Vaccine 23 (2005) 1312–1321

Interactions of proteoliposomes from serogroup BNeisseria meningitidiswith bone marrow-derived dendritic cells and macrophages: adjuvant

effects and antigen delivery

Tamara Rodrıgueza, Oliver Pereza, Nathalie Menagerb, Sanja Ugrinovicb,Gustavo Brachoa, Pietro Mastroenib,∗

a Department of Immunology, Finlay Institute, P.O. Box 16017, Havana, Cubab Centre for Veterinary Science, Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK

Received 7 January 2004; accepted 19 July 2004Available online 6 October 2004

Abstract

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Exposure to proteoliposomes from serogroup BNeisseria meningitidis(PL) induced up-regulation of MHC-II, MHC-I, CD40, CD80 aD86 expression on the surface of murine bone marrow-derived dendritic cells (DC). CD40, CD80 and CD86 were up-regulatearrow-derived macrophages (M�) upon stimulation with PL. Both DC and M� released TNF�, but only DC produced IL12(p70) in respon

o PL. A small increase in the expression of MHC-II, CD40 and CD86, as well as production of IL12(p70), was observed on the cef DC, but not M� from LPS-non-responder C3H/HeJ after exposure to PL. DC, but not M�, incubated with PL containing ovalbumPL-OVA) presented OVA-specific peptides to CD4+ and CD8+ OVA-specific T-cell hybridomas. These data clearly indicate that PLn immunomodulatory effect on DC and M�, with some contribution of non-LPS components besides the main role of LPS. The wohows the potential of PL as a general system to deliver antigens to DC for presentation to CD4+ and CD8+ T-cells.2004 Published by Elsevier Ltd.

eywords:Proteoliposomes; Delivery system; Dendritic cells; Adjuvant

. Introduction

Capture, processing and presentation of bacterial productsy antigen-presenting cells (APC) is one of the first crucialteps in the initiation of acquired immune responses duringnfection or vaccination. Dendritic cells (DC) actively inter-alize antigens in soluble or particulate form and migrate to

he draining lymph nodes where they can present antigen toaıve T-cells. The capacity of DC to internalize antigens de-reases after being exposed to antigenic stimuli and/or mat-ration signals such as IL1�, TNF�, or bacterial products.onversely, surface expression of co-stimulatory molecules

ncluding CD80 (B7-1), CD86 (B7-2), CD40, CD54 (ICAM-), major histocompatibility complex class I (MHC-I) and

∗ Corresponding author. Tel.: +44 1223 765800; fax: +44 1223 337610.E-mail address:pm274@cam.ac.uk (P. Mastroeni).

class II (MHC-II) molecules is up-regulated[1–3]. DC ex-press surface receptors that allow continuous monitorinthe environment and can trigger cell maturation upon bing of bacterial components. For example, Toll-like recep(TLRs) recognise conserved microbial molecular structknown as pathogen-associated molecular patterns (PAand contribute to the induction of inflammatory responsesdevelopment of antigen-specific adaptive immunity. At lten members of the TLRs family have been described,ognizing different PAMPs. For example, TLR4 is involvin recognition of Gram-negative bacterial lipopolysaccride (LPS), whilst TLR2 recognizes other PAMPs includporins ofNeisseria meningitidis[4].

Macrophages (M�) are actively phagocytic and endocycells, and constitutively express low levels of MHC-II andstimulatory molecules such as CD80 and CD86 unlessvated by mediators such as interferonγ (IFN�) [5]. Although

264-410X/$ – see front matter © 2004 Published by Elsevier Ltd.oi:10.1016/j.vaccine.2004.07.049

T. Rodrıguez et al. / Vaccine 23 (2005) 1312–1321 1313

M� can process and present antigens, they are generally be-lieved to be inferior to DC in their ability to initiate primaryT-cell responses[6].

Vaccine-associated immune responses are dependent onthe inherent immunogenicity of the antigens, the availabilityof immune adjuvants and on efficient delivery systems. Veryfew adjuvants have been approved for humans and new ap-proaches are being evaluated to increase the immunogenicityof antigens. Proteoliposomic structures from serogroup BN.meningitidishave been extensively used as vaccine compo-nents[7–13]. Proteoliposomes (PL) can induce productionof inflammatory cytokines and can elicit Th1 type immuneresponses[14]. The potential of PL as general adjuvants andas vectors for antigen delivery to the immune system is cur-rently being explored. However, little is known on the cellularevents triggered by the interaction of PL with APC involvedin the induction of immune responses. The cell surface recep-tors and the bacterial molecules involved in the interaction ofPL with immune cells are also unclear. PL contain phospho-lipids, proteins, lipoproteins from the outer membrane of Bserogroup meningococci, and also LPS. The relative impor-tance of LPS and non-LPS components during the interactionof PL with DC and M� is still unknown.

Antigenic structures can be incorporated into PL. Thispotentially makes PL an attractive adjuvant system to delivera abil-i ofT vantf

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2.2. OVA incorporation into proteoliposomes

Encapsulation of OVA within PL was performed by de-tergent disruption of vesicles followed by re-assembly in thepresence of OVA. PL were dissolved in 30 mM Tris buffer(Fluka, Switzerland) containing EDTA (2 mM) (BDH, Poole,UK) and 0.5% deoxycholate to give a final protein concentra-tion of 3 mg/ml. The concentration of deoxycholate in the PLsuspension was increased to 1.5% in order to disrupt the vesi-cle structure. OVA type V (Sigma, St. Louis, USA) was addedto give a final concentration of 2.6 mg/ml. The mixture wasdialyzed against five changes of Tris–EDTA for 24 h. Vesiclesentrapping OVA were purified by gel filtration chromatogra-phy on Sephacryl S-300 (Pharmacia). The vesicle structurewas confirmed by their chromatography profile that was sim-ilar to the one of untreated PL (Fig. 1). Also, SDS–PAGE andwestern blotting using OVA specific polyclonal serum con-firmed the presence of OVA in the vesicles. The amount ofencapsulated OVA was estimated by densitometry analysison polyacrylamide gels stained with Coommassie blue usingan ImageMaster® VDS (Pharmacia Biotech).

2.3. Animals

C57BL/6 (H-2b), C3H/HeN (H-2k) and C3H/HeJ (H-2k)f rlanO eri-n res htw

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nd target antigens to the immune system. However, thety of PL to deliver antigens to APC for the stimulation-cells and the possibility that PL might act as an adjuor other antigens have not been studied in detail.

In the present study we investigated: (a) the ability of Pnduce cytokine production and up-regulation of molecssential for antigen presentation in DC and M�; (b) whethe

he presence of a functional TLR4 (LPS recognition) isential for PL to exert their effects on APC; (c) whetheran deliver a model antigen (ovalbumin, OVA) to APC anduce its presentation to MHC-II restricted CD4+ and MHC-Iestricted CD8+ T-cell hybridomas.

. Materials and methods

.1. Preparation of proteoliposomes

PL were prepared from the outer membranes ofN. meninitidis serogroup B strain Cu 385-83 (B:4:P1.19.15;L3,y detergent extraction[15]. Briefly, PL were obtained from

ive bacteria by gentle extraction with 10% deoxychoMerck, Darmstadt, Germany). Bacterial debris was remy centrifugation and nucleic acids were eliminated byymatic treatment with deoxyribonuclease and ribonucl5�g/ml) (Merck). PL were purified by gel filtration chratography on Sephacryl S-300 (Pharmacia Fine Ch

als, Uppsala, Sweden) followed by precipitation with 9thanol. PL are membrane vesicles that contain majorembrane proteins (P1 and P3), a complex of proteins5 to 95 kDa, LPS and phospholipids.

emale mice, 6–8 weeks old, were purchased from Halac Ltd. (Blackthorn, Bicester, UK) and kept at the Vetary Medicine Animal Unit, Cambridge, UK. Animals weupplied food and waterat libitumand used when over eigeeks old.

.4. Cell lines and T-cell hybridomas

OT4H [16] and CD8OVA[17] T-hybridoma cells werindly donated by Dr. M.J. Wick, University of Goterborgweden and Dr. J. Kapp, Emory University Schooledicine, Atlanta, USA, respectively. All tissue cultu

eagents were obtained from Sigma (Poole, UK) unless oise stated. Cells were maintained at 37◦C in 5% CO2 in Is-ove’s modified Dulbecco’s medium supplemented withf foetal bovine serum (FBS), 2 mMl-glutamine, 0.05 mM 2ercaptoethanol (2-ME), 100 U/ml penicillin and 100�g/ml

treptomycin. CTLL-2 (IL2/IL4 dependent) cells were cured in RF10 (RPMI 1640 supplemented with 10% FmM l-glutamine, 0.05 mM 2-ME, and 10�g/ml gentam

cin).

.5. Cultures and antigenic stimulation of DC and M�

18]

DC were prepared by culturing bone marrow cellsdays at a density of 2× 105/ml in six-well plates usinF10 supplemented with 20% conditioned medium f63-Ag8 cells (kindly provided by Dr. A. Knight, ICAPBniversity of Edinburgh, UK). DC were uniformly CD11b+,D11c+, CD19−, TCR��− as confirmed by flow cytome

1314 T. Rodrıguez et al. / Vaccine 23 (2005) 1312–1321

Fig. 1. Typical chromatogram of the PL purification process. Proteoli-posomes entrapping OVA were purified by gel filtration chromatogra-phy on Sephacryl S-300. Column XK16, 1.6× 100 cm; eluent, 30 mMTris buffer containing EDTA (2 mM) and 0.5% deoxycholate; flow rate,0.6 ml/min.

try. M� were prepared by culturing bone marrow cells for6 days at 3× 105/ml in bacterial grade petri dishes in RPMI1640 supplemented with 10% FBS, 5% horse serum, 2 mMl-glutamine, 0.05 mM 2-ME, 10�g/ml gentamicin, 1 mMsodium pyruvate and 15% conditioned medium from L929cells. The adherent population was washed with PBS andremoved from the plastic with cold PBS containing 0.1%EDTA, and then washed twice with PBS. M� were uniformlyCD11b+, CD11c−, CD19−, TCR��− as confirmed by flowcytometry.

DC and M� were stimulated by addition of 0.1–10�g/mlof PL. Similar results were obtained within this dose range of

PL and therefore representative data relative to the 5�g/mldose are shown in the results section. Non-stimulated cellsand cells stimulated with 1�g/ml of Salmonella entericaserovar Typhimirium LPS (Sigma) were included in allassays, as negative and positive controls, respectively. Tomeasure expression of MHC-II, MHC-I and co-stimulatorymolecules (CD40, CD80 and CD86) the cells were stimu-lated for 18 h and maintained at 37◦C in the presence of 5%CO2. To measure TNF� and IL12 production, supernatantswere collected 4–6 h and 24–48 h after PL stimulation, re-spectively. Pilot experiments determined the optimal timepoints for measuring the expression of cell surface mark-ers and the production of either cytokine in our experimentalsystem.

2.6. Flow cytometry

Monoclonal antibodies (mAbs) specific for mouse cellsurface markers CD16/CD32, TCR��, CD19, CD11b,CD11c, CD80 (B7.1), CD86 (B7.2), CD40, MHC-II (I-A/I-E), MHC-I (K-b), as well as appropriate isotype con-trols, were purchased from BD PharMingen (Cowley, UK).Unless otherwise stated, antibodies were directly conju-gated to fluorescein isothiocyanate (FITC) or phycoerythin(PE).

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Cells were incubated at 4C for 25 min in 25�l of block-ng solution (anti-CD16/CD32 mAb diluted in Hank’s bnced salt solution (HBSS) containing 2% FBS, 10% merum, 0.01% sodium azide, and 1% HEPES). Cells weremmunostained with the appropriate fluorochrome-labentibody at 4◦C for 30 min in 25�l of HBSS containing% FBS, 0.01% sodium azide, and 1% HEPES). Cellsashed in FACS buffer and analyzed on a Becton Dicon FACSCalibur flow cytometer. Data were collected0,000 cells.

.7. TNF� and IL12(p70) ELISA

TNF� and IL12(p70) were measured by capture enzyinked immunosorbent assay (ELISA) with antibody pnd cytokine standards purchased from BD PharMinge

For TNF� determinations, 96-well ELISA plates (Masorp Nunc Immuno plate, Nunc, Roskilde, Denmark) woated overnight at 4◦C with 50�l per well of a capture ranti-mouse TNF�-mAb (clone G281-2626) diluted in 0.1odium phosphate buffer (pH 6.0) to give a final concenion of 5�g/ml. After blocking with PBS supplemented w0% FBS at 37◦C for 1 h, 100�l of supernatants from Dr M� stimulation assays were added in triplicate andlates were incubated at 37◦C for 2 h. Serial twofold dilu

ions of recombinant mouse TNF� ranging from 10 ng/mo 156 pg/ml were included as standards. Biotinylatednti-mouse TNF� mAb (clone MP6-XT3; 100�l/well) at�g/ml in PBS-10% FBS was then added for 1 h at 37◦C,fter which 100�l of peroxidase-labelled streptavidin pell at 2.5�g/ml (Sigma) in PBS-10% FBS was add

T. Rodrıguez et al. / Vaccine 23 (2005) 1312–1321 1315

for 45 min at room temperature.Ortho-Phenylenediamine(Sigma) (1 mg/ml in 0.2 M Na2HPO4–0.1 M citrate buffer)was used to develop the plates in the presence of H2O2. Thereaction was stopped with 15�l of 3 M H2SO4 per well andthe optical density of each well was read at 490 nm. The sen-sitivity of the TNF� ELISA was≥156 pg/ml.

IL12(p70) was measured by ELISA using a protocol sim-ilar to the one described above. The capture rat anti-mouseIL12(p70) mAb (clone 9A5) was diluted in 0.1 M sodiumphosphate buffer (pH 9.0) at 2�g/ml. Recombinant mouseIL12 ranging from 10 ng/ml to 156 pg/ml was included asstandard. Biotinylated rat anti-mouse IL12(p40/p70) mAb(clone C17.8; 100�l/well) at 1�g/ml was used as the de-tection antibody. The sensitivity of the IL12 ELISA was≥156 pg/ml.

2.8. ELISPOT assay

DC were stimulated with either PL, LPS or left unstim-ulated in ELISPOT 96 nitrocellulose-based plates (Multi-Screen HA, Millipore, Watford, UK) for 18 or 48 h beforemeasuring the production of TNF� or IL12, respectively. Forthe TNF� and IL12 ELISPOT assays, plates were coated with100�l of 4 �g/ml capture anti-mouse TNF� or anti-mouseIL12 mAbs in PBS overnight at 4◦C. Wells were washed withP ededas llssL con-t BS,ao edr Ex-t at3 evel-o ateds sw potsw ation× d asT owtt igen,c latedc

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ex-ct erei( reet ventp

omycin C (Sigma) was added to the cultures for 25 min fol-lowed by washing with HBSS. APC (1× 105 per well) wereseeded in 96-well plates and co-cultured for 24 h with 1× 105

of either OT4H or CD8OVA T-hybridoma cells. The OT4Hand CD8OVA T-cell hybridomas secrete IL2 upon specificrecognition of the OVA(265–277)/I-Ab or OVA(257–264)/Kb

complex, respectively.T-cell activation was determined by measuring the pro-

liferative response of the IL2/IL4 dependent CTLL-2 cells.Briefly, CTLL-2 cells were incubated for 24 h at 37◦C in ahumidified CO2 incubator in flat-bottom 96-well microtiterplates in the presence of the supernatants harvested from theantigen presentation assays described above. The prolifera-tion of CTLL-2 cells was measured by pulse-labelling trip-licate wells for the last 18 h of incubation with 0.37 MBq of[3H]thymidine/well (Amersham International, Little Chal-font, UK). [3H]thymidine incorporation was measured byharvesting cells onto glass-fibre mats using a Tomtek 96-wellharvester (Tomtek, USA) and counting by liquid scintillationin a Wallac Microbeta 1450 counter (Wallac, Perkin-Elmer,USA).

2.10. Statistical analysis

Significant differences between the levels of presentationo -c n’sm

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BS and saturated with RF10. The DC cultures were set serial doubling dilutions (starting at 1× 105 cells/well) andtimulated with 5�g/ml of PL. Non-stimulated cells and cetimulated with 1�g/ml ofS. entericaserovar TyphimuriumPS (Sigma) were included as negative and positive

rols, respectively. The plates were then washed in Pnd incubated with 1�g/ml of the biotinylated anti-TNF�r anti-IL12 mAbs for 2 h at 37◦C. The plates were washepeatedly with PBS and alkaline phosphatase-labeledrAvidin (1:1000 dilution; Sigma) was added for 1 h7◦C. The plates were washed again with PBS and dped with a chromogenic alkaline phosphatase-conjugubstrate (Sigma Fast®, Sigma). After 30 min, the plateere washed with water and air dried overnight. Sere counted using a dissecting microscope (magnific40). Only large spots with fuzzy borders were scoreNF� or IL12 spot-forming cells (SFC). The data sh

he number of cytokine producing SFC per 106 cells de-ected when DC were cultured in the presence of antorrected for background levels measured in unstimuultures.

.9. Antigen-presentation assays

DC or M� (APC) were cultured as described above,ept that M� were incubated for 24 h with IFN� (1�g/ml)o induce the expression of MHC molecules. The cells wncubated with PL-OVA (5�g/ml), PL (5�g/ml) or OVA1�g/ml) for 2 h at 37◦C. The cultures were washed thimes with HBSS and incubated for a further 4 h. To preroliferation of the APC in the T-cell assays, 25�g/ml of mit-

f specific OVA peptides in MHC-I and MHC-II by DC inubated with PL-OVA or OVA were determined by Duncaultiple comparison test with 95% confidence level.

. Results

.1. Expression of MHC and co-stimulatory moleculesn DC and M� after stimulation with proteoliposomes

We tested the changes in expression of MHC-II, MH, CD40, CD80 and CD86 on DC and M� following initro stimulation with 5�g/ml PL. Medium alone or LP1�g/ml) were included as negative and positive contespectively. After 18 h of incubation of DC with PL, txpression of MHC-II, CD40, CD80 and CD86 was gre

ncreased (Fig. 2A). A similar effect was seen after exposo LPS (data not shown). MHC-I expression was up-regun response to LPS and only a slight, but detectable incas seen upon exposure to PL (data not shown). Thuxposure results in up-regulation of MHC-II, MHC-I, CD4D80 and CD86 on DC, an event known to coincide waturation of these cells into APC[1–3].Incubation of M� with PL for 18 h resulted in increas

xpression of CD40, CD80 and CD86 (Fig. 2B) but did notlter the levels of MHC-II and MHC-I (data not shown)

he cell surface. Expression of MHC-II on M� was found toncrease when the cells were incubated with IFN�. In con-rast, simultaneous exposure of M� to IFN� and PL induce

decrease in the surface expression of MHC-II (datahown).

1316 T. Rodrıguez et al. / Vaccine 23 (2005) 1312–1321

Fig. 2. DC and M� up-regulate cell surface molecule expression after co-incubation with PL. DC and M� were incubated with PL (5�g/ml) for18 h and then stained with anti-MHC-II, -MHC-I, -CD40, -CD80, or -CD86antibodies. A and B show the results for DC and M�, respectively. Filledhistograms show the level of fluorescence using isotype control antibodiesthat was not affected by PL; the thin lines represent cell surface moleculeexpression on cells incubated in medium only; the thick lines show the cellsurface molecule expression following stimulation with PL. The data arerepresentative of three separate experiments yielding similar results.

3.2. Effect of proteoliposome stimulation on theexpression of co-stimulatory molecules on the cellsurface of DC and M� from C3H/HeJ and C3H/HeNmice

C3H/HeJ mice have a point mutation in TLR4 that makesthem unable to respond to bacterial LPS[19]. To test theinvolvement of LPS in the PL-induced up-regulation ofco-stimulatory molecules, we compared the ability of PLto modulate the expression of MHC-II, CD40, CD80 andCD86 in DC and M� from LPS-non-responder C3H/HeJmice and LPS-responder C3H/HeN mice. DC from C3H/HeJ

Fig. 3. LPS contained in PL plays an essential role in the induction of theup-regulation of cell surface molecule expression in DC and M�. (A) Theexpression of MHC-II, CD40, CD80, and CD86 on the cell surface of DCfrom C3H/HeN (left) or C3H/HeJ (right) mice after 18 h of incubation withPL. (B) The expression of CD40 on the surface of M�after 18 h of incubationwith PL. Filled histograms show the level of fluorescence using isotypecontrol antibodies that was not affected by PL; the thin lines represent thesurface molecule expression on cells incubated with medium only; the thicklines show the surface molecule expression on cells stimulated with PL. Thedata are representative of three separate experiments yielding similar results.

mice show only a slight up-regulation of CD40, CD86 andMHC-II and no changes in CD80 expression in response toPL (Fig. 3A). In contrast, the expression of all the surfacemolecules tested was highly increased in DC from C3H/HeN

T. Rodrıguez et al. / Vaccine 23 (2005) 1312–1321 1317

mice stimulated with PL (Fig. 3A). DC from C3H/HeJ micedid not show changes in either MHC-II or co-stimulatorymolecules in response to LPS (data not shown). Conversely,the expression profiles of MHC and co-stimulatory moleculesseen in DC from C3H/HeN in response to LPS were similarto those obtained when stimulating the cells with PL, thusshowing a marked up-regulation (data not shown).

PL exposure resulted in the up-regulation of CD40 onM� from C3H/HeN mice, with no changes in the levels ofCD80, CD86, and MHC II. Neither CD40 up-regulation norchanges in CD80, CD86 and MHC-II cell surface expressionlevels were observed on M� from C3H/HeJ mice (Fig. 3B).Similar results were obtained after LPS stimulation (data notshown).

Thus, LPS present in the PL is the principal inducer ofthe up-regulation of MHC-II and co-stimulatory moleculeson DC although non-LPS components of PL are also capa-ble of exerting minor stimulating effects on DC. LPS is alsoessential for the increase in the expression of CD40 seen inM� exposed to PL.

3.3. TNF� and IL12(p70) secretion by DC and M�after stimulation with proteoliposome

DC and M�were stimulated with PL or LPS as above. Par-aI 24 ha s ofTI )w ts effi-c uto

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Fig. 4. Cytokine production by DC from C57BL/6 mice after incubationwith PL. DC were stimulated with either PL or LPS. (A) TNF� production6 h after stimulation and (B) IL12(p70) production 24 h after stimulationas determined by ELISA. Data are presented as mean cytokine concentra-tions in supernatants (pg/ml)± standard deviation (S.D.) of three differentexperiments.

from C3H/HeJ stimulated with PL but not in the culturesstimulated with LPS. Few TNF� producing cells were foundin the DC cultures from C3H/HeJ stimulated with either PLor LPS.

Thus, LPS contained within PL plays a major role in theinduction of TNF� and IL12(p70) in murine DC and M�.Non-LPS components within PL contribute to the inductionof IL12, but not TNF� production.

Table 1TNF� production by DC and M� from C3H/HeN and C3H/HeJ mice afterincubation with PL

Mouse strain Stimulus TNF� (pg/ml)

DC M�

C3H/HeN Medium <156 <156PL 9103± 0.06 504± 0.03LPS 8979± 0.2 525± 0.04

C3H/HeJ Medium <156 <156PL <156 <156LPS <156 <156

Cells were stimulated with either PL (5�g/ml) or LPS (1 (g/ml). TNF�levels (pg/ml) in the supernatants were determined by ELISA 4 h after stim-ulation and expressed as mean± standard deviation of triplicate cultures.One representative experiment of three is shown.

llel cultures were incubated with medium alone. TNF� andL12(p70) were measured in the supernatants at 6 h andfter stimulation, respectively. DC produced high levelNF� and IL12(p70) after PL and LPS stimulation (Fig. 4).

n contrast, low concentration of TNF� and no IL12(p70ere detected after stimulation of M� with PL (data nohown). Thus, in our experimental conditions, PL caniently induce TNF� and IL12(p70) secretion from DC, bnly TNF� from macrophages.

.4. TNF� and IL12(p70) secretion by DC and M� in3H/HeJ and C3H/HeN mice

DC and M� from C3H/HeN or C3H/HeJ mice were stimlated with PL or LPS as above. Parallel cultures were iated with medium alone. Both DC and M� from C3H/HeNice produced TNF� and IL12(p70) after stimulation witL or LPS (Tables 1 and 2). No TNF� was detected in thultures of DC and M� from C3H/HeJ mice in responseither LPS or PL (Table 1). IL12(p70) was found in the supeatants of DC from C3H/HeJ mice 48 h after stimulation wL but not after stimulation with LPS (Table 2). TNF� and

L12(p70) production by DC was also analyzed by ELISPo further confirm the finding that DC from C3H/HeJ man produce IL12 in response to PL.Fig. 5shows the numbef TNF� (Fig. 5A) and IL12(p70) (Fig. 5B) producing cells

n DC cultures stimulated with either PL or LPS as descrbove. Exposure to either PL or LPS resulted in the presf TNF� and IL12(p70) producing cells in DC cultures fro3H/HeN mice as visualized as spot forming cells (SF

L12(p70) producing cells were present in the DC cultu

1318 T. Rodrıguez et al. / Vaccine 23 (2005) 1312–1321

Table 2IL12(p70) production by DC and M� from C3H/HeN and C3H/HeJ mice after incubation with PL

Mouse strain Stimulus IL12(p70) (pg/ml)

DC M�

24 h 48 h 24 h 48 h

C3H/HeN Medium <156 <156 <156 <156PL 3458± 0.006 2215± 0.004 197± 0.008 <156LPS 3177± 0.01 2051± 0.006 362± 0.002 455± 0.008

C3H/HeJ Medium <156 <156 <156 <156PL <156 573± 0.04 <156 <156LPS <156 <156 <156 <156

Cells were stimulated with either PL (5�g/ml) or LPS (1�g/ml). IL12(p70) levels (pg/ml) in the supernatants were determined by ELISA 24 and 48 h afterstimulation and expressed as mean± standard deviation of triplicate cultures. One representative experiment of three is shown.

3.5. DC but not M� exposed to proteoliposomescontaining OVA can present OVA peptides to T-cells

Given the immunostimulatory effect of PL on APC, wetested whether DC and M� pulsed with PL-OVA couldpresent peptides from OVA to OVA-specific T-cell hybrido-mas. We used MHC class II restricted CD4+ or MHC class Irestricted CD8+ T-hybridoma cells that are specific for OVA(257-264) and OVA (265-277) peptides, respectively. DC or

F essaIooa(e

M� were pulsed for 2 h with PL-OVA or soluble OVA andthen co-cultured with T-cell hybridomas. Supernatants werecollected after 24 h and tested for IL2/IL4 production us-ing [3H]thymidine incorporation by the IL2/IL4-dependentCTLL-2 cell line. IL2/IL4 production was used as a measureof the activation of the OVA-specific cell hybridomas.

ig. 5. Proportion of TNF� and IL12(p70) producing cells in DC cultur

timulated with PL. DC were stimulated with PL (5�g/ml), LPS (1�g/ml)s positive control or incubated with medium alone. (A) TNF� and (B)

L12(p70) producing cells in cultures of DC from C3H/HeN (black bars)r C3H/HeJ (white bars) mice were measured by ELISPOT. The numbersf SFC per 106 cells detected when DC were cultured in the presence ofntigen, corrected for background levels measured in the absence of antigenmedium only), are presented. The data are representative of three separatexperiments yielding similar results.

FshOppto

ig. 6. DC can process OVA peptides from OVA included in proteolipo-omes (PL-OVA) for MHC-I and MHC-II presentation. (A) The OT4H T-ybridoma cell response to DC from C57BL/6 mice co-incubated with PL,VA or PL-OVA. (B) The OVACD8+ T-hybridoma cells response. IL2/IL4roduction from the hybridomas was quantitated by [3H]thymidine incor-oration by the IL2/IL4-dependent CTLL-2 cell line. Data are presented as

he mean± S.D. of three different experiments.* Significantly different fromther groups (P< 0.05).

T. Rodrıguez et al. / Vaccine 23 (2005) 1312–1321 1319

Fig. 6 shows that DC pulsed with PL-OVA can presentOVA antigen to both CD4+ and CD8+ T-cell hybridomas. T-cell hybridomas were significantly activated when stimulatedwith DC that had been pulsed with PL-OVA as compared toDC pulsed with OVA alone. M� were unable to present OVAeither to CD4+ or CD8+ T-cell hybridomas in our assays (datanot shown). Thus, PL can deliver antigens to DC for efficientantigen presentation to T-cells.

4. Discussion

In the present study we found that exposure of murinebone marrow-derived dendritic cells (DC) and macrophages(M�) to PL from serogroup BN. meningitidisinduces theup-regulation of MHC-II, MHC-I, CD40, CD80, and CD86expression on the cell surface and the production of cytokinesimportant in T-cell activation. The use of LPS-non-responderC3H/HeJ mice has shown that LPS plays a major role in thebiological activity of PL. Other non-LPS components withinPL are also responsible for PL-mediated cell activation. Thework also showed that DC, but not M�, incubated with PLcontaining ovalbumin (PL-OVA) can present OVA-specificpeptides to CD4+ and CD8+ OVA-specific T-cell hybrido-mas. Taken together, the results indicate that exposure to PLc pre-s PLi inga lassI ana

v torym pona ofM o-d itht re-s e und trig-g rativea ed x-p at in-d ngeMi ells.T 40,C withP thath sioni

henc dt ,w w

and IL12(p70) was undetectable. The reasons for the lackof detectable IL12(p70) by PL-stimulated M� are unclear.However, it has been previously reported that exposure to bac-teria can result in substantial secretion of the 40-kDa subunitof IL-12 [IL12(p40)], but minimal secretion of IL12(p70)[24,25]. The marked effects of PL on DC as compared toM� suggest that DC may be one of the potential targets forthe immunostimulating activity of PL during the induction ofimmune responses. The present work is in line with previousreports showing the ability of PL to induce cytokine produc-tion in human peripheral blood mononuclear cells, humanneutrophils and mouse splenocytes[14,26].

In the present study we have established that LPS con-tained within the PL structure is one of the key molecules re-sponsible for the biological activity of PL. However, we foundthat CD40, CD86 and MHC-II expression and IL12(p70) pro-duction is increased in DC from LPS-non-responder micein response to PL. These data clearly indicate that some ofthe biological activity of PL on DC involves non-LPS struc-tures that interact with cell surface receptors different fromTLR4. The molecules and receptors involved in these in-teractions will be part of future studies. Our results are inline with observations indicating that bothNeisseriaLPS andnon-LPS components are important in the induction of co-stimulatory molecules and cytokines from DC[27–29] andw ac-t e ofL

anti-g C-Imp lt ine ofs b-sI DCa seda iblet DCr D4a -s thec om-p ex-p D8T d tos veryes kw sesa urs.

theia im-m vedi ates

an activate DC functions needed for efficient antigenentation. The results also show that incorporation intos an efficient way to deliver antigens to DC for processnd presentation in association with MHC class I and c

I molecules. The work highlights the potential of PL asdjuvant and as a delivery system.

One of the hallmarks of DC maturation and M� acti-ation is increased expression of MHC and co-stimulaolecules and the activation of cytokine production untigen stimulation[20,21]. The increase in expressionHC-II, MHC-I, CD40, CD80 and CD86 on DC and pruction of TNF� and IL12 in response to PL correlate w

he ability of PL to trigger T-cell dependent immuneponses. These data provide new evidence towards therstanding of the early immunological mechanismsered by proteoliposome-based adjuvants. The companalysis of phenotypic changes in DC and M� revealed thifferential effects of PL in the modulation of MHC-II eression on these two cell types. The same stimulus thuced increase of MHC-II expression in DC did not chaHC-II in M � and noticeably was able to inhibit IFN�-

nduced up-regulation of MHC-II expression in these che differential expression of class II compared with CDD80 and CD86 on the surface of macrophages treatedL is in agreement with a number of previous reportsave claimed no significant changes in MHC-II expres

n macrophages due to LPS treatment[22,23].Different effects of PL exposure were appreciated w

omparing cytokine production in DC and M�. DC exposeo PL released high amounts of both TNF� and IL12(p70)hilst TNF� secretion from M� in response to PL was lo

-

ith work that indicates the ability of Gram-negative beria to induce proinflammatory cytokines in the absencPS[30,31].

M� and immature DC can internalize and degradeens and present them in association with MHC-II or MHolecules to stimulate CD4+ and CD8+ T-cells [6,32]. Ex-osure of DC to soluble antigens (e.g. OVA) can resufficient presentation to CD4+ T-cells, whilst presentationoluble OVA to CD8+ T-cells is difficult to achieve in the aence of appropriate stimuli and/or delivery systems[33,34].n the present work, we found that PL induce changes onnd M� known to correlate with maturation and increantigen-presenting ability. We also found that it is poss

o deliver antigens incorporated within PL structures toesulting in efficient presentation of these antigens to C+

nd CD8+ T-cell hybridomas. Activation of the MHC-II retricted CD4+ T-cell hybridoma was more marked whenells were co-cultured with DC exposed to PL-OVA as cared to DC exposed to soluble OVA. Furthermore, DCosed to PL-OVA could stimulate the class I restricted C+

-cell hybridoma which was not primed by DC exposeoluble OVA. These results indicate that PL may be afficient general delivery system to stimulate CD8+ T-cell re-ponses and to enhance CD4+ T-cell activation. Future worill investigate the ability of PL to induce T-cell respongainst antigens from intracellular pathogens and tumo

In conclusion, this study describes, for the first time,nteraction of PL from serogroup BN. meningitidis, with DCnd M�. The data clearly indicate that PL have a strongunomodulatory activity over those immune cells invol

n the initiation of immune responses. The work also indic

1320 T. Rodrıguez et al. / Vaccine 23 (2005) 1312–1321

that PL have the potential to deliver antigens to the immunesystem in a form suitable for T-cell recognition.

Acknowledgments

This work was supported by The Royal Society, BBSRCand The Wellcome Trust. We are grateful to Dr. M.J. Wickand Dr. J. Kapp for providing the T-cell hybridomas and toA. Knight for providing the X63-Ag8 cells.

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