© 2003 Blackwell Publishing Ltd

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, 199–209

Blackwell Publishing Ltd.

ORIGINAL ARTICLE

DCs as efficient APCs in Leishmaniasis

Characterizing cellular immune response to kinetoplastid membrane

protein-11 (KMP-11) during

Leishmania (Viannia) panamensis

infection using dendritic cells (DCs) as antigen presenting cells (APCs)

GABRIELA DELGADO

1,2

*, CARLOS A. PARRA-LÓPEZ

1,2

*, LUIS EDUARDO VARGAS

1

, RUBÉN HOYA

1

, MÓNICA ESTUPIÑÁN

1

, FANNY GUZMÁN

1,2

, ANGELA TORRES

1

, CARLOS ALONSO

3

, IVÁN DARIO VELEZ

4

, CLARA SPINEL

2

& MANUEL E. PATARROYO

1,2

1

Fundación Instituto de Inmunología de Colombia (FIDIC),

2

Departamento de Farmacia, Facultad de Ciencios, Universidad Nacional de

Colombia,

3

Universidad Autonoma de Madrid, Spain and PECET, Universidad de Antioquia, Colombia

SUMMARY

In vitro peptide binding assays and DCs pulsed with recom-binant KMP-11 (rKMP-11) plus six 20-mer overlappingpeptides covering the entire protein of Leishmania (Viannia)panamensis (L(V)p) promastigotes were used to identify T-cell epitopes in this protein. Such in vitro binding assays, usingHLA DRB1* 0101, -0401, -0701 and -1101 alleles, demon-strated that two peptide sequences (

DEEFN

KKMQEQNAK-F

FADKP

and

FKHKFAEL

LEQQKAAQYPSK) exhibitedhigh HLA DRB1* 0401 allele binding capacity. rKMP-11specific T-cell proliferation and cytokine production, derivedfrom 13 volunteers exposed to the parasite, suggested thatusing autologous DCs as APCs becomes advantageous inuncovering T-cell epitopes promoting proliferation and differ-ences in IFN-

γ

and IL-4 production in T-cells from volunteerswith ACTIVE and CURED undetectable disease when otherAPCs were used. The two peptides which bound

in vitro

to theHLA DRB1* 0401 allele were immunogenic in HLA DRB1*04 volunteers, thus validating the use of

in vitro

binding assaysfor predicting epitopes in this protein. The experimentalapproach used here may prove useful for characterizing T-cellepitopes in a protein useful in designing peptide-based vaccinecandidates for Leishmania and other intracellular pathogens.

Abbreviations

:

(L(V)p),

Leishmania (Viannia) panamensis

;(DC), monocyte-derived Dendritic Cell; (APC), AntigenPresenting Cell; (rKMP-11), recombinant Kinetoplastid

Membrane Protein-11; (Th), T-helper; (LSA),

Leishmania

Soluble Antigen from the L(V)p promastigote membrane;(PBMC), Peripheral Blood Mononuclear Cell

INTRODUCTION

Protozoan parasites belonging to the

Leishmania (Viannia)panamensis

species (causing New World cutaneous ormucocutaneous leishmaniasis) are responsible for 86·7% ofthe reported clinical cases of leishmaniasis in affected areas(1,2). Recent attempts to find a vaccine for

Leishmania

infection have focused on studying major surface antigens,such as the metalloproteinase Leishmanolysin gp63, thePromastigote Surface Antigen (PSA) complex and lipopho-sphoglycans (LPG) (3–5). Special interest in LPG lies inits role in promoting binding to (6) and parasite survivalin macrophage phagolysosome (7,8). Some immunologicalstudies characterizing T-cell response to LPG in mice havedemonstrated that such responses are capable of protectingsusceptible mice against

Leishmania

(3,6,9), whereas otherstudies in humans have demonstrated that LPG is highlyimmunogenic (10).

Apart from its immunogenicity, LPG has been consideredas an attractive vaccine candidate because of its abundanceon the promastigote cell surface and its important physio-logical role in parasite–macrophage interaction (7,8,11). Morerecent studies have shown that the

Leishmania donovani

LPGcomplex is co-purified and is tightly, non-covalently asso-ciated with the protein denominated Kinetoplastid Mem-brane Protein 11 (KMP-11) (12). It has also been shown thatmouse lymphocytes primed with purified KMP-11 and pro-mastigotes from

L. (L.) donovani

respond to KMP-11 froma wide variety of Old World

Leishmania

species and Africantrypanosomes, suggesting widespread T-cell epitope conserva-tion in this protein (13,14).

Correspondence

: Carlos A. Parra-López, Fundación Instituto de Inmunología de Colombia (FIDIC), Carrera 50, no. 26-00, Bogotá, Colombia (e-mail: carlos_parra@fidic.org.co).*Both authors contributed equally to this work.

Received

: 11 March 2002

Accepted for publication

: 12 June 2003

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Different aspects of KMP-11 in New World

Leishmania

strains have been studied recently in our laboratory. A highKMP-11 mRNA transcription rate in axenic amastigotes,the mapping of a binding domain required for promastigoteinternalization in macrophages and high KMP-11 homologyand ubiquitous distribution throughout New World speciesmake this protein an excellent vaccine candidate. DespiteKMP-11 from Old World species having proved to beimmunogenic during

in vivo Leishmania

infection in dogs(15) and human plasma (16), studies in humans regardingthe immunogenicity of this protein in New World species(17,18) have been limited to characterizing B-cell epitopes.Identifying KMP-11 T-cell epitopes in New World

Leishmania

spp. has yet to be carried out.The use of

in vitro

peptide binding assays has recentlybeen explored with some success for predicting T-cell epitopesin a protein in our Institute (19). An attempt was thusmade (without success) to characterize T-cell epitopes inKMP-11 from L(V)p using irradiated PBMC as APCs fromvolunteers suffering from cutaneous leishmaniasis (controlsand subjects having active and cured disease), by using acombination of peptide binding assays and conventionalT-cell mapping studies. Based on these negative results, itwas predicted that more proficient APCs might be requiredfor mapping epitopes in this protein. It was decided to useautologous DCs as APCs to circumvent observed T-cell unres-ponsiveness when irradiated PBLs were used as APCs.This paper describes how DCs used as APCs led to thedetection of various T-cell epitopes in L(V)p KMP-11 thathave not been described until now. The genetic restrictionof two epitopes (identified in KMP-11 predicted through

in vitro

binding assays using purified DRB1* molecules andKMP-11 peptides) was confirmed in these studies, thusvalidating the use of

in vitro

binding assays for predicting T-cell epitopes in a protein. The results suggest an importantrole for DCs as APCs in uncovering epitopes triggeringcellular immune responses in

Leishmania

-infected volunteers.The results also suggest the advantage of the use of DCs inuncovering peptide epitopes in

Leishmania

parasite proteinsand other intracellular pathogens, for the development ofsubunit, synthetic vaccine candidates.

MATERIALS AND METHODS

Groups of volunteers studied

Volunteers were only enrolled in the study after they hadsigned the informed consent-form. After volunteers hadundergone DR typing (see below), they were classified intothree groups. The first group contained

Volunteers withACTIVE disease

(A1 to A4 shown in Tables 2, 3 and 4),having cutaneous leishmaniasis who had not received anti-

leishmanial treatment prior to the blood samples beingtaken.

CURED volunteers

(P1 to P9 shown in Tables 2, 3 and4), were people who had successfully overcome skin lesioncaused by L(V)p cutaneous leishmaniasis without relapse3 months after treatment with anti-leishmanial drugs.Evidence pointing to ACTIVE and CURED volunteers’ trueexposure to the parasite was: (i) blood samples taken fromvolunteers living in high L(V)p endemic areas; (ii) subjectsdiagnosed with leishmaniasis having active cutaneous lesion(ACTIVE); (iii) volunteers with scars left on their skinfrom cured lesions having a clinical history of cutaneousleishmaniasis treated by chemotherapy with meglumineantimoniate (CURED); and (iv) volunteers with positiveLeishmanin skin test.

CONTROLS

(C1 to C5 shown inTable 3) were people who had never lived in or visited areaswhere L(v)p is endemic and whose Leishmanin skin testwas negative.

PCR characterization of volunteers’ HLA-DRB haplotype

Genomic DNA from patients and controls was isolatedfrom whole blood by using a Wizard genomic DNA purifica-tion kit (Promega, Madison, Wisconsin), according to themanufacturer’s instructions. DNA samples were subjectedto polymerase chain reaction to amplify the DRB gene’ssecond exon using sense and anti-sense sequence-specificprimers for four groups of alleles (DRB1* 0101; DRB1*0401–0422; DRB1* 0701; and DRB1* 1102–1129 alleles) asdescribed by Olerup (20).

ELISA

Flat-bottomed microtitre plates (Nunc Brand Products,Nalge Nunc International, Denmark) were coated overnightwith a 100

µ

L volume of peptide, rKMP11 and LSA anti-gens (all at 10

µ

g /mL concentration), blocked with blockingbuffer (2·5% non-fat milk in PBS containing 0·05% Tween20) and then incubated with 1 : 100 plasma dilutions inblocking buffer, tested in duplicate. Plates were incubatedat room temperature with peroxidase-conjugated rabbitantihuman IgG (Amersham-Pharmacia, Buckinghamshire,UK) diluted at 1 : 2000 in blocking buffer and developedwith 100

µ

L tetramethyl-benzidine-peroxidase substrate(Kirkegaard & Perry Laboratories, Gaithersburg, MD) inPBS-Tween. Cut-off points were established as three standarddeviations above the mean OD

620

value of plasma fromCONTROL volunteers (each peptide calculated individu-ally). When quantifying cytokines by ELISA, the concentra-tion in pg/mL was found by using commercial kits OptEIAHuman IL-4 set, and IFN-

γ

sets (Pharmingen, San Diego,CA), following the manufacturer’s instructions. The detectionranges were worked out to assure a detection range of

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between 2 and 1000 pg/mL for IL-4 and between 2 and500 pg/mL for IFN-

γ

. A response was considered measur-able if the Stimulation Index was

≥

2 (SI: cytokine level insupernatant from antigen-stimulated culture/cytokine levelin supernatant from cultures without antigen).

Indirect Immunofluorescence Assay (IIFA)

Ten

µ

L containing 1

×

10

6

L(V)p promastigotes in log-arithmic phase were placed on a slide, fixed with cold acetoneand blocked with PBS solution (PBS supplemented with5% inactivated Foetal Calf Serum), followed by 30 minincubation in a humid chamber. The slides were then leftin plasma dilutions in PBS solution for 45 min in a humidchamber. Afterwards, they were incubated at room tem-perature, with enzyme-FITC conjugated to anti-human IgGproduced in rabbits (Amersham-Pharmacia, Buckingham-shire, UK), used 1 : 150 dilution in PBS solution for 45 min.The slides were stained with 1 : 50 Evans’ blue solution (todemonstrate contrast under UV microscope), dried at roomtemperature and inspected under a UV microscope (Nikon,Japan).

Parasites

L(V)p (M/HOM/PA/71/LS94) promastigotes were used.Parasites were maintained

in vivo

using Sirium GoldenHamsters (Instituto Nacional de Salud, Bogotá, Colombia)to preserve virulence. Animals were inoculated intra-nasallyand parasites collected from the lesions. Promastigotes werecultured at 24

°

C in RPMI-1640 (Gibco BRL-Life Techno-logies Inc., Grand Island, NY, USA) supplemented with 10%FCS (Foetal Calf Serum, Gibco BRL-Life Technologies),and harvested during stationary phase for

in vitro

assays asLSA source (see below). Since disperse parasites collected inlogarithmic phase facilitated monitoring parasite uptakein phagocytosis experiments, they were used preferably topulse DCs when whole parasite L(V)p pulsed APCs wereused. DCs were pulsed with L(V)p promastigotes by adding100-fold excess parasites over DC for 4 h, followed byseveral washes to remove non-internalized parasites.Parallel culture preparations of DC exposed to parasiteswere processed by Giemsa to monitor parasite uptake andimmunocytochemistry.

Immunocytochemistry

Surface MHC class I and class II antigen expression onDCs was evaluated by immunocytochemistry. The cells wereplaced on slides and fixed with 70% methanol. Class I andclass II molecules were detected by light microscope ana-lysis after staining cell preparations with reagents from a

commercial kit (Promega, Madison, WI, USA) containinganti-class I or class II mAbs coupled to alkaline phosphatase,using BCIP and NBT as substrate and colour developmentreagents, respectively.

Antigen-pulsed DC preparation

Dendritic cells used as APCs in phagocytosis and antigen-specific T-cell stimulation assays were obtained by thefollowing protocol: 10

µ

g/mL of purified human gammaglobulin (ICN Pharmaceuticals, Inc, Aurora, OH, USA)were left for 30 min in a polystyrene Petri dish (Nunc BrandProducts, Nalge Nunc International, Denmark). After theplates had been rinsed with PBS or RPMI 1640 (GibcoBRL, Life Technologies, Inc, Grand Island, NY), 5–20

×

10

6

PBMCs, previously separated by density gradient (Lym-phoprep, Nycomed Pharma AS, Oslo), were suspended in4 mL RPMI 1640 supplemented with 10% autologousplasma and placed in the dish. Non-adherent cells wereremoved 2 h later and adherent cells washed twice withpreheated RPMI 1640. Finally, 4 mL RPMI 1640 supple-mented with 10% autologous plasma, 250 ng/mL GM-CSF(Leucomax, Essex Chimie SA, Lucerne) and 100 ng/mL IL-4 (produced in supernatants by IL-2T6 transfectants kindlyprovided by Dr Antonio Lanzavecchia, Basel Institute forImmunology) were added to adhered cells. An equal amountof both transformation-inducing cytokines was added to thecultures every third day. On day 7 (48 h prior to adding T-cells), 10

4

immature dendritic cells per well were transferredinto round-bottomed, 96-well plates. The immature DCs weretreated with 2

µ

g /mL Poly I:C SIGMA, Sigma Chemical Co(St Louis, MO, USA) to enable DC maturation (21) and pulsedwith 10

µ

g /mL of each antigen (synthetic peptides, L(V)p orrKMP-11). A control culture was prepared in which noantigen was added at this step.

DC phenotype characterization by flow cytometry

Monocytes and DCs were characterized by flow cytometryusing 1 : 200 dilution of antibodies specific for CD1a-PE,CD1b-FITC, CD80-PE and HLA DR-FITC (Pharmingen,San Diego, CA, USA) and a flow cytometer (BecktonDickinson, San Jose, CA, USA). Histograms measuringrelative fluorescence intensity in FL1 (FITC) and FL2 (PE)channels were constructed and analysed after 10 000 eventshad been acquired.

Synthetic peptides

Six peptides, representing the complete

L. (Viannia) pana-mensis

KMP11 molecule amino acid sequence (GenBankaccession number AF219228), were synthesized by multiple

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solid-phase methodology (22–24): 19113 (MATTYEEFAAK-LDRL

DEEFN

), 19114 (

DEEFN

KKMQEQNAKF

FADKP

),19115 (

FADKP

DESTLSPEMK

EHYDK

), 19116 (

EHYDK

-FERMIKEHTE

KFNKK

), 19117 (

KFNKK

MHEHSEH

FKH-KFAEL

) and 19118 (

FKHKFAEL

LEQQKAAQYPSK);resides in bold correspond to overlapping amino acids. Biotin-tagged peptide controls used in peptide binding assays werelabelled using the Sulfo-NHS-LC-Biotin biotin derivative(Pierce Chemical, Rockford, IL, USA) as described (25).Peptide purity was assessed by reverse phase HPLC andbiotinylation efficacy monitored by the Kaiser-test (23) andstandard ELISA assays.

rKMP-11 and

Leishmania

Soluble Antigen (LSA) from L(V)p promastigote membranes

Leishmania (L.) infantum

rKMP-11 produced in

E. coli

aspreviously described (15) was used to prime T-cell lines,based on the high degree of homology between the L(V)pKMP-11 amino-acid sequence and that of

L. (L.) infantum

(96% identity at the amino-acid level) (17). Ten to fiftymillion L(V)p promastigote forms were placed in 1 mL lysissolution containing Triton X 114 and protease inhibitors(SIGMA, Sigma Chemical Co, St Louis, MO, USA) toproduce LSA. LSA was then obtained as described byMancianti

et al

. (26) following 10 freeze/thaw cycles.

Peptide binding assays

Peptide competition assays were conducted for measuringunlabelled KPM-11 peptide ability to compete with bio-tinylated indicator peptides for binding to purified HLA-DR molecules. EBV-transformed EKR and 9043 B-cell lineswere used as the sources for DRB1*0701 and DRB1*1101molecules, respectively. DR1 (DRB1*0101) and DR4(DRB1*0401) molecule experiments were performed withpurified recombinant molecules produced in S-2 cells, kindlyprovided by Dr Lawrence Stern (MIT, Boston, MA). Bio-tinylated peptides containing allele-specific binding motifsproviding optimal binding to each allele were used ascontrol peptides for these assays; the HA 306–318 peptidewas used for DR1 and DR4 and TT 830–843 and Gly-Phe-Lys-(Ala)

7

(GFK(A)

7

) were used for DR7 and DR11, res-pectively. Affinity purification of HLA-DR molecules fromB-cell lines and peptide binding assays (27) was per-formed as previously described (19,28). KMP-11 peptidebinding to DR molecules was determined by measuringthe OD in the presence or absence of competitor peptide.Binding inhibition was calculated as a percentage as fol-lows: 100

×

[1

−

(

∆

OD in the presence of competitor peptide/

∆

OD without competitor)]. Competitor peptide inhibitingbiotinylated indicator peptide binding by 50% or more was

taken as indicating that a peptide bound efficiently to thatparticular MHC-class II allele.

rKMP-11 T-cell specific T-cell line and stimulation assays

Volunteers’ PBMCs were separated by density gradient(Lymphoprep, Nycomed Pharma AS, Oslo, Norway); cellviability was established by Trypan blue exclusion. 2

×

10

6

PBMC in the presence of 10

µ

g /mL recombinant KMP-11were cultured in 1 mL complete medium (RPMI-1640 sup-plemented with 2 m

-glutamine, 1% non-essential aminoacids, 1 m

sodium pyruvate, 25 U/mL penicillin, 50 mg/mL streptomycin and 5

×

10

−

5

2-ME and 10% autologousplasma). The medium was supplemented with 100 U/mLrIL-2 (Pharmingen, San Diego, CA) after 3, 7 and 10 daysculture at 37

°

C and 5% CO

2

. T-cell stimulation assays werecarried out in triplicate using 1

×

10

5

T-cells and 1

×

10

4

autologous mature DCs pulsed with antigens, 48 h priorto being used as APCs. After DCs had been cultured withT-cells for 48 h, the supernatant was harvested and cryo-preserved at

−

70

°

C until used for detecting IL-4 and IFN-

γ

.100

µ

L/well fresh complete medium, containing 1 µCi [3H]-thymidine (Amersham-Pharmacia, Buckinghamshire, UK),were added to pulse the cultures for the last 16 h. The cellswere then harvested by liquid scintillation in a beta counterto determine specific incorporation. The results are expressedas stimulation index (S.I.), determined as the (mean c.p.m. inpeptide-stimulated culture)/(mean c.p.m. in control culturesincubated without antigen).

Statistics

Statistical significance (P-value) of the immune responseamongst different groups of volunteers was analysed by theKruskal–Wallis test.

RESULTS

In vitro HLA-DR binding assays

The results of in vitro studies aimed at evaluating the afore-mentioned six peptides’ ability to bind to four HLA DRB1*0101, 0401, 0701 and 1101 molecules, covering close to 75% ofthe population (29), demonstrated that peptides 19114 and19118 exhibited high HLA-DRB1* 0401 allele binding ability(Table 1). It was decided to assess KMP-11 peptide immuno-genicity in positive Leishmanin skin-test volunteers typedfor HLA DRB1* 01, 04, 07 and 11 allele groups; eight out of13 selected volunteers exposed to the parasite (ACTIVE andCURED) were identified as having the DRB1* 04 haplo-type. The correlation between in vitro immune response andpeptide binding assay predictions was also ascertained.

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Peptide mapping and antibody response to KMP-11 in selected volunteers

Table 2 shows serological activity against rKMP-11 andpeptides in plasma taken from patients and controls. Similarto previous experiments searching for anti-rKMP-11 anti-bodies, six peptides and LSA in plasma and whole parasiteantibody titres (assessed by ELISA for the first three andIIFA for the whole parasites) were low in selected volunteers,even in those with ACTIVE disease (Table 2 and data notshown). Most antigens revealed similar reactivity by ELISAto that of volunteers in the control group (see cut-off for

each antigen shown in Table 2). Limited antibody reactivity(as detected by IIFA) in the ACTIVE group waned as thedisease became cured (CURED volunteers). SignificantLeishmania antibody levels were not detected by IIFA incontrol volunteers (data not shown).

DCs efficiently stimulate proliferation of short-term, rKMP-11 specific T-cell lines

Autologous DCs pulsed with KMP-11 antigens were usedto study cellular immune response to this antigen. Table 3summarizes each volunteer’s yield of immature DC obtained

Table 1 Six overlapped KMP-11 peptides’ capacity for binding to purified HLA-DR molecules

Peptide code Sequence

HLA-DR molecules

DRB1* 0101 DRB1* 0401 DRB1* 0701 DRB1* 1101

19113 MATTYEEFAAKLDRLDEEFN 20a 7 18 4319114 DEEFNKKMQEQNAKFFADKP 23 57 −8 2119115 FADKPDESTLSPEMKEHYDK 12 13 −20 3819116 EHYDKFERMIKEHTEKFNKK 20 35 −36 4019117 KFNKKMHEHSEHFKHKFAEL 8 1 −12 2419118 FKHKFAELLEQQKAAQYPSK 4 60 3 62

aRelative binding efficacy of the peptide to the DRB1* allele. DR1 and DR4 were recombinant empty DR molecules (S-9 cells), DR7 and DR11 were purified from EBV transformed B cells.

Table 2 Characteristics of 15 subjects participating in this study and serological activity against rKMP-11 and peptides therefrom in plasma of volunteers studied

Group Patient Code HLA-DRB1* Sex DTH (mm) IIFAa title

Antigens

LSA rKMP11

Individual peptides

19113 19114 19115 19116 19117 19118

Active A1 04 M 4 20 0·246b 0·237 0·201 0·290 0·339 0·218 0·149 0·136A2 04 M 15 320 0·322 0·275 0·091 0·097 0·110 0·055 0·078 0·053A3 F 20 40 0·138 0·100 0·060 0·149 0·081 0·036 0·100 0·037A4 04 F 10 40 0·261c 0·151 0·083 0·124 0·088 0·065 0·074 0·057A5 M ND 160 0·226 0·110 0·077 0·085 0·093 0·044 0·048 0·056A6 M ND 80 0·179 0·172 0·082 0·105 0·107 0·058 0·051 0·064

Cured P1 04 M 17 20 0·129 0·224 0·211 0·190 0·240 0·131 0·120 0·072P2 04 F 8 20 0·125 0·165 0·145 0·205 0·188 0·089 0·089 0·092P3 M 9 10 0·077 0·173 0·177 0·334 0·299 0·151 0·173 0·159P4 04 M 9 10 0·193 0·382 0·074 0·203 0·183 0·054 0·068 0·053P5 01 M 8·5 20 0·162 0·165 0·105 0·143 0·201 0·072 0·054 0·102P6 F 7 10 0·302 0·114 0·100 0·115 0·143 0·092 0·114 0·108P7 04 M ND 10 0·180 0·084 0·068 0·112 0·109 0·025 0·043 0·036P8 04 M 6 10 0·261 0·389 0·248 0·305 0·266 0·137 0·101 0·101P9 F ND 10 0·269 0·195 0·147 0·218 0·143 0·059 0·060 0·079Cut off – – ≥ 4 20 0·250 0·180 0·400 0·390 0·240 0·230 0·100 0·130

aPlasma dilution up to specific fluorescence can be observed. bOptical density value (OD620). cValues in bold represent values considered

positive.

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from a given number of PBMC-derived monocytes treatedwith IL-4 and GM-CSF. Figure 1 shows the characterizationby immuno-histochemistry and flow cytometry of represent-ative immature and mature DC samples obtained from thosevolunteers studied. A population of partially sticky veiledcells (very different morphologically from their monocyteprecursors) was revealed by Giemsa smear following 7 days’treatment of the monocyte-enriched fraction with cytokines(Figure 1a–c). Treatment of immature DC with Poly I:C did

Table 3 Immature DC yields following 7 days monocyte treatment with IL-4 and GM-CSF

Patient codePBMC(× 106)

Immature DCs(× 106)

A1 12·8 0·8a

A2 3·0 0·7A3 16·5 1·9A4 8·6 1·4

P1 12·3 3·6P2 13·6 1·5P3 5·0 2·7P4 6·9 1·3P5 5·8 0·6P6 18·0 1·9P7 8·6 2·6P8 29·4 1·8P9 32·0 2·6

C1 15·2 2·9C2 12·3 1·5C3 18·9 0·9C4 20·5 1·5C5 10·9 1·0

aNumber of sticky veiled viable cells established by trypan blue exclusion.

Figure 1 DC characterization by light microscopy and flow cytom-etry. Microscope and flow cytometry analysis of monocyte-derived DCs. Pictures (a) to (g) and (l) to (m) and histograms (h) to (k) are representative of numerous experiments performed on monocyte-derived DCs on each volunteer studied. Giemsa staining of: (a) monocytes recovered after panning on PBMC Ig-G coated plates and (b) immature DCs 7 days after monocyte treatment with IL-4 and GM-CSF. (c) Light microscopy of mature DCs after 48 hours’ treatment of immature DCs with Poly I:C. Immuno-histochemistry of immature DCs (d) and (f) and mature DCs (e) and (g) stained with anti-human Class I (d), (e) and anti-human DR (f), (g). Histograms (h) to (k) show up-regulation of (h) CD1a (i) CD1b ( j ) CD80 and (k) HLA-DR surface molecules detected by flow cytometry. Continuous lines represent each marker expression on mature DC cell surface. Dotted lines correspond to base expression of these molecules on freshly isolated monocytes. (l), (m) Representative DC preparations of those performed on each volunteer showing the phagocytic capacity of immature Giemsa-stained DCs. The parasite load in (l) immature (shown with arrows) and (m) mature DCs after 4 h incubation with viable L(V)p promastigotes.

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induce strong maturation of these cells revealed in up-regulation of surface CD1, Class II and co-stimulatorymolecules (Figure 1h–k). The phagocytic activity character-istic of immature DCs became evident in experiments inwhich whole live parasites were rapidly internalized byimmature DCs after 4 h in vitro exposure to promastigotes.Figure 1(l,m) shows an experiment representing the con-sistently higher parasite internalization in immature DCsfollowing their exposure to L(V)p promastigotes.

Morphological and flow-cytometry analysis showed that,in the culture conditions used here, monocytes purified fromPBMC efficiently transformed into DCs in all those volun-teers examined (Figure 1 and data not shown). Table 4(a)presents the efficacy of DCs pulsed with various antigens

(whole L(V)p parasites, rKMP-11 or six synthetic peptides)in promoting cellular proliferation by rKMP-11 specific T-cells. All antigens poorly elicited T-cell proliferation (shownas stimulation index in panel a) in three out of four volunteershaving ACTIVE disease. The same unresponsiveness wasobserved in the CONTROL group (data not shown). Inclear contrast, T-cell proliferation from volunteers in whomthe disease had been cured (P subjects in Table 4) was specificfor at least one peptide in six out of nine volunteers. MostCURED volunteers’ T-cells specifically proliferated topeptides 19113 and 19118 (six and five out of nine, respect-ively). Peptide 19114 induced proliferation in five out ofthe 13 volunteers examined whereas 19118 did so in six. Thedifference in cellular proliferation to peptides observed

Table 4 DCs pulsed with rKMP-11, KMP-11 peptides and L(V)p promastigotes efficiently stimulate rKMP-11 specific T-cells

Patient codeHLA-DRB1*

A104

A204

A3 A404

P104

P204

P3 P404

P5 P601

P704

P804

P9 Total no. of responsive patients

(a) Proliferationa

Antigen19113 5·8 – – – 2·1 3·9 – 7·6 5·2 – 2·4 – 2·8 719114 24·0 – – – – – – 8·6 3·6 – 3 – 2 519115 4·3 – – – – – – 4 3·1 – 3·1 – – 419116 6·7 – – – – 3·1 – 2·3 5·6 – – – – 419117 3·0 – – – – 3·3 – 2·9 3·2 – – – – 419118 4·6 – – – – 3·3 – 5·6 4·5 – 2·7 – 2·9 6KMP-11 3·8 – – – – 2·9 – 5·2 4·7 – 3·3 – – 5L(V)p – – – – ND 4 ND ND ND 8 – 3·5 – 3PHA 10·7 5·7 3·8 6·8 2·0 6·0 2·2 13·5 9·0 9·9 6·5 3·8 4·8 13

(b) IL-4b

19113 – – – 16 8·8 – 22 – – – – – – 319114 – – – 19 – – – – – – – 13 – 219115 27 – – 22 – – – – – – – – – 219116 – – – 20 22 – – – 22 – – – – 319117 – – 2 – – 3 – – 21 – – – – 319118 2 – 23 – – – – – – – – – – 2KMP-11 – – ND – – – – – – – – – – 0L(V)p – – 7 – ND – ND ND ND – – – – 1Poly I:C only 0 22 8·8 0 0 6 7 14 0 0 – 0 22 6

(c) IFN-γ c

19113 – – 24 3 – 16 2 – – – 5 – – 519114 – – 2 – – 3 – 2 2 – 9 – – 519115 – – 2 7 – 2 3 – – – 12 – – 519116 – – 3 – – 16 – 2 – – – – – 319117 – – 4 – – – – – – – – 27 – 219118 – – 3 – – 3 – 121 25 – – 15 – 5KMP-11 – – – – – – – – – 5 8 2 – 3L(V)p – – – – ND 6 ND ND ND – 21 – – 2Poly I:C only 7 2 2 0 0 3 7 2 30 3 5 8 12 11

aStimulation index determined as (the mean of c.p.m. in peptide-stimulated DCs matured with Poly I:C)/(mean c.p.m. in control matured DC cultures without antigen). Counts ranged from 1676 to 20 523 c.p.m. in non-stimulated and antigen-stimulated cultures, respectively. Stimulation index of bIL-4 and cIFN-γ is (the production of T cells lines stimulated with antigen)/(cytokine level in supernatant from the line cultured without antigen). L(V)p: Leishmania (Viannia) panamensis promastigotes. PHA: phyto-haemaglutinin. N.D.: not done. HLA DRB1*: HLA DRB1* haplotype of volunteers; A: ACTIVE and P: CURED subjects.

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between volunteers exposed to the parasite (ACTIVE andCURED) and CONTROL volunteers reached statisticalsignificance (P < 0·01). Poor T-cell proliferation in mostvolunteers was observed when DCs pulsed with proma-stigotes were used as APCs (see proliferation values in res-ponse to L(V)p in Table 4a).

Cytokine profile of rKMP-11 specific T-cells in volunteers with ACTIVE and CURED disease

The level of antigen-specific cytokine production by rKMP-11 specific T-cells elicited from ACTIVE and CUREDvolunteers was ascertained to study the correlation betweenT-cell IL-4 and IFN-γ cytokine production and the evolutionof the disease. Panels b and c in Table 4 show T-cell IL-4 andIFN-γ production after stimulation with various antigens.The numbers correspond to IL-4 and IFN-γ increase inT-cell cultures with DCs pulsed with antigens, regardingcontrol stimulated with autologous DCs but without antigen.T-cell IL-4 production in response to mature DCs pulsedwith peptides was predominantly observed in ACTIVEvolunteers; it was not dominated by a single peptide in allvolunteers. Significant IL-4 production was not detected inmost volunteers in response to DCs pulsed with rKMP-11or with L(V)p promastigotes. IL-4 production was morefrequent in ACTIVE than in CURED volunteers (Table 4b)or CONTROLS (data not shown). The difference in IL-4 production between ACTIVE and CURED volunteerswas statistically significant (P < 0·05). On the other hand,although IFN-γ production did not reach a statistically signi-ficant difference between ACTIVE and CURED volunteers,IFN-γ response showed: (i) antigen-induced IFN-γ produc-tion was widely distributed in volunteers in whom the dis-ease had been CURED; and (ii), with few exceptions, noIL-4 was found in subjects in which IFN-γ production wasdetected.

DISCUSSION

Attempts to find a human leishmaniasis vaccine have per-sisted in characterizing T-cell epitopes because protectionagainst leishmaniasis is a T-cell mediated event (10,30).Diverse parasite major surface components have beenexplored in vitro to determine the cellular protective profileof epitopes in various Leishmania proteins (10,31–34).Widespread T-cell epitope conservation shown in animalmodels (13–14), together with high KMP-11 homology andubiquitous distribution throughout Old World Leishmaniaspecies, make this protein an excellent vaccine candidate.Although immunological studies with purified KMP-11from different species have suggested great T-cell epitopeconservation within Old World species (13,15–16,35),

immunological analysis of KMP-11 in New World strainshas been limited (17,18) and the study of T-cell epitopes hasyet to be undertaken.

Peptide mapping and humoral immune response to KMP-11

Because of their simplicity in terms of manufacture andformulation, their low cost and ability to elicit strongimmune responses against complex pathogens, peptide-based vaccines represent an important alternative for vaccinedevelopment. Identifying peptide antigens that bind in vitroto HLA DR alleles has been successfully used for predictingpeptide-based vaccine candidates by ourselves (19) andothers (27). This study was conducted to identify T-cellepitopes in KMP-11 protein.

The antibody response of volunteers to rKMP-11 andits related peptides showed that these antigens did not elicita vigorous humoral immune response in volunteers withcutaneous leishmaniasis. This observation was consistentwith previous experiments in which (despite numeroussera examined) low antibody reactivity to this antigen wasalways observed (this paper and data not shown). Although40% of volunteers infected with L(V)p (ACTIVE andCURED volunteers) studied here exhibited antibodyreactivity against rKMP-11 by ELISA, the reactivity to thisrecombinant protein and those peptides derived from itwas low. Furthermore, the level of antibodies producedagainst the whole parasite detected by IIFA was low inACTIVE volunteers and waned as the disease became cured.Such low sera reactivity against Leishmania antigens agreeswith Isaza’s previous observations that (i) IIFA antibodytitres hardly correlated with the quantity of parasitesdetected in the wound or the clinical characteristics of thedisease at the time of diagnosis and (ii) that they wereusually negative in post-treatment samples (36). Conforma-tional B-cell epitopes have been suggested in KMP-11 (35),whereas others have reported that linear epitopes in thisantigen were reactive solely in volunteers with visceralleishmaniasis (16). Although these two possibilities mayexplain our failure to detect anti-peptide antibodies in thegroup of volunteers studied here, the discrete antibody titresto peptides or whole KMP-11 protein evidenced by ELISAand IIFA in our studies led us to argue that B-cell epitopesin KMP-11 seem to be poorly reactive in volunteers withcutaneous leishmaniasis caused by L(V)p.

Peripheral blood monocyte DC production in those volunteers studied

Besides studying the humoral response to KMP-11, theimmunological characterization of T-cell epitopes in L(V)p

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KMP-11 protein using DCs as APCs has also been presented.Preliminary attempts to address the immunological relevanceof this protein and its pertinent peptides by using irradiatedautologous PBMCs as APCs were discouraging (data notshown). Neither substantial proliferation nor cytokineproduction was elicited in vitro on antigen-stimulated T-celllines, despite numerous volunteers being examined whopresented active cutaneous leishmaniasis or were recoveringfollowing treatment (data not shown). These negative resultsled to the hypothesis that more efficient APC should be usedto map T-cell peptide epitopes in this protein; hence DCsderived from peripheral blood monocytes as APCs wereused to circumvent this problem.

Variable yields of immature DCs depended largely oneach volunteer’s monocyte count as established by volunteerhaemogram (data not shown). Even though monocyte-derived DCs from numerous healthy volunteers and patientswere examined, no alterations were detected in the expres-sion of maturation markers monitored by flow cytometry inDCs from ACTIVE and CURED volunteers compared toCONTROLS, when immature DCs from all volunteers werepulsed with live promastigotes (data not shown).

DCs used as APC favoured identifying T-cell epitopes in KMP-11 in volunteers exposed to the parasite (ACTIVE and CURED volunteers)

The high density of MHC class I and II molecules on thecell surface and their longevity there, the proficient machineryfor antigen processing and presentation together with thehigh density of co-stimulatory molecule expression found inmonocyte-derived DCs, have led investigators to use DCsas APC for successfully exerting such functions both in vivo(37) and in vitro (38–40). Indeed, as shown here, the useof DCs pulsed with KMP-11 resulted in profound T-cellproliferation and cytokine production by rKMP-11 specificT-cells (and the peptides derived from it) in most volunteersstudied. The responsiveness of T-cells from CUREDsubjects compared to that from ACTIVE and CONTROLvolunteers contrasted with our negative results when usingirradiated PBMC as APC (data not shown). Proper prolifer-ation of cells in response to mitogen was observed in allthose volunteers examined (PHA in Table 4a). This resultsuggests that differences in responsiveness to rKMP-11 andpeptides detected in volunteers exposed to the parasite(ACTIVE and CURED) was antigen-dependent whencompared to CONTROLS. The results reported concerningKMP-11 epitope mapping using DCs as APC demonstrate:(i) a remarkably different responsiveness to peptides inthis protein between ACTIVE and CURED volunteers;(ii) a profound capacity of the culture system described hereto detect subtle differences in T-cell responses elicited by

various peptides in CURED volunteers; (iii) that antigen-specific stimulation of T-cells specific for this antigen maybe difficult to elicit if less efficient APC are used; and (iv)that proliferation to various peptides in some volunteersindicates that T-cell precursors to various KMP-11 epitopescan be expanded in a single volunteer, as has been demon-strated for other Leishmania antigens (33). The proliferationinduced by mature and immature DCs (not shown) pulsedwith various peptides in most CURED volunteers may beexplained by immature DCs’ ability to present peptideantigens due to abundant empty MHC class II moleculeson the cell surface, as has been shown in immature mouseDCs (41,42). Empty DR molecules in human DCs (althoughyet to be demonstrated) represent an intriguing possibilityoperating in human immature DCs, possibly explainingthe immunogenicity of peptide-pulsed DCs used as APC inour experiments. DCs were able to sustain T-cell stimulationeven though excess peptide antigen was removed several daysprior to their contact with T-cells. Interestingly, this ‘memory’capacity of DCs for stimulating responding T-cells up to3 days after being pulsed with the antigen, demonstratedfirst by others working with T-cell clones (39), could be seenin our culture system.

DC used as APC revealed Th response dichotomy in ACTIVE and CURED volunteers

Cellular immune response by T-helper CD4+ T-cells is ofparamount importance in the immune response to Leishma-nia spp. Whereas most individuals living in endemic areasfor cutaneous leishmaniasis become cured of the infection(eliciting T-cells protecting them from re-infection), a lowpercentage of individuals are unable to control the diseaseand develop skin lesions (43). Experimental evidence hassuggested that similar to the Leishmania major mouse modelin susceptible and resistant mice (34), T-cell activation byinfected macrophages leads T-helper cells to produce theIFN-γ necessary to foster TNF-α and nitric oxide produc-tion, making macrophages a harsh environment for thisparasite in those humans resistant to Leishmania infection.By contrast, cytokine deactivating macrophage functions(IL-4, IL-10 and IL-13) in susceptible individuals seem toperpetuate macrophage infection and render the hostsusceptible (44). Interestingly, the delicate balance of Th1vs. Th2 cytokines handling T-helper immune response dif-ferentiation in volunteers infected with Leishmania becameevident in this study through using DCs as APC when IL-4production by T-cells was compared to that of IFN-γ. Reci-procity in T-cell IL-4 production vs. IFN-γ was noteworthyin ACTIVE and CURED volunteers, in the sense thatwhereas IFN-γ production was more frequent in CUREDvolunteers’ T-cells, IL-4 production was characteristic of

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T-cells from volunteers in whom the disease was ACTIVE.These results agree with those reported by others (44,45).Finally, despite DCs pulsed with KMP-11 peptides beingable to elicit T-cell responses in KMP-11 specific cells, DCspulsed with promastigotes failed to do so. This result sug-gests that Leishmania promastigote infection of DCs mayabrogate their ability to process and present this antigenduring natural infection. The alteration in natural processingby DCs of a Leishmania immuno-relevant antigen suchKMP-11, may be important for the pathogenesis of humanleishmaniasis. This observation agreed with alterations inantigen processing and presentation of protein antigensproposed by others using Leishmania infected macrophagesas APC (9,46,47).

Although the fact that most T-cells from HLA-DRB1* 04CURED volunteers proliferated or produced IFN-γ inresponse to two peptides (19114 and 19118) suggests a cor-relation between these two phenotypes and HLA-DRB1* 04restriction, some peptides that did not bind to HLA-DRB1*04 did induce proliferation. This result may suggest that thebiological rather than the biophysical (T-cell response overpeptide binding) readout is more sensitive for identifyingepitopes. However, some peptides are not recognized byindividuals classified as DRB1* 04 by PCR typing, as theymight be binding to a different haplotype from the DRB1*0401 molecule used to perform the binding assays. Despitethe possibly limiting threshold of the peptide binding assaysto identifying peptides suggested above, the correlationfound between peptide binding capacity and Th1 phenotypeelicited in KMP-11 specific T-cells responding to 19114 and19118 peptides in HLA-DRB1* 04 CURED individualsvalidated the usefulness of peptide binding assays for iden-tifying peptide candidates for a vaccine against cutaneousleishmaniasis and other intracellular pathogens.

ACKNOWLEDGEMENTS

This research project was supported by the ColombianPresident’s office, the Colombian Ministry of Public Healthand the Cundinamarca Department’s health service (we aregrateful to Dr José Fernando Sánchez and Dr Jorge Monroyfor their active search for patients in endemic areas). Wewould like to thank personnel from the National Institute ofHealth for their technical support in some experiments andalso Luz Mery González for reviewing the statistical analysis.Finally, we would like to thank Jason Garry for patientlyreviewing the manuscript.

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