METHOD FOR ACTIVATING DENDRITIC CELLS

Abstract

It is provided a method for activating dendritic cells in vitro, comprising the following steps: a) providing a substrate, wherein a surface of the substrate comprises at least one poly(glycidyl ether) derivative according to general formula (i); b) contacting the substrate with a dendritic cell in an isosmotic aqueous solution or buffer.

Claims

1. A method for activating dendritic cells in vitro or in vivo, comprising the following steps: a) providing a substrate, wherein a surface of the substrate comprises at least one poly(glycidyl ether) derivative according to general formula (I) ##STR00009## wherein R.sup.1 and R.sup.2=independently from each other H, CH.sub.3, CH.sub.2CH.sub.3, CHCH.sub.2, CH.sub.2CH.sub.2CH.sub.3, CH(CH.sub.3).sub.2, CH.sub.2CHCH.sub.2, CH.sub.2CCH, CH.sub.2CH.sub.2CH.sub.2CH.sub.3, C(CH.sub.3).sub.3, CH.sub.2CH(CH.sub.2CH.sub.3)CH.sub.2CH.sub.2CH.sub.2CH.sub.3, CH.sub.2(CH.sub.2).sub.6CH.sub.3, CH.sub.2(CH.sub.2).sub.8CH.sub.3, C.sub.6H.sub.5, CH.sub.2C.sub.6H.sub.5, C.sub.6H.sub.4CH.sub.3, or C.sub.6H.sub.4OCH.sub.3, R.sup.3?H, Br, Cl, OC.sub.1-C.sub.20-alkyl, or OC.sub.6-C.sub.20-aryl, R.sup.4=a photo-reactive compound according to any of general formulae (II) to (V), wherein the photo-reactive compound is linked to the oxygen atom next to residue R.sup.4 directly or via a linker molecule: ##STR00010## wherein R.sup.5 is any of the following residues covalently bound to a carbon atom of the structures having general formulae (II) to (V): ##STR00011## x=0 to 1000, y=1 to 1000, and z=1 to 100; and b) contacting the substrate with a dendritic cell in an isosmotic aqueous solution or buffer.

2. The method according to claim 1, wherein the poly(glycidyl ether) derivative forms a coating on the substrate, wherein the coating fulfils at least one of the following criteria a) a dry thickness lying in range of from 2 nm to 50 nm; b) a static water contact angle lying in a range of from 65? to 85?; c) a roughness lying in a range of from 0.5 nm to 10 nm.

3. The method according to claim 1, wherein R.sup.1 is CH.sub.3 and R.sup.2 is CH.sub.2CH.sub.3.

4. The method according to claim 3, wherein a ratio between x and y lies in a range of from 50:50 to 0:100, wherein a ratio between z and a sum of x and y lies in a range of from 1:100 to 5:100.

5. The method according to claim 1, wherein the repeating units carrying residue R.sup.4 are statistically distributed over the poly(glycidyl ether) derivative.

6. The method according to claim 1, wherein the repeating units carrying residue R.sup.4 are present in the poly(glycidyl ether) derivative as at least one block.

7. The method according to claim 1, wherein the poly(glycidyl ether) derivative is present on the surface of the substrate in form of a gel or a brush.

8. The method according to claim 1, wherein the dendritic cell is chosen from the group consisting of monocyte-derived dendritic cells, myeloid dendritic cells, plasmacytoid dendritic cells, Langerhans cells, Kupffer cells, and subpopulations of the before-mentioned dendritic cells.

9. A method for activating dendritic cells in vitro, the method comprising contacting dendritic cell with a substrate, wherein a surface of the substrate comprises at least one poly(glycidyl ether) derivative according to general formula (I) ##STR00012## wherein R.sup.1 and R.sup.2=independently from each other H, CH.sub.3, CH.sub.2CH.sub.3, CHCH.sub.2, CH.sub.2CH.sub.2CH.sub.3, CH(CH.sub.3).sub.2, CH.sub.2CHCH.sub.2, CH.sub.2CCH, CH.sub.2CH.sub.2CH.sub.2CH.sub.3, C(CH.sub.3).sub.3, CH.sub.2CH(CH.sub.2CH.sub.3)CH.sub.2CH.sub.2CH.sub.2CH.sub.3, CH.sub.2(CH.sub.2).sub.6CH.sub.3, CH.sub.2(CH.sub.2).sub.8CH.sub.3, C.sub.6H.sub.5, CH.sub.2C.sub.6H.sub.5, C.sub.6H.sub.4CH.sub.3, or C.sub.6H.sub.4OCH.sub.3, R.sup.3?H, Br, Cl, OC.sub.1-C.sub.20-alkyl, or OC.sub.6-C.sub.20-aryl, R.sup.4=a photo-reactive compound according to any of general formulae (II) to (V), wherein the photo-reactive compound is linked to the oxygen atom next to residue R.sup.4 directly or via a linker molecule: ##STR00013## wherein R.sup.5 is any of the following residues covalently bound to a carbon atom of the structures having general formulae (II) to (V): ##STR00014## x=0 to 1000, y=1 to 1000, and z=1 to 100.

10. The method according to claim 9, wherein the activated dendritic cells are used as growth factor source.

11. A method for treatment of a patient in need thereof, wherein the method comprises extracting dendritic cells from the patient, activating the extracted dendritic cells by contacting the extracted dendritic cells with a substrate, and re-implanting the activated dendritic cells to the patient in their activated state, wherein a surface of the substrate comprises at least one poly(glycidyl ether) derivative according to general formula (I) ##STR00015## wherein R.sup.1 and R.sup.2=independently from each other H, CH.sub.3, CH.sub.2CH.sub.3, CHCH.sub.2, CH.sub.2CH.sub.2CH.sub.3, CH(CH.sub.3).sub.2, CH.sub.2CHCH.sub.2, CH.sub.2CCH, CH.sub.2CH.sub.2CH.sub.2CH.sub.3, C(CH.sub.3).sub.3, CH.sub.2CH(CH.sub.2CH.sub.3)CH.sub.2CH.sub.2CH.sub.2CH.sub.3, CH.sub.2(CH.sub.2).sub.6CH.sub.3, CH.sub.2(CH.sub.2).sub.8CH.sub.3, C.sub.6H.sub.5, CH.sub.2C.sub.6H.sub.5, C.sub.6H.sub.4CH.sub.3, or C.sub.6H.sub.4OCH.sub.3, R.sup.3=H, Br, Cl, OC.sub.1-C.sub.20-alkyl, or OC.sub.6-C.sub.20-aryl, R.sup.4=a photo-reactive compound according to any of general formulae (II) to (V), wherein the photo-reactive compound is linked to the oxygen atom next to residue R.sup.4 directly or via a linker molecule: ##STR00016## wherein R.sup.5 is any of the following residues covalently bound to a carbon atom of the structures having general formulae (II) to (V): ##STR00017## x=0 to 1000, y=1 to 1000, and z=1 to 100

12. The method according to claim 11, wherein said treatment is a treatment for regeneration of skin, heart, cartilage, joint, liver and/or brain, or for wound healing.

13. The method according to claim 11, wherein said treatment is a treatment in enhancing the patient's natural tissue repair mechanism or for enhancing or regulating the patient's natural immune response.

14. The method according to claim 11, wherein the substrate is applied in form of an implant.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Further details of aspects of the proposed solution will be explained in the following with respect to exemplary embodiments and accompanying Figures.

[0054] FIG. 1A shows the general chemical structure of poly(glycidyl ether) (PGE) copolymers and schematic structures of PGE brush and PGE gel coatings.

[0055] FIG. 1B shows the dry thickness of PGE coatings on PS coated silicon wafer model substrates determined by spectroscopic ellipsometry.

[0056] FIG. 1C shows static water contact angle (CA) of PGE coatings on PS coated silicon wafer model substrates determined by goniometry.

[0057] FIG. 2A shows a representative surface roughness profile of a PGE brush 1:3 on PS coated silicon wafer model substrates measured by atomic force microscopy (AFM) in water at standard cell culture temperature of 37? C.

[0058] FIG. 2B shows a representative surface roughness profile of a PGE gel 1:3 on the same substrate under the same measuring conditions as in FIG. 2A.

[0059] FIG. 2C shows a representative surface roughness profile of a PGE gel 1:7 (C) on the same substrate under the same measuring conditions as in FIG. 2A.

[0060] FIG. 3A shows a quantitative histogram of the coating deformation of a PGE gel 1:3 and a PGE brush 1:3 coating on PS coated silicon wafer model substrates measured by atomic force microscopy (AFM) in water at standard cell culture temperature of 37? C.

[0061] FIG. 3B shows a quantitative histogram of the elastic DMT modulus of the PGE gel and the PGE brush coating of FIG. 3A under the same measuring conditions as in FIG. 3A.

[0062] FIG. 3C shows a quantitative histogram of the adhesive force of the PGE gel and the PGE brush coating of FIG. 3A under the same measuring conditions as in FIG. 3A.

[0063] FIG. 4A shows dot plots of FACS analyses of DCs generated from primary monocytes in PBMCs of n=5 healthy donors indicating an increase of DC-specific markers by polymers in flow cytometry.

[0064] FIG. 4B shows the mean fluorescence intensity (Fl) and percentage of positive cells of one representative donor of the samples of FIG. 4A.

[0065] FIG. 5A shows an upregulation of CD86 on the surface of human DCs by gel polymers after blood derived DCs were cultured on different polymer-coated plates.

[0066] FIG. 5B shows an upregulation of CD40 by the same gel polymers as in FIG. 5A.

[0067] FIG. 5C shows an upregulation of HLA-DR by the same gel polymers as in FIG. 5A.

[0068] FIG. 5D shows an upregulation of CD14 by the same gel polymers as in FIG. 5A.

[0069] FIG. 6 shows the release of transforming growth factor (TGF)-?1 by DCs cultured on different polymer-coated plates.

[0070] FIG. 7 shows the release of epidermal growth factor (EGF) by DCs cultured on different polymer-coated plates.

DETAILED DESCRIPTION

[0071] The Figures will be explained with respect to an exemplary embodiment.

Polymer Synthesis

[0072] Reports on the activation of immune cells, particularly dendritic cells (DCs), through natural or synthetic culture substrate materials are rather scarce. Most notably, chitosan- and PLGA-based culture substrates have shown to promote the differentiation and activation of DCs, whereas most conventional culture materials do not significantly affect DC maturation. As fully synthetic polymer materials, poly(glycidyl ether) (PGE) coatings are non-toxic, biocompatible and have already demonstrated to facilitate the adhesion and proliferation of mammalian as well as primary human cells.

[0073] To study their potential for DC activation, the inventors prepared PGE brush and gel coatings on polystyrene (PS) tissue culture substrates using the grafting-to method. The monomer-activated anionic ring-opening polymerization (MA-AROP) of the two comonomers glycidyl methyl ether (GME) and ethyl glycidyl ether (EGE) allows for the synthesis of PGEs with different hydrophilicities via the adjustment of the GME:EGE comonomer ratio. As illustrated in Scheme 1, the inventors synthesized two types of photo-reactive PGE terpolymers comprising benzophenone (BP) moieties, which can be utilized to covalently immobilize and/or crosslink PGEs on PS culture substrates via convenient UV irradiation. PGE block copolymers bearing a short BP anchor block were synthesized via the sequential MA-AROP of GME, EGE and the photo-reactive comonomer 4-(2,3-epoxypropoxy) benzophenone (EBP) (Scheme 1a). In contrast, statistical terpolymers bearing BP moieties along the PGE backbone were synthesized via the two-step post-functionalization of allyl-bearing terpolymers composed of GME, EGE and allyl glycidyl ether (AGE) (Scheme 1b).

##STR00007## ##STR00008##

[0074] The targeted as well as experimentally determined composition and molecular weight data of all synthesized PGE terpolymers are summarized in Table 1. For the fabrication of brush coatings, PGE block copolymers B1 and B2 with GM E:EGE comonomer ratios of 1:1 and 1:3, respectively, and in each case an average of 5 EBP repeating units as well as a number average molecular weight (M.sub.n) of 30 kDa were targeted (Table 1, entry 1-2). Statistical PGE terpolymers G1, G2 and G3, with GME:EGE comonomer ratios of 1:1, 1:3 and 1:7, respectively, and in each case an average of 1.5 mol-% AGE repeating units as well as an M.sub.n of 40 kDa were targeted to fabricate hydrogel coatings (Table 1, entry 3-5).

TABLE-US-00001 TABLE 1 Targeted (theoretical) and experimental molecular weight data and comonomer composition of PGE block copolymers B1 and B2 and statistical PGE terpolymers G1, G2 and G3 determined by gel permeation chromatography (GPC) and .sup.1H-NMR spectroscopy. M.sub.n, theor. M.sub.n, GPC GME:EGE GME:EGE BP units BP units PGE [kDa] [kDa] PDI.sub.GPC (theor.) (NMR) (theor.) (NMR) B1 30 28.4 1.21 1:1 1.1:1.0 5.0 4.2 B2 30 27.1 1.20 1:3 1.0:2.8 5.0 4.8 G1 40 47.5 1.19 1:1 1.0:1.0 1.5% 1.2% G2 40 45.1 1.28 1:3 1.0:2.8 1.5% 1.2% G3 40 46.9 1.25 1:7 1.0:6.0 1.5% 1.1%

[0075] As shown in Table 1, the molecular weights of polymers B1-G3 determined by gel permeation chromatography (GPC) are close to the targeted values and all polymers display adequately narrow polydispersity indices (PDI), which indicates the controlled nature of the MA-AROP process. Further, the comonomer compositions determined via .sup.1H-NMR spectroscopy closely resemble the targeted comonomer ratios. As compared to the aimed at contents, slightly lower experimental yields in terms of BP functionalization can be attributed to the decreased reactivity of the EBP monomer in the sequential MA-AROP of PGE block copolymers (B1-B2) as well as to the incomplete, added up yields during the two-step post-functionalization process of statistical PGE terpolymers (G1-G3). Representative .sup.1H-NMR spectra of the PGE block copolymers and the statistical PGE terpolymers have also been recorded (not shown).

Coating and Surface Characterization

[0076] Two different grafting-to protocols were utilized to immobilize PGE coatings onto PS culture substrates. PGE brushes were fabricated via the adsorption of PGE block copolymers onto PS substrates from dilute aqueous-ethanolic solution (c.sub.EtCH=32% (B1) and 45% (B2) (v/v)) at a polymer concentration of 62.5 ?g mL.sup.?1. In general, PS substrates were incubated in PGE solutions for 1 h, washed with water and irradiated with UV light in the dry state to covalently immobilize the self-assembled brushes on the PS surfaces via their EBP anchor block. After extraction of unbound polymer chains with ethanol overnight, stable PGE brush layers were obtained. PGE gels were fabricated by spin coating statistical PGE terpolymers onto PS substrates from ethanolic solution at a polymer concentration of 1% (w/w). The layers were immobilized and crosslinked via UV irradiation and extracted with ethanol for 5 days until the thickness of the coatings adopted steady values. The thickness and wettability of the coatings, which were determined on PS-coated silicon wafer model substrates using spectroscopic ellipsometry (SE) and static water contact angle (CA) measurements, respectively, are summarized in FIG. 1.

[0077] FIG. 1A shows the general chemical structure of poly(glycidyl ether) (PGE) copolymers and schematic structures of PGE brush and PGE gel coatings. FIG. 1B shows the dry layer thickness of PGE brush and gel coatings determined by spectroscopic ellipsometry (SE) on PS-coated silicon wafer model substrates. FIG. 1C shows the wettability of PGE brush and gel coatings determined by static water contact angle (CA) measurements on PS-coated silicon wafer model substrates.

[0078] As shown in FIG. 1B, PGE brush coatings B1 and B2 with an average dry thickness of about 3.5 nm were obtained. Under aqueous conditions, the grafting density of PGE chains (M.sub.n=30 kDa) is high enough for the polymers to adopt a brush-like conformation and to fully cover the PS substrate surface. In contrast, with average values of around ?15 nm, the dry thickness values of PGE gels G1, G2 and G3 were significantly higher than for the PGE brushes (FIG. 1B). As illustrated in FIG. 1C, the static water CAs of the coatings range from about 65 to 80? and reflect the hydrophilicity of the coatings, which was adjusted via the GME:EGE comonomer ratio of the statistical as well as block copolymers. Notably, the wettability of PGE coatings with comparable comonomer compositions is higher for PGE gels, which is reflected in the slightly lower water CAs. This is likely due to the higher swelling capability and water uptake capacity of the significantly thicker PGE gels. Nevertheless, the CAs of the two types of PGE coatings are in the same realm and, in general, CA differences are not necessarily commensurable for coatings with different architectures, such as assembled brushes and gel-like networks.

[0079] To determine the morphology and mechanical properties of the coated culture substrates, the PGE coatings were characterized by atomic force microscopy (AFM) using quantitative nanomechanical mapping (QNM). Representative topological images of PGE brush and PGE gel coatings on PS-coated silicon wafer model substrates are illustrated in FIG. 2A to 2C.

[0080] FIG. 2A shows a height cross section profile of brush B2 coatings on PS-coated silicon wafer model substrates. FIG. 2B shows a comparable height cross section profile for PGE gel G2 coatings, and FIG. 2C for PGE gel G3 coatings. All height cross section profiles were measured in water at 37? C. via AFM in quantitative nanomechanical mapping (QNM) mode.

[0081] Whereas PGE brushes exhibit a very homogeneous and flat morphology forming a well-ordered layer with a roughness in the range of 1 nm, PGE gels present a slightly rougher and laterally more inhomogeneous surface topology. The slightly higher roughness of PGE gels is due to their higher thickness and crosslinked structure. The more hydrophilic and, hence, swollen nature of PGE gels with a comonomer ratio of 1:3 (G2) (FIG. 2B) is further reflected in a morphologically more inhomogeneous, patch-like structure, as compared to PGE gels with a comonomer ratio of 1:7 (G3) (FIG. 2C).

[0082] It is worth noting that the PS-coated model substrates are very flat compared to the rather rough commercial PS tissue culture substrates. The reason for this is the spin coating process, which was applied for the manufacture of PS-coated silicon wafers. The morphology of the bare PS substrates and PGE-coated silicon wafers as well as common PS culture dishes, respectively, has been further analyzed (not shown).

[0083] A comparison between the mechanical properties of the PGE brushes and gels, more precisely, the deformation and elastic modulus of the coatings as well as the adhesive force between the coatings and the AFM tip, is shown in FIGS. 3A to 3C.

[0084] FIG. 3A shows representative deformation depth histograms, FIG. 3B representative elastic modulus histograms, and FIG. 3C representative adhesion histograms of PGE gel G2 and PGE brush B2 coatings measured in water at 37? C. via AFM in quantitative nanomechanical mapping (QNM) mode.

[0085] As indicated by the deformation histograms in FIG. 3A, PGE gels exhibit a higher deformability than PGE brushes with a similar comonomer composition. Considering that the same indentation force was applied on both surfaces, this is also reflected in a lower elastic modulus of PGE gels, as illustrated in FIG. 3B.

[0086] Both results on deformation and elastic modulus affirm the brush- and gel-like architecture of the two types of coatings, respectively. Furthermore, the higher adhesive forces between the gel coatings and the AFM tip, which are depicted and compared to PGE brushes in FIG. 3C, indicate a more efficient adhesion of the PGE gels to the AFM tip, which is presumably due to its enhanced hydrophobic interactions with the thicker network-like PGE gel coatings.

[0087] Summarizing, PGE gels are more deformable (FIG. 3A) and exhibit a lower elastic modulus (FIG. 3B) than PGE brushes, which is due to the crosslinked, thicker, and, hence, softer structure of PGE gels. The elastic moduli of the coatings are further about one order of magnitude lower than the elastic modulus of standard PS or TOPS culture substrates, making PGE coatings rather soft cell culture substrates. The higher roughness and network-like structure of the gels further increases the adhesive force between the surface and the AFM tip through hydrophobic polymer-tip interactions (FIG. 3C).

[0088] In general, all investigated brush and gel coatings, respectively, exhibit comparable mechanical properties, which are independent on their comonomer composition within the investigated range. Detailed comparisons between the morphology and the mechanical properties of PGE brush and gel coatings with different comonomer compositions have been established (not shown). In summary, the inventors successfully fabricated PGE-based self-assembled brush as well as crosslinked gel coatings on PS culture substrates. In the following, the potential of these coatings for the activation of DCs will be examined in order to discern their applicability in the field of immunoengineering.

Activation of Dendritic Cells (DCs)

[0089] In a first series of cell culture experiments, DCs were generated on culture plates coated with different polymers. To evaluate the potential of the synthetic polymer coatings to activate immune cells in general, the crucial immune cell, the DC, which controls the innate immune response and initiates the adaptive immune response, was selected. The range of activation in DCs is known to be measured by the level of cell surface marker expression involved in the antigen presentation processes.

[0090] Thus, the costimulatory molecules CD40 and CD86, the maturation marker CD14 for the DC precursor monocyte and the human MHC-II associated molecule HLA-DR were detected. For this purpose, human blood derived DCs were cultured on different polymer-coated plates and analyzed for surface expression of costimulatory CD40 and CD86, HLA-DR and monocyte marker CD14

[0091] The results are summarized in FIGS. 4A and 4B. After generation of DCs in presence of GM-CSF and IL-4, according to a scientifically accepted protocol, DCs showed different signs of maturation. After having been cultured on PGE coatings, DCs exhibit morphological characteristics of DCs, such as an enlarged surface area and numerous DC protrusions. Compared to DCs harvested from commercially available cell culture plate controls (Nunclon Delta), activated DCs enhanced cell surface marker expression more pronounced as detected by fluorescence-activated cell scanning (FACS).

[0092] Both parameters, fluorescence intensity (Fl) and % positive cells, were upregulated in both PGE brushes and gels (FIGS. 4A and 4B). For CD86, the range of upregulation in fluorescence intensity was higher than for CD40 and HLA-DR. Since the cultured cells resembled a more monocytic phenotype on thin crosslinked gels with a comonomer ratio of 1:1 (G1) and a thickness of 2.5 nm, as evidenced by CD14 values, these conditions were omitted in the next series of experiments.

[0093] In the next set of cell culture experiments, samples from different human donors were analyzed. The fold change of the mean fluorescence intensity (MFI) revealed that PGE gels increase DC specific markers more than PGE brushes and control plates do. This is summarized in FIGS. 5A to 5D showing mean fold change in expression (MFI) of costimulatory molecules CD86, CD40 and HLA-DR as well as monocyte marker CD14 of DCs cultured on B2 brush and G2 and G3 gel coatings determined by flow cytometry. Average MFIs of DCs generated from primary monocytes in PBMCs of healthy donors (n=5) are plotted together with their standard deviations (error bars). Differences to DCs cultured on control substrates (Nunclon) were analyzed for statistical significance (*, p<0.05; **, p<0.01).

[0094] Besides Nunclon, also Corning denotes a non-coated surface serving as negative control. The antigen densities of CD86, CD40 and HLA-DR were strongly enhanced by PGE gels. Moreover, the expression of the maturation marker CD14 was not significantly altered compared to control substrates. Hence, all conditions could be regarded as DC generating. Under in vivo conditions, the activation of DCs is only achieved by exposure to pathogenic organisms, such as bacteria, protozoa, viruses or fungi, or by exposure to xenobiotica. Numerous publications exist in literature, which deal with the secretion of cytokines, chemokines and growth factors in DCs after exposure to total pathogens, pathogen associated molecular pattern (PAMPS), and chemicals.

[0095] However, the present embodiment shows that also synthetic polymers can modulate the expression of immune factors cytokines and chemokines. As only little is known on the release of growth factors by DCs in contact with synthetic polymers, these soluble factors released by surface activated DCs were analyzed in the next series of experiments.

[0096] In parallel to conditions identified in the FACS study, it was possible to show that DCs cultured on B2 brush and G2 and G3 gel coatings released transforming growth factor (TGF)-?1 (FIG. 6) and epidermal growth factor (EGF) (FIG. 7). The proteins in the supernatant of DCs generated from primary monocytes in PBMCs of healthy donors (n=5) were analyzed by enzyme-linked immunosorbent assay (ELISA) after 4 days of culture and are plotted together with their standard deviations (error bars). Differences in TGF-?1 and EGF expression compared to DCs cultured on control substrates (Nunclon or Nunc) were analyzed for statistical significance (*, p<0.05; **, p<0.01).

[0097] For TGF-?1 and EGF, the amounts found in the supernatant of DCs were significantly enhanced compared to control plates. Moreover, the amounts of both growth factors were evidently higher when DCs were cultured on PGE gels than on PGE brushes (FIGS. 6 and 7). These results clearly indicate that DCs are efficiently activated by PGE-based cell culture coatings and that their activation is induced by the synthetic PGE polymer material. There are no reports so far on polyether-based materials, such as PGE coatings, being able to activate DCs. Hence, PGE coatings are very promising materials in the field of immunoengineering.

Experimental Details

Materials

[0098] Glycidyl methyl ether (GME, ?85%), ethyl glycidyl ether (EGE, ?98%), 4-hydroxybenzophenone (4-HBP, ?98%) and cysteamine hydrochloride (Cys-HCl, ?98%) were purchased from TCI GmbH (Eschborn, Germany). Allyl glycidyl ether (AGE, ?99%), tetraethylammonium bromide (N(Oct).sub.4Br, 98%), triisobutylaluminum (Al(i-Bu).sub.3, 1.0 M in hexanes), 4-benzoylbenzoic acid (4-CBP, 99%), 2,2-dimethoxy-2-phenylacetophenone (DMPA, 99%), N,N-dimethylformamide (DMF, 99.8%, anhydrous), benzophenone (BP, 98%), sodium lumps (Na), toluene (99.8%), methanol (99.8%) and ethanol (tech.) were supplied by Merck KGaA/Sigma Aldrich (Darmstadt/Steinheim, Germany). Epichlorohydrin (ECH, 98%) was purchased from abcr GmbH (Karlsruhe, Germany). N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC-HCl, ?99%), sodium hydroxide (NaOH, ?98%), sodium sulfate (Na.sub.2SO.sub.4, 99%), molecular sieve (3 ?) and calcium hydride (CaH.sub.2, 93%) were supplied by Carl Roth GmbH+Co. KG (Karlsruhe, Germany). Diethyl ether (Et.sub.2O, 99%) was purchased from VWR Chemicals (Leuven, Belgium). Silicon wafers were supplied by Silchem GmbH (Freiberg, Germany). Toluene was pre-dried via the solvent system MB SPS-800 from M. Braun GmbH (Garching, Germany), refluxed with elemental sodium and a pinch of BP and subsequently distilled on activated molecular sieve directly before use. GME, EGE and AGE were dried over CaH.sub.2, distilled and stored over activated molecular sieve before use. Et.sub.2O and ethanol were distilled before use to remove impurities.

Methods

[0099] .sup.1H and .sup.13C NMR spectra were recorded on a Joel ECX at 400 or 500 and 100 or 126 MHz, respectively, and processed with the software MestReNova (version 7.1.2). Chemical shifts were reported in ? (ppm) and referenced to the respective deuterated solvent peak (CDCl.sub.3). GPC was conducted on an Agilent 1100 Series instrument in THE as the eluent at concentrations of 3.5 mg mL.sup.?1 and a flow rate of 1 mL min.sup.?1 at 25? C. Three PLgel mixed-C columns (Agilent, Waldbronn, Germany) with dimensions of 7.5?300 mm and a particle size of 5 ?m were used in-line with a refractive index detector. Calibration was performed with polystyrene (PS) standards from PSS (Mainz, Germany) and calculation was performed with PSS Win-GPC software. ESI-ToF mass spectral data were obtained on an Agilent 6210 ESI-TOF (Agilent Technologies, Santa Clara, CA, USA) spectrometer at flow rates of 4 mL min.sup.?1 and a spray voltage of 4 kV.

[0100] Spin-coating was performed using a WS-650-23 spin-coater from Laurell Technologies (North Wales, PA, USA). Silicon wafers (11?11 mm) were coated at 3000 rpm for 60 s using 50 ?L of a 1% (w/w) solution of PS in toluene. Static water contact angles (CAs) were measured with an OCA contact angle system from DataPhysics Instruments GmbH (Filderstadt, Germany) and fitted with the software package SCA202 (version 3.12.11) using the sessile drop method. CAs were determined before and after surface functionalization at 20? C. A drop of Milli-Q water (2 ?L) was placed onto the respective surface and CAs were determined with the Young-Laplace model. For each substrate, CAs were measured on at least five different spots to test for the homogeneity of the sample and at least six independent substrates (n=6) to test for reproducibility. The dry layer thickness of the polymer coatings was determined by spectroscopic ellipsometry (SE) at an incident angle of 70? with a SENpro spectroscopic ellipsometer from Sentech Instruments GmbH (Berlin, Germany). The thickness of the SiO.sub.2 layer before spin coating and the additional thickness of the spin-coated PS layers were determined separately using a Cauchy layer for modelling and respective average values of at least five different spots on the surfaces were taken as fixed values for the subsequent modeling of the adsorbed PGE layers. The PGE thickness was measured at wavelengths from 370 nm to 1050 nm and was fitted using a model consisting of the previously measured layers with fixed parameters, a PGE layer with a fixed refractive index of n=1.45 and air as the surrounding medium. For photo-immobilization, samples with adsorbed PGE layers were irradiated with UV light using a UV-KUB 2 (?=365 nm, irradiance=25 mW cm.sup.?2) from Klo? (Montpellier, France) for 160 s, which corresponds to a radiant exposure of 4.0 J cm.sup.?2.

Polymer Synthesis

[0101] The sequential monomer-activated anionic ring-opening polymerization (MA-AROP) of poly(GME-ran.-EGE)-block-poly(EBP) block copolymers B1 and B2 was described in our previous report. [13] The initiator N(Oct).sub.4Br was melted and dried in a flame dried Schlenk flask (100 mL) under high vacuum at 103? C. and dissolved in dry toluene at room temperature. The solution was cooled to 0? C. with an ice bath and the dry monomers GME and EGE were added. The polymerization was initiated via rapid addition of dry Al(i-Bu).sub.3 activator solution. After stirring for 15 min at 0? C., the photo-reactive comonomer EBP was added and the reaction was allowed to proceed for 3 h while warming up to room temperature. The reaction was quenched by adding Milli-Q water (?0.5 mL), stirred for 1 h, dried over Na.sub.2SO.sub.4 for 1 h under stirring and filtered. After removing toluene under reduced pressure, the crude polymers were dissolved in Et.sub.2O and residual initiator salts were precipitated by centrifugation at 0? C. After decanting, Et.sub.2O was evaporated and the block copolymers were dissolved and dialyzed against MeOH for 3 d. PGE block copolymers comprising photo-reactive EBP anchor blocks were protected against light throughout synthesis and workup and stored in stock solutions in ethanol (10 mg mL.sup.?1) until further use.

Poly(GME-ran.-EG E)-block-poly(EBP) (B1)

[0102] N(Oct).sub.4Br=116 mg (0.21 mmol, 1 eq), GME=3.00 mL (33.44 mmol, 157.7 eq), EGE=3.63 mL (33.44 mmol, 157.7 eq), Al(i-Bu).sub.3=0.85 mL (0.85 mmol, 4 eq), EBP=270 mg (1.06 mmol, 5 eq), toluene=30 mL, yield=97%; .sup.1H NMR (500 MHz; CDCl.sub.3): ?(ppm)=7.71 (m, 4H, BP); 7.52-7.43 (m, 3H, BP); 6.92 (m, 2H, BP); 4.19 (m, 1H, BPOCH.sub.2); 4.05 (m, 1H, BPOCH.sub.2); 3.61-3.45 (m, polymer backbone+OCH.sub.2CH.sub.3); 3.33 (s, OCH.sub.3); 1.16 (t, OCH.sub.2CH.sub.3); .sup.13C NMR [.sup.1H] (126 MHz; CDCl.sub.3): ?(ppm)=197.5 (C?O); 165.7 (C(BP)OCH.sub.2-polymer backbone); 138.1 115.4 (BP); 78.9-78.6 (CH.sub.2CH(CH.sub.2OR)O+BPOCH.sub.2), 72.8 (CH.sub.2OCH.sub.3), 70.6 (CH.sub.2OCH.sub.2CH.sub.3), 70.2-69.6 (CH.sub.2CH(CH.sub.2OR)O), 66.7 (OCH.sub.2CH.sub.3); 59.2 (OCH.sub.3); 15.3 (OCH.sub.2CH.sub.3); GPC: M.sub.n=28.4 kg mol.sup.?1, PDI=1.21.

Poly(GME-ran.-EG E)-block-poly(EBP) (B2)

[0103] N(Oct).sub.4Br=120 mg (0.22 mmol, 1 eq), GME=1.50 mL (16.72 mmol, 76.1 eq), EGE=5.45 mL (50.15 mmol, 228.2 eq), Al(i-Bu).sub.3=0.88 mL (0.88 mmol, 4 eq), EBP=279 mg (1.10 mmol, 5 eq), toluene=30 mL, yield=98%; .sup.1H NMR (500 MHz; CDCl.sub.3): ?(ppm)=7.71 (m, 4H, BP); 7.52-7.43 (m, 3H, BP); 6.92 (m, 2H, BP); 4.19 (m, 1H, BPOCH.sub.2); 4.05 (m, 1H, BPOCH.sub.2); 3.61-3.45 (m, polymer backbone+OCH.sub.2CH.sub.3); 3.33 (s, OCH.sub.3); 1.16 (t, OCH.sub.2CH.sub.3); .sup.13C NMR [.sup.1H] (126 MHz; CDCl.sub.3): ?(ppm)=197.5 (C?O); 165.7 (C(BP)OOH.sub.2-polymer backbone); 138.1-115.4 (BP); 78.9-78.6 (CH.sub.2CH(CH.sub.2OR)O+BPOCH.sub.2), 72.8 (CH.sub.2OCH.sub.3), 70.6 (CH.sub.2OCH.sub.2CH.sub.3), 70.2-69.6 (CH.sub.2CH(CH.sub.2OR)O), 66.7 (OCH.sub.2CH.sub.3); 59.2 (OCH.sub.3); 15.3 (OCH.sub.2CH.sub.3); GPC: M.sub.n=27.1 kg mol.sup.?1, PDI=1.20.

[0104] Statistical poly(GME-stat.-EGE-stat.-AC-BP) terpolymers G1, G2 and G3 were synthesized via the MA-AROP and a subsequent 2-step post-modification via thiol-ene chemistry and amidation. First, allyl-functional poly(GME-stat.-EGE-stat.-AGE) terpolymers were synthesized according to the following general procedure. The initiator N(Oct).sub.4Br was melted and dried in a flame dried Schlenk flask (100 mL) under high vacuum at 103? C. and dissolved in dry toluene at room temperature. The solution was cooled to 0? C. with an ice bath and the dry monomers GME, EGE and AGE were added. The polymerization was initiated via rapid addition of dry Al(i-Bu).sub.3 activator solution and stirred for 3 h at 0? C.-rt. The reaction was quenched by adding Milli-Q water (?0.5 mL), stirred for 1 h, dried over Na.sub.2SO.sub.4 for 1 h under stirring and filtered. After removing toluene under reduced pressure, the crude polymers were dissolved in Et.sub.2O and residual initiator salts were precipitated by centrifugation at 0? C. After decanting, Et.sub.2O was evaporated and the block copolymers were dissolved and dialyzed against MeOH for 3 d.

Poly(GME-stat.-EGE-stat.-AGE) (1a)

[0105] N(Oct).sub.4Br=174 mg (0.32 mmol, 1 eq), GME=4.50 mL (50.15 mmol, 157.7 eq), EGE=5.45 mL (50.15 mmol, 157.7 eq), Al(i-Bu).sub.3=1.27 mL (1.27 mmol, 4 eq), AGE=0.26 mL (2.23 mmol, 7 eq), toluene=50 mL, yield=96%; .sup.1H NMR (500 MHz; CDCl.sub.3): ?(ppm)=5.87 (m, OCH.sub.2CHCH.sub.2); 5.27-5.13 (m, OCH.sub.2CHCH.sub.2); 3.98-3.97 (m, OCH.sub.2CHCH.sub.2); 3.61-3.47 (m, polymer backbone+OCH.sub.2CH.sub.3); 3.33 (s, OCH.sub.3); 1.17 (t, OCH.sub.2CH.sub.3); GPC: M.sub.n=30.1 kg mol.sup.?1, PDI=1.15.

Poly(GME-stat.-EGE-stat.-AGE) (2a)

[0106] N(Oct).sub.4Br=184 mg (0.34 mmol, 1 eq), GME=2.30 mL (25.63 mmol, 79.1 eq), EGE=8.40 mL (76.90 mmol, 228.2 eq), Al(i-Bu).sub.3=1.35 mL (1.35 mmol, 4 eq), AGE=0.28 mL (2.36 mmol, 7 eq), toluene=50 mL, yield=94%; .sup.1H NMR (500 MHz; CDCl.sub.3): ?(ppm)=5.87 (m, OCH.sub.2CHCH.sub.2); 5.27-5.13 (m, OCH.sub.2CHCH.sub.2); 3.98-3.97 (m, OCH.sub.2CHCH.sub.2); 3.62-3.47 (m, polymer backbone+OCH.sub.2CH.sub.3); 3.33 (s, OCH.sub.3); 1.17 (t, OCH.sub.2CH.sub.3); GPC: M.sub.n=29.2 kg mol.sup.?1, PDI=1.17.

Poly(GME-stat.-EGE-stat.-AGE) (3a)

[0107] N(Oct).sub.4Br=174 mg (0.32 mmol, 1 eq), GME=4.50 mL (50.15 mmol, 157.7 eq), EGE=5.45 mL (50.15 mmol, 157.7 eq), Al(i-Bu).sub.3=1.27 mL (1.27 mmol, 4 eq), AGE=0.26 mL (2.23 mmol, 7 eq), toluene=50 mL, yield=96%; .sup.1H NMR (500 MHz; CDCl.sub.3): ?(ppm)=5.87 (m, OCH.sub.2CHCH.sub.2); 5.27-5.13 (m, OCH.sub.2CHCH.sub.2); 3.98-3.97 (m, OCH.sub.2CHCH.sub.2); 3.61-3.47 (m, polymer backbone+OCH.sub.2CH.sub.3); 3.33 (s, OCH.sub.3); 1.17 (t, OCH.sub.2CH.sub.3); GPC: M.sub.n=30.1 kg mol.sup.?1, PDI=1.15.

[0108] Statistical poly(GME-stat.-EGE-stat.-AC-BP) terpolymers 1a, 2a and 3a were functionalized with photo-reactive BP units via a two-step post-polymerization protocol according to our previous report. [13] In the first step, the allyl groups were functionalized with cysteamine groups via thio-ene chemistry. In brief, PGE terpolymers 1a, 2a or 3a, Cys-HCl and the photo-initiator DMPA were dissolved in MeOH in a 50 mL reaction vial and purged with Ar for 15 min while protected against light. The reaction mixtures were then irradiated with UV light (Hg lamp, 90 W) for 2 h. The crude products were dialyzed against MeOH for 3 d. After removing the solvent under reduced pressure, the products 1 b, 2b and 3b were obtained as highly viscous pale-yellow solids. In the second step, the amine group bearing PGE block copolymers were functionalized with BP groups via amide coupling. In brief, to solutions of 1b, 2b or 3b and 4-CBP in DMF (10 mL) in a 100 mL round bottom flask were added solutions of EDC-HCl in DMF (5 mL). The mixtures were stirred at room temperature for 24 h. The crude product solutions were diluted with MeOH (20 mL) and dialyzed against MeOH for 3 d. After removing the solvent under reduced pressure, G1, G2 and G3 were obtained as highly viscous pale-yellow liquids. The polymers were subsequently stored in stock solutions in ethanol (10 mg mL.sup.?1) until further use.

Poly(GME-stat.-EGE-stat.-AC) (1b)

[0109] 1a=2 g (6.7 AGE/chain, 1 eq), Cys-HCl=254 mg (2.24 mmol, 5 eq), DMP?=23 mg (0.09 mmol, 0.2 eq), MeOH=10 mL, conversion (AGE)=100%, yield=90%; .sup.1H NMR (500 MHz; CDCl.sub.3): ?(ppm)=3.62-3.42 (m, polymer backbone+OCH.sub.2CH.sub.3); 3.34 (s, OCH.sub.3); 3.12 (m, SCH.sub.2CH.sub.2NH.sub.2); 2.94 (m, SCH.sub.2CH.sub.2NH.sub.2); 2.68 (m, OCH.sub.2CH.sub.2CH.sub.2S); 1.85 (m, OCH.sub.2CH.sub.2CH.sub.2S); 1.17 (t, OCH.sub.2CH.sub.3).

Poly(GME-stat.-EGE-stat.-AC) (2b)

[0110] 2a=2 g (7.1 AGE/chain, 1 eq), Cys-HCl=269 mg (2.37 mmol, 5 eq), DMP?=24 mg (0.09 mmol, 0.2 eq), MeOH=10 mL, conversion (AGE)=100%, yield=93%; .sup.1H NMR (500 MHz; CDCl.sub.3): ?(ppm)=3.61-3.42 (m, polymer backbone+OCH.sub.2CH.sub.3); 3.33 (s, OCH.sub.3); 3.12 (m, SCH.sub.2CH.sub.2NH.sub.2); 2.94 (m, SCH.sub.2CH.sub.2NH.sub.2); 2.68 (m, OCH.sub.2CH.sub.2CH.sub.2S); 1.85 (m, OCH.sub.2CH.sub.2CH.sub.2S); 1.17 (t, OCH.sub.2CH.sub.3).

Poly(GME-stat.-EGE-stat.-AC) (3b)

[0111] 3a=2 g (6.7 AGE/chain, 1 eq), Cys-HCl=254 mg (2.24 mmol, 5 eq), DMP?=23 mg (0.09 mmol, 0.2 eq), MeOH=10 mL, conversion (AGE)=100%, yield=90%; .sup.1H NMR (500 MHz; CDCl.sub.3): ?(ppm)=3.62-3.42 (m, polymer backbone+OCH.sub.2CH.sub.3); 3.34 (s, OCH.sub.3); 3.12 (m, SCH.sub.2CH.sub.2NH.sub.2); 2.94 (m, SCH.sub.2CH.sub.2NH.sub.2); 2.68 (m, OCH.sub.2CH.sub.2CH.sub.2S); 1.85 (m, OCH.sub.2CH.sub.2CH.sub.2S); 1.17 (t, OCH.sub.2CH.sub.3).

Poly(GME-stat.-EGE-stat.-AC-BP) (G1)

[0112] 1b=1.5 g (5.6 Cys-HCl/chain, 1 eq), 4-CBP=318 mg (1.41 mmol, 5 eq), EDC-HCl=270 mg (1.41 mmol, 5 eq), DMF=15 mL, conversion (Cys-HCl)=91%, yield=96%; .sup.1H NMR (500 MHz; CDCl.sub.3): ?(ppm)=7.91 (m, 2H, BP); 7.79-7.75 (m, 4H, BP); 7.58 (m, 1H, BP); 7.46 (m, 2H, BP); 3.61-3.42 (m, polymer backbone+OCH.sub.2CH.sub.3); 3.33 (s, OCH.sub.3); 2.77 (m, SCH.sub.2CH.sub.2NHCOBP); 2.62 (m, OCH.sub.2CH.sub.2CH.sub.2S); 1.82 (m, OCH.sub.2CH.sub.2CH.sub.2S); 1.16 (t, OCH.sub.2CH.sub.3); GPC: M.sub.n=29800 g mol.sup.?1, PDI=1.20.

Poly(GME-stat.-EGE-stat.-AC-BP) (G2)

[0113] 2b=1.5 g (5.5 Cys-HCl/chain, 1 eq), 4-CBP=312 mg (1.38 mmol, 5 eq), EDC-HCl=265 mg (1.38 mmol, 5 eq), DMF=15 mL, conversion (Cys-HCl)=96%, yield=97%; .sup.1H NMR (500 MHz; CDCl.sub.3): ?(ppm)=7.91 (m, 2H, BP); 7.79-7.75 (m, 4H, BP); 7.58 (m, 1H, BP); 7.46 (m, 2H, BP); 3.61-3.42 (m, polymer backbone+OCHCH.sub.3); 3.33 (s, OCH.sub.3); 2.77 (m, SCH.sub.2CH.sub.2NHCOBP); 2.62 (m, OCH.sub.2CH.sub.2CH.sub.2S); 1.82 (m, OCH.sub.2CH.sub.2CH.sub.2S); 1.16 (t, OCH.sub.2CH.sub.3); GPC: M.sub.n=28100 g mol.sup.?1, PDI=1.18.

Poly(GME-stat.-EGE-stat.-AC-BP) (G3)

[0114] 3b=1.5 g (5.6 Cys-HCl/chain, 1 eq), 4-CBP=318 mg (1.41 mmol, 5 eq), EDC-HCl=270 mg (1.41 mmol, 5 eq), DMF=15 mL, conversion (Cys-HCl)=91%, yield=96%; .sup.1H NMR (500 MHz; CDCl.sub.3): ?(ppm)=7.91 (m, 2H, BP); 7.79-7.75 (m, 4H, BP); 7.58 (m, 1H, BP); 7.46 (m, 2H, BP); 3.61-3.42 (m, polymer backbone+OCH.sub.2CH.sub.3); 3.33 (s, OCH.sub.3); 2.77 (m, SCH.sub.2OH.sub.2NHCOBP); 2.62 (m, OCH.sub.2OH.sub.2OH.sub.2S); 1.82 (m, OCH.sub.2OH.sub.2OH.sub.2S); 1.16 (t, OCH.sub.2CH.sub.3); GPC: M.sub.n=29800 g mol.sup.?1, PDI=1.20.

Surface Preparation and Characterization

[0115] Silicon wafers (?11?11 mm) were equipped with thin films (?50 nm) of the PS culture substrate material via spin coating. The native SiO.sub.2 layer on the silicon wafers and the thickness of the substrate material films were determined separately by spectroscopic ellipsometry (SE) prior to coating. PGE brush coatings were fabricated by incubation of the coated silicon wafer substrates in dilute (0.25 mg mL.sup.?1) aqueous/ethanolic solutions of the block copolymers for 1 h. The polymer solutions were subsequently discarded and the surfaces briefly (?30 s) immersed in water to remove excess polymer solution. After drying under a stream of N.sub.2, the substrates were irradiated with UV light (LED, ?=365 nm) to covalently immobilize the physically adsorbed PGE layers via their photo-reactive BP anchor blocks. PGE gel coatings were fabricated by spin coating PS-coated silicon wafers with 1% (w/w) solutions of G1, G2 and G3 in ethanol. The substrates were subsequently irradiated with UV light (LED, ?=365 nm) to covalently immobilize and crosslink the PGE layers via their photo-reactive BP units. All surfaces were then washed with ethanol to extract non-immobilized PGE chains until the thickness of the coatings was constant (?1-3 d).

[0116] The dry thickness of the PGE coatings was measured by SE before and after UV irradiation as well as during extraction with ethanol. The wettability of the obtained PGE coatings was characterized by static water contact angle (CA) measurements at 20? C. AFM measurements were performed on a Nanoscope MultiMode 8 equipped with a fluid cell and a thermal application (TA) controller from Bruker (Billerica, MA, USA). The morphology and material properties of the PGE coatings were measured in PeakForce QNM (Quantitative Nanomechanical Mapping) mode. Coated silicon wafers were mounted on the AFM head, degassed Milli-Q water was inserted into the liquid cell and the TA controller was set to the desired temperature (37? C.) and equilibrated for at least 10 min prior to each measurement. To obtain high resolution images with reduced sample damage, SNL-10 cantilevers from Bruker (Billerica, MA, USA) with a nominal spring constant of 0.3 N m.sup.?1 and a tip radius of 2-12 nm were used, and images were recorded with a loading peak force of 0.5 to 1 nN, 512 points per line and scan rates of 1.0 Hz. Obtained images were analyzed with the Nanoscope analysis software (version 1.4) and processed using 1.sup.st order flattening.

Cell Culture and Characterization

Ethical Approval

[0117] For whole blood samples, approval by the ethics committee of the Charit?-Universit?tsmedizin Berlin was obtained. Anonymized blood samples were obtained from the German Red Cross blood donation service Berlin with informed written consent from all participants. All studies were in accordance to the Helsinki guidelines. No part of these studies was conducted outside of Germany.

Preparation of Blood Derived Monocytes and Generation of DCs

[0118] Peripheral human blood of normal donors was obtained by buffy coats from the German Red Cross blood donation service, Berlin. All studies performed were in adherence to the Helsinki guidelines. By depletion of contaminating cells monocytes were magnetically isolated from PBMC (monocyte isolation kit II, Miltenyi Biotec). Cells were cultured in RPMI 1640 supplemented with 2 mM L-glutamine, 100 IU/ml penicillin, 100 ?g/ml streptomycin and 10% (v/v) heat-inactivated FCS (Biochrom) for 6 days with human recombinant GM-CSF (100 ng/ml), IL-4 (10 ng/ml, both R&D Systems).

Flow Cytometry

[0119] Cells were incubated with anti-CD86 BB515 (2331), CD40 APC (5C3), HLA-DR PerCPCy5 (G46-6) and CD14 PE (M5E2) for 30 min on ice. After washing, cells were analyzed by FACS Accuri (BD Biosciences) flow cytometer using BD Accuri C6 software.

ELISA

[0120] Culture supernatants were analyzed for TGF-?1 and EGF (DuoSet, R&D Systems). Protein was determined by sandwich ELISA after 48 h in the supernatants of 1?106 DCs per ml.

Statistical Analysis

[0121] Student's two-tailed unpaired t test was used to calculate statistical significance. P values below 0.05 were considered significant.

LIST OF REFERENCES CITED IN THE PRECEDING SECTIONS

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