept concentration exceeded 100 nm linearity problems were detected with FITC standard beads. To achieve higher concentration of alefacept we then added unlabeled alefacept while keeping the FITC-alefacept at 100 nm. The binding was allowed to reach equilibrium for 5 min at room temperature. Nonspecific binding was assessed after saturating CD2 on the cells with 100 μg/ml of TS2/18. We found that TS2/18 did not dissociate even with high concentrations of alefacept. The fluorescence standard beads were read in parallel at the same concentration of free alefacept to generate a standard curve to determine the number of alefacept molecules bound per cell. Planar Bilayers—All phospholipids were obtained from Avanti Polar Lipids (Alabaster, AL). Human CD58 was purified from human red blood cells and labeled with FITC while bound to the anti-CD58 antibody TS2/9 (9Dustin M.L. Ferguson L.M. Chan P.Y. Springer T.A. Golan D.E. J. Cell Biol. 1996; 132: 465-474Crossref PubMed Scopus (199) Google Scholar, 17Dustin M.L. Sanders M.E. Shaw S. Springer T.A. J. Exp. Med. 1987; 165: 677-692Crossref PubMed Scopus (195) Google Scholar). Mouse CD48 was labeled with Cy5 (Amersham Biosciences), by a similar procedure, but with OX78 mAb (rat anti-mouse CD48) (18Kato K. Koyanagi M. Okada H. Takanashi T. Wong Y.W. Williams A.F. Okumura K. Yagita H. J. Exp. Med. 1992; 176: 1241-1249Crossref PubMed Scopus (215) Google Scholar, 19Dustin M.L. J. Biol. Chem. 1997; 272: 15782-15788Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). CD16B was purified from CD16B transfected Jurkat cells using 3G8 mAb (IgG was purified from the hybridoma) and was labeled with Cy3 (Amersham Biosciences) while attached to 3G8 agarose. The CD16B was eluted at pH 3 in a solution with 1% octyl-β-d-glucopyranoside. Planar bilayers composed of 0.4 mm egg phosphatidylcholine (PC) were prepared by detergent dialysis as previously described (9Dustin M.L. Ferguson L.M. Chan P.Y. Springer T.A. Golan D.E. J. Cell Biol. 1996; 132: 465-474Crossref PubMed Scopus (199) Google Scholar). Bilayers were formed on clean glass coverslips (chromic sulfuric acid for 15 min then rinsed with deionized water, high purity acetone, and dried under a nitrogen stream) in a FCS II flow cell (Bioptechs, Butler, PA). Bilayer formation was initiated by trapping a 1-μl drop or liposome suspension between the coverslip and microaqueduct slide separated by a 250-μm gasket. After 20 min, the flow cell was perfused with 5 ml of Hepes-buffered saline 1% human serum albumin (HBS/HSA; 20 mm Hepes, 1.7 mm K2HPO4, 137 mm, NaCl, 5 mm KCl, 5 mm glucose, 1% clinical grade human serum albumin, pH 7.4.). The surface was then blocked with 5% casein in phosphate-buffered saline. After 20 min, the flow cell was again perfused with HBS/HSA. Imaging—The microscope used to acquire images was equipped with a cooled CCD camera and excitation and emission filter wheels (Yona Microscopes, Silver Spring, MD) (19Dustin M.L. J. Biol. Chem. 1997; 272: 15782-15788Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The filter set was the XF93 triple band dichroic mirror and individual excitation and emission filters recommended by the manufacturer for FITC, Cy3, and Cy5 dyes (Omega, Burlington, VT). Using this filter set there was no detectable cross-detection of the different dyes in each others filter combinations. A Zeiss ×100 Plan-Neofluar or Neofluar 1.3 NA objective was used for all experiments. Digital images were acquired using IP-Lab software and processed by background subtraction and flat field correction prior to segmentation and analysis of adhesion areas. Bleaching was performed with the focused beam from a Spectraphysics 2.5 W Krypton-Argon laser with wavelength selection using a acoustooptic tunable filter (Solamere Technologies, Salt Lake City, UT). Single Molecule Imaging of GFP-CD2—Glass-bottomed observation dishes (MatTek Inc., Ashland, MA) were cleaned thoroughly with a 2:1 mixture of concentrated sulfuric acid and 30% hydrogen peroxide prior to use. Cells expressing low levels of CD2-GFP were imaged using objective-type total internal reflection fluorescence excitation on a Zeiss Axiovert 200 m microscope equipped with a Mega-10 ICCD camera (Stanford Photonics, Palo Alto, CA). Cells were treated with 0.5 μm alefacept in HBS, or with HBS alone, and allowed to adhere to glass-bottom observation dishes for 10 min prior to imaging. Movies were then acquired at 30 frames per second in 200-frame bursts during a 10-min period. A suite of particle tracking functions written in the IDL language by John Crocker and colleagues was used to identify trajectories in each image. Short-range diffusion coefficients were determined using Mat-lab (The MathWorks, Inc., Natick, MA) scripts that perform mean-squared displacement analyses of overla" @default.
• W2167079782 created "2016-06-24" @default.
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• W2167079782 date "2007-11-01" @default.
• W2167079782 modified "2023-10-16" @default.
• W2167079782 title "Quantification and Modeling of Tripartite CD2-, CD58FC Chimera (Alefacept)-, and CD16-mediated Cell Adhesion" @default.
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