Characterization of poly(d ,l -lactic-co-glycolic acid) based nanoparticulate system for enhanced delivery of antigens to dendritic cells

Vaccine 22 (2004) 2406–2412

Characterization of poly(d,l-lactic-co-glycolic acid) based nanoparticulate system for enhanced delivery of antigens to dendritic cells Praveen Elamanchili, Manish Diwan, Min Cao, John Samuel∗ Faculty of Pharmacy and Pharmaceutical Sciences, 3118 Dentistry/Pharmacy Centre, University of Alberta, Edmonton, Alberta, Canada T6G 2N8 Received 5 September 2003; accepted 1 December 2003 Available online 8 April 2004

Abstract Biodegradable nanoparticles made of poly(d,l-lactic acid-co-glycolic acid) (PLGA) copolymer were characterized for enhanced delivery of antigens to murine bone marrow derived dendritic cells (DCs) in vitro. PLGA nanoparticles were efficiently phagocytosed by the DCs (CD11c+ , MHC class II+ , CD86+ ) in culture, resulting in their intracellular localization. The efficiency of the uptake was influenced by the incubation time and nanoparticle concentration. DCs pulsed with PLGA nanoparticles containing an immunomodulator, monophosphoryl lipid A (MPLA), showed upregulation of surface expression of MHC class II and CD86 molecules. Delivery of a cancer-associated antigen (MUC1 mucin peptide: BLP25) and MPLA in PLGA nanoparticles was shown to be superior to their delivery in the soluble form for activation of na¨ıve T cells of normal and MUC1-transgenic mice. These results strongly suggest that PLGA nanoparticles provide an efficient vaccine delivery system for targeting DCs and the development of DC based cellular vaccines. © 2004 Elsevier Ltd. All rights reserved. Keywords: Dendritic cells; Vaccine delivery; Nanospheres

1. Introduction Dendritic cells (DCs) are potent antigen presenting cells capable of activating na¨ıve T cells and initiating antigenspecific immune responses against pathogens [1]. This is due to their high capability for antigen capture, abundant expression of MHC class I, class II, co-stimulatory, and other accessory molecules that are required for efficient antigen presentation to T cells. Immature DCs reside in the non-lymphoid tissues and constantly sample the environment for pathogens and foreign substances. Antigen uptake and processing triggers complex signaling networks resulting in activation and maturation of DCs characterized by increased expression of costimulatory (e.g., CD80, CD86, CD40) and MHC molecules, secretion of cytokines (e.g.,

Abbreviations: PLGA, poly(d,l-lactic acid-co-glycolic acid); TMRdextran, tetramethylrhodamine conjugated dextran; BLP25, MUC1 lipopeptide; MPLA, monophosphoryl lipid A; MUC1.Tg, MUC1transgenic ∗ Corresponding author. Tel.: +1-780-492-7469; fax: +1-780-492-1217. E-mail address: [email protected] (J. Samuel). 0264-410X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2003.12.032

IL-12) and chemokines (e.g., CCL19 and CCL22), and expression of chemokine receptors (e.g., CCR7). DCs finally migrate to secondary lymphoid organs and present processed antigens to na¨ıve T cells in an MHC restricted fashion [2]. DCs also capture ‘self-antigens’ and present them to T cells which usually leads to induction of peripheral tolerance or anergy against ‘self-antigens’ [3]. DCs distinguish pathogens from host cells based on pathogen-associated molecular patterns (PAMPs) on microbes through Toll-like receptors (TLRs). The choice between T cell activation and tolerance is believed to be largely controlled by the microenvironment of antigen capture and presentation by DCs [4]. DCs have been loaded with aqueous solutions of proteins, peptides, cell lysates, and RNA or DNA to induce antigen-specific immunity in vivo. DCs can internalize soluble antigens by pinocytosis and particulate antigens by phagocytosis, where the latter is considered to be more efficient in induction of immune responses [5,6]. Cross presentation of exogenous antigens by MHC class I molecules is possible using particulate delivery of antigens to phagosomes through the phagosome-to-cytosol pathway of antigen trafficking [7]. DCs have been reported to internalize a

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variety of particulate materials including apoptotic bodies, latex particles, and liposomes. More importantly, particulate antigens have been shown to be more efficient than soluble antigens for induction of CTL responses [7,8]. Poly(d,l-lactic-co-glycolic acid) (PLGA) is a biodegradable polymer that is approved for human use [9]. We have shown that PLGA particles are suitable vehicles for the delivery of recombinant proteins [10,11], peptides [12–14] and plasmid DNA [15] to generate immune responses in vivo. We have also observed that PLGA nanoparticles following intradermal administration in mice are taken up predominantly by DCs [16]. More visual proofs of the uptake of micro- and nano-particles by human DCs after in vitro pulsation were reported recently [17]. Here we describe the systematic characterization and in vitro optimization of this delivery approach for activation of the T cells using murine bone marrow derived dendritic cells (BMDCs). Murine BMDCs were chosen here as an in vitro model, due to their similarities with human myeloid DCs and ease of generation in large numbers. The significance of co-delivery of antigens and TLR ligands to DCs using PLGA nanoparticles was investigated for a cancer-associated antigen (MUC1 mucin peptide) and monophosphoryl lipid A (MPLA). This approach was found to be superior to soluble antigen delivery to DCs for activation of unprimed T cells from normal and MUC1 transgenic (MUC1.Tg) mice in vitro. 2. Materials and methods 2.1. Preparation of PLGA nanoparticles PLGA nanoparticles containing a human MUC1 lipopeptide (BLP25; STAPPAHGVTSAPDTRPAPGSTAPP-K(εPalmitoyl)G; m.w., 2765 Da; kindly provided by Biomira Inc.) were prepared by a single emulsion solvent evaporation technique. Briefly, BLP25 solution (100 ␮l, 1% (w/v) in 1:4, methanol:chloroform) and MPLA (100 ␮l, 0.2% (w/v) in 1:4, methanol:chloroform) were added to PLGA (monomer ratio 50:50; m.w., 7,000; Birmingham Polymers, Birmingham, AL) solution in chloroform (200 ␮l, 50% (w/v)). The above solution was emulsified in polyvinyl alcohol (PVA, m.w., 31 to 50,000; Aldrich Chemicals, Milwaukee, WI) solution in PBS (2 ml, 9% (w/v)). The organic solvent was removed by evaporation and BLP25 nanoparticles were collected, washed, freeze dried and stored at −20 ◦ C. PLGA nanoparticles containing a fluorescent probe (tetramethylrhodamine conjugated dextran, TMR-dextran) were prepared by a double emulsion solvent evaporation technique as described earlier [16]. 2.2. Characterization of PLGA nanoparticles PLGA nanoparticles were analyzed for particle size by dynamic light scattering technique using a Zetasizer 3000 (Malvern, UK). The peptide content in BLP25 loaded

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nanoparticles was determined by tetrahydrofuran/water peptide extraction followed by reverse phase HPLC (manuscript in preparation). The quantification of TMR-dextran in nanoparticles was done as described earlier [16]. 2.3. Culture of DCs BMDCs were generated from normal C57BL/6 or MUC1.Tg mice (C57BL/6 background; bred in Health Sciences Laboratory Animal Services, University of Alberta, Edmonton, Canada; MUC1 transgene confirmed by PCR analysis of ear) following the method described by Lutz et al. [18]. Purity of DCs in the cultures was more than 70% on day 8 (CD11c+ ). 2.4. Uptake of PLGA nanoparticles by DCs 2.4.1. Characterization of nanoparticle uptake by fluorescence activated cell sorting (FACS) To determine the maximal uptake of PLGA nanoparticles, 6–9 days old DC primary cultures were co-incubated up to 24 h with TMR-dextran containing nanoparticles (2 mg/107 cells in 20 ml supplemented RPMI in 100 mm bacteriological grade petridishes) TMR-dextran. In a parallel experiment, the amount of nanoparticles was varied from 0.5 to 8 mg and the uptake determined over a 24 h incubation period. The extent of particle uptake was determined by analyzing for TMR-dextran+ cells in the harvested non-adherent and semi-adherent populations expressing CD11c. After establishing the conditions for optimal uptake of nanoparticles, the particulate and soluble modes of delivery to DCs were compared. Suspensions of nanoparticles equivalent to 10 ␮g TMR-dextran with and without MPLA, empty nanoparticles or soluble TMR-dextran (10–50 ␮g) in HBSS (0.5 ml) were added separately to day 7 DC cultures. Parallel DC cultures were treated either with Cytochalasin B (5 ␮g/ml; Sigma-Aldrich, ON, Canada), mannose (2 mg/ml; Sigma-Aldrich) or dextran (1 mg/ml; m.w., 400 kDa; Sigma-Aldrich) for 30 min prior to the addition of TMR-dextran formulations. Control cell cultures received HBSS. After 24 h, cells were harvested, washed twice with PBS, and incubated with titrated amounts of either anti-CD11c, MHC class II, and CD86 (respective isotype) with FITC conjugated second antibodies. Samples were acquired on a Becton–Dickinson FACS sort flow cytometer (Franklin Lakes, NJ) and data analyzed with CELLQuest software (Becton–Dickinson). All mAbs were purchased from BD Pharmingen, Mississauga, ON, Canada. 2.4.2. Confocal laser scanning microscopy (CLSM) Day 7 DC cultures were transferred into Lab-Tek II eight well chamber slides (2 × 105 cells/well; Nalgene Nunc Int., IL) and incubated for 6 h at 37 ◦ C followed by addition of TMR-dextran containing nanoparticles (30 ␮g in 50 ␮l HBSS/well). Control wells were treated with Cytochalasin B (5 ␮g/ml) for 30 min prior to the addition of nanoparticles.

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3. Results 3.1. Antigen loaded PLGA nanoparticles Nanoparticles loaded with TMR-dextran showed a Guassian distribution (200−650 nm) with a mean size of 357 nm. Nanoparticulate loading of the TMR-dextran probe was 0.5% (w/w). The mean particle size for nanoparticles containing BLP25 was found to be 287 nm with a size distribution of 150 − 580 nm and a peptide loading of 1% (w/w). 3.2. Optimization of parameters affecting nanoparticles uptake Six to nine day old DC cultures showed similar particle uptake. Day 7 was considered as optimum culture age and used for further experiments. The extent of nanoparticle uptake by DCs was increased from 58±2% to 75±2% with an increase in the amount of nanoparticles (0.5–2 mg/20 ml culture medium) (Fig. 1A). No enhancement in the total number of cells, taking up particles was observed when nanoparticle concentration was further increased to 8 mg/20 ml. However, the number of particles taken up per cell showed an increase as indicated by the change in mean fluorescence intensity (MFI) i.e., from 20 ± 5 to 62 ± 2. Fig. 1B shows 24 h incubation period to be optimal. In the subsequent uptake experiments, optimized incubation conditions of 2 mg nanoparticles per 1 × 107 DCs (day 7 cultures) in 20 ml medium for 24 h in a bacteriological grade 100 mm Petri dish were used.

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DCs from normal or MUC1.Tg mice were incubated with either of the following antigen formulations for 24 h: (a) nanoparticles containing BLP25 and MPLA; (b) nanoparticles containing BLP25; (c) nanoparticles containing MPLA; (d) empty nanoparticles; (e) BLP25 and MPLA in solution. The DCs were then irradiated with 3000 rads using a 137 Cs irradiator, washed thoroughly, and plated in triplicates in 96 well flat bottomed microtiter plates (Costar, Cambridge, MA) in complete RPMI. Syngenic nylon wool purified T cells from unimmunized mice were co-incubated at 2 × 105 cells per well, with DC to T cell ratios of 1:5 and 1:10. After 72–96 h of incubation, T cell proliferation was assessed by [3 H]-thymidine incorporation following a final 24 h pulse (1 ␮Ci/well; Amersham, Oakville, ON, Canada).

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Fig. 1. Optimization of the experimental conditions for PLGA nanoparticle uptake by DCs. Nanoparticle formulation (2 mg unless otherwise stated) containing TMR-dextran was incubated with DC cultures (1 × 107 cells, day 7) in Petri dishes containing 20 ml medium at 37 ◦ C for 24 h. This figure shows the effect of (A) nanoparticle amount, and (B) incubation period, on the particle uptake efficiency of DCs. The data are presented as the mean±S.D. (error bars) of the values from three separate experiments.

3.3. Increased cellular granularity after nanoparticle uptake After internalization of nanoparticles, DCs apparently became more granular and were seen as a distinct population in the FACS dot plot with a higher side scatter (data not shown). Cell cluster with altered granularity showed 97% positivity for TMR-dextran. No change in the cellular granularity was observed when DCs were pulsed with equivalent amount of fluorescent probe in solution (10 ␮g). Less than 5% of the cells showed uptake of soluble TMR-dextran. Double color FACS analysis indicated that 71% of TMR-dextran+ cells were CD11c+ , i.e., DCs. These cells were also found to be MHC class IIhi , CD86hi , and CD8␣+ (myeloid origin). 3.4. Intracellular localization of nanoparticles after uptake by DCs CLSM was used to examine whether nanoparticles were indeed taken up and localized intracellularly or were simply adsorbed onto the cell surface. Chamber slide cultures of cells exhibited typical DC morphology with the characteristic long dendrites or veils as visualized by staining with anti-CD11c mAb (Fig. 2A). The non-diffused red signal inside the cells showed localization of the TMR-dextran containing nanoparticles inside the cells (Fig. 2B). The DC cultures treated with Cytochalasin B, prior to the addition

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Fig. 2. CLSM images of DCs pulsed with nanoparticles. Day 7 DC cultures were incubated in chamber slides with (B) or without (A) TMR-dextran containing nanoparticles for 24 h and surface stained using CD11c mAb. Phagocytosis inhibition can be seen after pretreatment of DC cultures with Cytochalasin B for 30 min prior to addition of TMR-dextran nanoparticles (C). White bar represents 15 ␮m.

of nanoparticles, remained TMR-dextran− (Fig. 2C). Membrane staining performed with Alexa Fluor® 488 further confirmed the intracellular localization of the TMR-dextran nanoparticles by visualization of the cells at different planar sections (data not shown). 3.5. Increased expression of co-stimulatory and MHC molecules in DCs after nanoparticle uptake Immature, day 7 DC primary cultures were pulsed with nanoparticles with or without MPLA for 24 h and the expression of MHC class II and CD86 determined by flow cytometry (Fig. 3). Compared to untreated cells, nanoparticle pulsed DCs showed a two-fold increase in MFI corresponding to MHC class II expression (from 83 to 180), which was further increased (from 83 to 300) following MPLA incorporation in the nanoparticles. The percentage of cells expressing MHC class II was however not affected. The number of CD86+ cells was increased by two folds (25 to 50%) following nanoparticle pulsing (MFI changed from 48 to 60). In the presence of MPLA containing nanoparticles, the total number of CD86+ cells was further increased to 68% with a corresponding increase in MFI upto 73. Notably, these particles were endotoxin free when tested using a commercial endotoxin testing kit (QCL-1000, Bio-Whittaker, MD, USA). 3.6. Uptake of PLGA nanoparticles by DCs is not mannose receptor mediated DCs express mannose receptors, which play an important role in receptor mediated endocytosis of molecules containing mannose or dextran [19]. To investigate the possibility of TMR-dextran containing nanoparticles being taken up by DCs through mannose receptor pathway, uptake studies were performed in the presence of soluble dextran and mannose as competitive inhibitors. Pre-incubation of DC cultures with either mannose or dextran prior to nanoparticle pulsing showed no significant inhibition in the uptake (Fig. 4). The uptake of soluble probe (∼5%) was however completely inhibited. For the sake of clarity, the inhibitory effects on a five-fold higher concentration (50 ␮g) of soluble TMR-dextran are given in Fig. 4.

Fig. 3. Enhanced expression of MHC class II and CD86 on DCs after uptake of PLGA nanoparticles. DCs were pulsed with nanoparticles (with or without MPLA) for 24 h and analyzed for expression levels of MHC class II and CD86. Control groups were treated with HBSS. Thin black line in the histogram plot represents the background staining with isotype controls. Figures showing the influence of soluble TMR-dextran are not given as there was not any detectable change in the expression of the markers. Numbers in the histogram plot indicate MFI values for the respective markers. Similar results were obtained in two separate experiments.

3.7. In vitro immunization of DCs with particulate antigen formulation of MUC1 peptide primes T cells from unimmunized normal and MUC1.Tg mice In vitro antigen presentation assays were performed to determine the ability of DCs to present antigen to na¨ıve T cells. DCs pulsed with PLGA nanoparticles containing a MUC1 lipopeptide (BLP25) and an immunomodulator (MPLA), primed na¨ıve T cells derived from normal and MUC1.Tg mice (Fig. 5). In contrast, the same was not observed when DCs were pulsed with BLP25 nanoparticles alone, which indicated MPLA was needed in the formulation for MUC1 specific T cells to proliferate. BLP25 and MPLA in solution could not prime T cells. MPLA by itself in nanoparticles was able to stimulate na¨ıve T cells to a small extent, possibly due to its non-specific mitogenic activity [20]. No significant T cell proliferation could be observed even when a three-fold higher concentrations of peptide and MPLA in solution were employed or even when empty nanoparticles were randomly mixed with soluble BLP25 and MPLA (data not shown). These results indicate the requirement for the antigen and the immunomodulator to be present in the particulate formulation for efficient uptake and presentation to

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Fig. 4. Effect on nanoparticle uptake by DCs in the presence of mannose receptor substrates (dextran and mannose) and phagocytosis inhibitor (Cytochalasin B). DC cultures were pre-treated with either mannose (2 mg/ml), dextran (1 mg/ml) or Cytochalasin B (5 ␮g/ml) for 30 min prior to the addition of TMR-dextran formulations and incubated for 24 h. The TMR-dextran+ cell population was analyzed by flow cytometry. Only a weak signal (<5% DCs) was obtained for the soluble probe at which it was difficult to study any substrate effects. The figures shown here are for studies where five folds higher amount of soluble TMR-dextran was used in order to enhance the signal. Solid black areas in the histogram plot indicate the background fluorescence after pulsing the cells either with empty nanoparticles (for nanoparticle uptake studies) or HBSS (for soluble formulation uptake studies). Similar results were obtained in three separate experiments.

T cells. The single in vitro immunization of BLP25 and MPLA containing nanoparticles was also able to stimulate na¨ıve T cells when both the cell types were derived from MUC1.Tg mice (Fig. 5B). These preliminary observations suggest that delivery of MUC1 antigen in the nanoparticle delivery system, but not its soluble form, was able to break self-tolerance against MUC1 antigen.

4. Discussion DC vaccination is a promising approach to generate strong T cell responses against both ‘foreign’ and ‘self-antigens’. We are currently investigating the potential of a PLGA

based nanoparticulate system for delivering MUC1-based cancer vaccines, hepatitis B antigens, and other antigens, using both in vitro and in vivo models [13–15]. The biodegradability of PLGA nanoparticles as compared to non-biodegradable latex and polystyrene beads, makes these a better carrier material for in vivo applications. The proposed nanoparticle-mediated antigen delivery to DCs offers distinct advantages over the use of antigens in soluble form. The foremost benefits include: (i) prevention of proteolytic degradation of antigens, (ii) limiting the distribution of antigens to mostly APCs (phagocytic cells) and avoiding their entry to systemic circulation, (iii) higher efficiency of antigen loading, and (iv) prolonged antigen presentation by DCs [21]. Also, co-delivery of immunomodulators like TLR ligands along with the antigen in the formulation permits manipulation of the microenvironment of interactions between antigen loaded DCs and T cells. The maturation stage of DCs has a direct effect on their ability to process and present antigens to T cells. Our results show that after PLGA nanoparticle pulsing, DCs exhibit a modest increase in the expression of MHC class II and CD86 compared to unpulsed controls, which was further increased by incorporation of MPLA. Similar observations were made in our recent studies with PLGA nanoparticle formulations and cord blood derived DCs [22], or others, with monocytes derived DCs using non-biodegradable, polystyrene-beads [23]. However, Sun et al. [24] have recently reported that microparticles (size ∼10 ␮m) used to pulse murine DCs did not increase the expression levels of MHC class II and CD86. As our nanoparticle formulations were endotoxin free, we speculate that their relative smaller size in the nanometer range, could be critical for uptake and hence, to modulate maturation of DCs. An inverse relationship between particle size and their extent of uptake by DCs has also been reported by Reece et al. [25]. MPLA is a non-toxic analog of lipid A, a component of LPS, which is a ligand for Toll-like receptor 4 (TLR4). Although the ability of MPLA to engage TLR4 and consequent activation of signaling mechanisms have not yet been fully characterized, our results are consistent with the view that this immunomodulator acts as ‘danger signal’ for DCs. While our current study focused on the use of MPLA to achieve this purpose, the PLGA-based nanoparticulate system offers the flexibility for incorporation of broad range of TLR ligands including CpG oligonucleotides and mannan-like molecules [14,22]. DCs in cancer and chronic viral infections are functionally defective. In addition, in vivo antigen capture by DCs in the immunosuppressive milieu of such diseases may generate tolerogenic DCs, which might contribute to further exacerbation of the disease. Ex vivo culture of large number of DCs followed by in vitro antigen loading and immunization is an important approach for generation of functionally potent DCs. The current studies demonstrated that PLGA nanoparticles offer an excellent delivery system for generating antigen-loaded functionally potent DCs. The

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Fig. 5. The ability of DCs, pulsed with MUC1 lipopeptide encapsulated in PLGA nanoparticles, to prime na¨ıve T cells derived from normal and MUC1.Tg mice in vitro. DCs were pulsed for 24 h with nanoparticles containing either MUC1 lipopeptide + MPLA (BLP25 + MPLA), MUC1 lipopeptide (BLP25), or MPLA (MPLA). DCs were also pulsed with empty nanoparticles (EMPTY), or soluble BLP25 + MPLA (SOL BLP25 + MPLA). Pulsed DCs were irradiated and co-cultured with syngenic na¨ıve T cells at 1:5 (black bars) and 1:10 (hollow bars) cell ratios with the T cell number kept constant (2 × 105 cells/well) for 72 h and the proliferation measured by 3 H incorporation. Positive control wells received concanavalin A mitogen (ConA). (A) DCs and T cells derived from normal C57BL/6 mice and (B) DCs and T cells derived from MUC1.Tg mice. The results are shown as mean cpm ± S.D. (error bars). Similar results were obtained in 3 independent experiments.

proposed antigen delivery approach, involving co-delivery of antigens and immunomodulators in the same particles, provides an effective way to overcome peripheral tolerance against ‘self-antigens’ like MUC1. Although alternate approaches like transfection of DCs with cancer-associated antigens such as MUC1 have also been shown to overcome peripheral tolerance [26], the PLGA nanoparticulate delivery holds promise for broader clinical applications due to the simplicity of the method and ease of large scale pharmaceutical production. We believe that this system will have broad applications to a number of vaccines designed to activate potent T cell responses including cancer vaccines, vaccines for chronic viral diseases such as hepatitis B, C, and human immunodeficiency virus infections.

Acknowledgements This work was supported by research grants from Canadian Institutes of Health Research (MOP 42407) and Natural Sciences and Engineering Research Council of Canada

(STPGP 234866 and STPGP 258032) to J.S., and a graduate research scholarship in pharmacy from the CIHR/Research based Pharmaceutical Companies (Rx&D) of Canada and Walter H. Johns Fellowship to P.E. The authors thank Dr. Conrad Coester for providing technical assistance with CLSM and Juanita Wizniak for her help with flow cytometry. References [1] Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998;392(6673):245–52. [2] Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, et al. Immunobiology of dendritic cells. Annu Rev Immunol 2000;18:767– 811. [3] Belz GT, Heath WR, Carbone FR. The role of dendritic cell subsets in selection between tolerance and immunity. Immunol Cell Biol 2002;80(5):463–8. [4] Walker LS, Abbas AK. The enemy within: keeping self-reactive T cells at bay in the periphery. Nat Rev Immunol 2002;2(1):11–9. [5] Scheicher C, Mehlig M, Dienes HP, Reske K. Uptake of bead-adsorbed versus soluble antigen by bone marrow derived dendritic cells triggers their activation and increases their antigen presentation capacity. Adv Exp Med Biol 1995;378:253–5.

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