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Surfactant-Free Poly(styrene-co-glycidyl methacrylate) Particles with Surface-Bound Antibodies for Activation and Proliferation of Human T Cells Katja Thu¨mmler,*,† Nadine Ha¨ntzschel,‡ Alla Skapenko,† Hendrik Schulze-Koops,† and Andrij Pich§ Division of Rheumatology, Medizinische Poliklinik, Ludwig-Maximilians-Universita¨t Munich, D-80336 Munich, Germany, and Department of Macromolecular Chemistry and Textile Chemistry, Technische Universita¨t Dresden, D-01062 Dresden, Germany. Received September 14, 2009; Revised Manuscript Received March 18, 2010
In this article, we present our results on the design of new polymeric carriers for antibodies. Polymer colloids based on poly(styrene-co-glycidyl methacrylate) were synthesized by surfactant-free emulsion polymerization. Obtained polymer particles stabilized by grafted poly(ethylene glycol) (PEG) chains and carrying active epoxy groups were used for the covalent immobilization of activating antibodies against the human surface proteins CD (cluster of differentiation) 3 and CD28. The particle-antibody conjugates were employed for the stimulation of human CD4 memory T cells. This was analyzed by the up-regulation of the activation markers CD69 and CD25 on T cells and T cell proliferation as assessed by the dilution of a fluorescent dye on dividing daughter T cells. The particle-antibody conjugates were able to stimulate T cells at least as efficiently as conventional methods, e.g., surface-immobilized antibodies. Furthermore, an increase of the PEG chain length of the particles decreased the efficiency of the particle-antibody conjugates to activate T cells.
INTRODUCTION Polymeric particles are very versatile objects whose properties can be tailor-made during synthesis by varying different parameters such as the nature of the monomer, the polymerization procedure, or additives. In biomedicine, for example, particles can be used as carriers in drug delivery systems, as well as for agglutination tests and in the area of biosensors. However, due to the complexity of biological systems there is still a need for the development of new reactive polymeric carriers. Most synthesis strategies employ surfactants that are noncovalently bound. Therefore, they can desorb under certain circumstances like high ionic strength, freezing, or mechanical stress leading to unstable dispersions and undesired interactions with other substances. One aim of this work was the synthesis of highly reactive particles avoiding classical surfactants by using surfactant-free heterophase polymerization. This technique employs functional monomers such as acetoacetoxyethyl methacrylate (1) or poly(ethylene glycol) methacrylate (2) that are able to stabilize the prepared particles. Furthermore, PEG derivatives are known for their biocompatibility and the reduction of unspecific protein adsorption and cell adhesion (3-5). The covalent attachment of biomolecules can be realized by a posterior grafting of molecules with desired functionalities or by direct functionalization during polymerization (6). The * Author to whom correspondence should be addressed. E-mail:
[email protected], Ludwig-Maximilians-Universita¨t Munich, Medizinische Poliklinik, Division of Rheumatology, Pettenkoferstr. 8a, D-80336 Munich, Germany. Tel.: +49 89 516 034 53. Fax: +49 89 516 034 74. † Ludwig-Maximilians-Universita¨t Munich. ‡ Technische Universita¨t Dresden. Current address: Centre for BioNano Interactions, School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland. § Technische Universita¨t Dresden. Current address: Deutsches Wollforschungsinstitut, RWTH Aachen University, Pauwelsstrasse 8, D-52056 Aachen, Germany.
use of glycidyl methacrylate (GMA) for immobilization of biomolecules has been known for a long time because its epoxy groups are able to react with several functional groups such as thiol, amino, or hydroxy groups without further activation. A lot of work in this area has been done by Hora´k and co-workers (7, 8) who synthesized magnetic particles containing GMA and used these for the immobilization of biomolecules. Several other publications deal with copolymers of GMA and 2-hydroxyethyl methacrylate and their use for the immobilization of enzymes (9-12). In contrast, little research has so far been done in the field of immobilization of antibodies using GMA. Faure et al. (13) prepared miniaturized immunoextraction columns by bonding of anti-ochratoxin A antibodies on poly(glycidyl methacrylate-co-ethylene dimethacrylate) monoliths. Iwata et al. (14) immobilized goat anti-mouse immunglobulin(Ig) G Fab′ fragments on GMA-containing polymer brushes in a defined orientation. To explain processes of the human immune system, it is necessary to examine single steps of an immune response in vitro. Furthermore, the amount of primary cells from blood donations is limited. Analysis of immunological systems in vitro frequently requires expansion and activation of the desired cell species, e.g., T cells. Another application for T cell expansion is for therapeutic purposes, e.g., for the treatment of cancer (15) or autoimmune diseases (16-18). Stimulation of T cells with immobilized antibodies to surface proteins, such as the polymorphic T cell receptor (TCR) or the nonpolymorphic CD (cluster of differentiation) 3 complex, mimics binding of the major histocompatibility class II/peptide complex (MHC class II/peptide) on the surface of antigenpresenting cells to T cells and hence initiates the T cell activation program (19). To stimulate T cells effectively, two activation signals are required. The first signal occurs via the T cell receptor complex, whereas the second, so-called co-stimulatory signal, can be provided by engaging the nonpolymorphic surface receptor CD28. A conventional method for polyclonal stimulation of memory T cells (Tmem) in vitro employs antibodies to CD3 adsorbed on plastic cell culture plates in combination with
10.1021/bc900402d 2010 American Chemical Society Published on Web 04/28/2010
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antibodies to CD28. The immobilization of the antibodies to CD3 is necessary, as it effectively permits cross-linking of the CD3 complexes on the surface of T cells, a prerequisite for T cell activation. Soluble antibodies to CD3, in fact, are not sufficient to induce T cell activation in most experimental settings (20-22). In contrast, CD28 mediated signals are rather independent of cross-linking, and stimulation of CD28 can be provided by soluble or immobilized antibodies to CD28. As cross-linking of CD3 can be employed upon contact with any carrier on which antibodies to CD3 can be immobilized in adequate densities, effective T cell activation can be initiated by CD3 and CD28 antibodies immobilized on polymeric particles. The aim of the present work was the development of a simple and cheap polymeric carrier system for antibodies. The important challenge was a proper design of the particle surface, e.g., the presence of reactive functional groups able to bind antibodies, the presence of hydrophilic polymer chains for enhanced colloidal stability to avoid particle aggregation, as well as adjustment of particle size in submicrometer range. In the present study, we used particles immobilized with anti-CD3 and anti-CD28 antibodies to stimulate T cells and compared this method to the activating effect of plate bound antibodies to CD3 in concert with soluble anti-CD28 (control sample). Furthermore, we assessed whether particle-antibody conjugates without poly(ethylene glycol) methyl ether methacrylate (PEGMEMA) chains or with PEGMEMA chains of different length vary in their capacity to stimulate T cells.
EXPERIMENTAL PROCEDURES Materials. Styrene (S, Fluka) was purified using an inhibitor remover (Aldrich) according to the manufacturer’s description. Glycidyl methacrylate (GMA, Fluka) was purified by vacuum distillation. Poly(ethylene glycol) methyl ether methacrylate (PEGMEMA, Aldrich), 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AMPA, Aldrich), dimethyl sulfoxide (Merck), 5-(and 6-) carboxyfluoresceine-diacetate-succinimidylester (CFSE, Molecular Probes), tris(hydroxymethyl)-aminomethane (Tris, Merck), ethanol (Aldrich), sodium azide (Merck), cresol red (Chemapol Praha), 1,4-dioxane (Riedel-de Hae¨n), hydrogen chloride dioxane solution (Fluka), potassium chloride solution in methanol (Gru¨ssing), tris(hydroxymethyl)-aminomethane (Merck), Hanks’ solution (Apotheke Innenstadt, LMU Munich), phosphate buffer solution (PBS, Invitrogen), Ficoll-Hypaquesolution/Lymphoflot (Biotest), fetal calf serum (FCS, Invitrogen), sheep erythrocytes (SBRC, Fiebig-Na¨hrstofftechnik), Penicillin G/Streptomycin (Invitrogen), L-glutamine (Invitrogen), Interleukin 2 (Chiron), and RPMI 1640 (Invitrogen) were used as received. For the preparation of FACS (fluorescence activated cell sorting)-PBS, 500 mL PBS, 10 mL FCS, and 0.5 mL of NaN3 solution in water (10%) were mixed. Deionized water was employed as reaction medium for emulsion polymerization. The following monoclonal antibodies (mAbs) were used for cell purification and cell culture: anti-CD3 (OKT3), anti-CD8 (OKT8), anti-CD16 (3g8FcIII), anti-HLA-DR (L243; all from American Type Culture Collection), anti-CD28 (28.2; BD Pharmigen), anti-CD19 (HD37, Dako Diagnostika), antiCD45RA (111.1 C5; a gift from Dr. R. Vilella, Barcelona, Spain), anti-CD56 (MOC-1, Dako Cytomation), and polyclonal antimouse IgG (MP Biomedicals). For cell staining, fluorescein isothiocyanate (FITC)-conjugated or phycoerythrin (PE)-conjugated mAbs were applied: FITC-anti-CD3 (UCHT1), PE-anti-CD4 (Q4120), FITC-antiCD4 (Q4120, all from Sigma), and PE-anti-CD3 (UCHT1), PEanti-CD28 (CD28.2), PE-anti-CD25 (M-A251), FITC-anti-CD27 (M-T271), PE-anti-CD45RO (UCHL1), FITC-anti-CD69 (FN50, all from BD Pharmigen).
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Synthesis of Polymeric Particles. A monomer mixture containing appropriate amounts of styrene and GMA was prepared. A double-walled glass reactor equipped with stirrer was filled with 170 mL distilled water. For the preparation of PEGMEMA containing particles, appropriate amounts of PEGMEMA were previously dissolved in the water. Then, the monomer mixture was added to the water phase under stirring. The obtained pre-emulsion was heated to 60 °C and stirred for 1 h while purging with nitrogen. To start the polymerization process, 10 mL of water with 1.5 wt % of initiator was added. Latexes were prepared at 10% solid content. The reaction time was 8 h at 60 °C. Titration of Epoxy Groups. Water-based particle dispersions were freeze-dried. 0.5 g of dried particles were redispersed in 20 mL 1,4-dioxane. Then, 5 mL of a solution of hydrogen chloride in dioxane were added. The mixture was stirred for 8 h at room temperature. Remaining hydrogen chloride was determined by titration with a solution of potassium hydroxide in methanol. The equivalence point was determined by either potentiometric titration or with 1 mL of a solution of 0.1 g cresol red in 100 mL ethanol-water mixture (volume ratio ) 1:1) as color indicator. Attachment of Antibodies to Polymeric Particles. Fluorescence-Labeled Antibodies. The particle dispersions were diluted 1:100 with 50 mM Tris-HCl (pH 9.5). 25 µL of fluorescence-labeled antibodies were added to 100 µL of diluted particle dispersion. Then, the samples were incubated at 37 °C for 2 h. After washing with 50 mM Tris buffer, the particle-antibody conjugates were resuspended in 150 µL FACS-PBS and analyzed by flow cytometry. Antibodies for Cell ActiVation. The particle dispersions were sterilized by washing with ethanol twice and diluted with 50 mM Tris (pH 9.5) 1:500. 150 µL of the antibody solution in 50 mM Tris with the desired antibody concentrations was added. The final concentration of antibodies was 1 µg/mL. The samples were incubated at 37 °C for 2 h, afterward washed twice with deionized water, and transferred to the cell culture medium. The particle amount in each sample corresponded to the surface area of one well of the cell culture plate. Attachment of Antibodies to Cell Culture Plates (Control Sample). Each well of a 48-well plate was filled with 150 µL of an antibody solution of appropriate concentration (1 µg/mL in 50 mM Tris-HCl) and incubated at 37 °C in a humidified atmosphere containing 5% CO2 for 2 h. The plates were washed twice with Hanks’ solution to remove unbound antibodies and obtain a neutral pH and used for further cell culture. Cell Purification. Mononuclear cells were obtained from heparinized peripheral blood of one healthy individual. Ethical approval was obtained by the local Institutional Review Board, and all subjects enrolled gave their written informed consent. After gradient centrifugation (20 min, 400g) over a FicollHypaque layer followed by washing with PBS, cells were incubated with sheep red blood cells (rosetting) (23). The rosettepositive cells were further purified by negative selection using saturating amounts of mAbs against CD8, CD16, and CD19, followed by panning on plastic Petri dishes coated with goat antimouse IgG for 15 min at room temperature as previously described (24, 25). CD4 T cells were harvested by gently washing the dishes with a solution of 4% NHS (normal human serum)/RPMI 1640 in PBS and centrifugation. In the last step, resting CD4 Tmem were isolated by negative selection using antiHLA-DR, anti-CD45RA, anti-CD56, and anti-CD8 antibodies. Recovered cells were washed with PBS and resuspended in 10% NHS/RPMI 1640. The homogeneity and purity of isolated T cells were assessed by flow cytometry. Routinely, g90% T cells were positive for CD3 and CD4.
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Table 1. Ingredients Used in Emulsion Polymerization (Reaction Temperature: 60 °C) N
S [g]
GMA [g]
GMA [wt%]
PEGMEMA [g]
PEGMEMA [wt%]
Mn, [g/mol] PEGMEMA
initiator [g]
water [g]
1 2 3 4
19 19 19 19
1 0.5 0.5 0.5
5 2.5 2.5 2.5
0.5 0.5 0.5
2.5 2.5 2.5
∼475 ∼1100 ∼2080
0.3 0.3 0.3 0.3
180 180 180 180
Cell Culture. Culturing of the cells was carried out in RPMI 1640 medium supplemented with penicillin G/streptomycin (50 U/mL), L-glutamine (2 mM), 10% NHS, and 10 ng/mL IL-2. Cell cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Memory CD4 T cells were aliquoted at a concentration of 0.75 × 106 cells/mL in the cell culture medium. 300 µL of the cell suspension were pipetted in each well of a 48-well cell culture plate (Costar) and diluted with the same volume of cell culture medium containing the particle-antibody conjugates. Alternatively, the same amount of T cells was stimulated with Dynabeads CD3/CD28 T Cell Expander beads (Dynal Biotech) according to the manufacturer instruction at a 3:1 beads per T cell ratio. Flow Cytometry. 1 ×105 T cells/sample were washed with FACS-PBS, resuspended in 50 µL FACS-PBS, and incubated with saturating amounts of directly fluorochrome labeled mAb against different surface molecules (CD25 or CD69) for 15 min at 4 °C in the dark. Afterward, cells were washed twice with 1 mL FACS-PBS and resuspended in 300 µL FACS-PBS. To obtain quantitative data with very high reliability, at least 1 × 104 T cells/sample were analyzed as recommended (26) and the mean fluorescence intensity (MFI) and standard deviation were calculated for the entire population of 1 × 104 T cells. Analysis of Cell Division. 1 µL of the CD4 Tmem cell suspension (10 × 106 cells/mL) in PBS was incubated with a 10 mM solution of CFSE in dimethyl sulfoxide for 8 min at room temperature, with a final CFSE concentration of 10 µM. Labeling was stopped by adding of 1 mL NHS. Cells were washed twice with RPMI 1640 supplemented as described above. At indicated time points of cell culture, cells were harvested, washed with FACS-PBS, resuspended with 250 µL FACS-PBS, and analyzed by flow cytometry. The proliferation index of the cells was calculated as 1/MFI × 100%. Characterization Methods. SEM images were taken with a Gemini microscope (Zeiss, Germany). For sample preparation, particle dispersions were diluted with deionized water, dropped onto aluminum support, and dried at room temperature. Samples were coated with a thin Au/Pd layer to increase the contrast and quality of the images. Images were obtained at a voltage of 4 kV. Dynamic light scattering measurements were performed with a Zetasizer Nanoseries ZEN3600 (Malvern Instruments, UK). Stability measurements of particle dispersions were performed with the separation analyzer LUMiFuge 114 (LUM GmbH, Germany). Measurements were made in glass tubes at an acceleration velocity of 3000 rpm. The slopes of the
Figure 1. Sedimentation velocities (a) and hydrodynamic radius (b) of polymer particles prepared with PEGMEMA macromonomers of different molecular weights.
sedimentation curves were used to calculate the sedimentation velocities and to get information about the stability of the samples. Flow cytometry analysis was done with a Cytomics FC500, Beckman Coulter. Fluorescence was excited with an Ar-ion laser at a wavelength of 488 nm. Fluorescence signals were determined using appropriate electronic compensation to exclude emission spectra overlap. For extracellular staining, 0.1 × 106 cells/staining were washed with 1 mL FACS-PBS, resuspended in 50 µL FACS-PBS, and incubated with saturating amounts of PE- or FITC-labeled antibodies for 15 min at 4 °C in the dark. Afterward, the cells were washed twice with 1 mL FACS-PBS, resuspended with 250 µL FACS-PBS, and analyzed by flow cytometry. Light microscopy was performed with an Axiovert 25 microscope (Zeiss, Germany).
RESULTS AND DISCUSSION Particle Size and Morphology. Poly(styrene-co-glycidyl methacrylate) (PS-PGMA) and PS-PEGMEMA-PGMA particles were prepared by surfactant-free heterophase polymerization. Interestingly, this technique could be used although GMA is regarded as a rather hydrophobic monomer. But in comparison to styrene, the difference in hydrophobicity is big enough for sufficient particle stabilization. Hence, the obtained dispersions were stable and showed no precipitation during storage. In all runs, the final monomer conversion was between 80% and 85%. An overview of the reaction recipes for emulsion polymerization is given in Table 1. For further evaluation of colloidal stability, sedimentation experiments with an analytical centrifuge were performed (Figure 1a). As expected, the best stability (lowest sedimentation velocity) was observed for dispersions with the highest molecular weight of PEGMEMA, because poly(ethylene glycol) and its derivatives are known as effective sterical stabilizers (27, 28). Figure 1b shows results of dynamic light scattering indicating that the molecular weight of the PEGMEMA macromonomer has a strong influence on the final size of polymer colloids. It is obvious that the particle size decreases with an increase of the PEGMEMA molecular weight. Obtained polymer particles are monodisperse and possess a spherical shape as shown in SEM images (Figure 2). Furthermore, it is believed that the particles possess a core-shell structure due to different hydrophilicity and reactivity of monomers. In the presence of a water-soluble initiator, polymerization in the water phase likely involves a homogeneous nucleation mechanism because the functional monomer has a low ability to form defined micelles in water. As both functional monomers, PEGMEMA and GMA, are more reactive than styrene, formed oligoradicals will contain more GMA and PEGMEMA units leading to enhanced heterogeneity of copolymers. Addition of styrene leads to the formation of amphiphilic polymer chains which can act as stabilizers during polymerization of residual styrene. Thus, final particles probably consist of a polystyrene-rich dense core and a more diffuse shell containing PEGMEMA and GMA units, respectively, as confirmed for similar particles (2). This leads to the assumption that the epoxy groups should be mainly located in the outer region of synthesized particles. The amount of reactive epoxy groups of polymerized particles was determined by titration using the HCl-dioxane method (29). About 85% of the epoxy groups were still intact and available
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Figure 2. SEM images of polymer colloids (numbers correspond to sample numbers in Table 1).
for further reactions, e.g., immobilization of antibodies after polymerization. Immobilization of Antibodies. Most standard methods bind proteins only by adsorption which usually leads to a high degree of protein denaturation (30, 31). In order to enhance the activity of bound antibodies and therefore minimize the amount needed for a certain cell activation, we chose to immobilize antibodies by covalent bonding via epoxy groups. In this context, PEGMEMA was incorporated into the particles not only for enhanced stability, but also for the prevention of protein adsorption. The ability of the synthesized polymeric particles to bind antibodies was demonstrated by immobilization of fluorescence-labeled antibodies (FITC labeled anti-CD3 and PE labeled anti-CD28) followed by flow cytometry analysis. As shown in Figure 3a, light scattering provides information about the size (forward scatter) and the surface characteristics (sideward scatter) of the particles. The scatter plots are then used to gate on the particle population of interest, and fluorescence signals of this population are measured in the different channels according to the fluorescent dye. Figure 3b,c demonstrates the corresponding flow cytometry plots of pure PS-PGMA particles. The particles did not show signals themselves, in either the FITC or the PE channel, verifying that the particles have no significant autofluorescence. In contrast, Figures 3d,e presents the flow cytometry plots for PS-PGMA particles after immobilization of saturating amounts of fluorescence-labeled antibodies to CD3 (FITC) and to CD28 (PE), respectively. The peaks in Figure 3d,e are shifted to higher fluorescence signals in FITC/PE channels compared to Figure 3b,c indicating fluorescent particle-antibody conjugates. FACS analysis showed that 99.5% of the particles were FITC-positive and 98.7% of the particles were positive for PE. Therefore, PS-PGMA particles are able to immobilize both antibodies to CD3 and CD28 effectively. Similar results were obtained for PS-PEGMEMA-PGMA particles (data not shown). Light Microscopy Investigation of Cell Culture. Since the particles effectively bind fluorescending antibodies as demonstrated by flow cytometric analysis, we immobilized particles
with activating antibodies (1 µg/mL) against human CD3 and CD28 and analyzed their effect on human T cells by light microscopy. As shown in Figure 4a, purified T cells alone exhibit a round shape and an equal distribution throughout the cell culture plate. The conventional T cell stimulation by platebound antibodies in the control sample results in the characteristic pattern of activated T cells (32) (Figure 4b). T cell activation induces the formation of cell clusters and changed cell morphology. Upon contact with particle-antibody conjugates (Figure 4c), the same effects can be observed as compared with conventional plate-bound antibodies confirming a proper T cell activation by particle-antibody conjugates. Interestingly, endocytosis of the particles by the human T cells was not observed in contrast to human monocytes, which show strong endocytotic activity when incubated with the particles (data not shown). Assessment of T Cell Stimulation. After antibody immobilization on the particles, we furthermore studied the influence of the particle surface properties on the ability of the antibodies to stimulate human CD4 memory T cells as monitored by their expression of the activation markers CD69 and CD25 and by their proliferation upon contact with the particle-antibody conjugates. The amount of activated T cells expressing the T cell activation markers CD69 and CD25 was determined by specific labeling of the T cells with fluorescent antibodies and further flow cytometric analysis of T cells on days 2, 3, and 4 after contact with particle-antibody conjugates. As already shown in other studies, expression of CD69 in activated T cells starts earlier than that of CD25 (33, 34). Figure 5 confirms successful cell activation in case of samples 1 and 2, whereas samples 3 and 4 (higher molecular weight of PEGMEMA) comprise significantly less activated cells. As all different particles (samples 1-4) were immobilized with the same amounts of antibodies, a comparable antibody density on the surface of the different particle is supposed. Therefore, the lower capacity of samples 3 and 4 to activate T cells possibly occurs due to restricted antibody-cell contact because longer PEGMEMA chains on the particle surface may prevent contact
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Figure 3. Flow cytometry analysis was performed for PS-PGMA particles (sample 1). (a) Forward scatter (FF) and sideward scatter (SS) of the PS-PGMA particles are plotted on a logarithmic scale, and the region of interest was selected based on the size and surface structure of the particles. Fluorescence signals in the FITC (b) or PE (c) channel of PS-PGMA particles alone or for PSPGMA particles with immobilized fluorescence-labeled anti-CD3-FITC (d) and anti-CD28-PE (e) are shown for the population selected in (a).
with the T cells. Of note, particles alone without antibodies on the surface have no influence on T cell activation or proliferation at all, because the measured data for CD69, CD25, and proliferation were similar to the data observed for T cells alone (blank). T cell activation by PS-PGMA particle-antibody conjugates (sample 1) is at least as sufficient as activation by conventional methods. In comparison to plate-bound antibodies (control sample), the frequencies of activated T cells were slightly enhanced after stimulation with particle-antibody conjugates especially at early time points of T cell activation. Since the surface areas of the particles and the cell culture plate in the control samples are identical, the applied polymeric particles may possess a higher density or better cross-linking of CD3 compared to the conventional method. For our study, we used the same antibody concentration that is routinely used for the immobilization of activating antibodies on plastic (1 µg/mL) (25, 35). However, our interest was also to evaluate if different amounts of antibodies on the surface of the particles or on the cell culture plate may have an influence on the activation of the T cells. As shown in Supporting Information Figure S1, the chosen antibody concentration of 1 µg/mL is in the saturating region for an appropriate activation of T cells. Interestingly, higher amounts of antibody did not increase cell activation, but rather showed a tendency to decrease the activation of memory T cells. This observation is in correlation with reports from the literature, that high levels of anti-CD3 antibodies and prolonged T cell receptor stimulation
Figure 4. Light microscopy image of T cell culture: (a) purified nonstimulated T cells, (b) T cells conventionally stimulated with platebound antibodies (control sample), (c) T cells after contact with PSPGMA particles with bound antibodies (note that particle-antibody conjugates are too small to be visualized by light microscopy).
may reduce T cell expansion and increase activation-induced apoptosis (36, 37). Cell proliferation was analyzed by equal distribution of the dye CFSE to daughter cells after cell division. The corresponding fluorescence signal was investigated by flow cytometry. Analysis of cell proliferation revealed similar tendencies (Figure 6) and, therefore, is in line with the results from the determination of activation markers. In addition, we compared the proliferation of T cells stimulated with plate-bound antibodies to CD3 or with antibodies bound to different particles with the effects of commercially available Dynabeads CD3/CD28 T Cell Expander beads on T cell proliferation. Dynabeads are 4.5 µm
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Figure 5. Mean fluorescence intensity of cells exhibiting the T cell activation markers CD69 (a) and CD25 (b) after contact with particlebound antibodies. Sample numbers correspond to Table 1. The control represents conventional activation of T cells with plate-bound antibodies to CD3 and soluble anti-CD28. The blank denotes nonstimulated T cells without contact to antibodies or particles.
Figure 6. Proliferation of cells after contact with particle-bound antibodies. Sample numbers correspond to Table 1. The control represents conventional activation of T cells with plate-bound antibodies to CD3 and soluble anti-CD28. Blank denotes nonstimulated T cells without contact to antibodies or particles. Dynabeads represents T cells stimulated with Dynabeads CD3/CD28 T Cell Expander beads.
superparamagnetic microparticles with covalently bound antibodies available for different applications. Here, we used CD3/ CD28 T Cell Expander Dynabeads for the expansion of human T cells, with antibodies to CD3 and CD28 coupled to the beads. Of note, the Dynabeads indeed showed an increased proliferation at early time points but reaching a plateau at later time points.
CONCLUSIONS Up to now, most methods for T cell expansion are timeconsuming, and large amounts of cells are needed. Improvements were achieved by using Dynabeads for the expansion of human T cells (38), but one potential disadvantage of these particles are surfactant residues. With regard to an application in complex biological systems, the negative effects of surfactants on skin permeability (39), on cell membrane structure (40), or on their cytotoxic effects (41) should be considered. Besides, normal microparticles must be separated from the cell population before flow cytometry analysis due to their large size. The benefit of Dynabeads for the expansion of T cells is undoubted, and this technique is now routinely used for the expansion of distinct T cell subsets with regulatory functions (42). We could also observe a robust proliferation induced by the Dynabeads, but excessive proliferation is often linked to a decreased
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differentiation of cells (43). Indeed, we observed reduced cytokine levels in T cells stimulated with Dynabeads (data not shown). Therefore, other tools for specific applications such as expansion of highly differentiated effector T cells would be desirable. To overcome those limitations, surfactant-free emulsion polymerization was used to synthesize poly(styrene-coglycidyl methacrylate) particles. Employing this technique, particles with size in the submicrometer range could be obtained. The use of glycidyl methacrylate as a functional co-monomer ensured incorporation of reactive epoxy-groups into particle surface. The use of poly(ethylene glycol) methyl ether methacrylate (PEGMEMA) macromonomers in the polymerization process improved the colloidal stability of particles. Additionally, the size of polymer colloids could be adjusted by varying the PEGMEMA molecular weight. Active epoxy groups on the particle surface were applied for immobilization of antibodies to CD3 and CD28 by covalent bonding. By flow cytometric analysis, we could demonstrate that more than 95% of the particles effectively bound the antibodies. These particle-antibody conjugates were used for stimulation of human CD4 memory T cells. Effective T cell responses after contact with particle-antibody conjugates were assessed by light microscopy of the T cells showing a characteristic pattern of activation. Furthermore, T cell activation was observed by evaluating the expression of the activation markers CD69 and CD25 and cell proliferation. For the PGMA particle-antibody conjugates, T cell activation is at least as sufficient as activation by conventional methods. Interestingly, at early time points frequencies of activated T cells were enhanced after stimulation with PGMA particle-antibody conjugates as compared with conventional activation (25 MFI vs 17.6 MFI for CD69 and 82.8 MFI vs 72 MFI for CD25). At higher PEG chain lengths, cell activation with particle-antibody conjugates was less efficient, possibly due to restricted antibody-cell contact. In summary synthesized particle-antibody conjugates can be used as an alternative for other techniques of T cell activation and therefore may have potential for therapeutic applications.
ACKNOWLEDGMENT Katja Thu¨mmler and Nadine Ha¨ntzschel contributed equally to this work. The authors thank Dr. Iryna Prots and Dr. Wolfgang Kuon for helpful discussions and comments. This work was supported by Deutsche Forschungsgemeinschaft (DFG: SCHU 786/8-1, SK 59/4-1 and SFB 287). Supporting Information Available: T cell activation (CD25 expression) is compared in response to stimulation with different amounts of antibodies bound to the particles (sample 1) or to the cell culture plate (control). This material is available free of charge via the Internet at http://pubs.acs.org.
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