INFECTION AND IMMUNITY, Oct. 2005, p. 6620–6628
Vol. 73, No. 10
Antigen Requirements for Efficient Priming of CD8?T Cells by
Leishmania major-Infected Dendritic Cells
Sylvie Bertholet,1† Alain Debrabant,2† Farhat Afrin,1‡ Elisabeth Caler,1Susana Mendez,1§
Khaled S. Tabbara,1¶ Yasmine Belkaid,1and David L. Sacks1*
Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health,
Bethesda, Maryland 20892,1and Division of Emerging and Transfusion Transmitted Diseases,
OBRR, CBER, U.S. Food and Drug Administration,
Bethesda, Maryland 208922
Received 2 December 2004/Returned for modification 21 February 2005/Accepted 14 June 2005
CD4?and CD8?T-cell responses have been shown to be critical for the development and maintenance of
acquired resistance to infections with the protozoan parasite Leishmania major. Monitoring the development
of immunodominant or clonally restricted T-cell subsets in response to infection has been difficult, however,
due to the paucity of known epitopes. We have analyzed the potential of L. major transgenic parasites,
expressing the model antigen ovalbumin (OVA), to be presented by antigen-presenting cells to OVA-specific
OT-II CD4?or OT-I CD8?T cells. Truncated OVA was expressed in L. major as part of a secreted or
nonsecreted chimeric protein with L. donovani 3? nucleotidase (NT-OVA). Dendritic cells (DC) but not
macrophages infected with L. major that secreted NT-OVA could prime OT-I T cells to proliferate and release
gamma interferon. A diminished T-cell response was observed when DC were infected with parasites expressing
nonsecreted NT-OVA or with heat-killed parasites. Inoculation of mice with transgenic parasites elicited the
proliferation of adoptively transferred OT-I T cells and their recruitment to the site of infection in the skin.
Together, these results demonstrate the possibility of targeting heterologous antigens to specific cellular
compartments in L. major and suggest that proteins secreted or released by L. major in infected DC are a major
source of peptides for the generation of parasite-specific CD8?T cells. The ability of L. major transgenic
parasites to activate OT-I CD8?T cells in vivo will permit the analysis of parasite-driven T-cell expansion,
differentiation, and recruitment at the clonal level.
Leishmania major is an obligate intracellular protozoan par-
asite establishing itself within the phagolysosome of host
phagocytic cells, primarily macrophages (Mø) and dendritic
cells (DC). In experimental infection models of L. major, mice
of the resistant background (C57BL/6) typically develop a pre-
dominant T-helper type 1 immune response. Acquired resis-
tance in this model relies on activation of CD4?T cells, re-
sulting in secretion of high levels of gamma interferon (IFN-?)
that induce NO-dependent parasite killing by infected macro-
phages (33, 35, 36). Using low parasite dose and intradermal
injection (ear dermis), it was revealed that CD8?T cells are
also required for the control of primary infection in the skin (4,
42), complementing existing observations on their role in re-
sistance to secondary challenge (6, 14, 25–27, 41). These ob-
servations are consistent with clinical studies of cutaneous
leishmaniasis, where efficient priming of CD8?T cells and
their presence within healing lesions have been reported (2, 5,
8, 15, 28).
Whereas the analysis of T-cell responses to many viral, bac-
terial, and some parasitic infections have benefited from the
description of immunodominant epitopes that have made it
possible to study the expansion, differentiation, and recruit-
ment of pathogen-specific T cells in vivo, in leishmaniasis only
a few major histocompatibility complex (MHC) class II (24, 38)
and no MHC class I-restricted parasite epitopes have been
mapped, making further characterization of specific T-cell re-
sponses more difficult. The model antigen ovalbumin (OVA)
has been successfully used with viruses, bacteria, and parasites
to provide important information ranging from antigen pro-
cessing and presentation to development and maintenance of
memory T cells (17, 21, 30–32, 44). In the present study, L.
major parasites expressing intracellular or secreted forms of
OVA bearing MHC class II and/or class I-restricted epitopes
were engineered and characterized as to their ability to prime
OVA-specific CD4?or CD8?T cells in vitro and in vivo,
including their ability to elicit the recruitment of effector T
cells to the site of infection in the skin.
MATERIALS AND METHODS
Mice. C57BL/6 mice were purchased from the Division of Cancer Treatment,
National Cancer Institute (Frederick, MD). B6.SJL congenic mice, OT-II CD4?
T-cell receptor (TCR) transgenic mice, and RAG1-deficient OT-I CD8?TCR
transgenic mice were purchased from Taconic Farms (Germantown, NY). All
mice were maintained in the National Institute of Allergy and Infectious Dis-
eases animal care facility under specific pathogen-free conditions.
Parasite preparation, intradermal inoculation, and estimation of parasite
load. L. major clone V1 (MHOM/IL/80/Friedlin) promastigotes were grown as
previously described (4), and infective-stage metacyclic promastigotes were iso-
* Corresponding author. Mailing address: Laboratory of Parasitic
Diseases, NIAID, Bldg. 4, Room 126, 4 Center Dr., MSC 0425, Be-
thesda, MD 20892-0425. Phone: (301) 496-0577- Fax: (301) 480-3708.
† These two authors contributed equally to this work.
‡ Present address: Centre for Biotechnology, Jamia Hamdard, Ham-
dard University, New Delhi, India.
§ Present address: Department of Microbiology and Tropical Med-
icine, George Washington University, Washington, D.C.
¶ Present address: Arabian Gulf University, College of Medicine,
lated from stationary cultures (4- to 5-days old) by density centrifugation on a
Ficoll gradient (39). Metacyclic promastigotes (5 ? 103) were inoculated intra-
dermally into the ear dermis using a 27.5-gauge needle in a volume of ?5 ?l. The
evolution of the lesion was monitored by measuring the diameter of the indu-
ration of the ear lesion with a direct-reading Vernier caliper (Thomas Scientific,
Swedesboro, NJ). Parasite titrations were performed with ear tissue homoge-
nates obtained as previously described (4). The number of viable parasites in
each sample was determined from the highest dilution at which promastigotes
could be grown out after 7 days of incubation at 26°C. Lesion scores and number
of parasites per ear measured from mice infected with the transgenic parasites
did not differ compared to wild-type L. major (data not shown).
Plasmid constructs and transfection of Leishmania parasites. The pKS NEO
plasmid was used to express several chimeric proteins involving the Leishmania
donovani 3? nucleotidase-nuclease (Ld3?NT/NU) (10) and chicken ovalbumin in
pKS NEO NT::OVA. A portion of the ovalbumin gene encoding amino acids
232 to 288 containing the MHC class I-restricted OVA257-264 (SIINFEKL)
epitope was amplified by PCR from an OVA-containing plasmid template
(kindly provided by S. M. Beverley, Washington University, St. Louis, and R.
Germain, National Institute of Allergy and Infectious Diseases, Bethesda, Md.)
using the forward (5?-TGGTGGAGCGCTCTGGAGCTTCCATTTGCC-3?)
and reverse (5?-CCAAGCGCTAGCCTATTACTCCATCTTCATGCGAGG-
3?) primers, both containing an Eco47III restriction site. The PCR product was
cloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA). The Eco47III OVA
insert was subsequently cloned into pCR2.1 3?NT/NU (9). The SpeI insert of
pCR2.1 NT::OVA was finally cloned into the pKS NEO plasmid.
pKS NEO iNT::OVA17. The nucleotide sequences encoding the OVA chi-
meric protein expressed by this plasmid were amplified by PCR using pCR2.1
NT::OVA as a template and the forward 5?-TAATAAACTAGTATGAGCAA
GGGCCACATGTCCGTG-3? and reverse 5?-TAATAAACTAGTAGCGCTA
GCCTATTACTCCAT-3? primers. The PCR product was cloned into pCR2.1,
and the SpeI insert was subsequently cloned into pKS NEO.
pKS NEO iNT::OVA4. This expression plasmid was constructed as the pKS
NEO iNT::OVA17 above, with the exception of the forward primer OVA-5
pKS NEO SP::OVA. A portion of the ovalbumin gene encoding amino acids
139 to 386 containing MHC class I OVA257-264- and class II OVA323-339-
restricted epitopes was amplified by PCR using the forward 5?-TGGTGGGCT
GAGCCGGCAGATCAAGCCAGAGAGCTC-3? and reverse 5?-CCACCAAG
CGCTCTATTAAGGGGAAACACATCTGCC-3? primers, containing EspI and
Eco47III restriction sites, respectively. The PCR product was first cloned into the
pCR2.1 vector, and the EspI/Eco47III OVA insert was subsequently cloned into
the corresponding sites of pCR2.1 3?NT/NU. The SpeI insert of the resulting
pCR2.1 SP::OVA plasmid was finally cloned into pKS NEO.
L. major clone V1 promastigotes were transfected with each of the expression
plasmids by electroporation and selected for growth in presence of Geneticin
(G418) (Sigma, St. Louis, MO) as previously described (9).
SDS-PAGE and Western blotting. For total cell analysis, either log-phase L.
major promastigotes or tissue-derived amastigotes were washed twice in ice cold
PBS and lysed at 2 ? 108cells/ml in sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) sample buffer. For analysis of parasite proteins
released into culture medium during growth in vitro, log-phase promastigotes
were harvested by centrifugation, washed once in culture medium, suspended at
107cells/ml into fresh culture medium, and incubated at 26°C. After 6 h, 24 h,
and 48 h of culture, 1 ml of each culture was collected and centrifuged at 1,000
? g for 10 min. Supernatants were further centrifuged at 15,000 ? g for 15 min
at 4°C. One volume of 2? SDS-PAGE sample buffer was subsequently added to
1 volume of cleared culture supernatants. Total parasite cell lysates and culture
supernatants were analyzed by SDS-PAGE and Western blotting with rabbit
polyclonal anti-Ld3?NT/NU (no. 1336) or anti-OVA (Sigma) antibodies as pre-
viously described (9).
Metabolic labeling and immunoprecipitation. Log-phase promastigotes (1 ?
107to 3 ? 107cells/ml) were harvested from parasite cultures by centrifugation,
washed three times at ambient temperature in RPMI medium lacking methio-
nine (Invitrogen), and resuspended in that medium to 2 ? 108cells/ml.35S-
methionine (NEN Life Science Product) was added to a final concentration of
100 ?Ci/ml, and the cells were incubated at 26°C for 30 min (pulse labeling).
Subsequently, cells were centrifuged at 5,000 ? g for 3 min, suspended in parasite
culture medium at 0.5 ? 108cells/ml, and further incubated at 26°C for up to 18 h
(chase). After 0, 4, and 18 h of chase, culture aliquots were collected and
centrifuged at 5,000 ? g for 3 min. The cell pellets were washed once in ice-cold
phosphate buffer (PBS; 50 mM Na2HPO4–150 mM NaCl, pH 7.4) and lysed at
108cells/ml in lysis buffer (50 mM Tris, 150 mM NaCl, 1% [vol/vol] NP-40, 5 mM
EDTA, 0.5% deoxycholic acid, 0.1% [wt/vol] SDS, 10 ?g/ml leupeptin, 4 ?g/ml
aprotinin, pH 7.5) for 30 min on ice. Cell lysates were subsequently centrifuged
at 10,000 ? g for 30 min at 4°C, and the resulting cleared cell lysates were
collected. The labeled cell-free culture supernatants were centrifuged at 10,000
? g for 15 min to eliminate remaining cell debris. The cell lysates and culture
supernatants were subsequently processed for immunoprecipitation with anti-
OVA antibody or control rabbit serum and analyzed by SDS-PAGE and fluor-
ography as previously described (11).
Preparation of APC, T cells, and coculture. Dendritic cells and macrophages
were generated from the marrow of C57BL/6 mice femurs. Dendritic cells were
expanded in RPMI 10% fetal calf serum (FCS) in the presence of 20 ng/ml
granulocyte-macrophage colony-stimulating factor (PeproTech, Inc., Rocky Hill,
NJ) for 1 week. Nonadherent immature cells were collected and used as antigen-
presenting cells (APC). Macrophages were grown for 10 days in RPMI 10%–FCS
30% L929 supernatant as a source of macrophage colony-stimulating factor (4).
APC were incubated for 18 h with live or heat-killed (10 min, 56°C) Leishmania
metacyclic promastigotes opsonized by incubation for 30 min at 37°C in 5% fresh
normal mouse serum (four parasites per APC) for 4 h with OVA-coated latex
beads (5:1) (19) or for 1 h with 40 to 1,000 pM of SIINFEKL. Parasites were
centrifuged down onto DC at 1,000 ? g for 3 min. Aliquots of cells were prepared
in a cytospin and stained with Diff Quick (Dade Behring, Dudingen, Switzerland)
to evaluate the level of infection. CD8?T lymphocytes from RAG1 KO OT-I
TCR transgenic mice and CD4?T cells from OT-II TCR transgenic mice were
negatively selected by magnetic separation (MACS system; Miltenyi Biotec,
Bergisch Gladbach, Germany) according to the manufacturer’s indications. The
purity of either CD8?or CD4?T lymphocytes was ?95%. Purified T cells were
labeled with the intracellular fluorescent dye carboxyfluorescein diacetate suc-
cinimidyl ester (CFSE; Molecular Probes, Eugene, OR). The cells were incu-
bated at 2.5 ? 107to 5 ? 107cells/ml in PBS with 0.5 ?M CFSE for 10 min at
37°C, the reaction was stopped with 10% normal mouse serum, and the cells
were washed twice with cold PBS–0.1% bovine serum albumin. CFSE-labeled T
cells were plated at 106cells/well in 24-well plates (Costar; Corning, Inc., Corn-
ing, NY) in RPMI 10% FCS and 3 ? 105uninfected, infected, or antigen-pulsed
APCs were added for 72 h, at which time the cells were fixed in 4% paraformal-
dehyde. To investigate the contribution of MHC class I peptide complexes
generated as a result of peptide regurgitation, a transwell assay was set up in
which 106CFSE-labeled CD8?OT-I T cells and 3 ? 105DC were plated in the
lower chamber of a 24-well transwell plate (Costar), and 3 ? 105L. major 3?NTs-
or NT-OVA-infected DC or DC with added peptide (1 nM SIINFEKL) were
plated in the upper chamber. Proliferation of CD8?OT-I T cells was analyzed
72 h later. To evaluate antigen presentation by L. major NT-OVA-infected
macrophages, IFN-? secretion by primed OT-I T cells was used as a more
sensitive assay of CD8?T-cell activation. Briefly, primed OT-I T cells were
obtained by in vitro stimulation with peptide-pulsed DC in the presence of 10
ng/ml interleukin 2 (PeproTech, Inc.), and used as effector cells at day 5 post-
stimulation. Primed T cells (2 ? 105) and infected APCs (2 ? 105) were plated
in 96-well plates in RPMI–10% FCS for 24 h. Cell culture supernatants were
collected and analyzed for cytokine content.
Adoptive transfer and in vivo activation. B6.SJL congenic mice received in-
travenously (i.v.) 2 ? 106to 5 ? 106CFSE-labeled OT-I total splenocytes. The
mice were challenged the same day in the pinea of the ear with 105metacyclic
promastigotes or 5 ?g of SIINFEKL peptide. Five days later, the draining lymph
nodes were removed and analyzed by flow cytometry. Recruitment of T cells to
the ear dermis of B6.SJL congenic mice that received 2 ? 106to 5 ? 106OT-I
and OT-II splenocytes i.v. was followed for 3 to 4 weeks upon infection with 104
L. major NT-OVA, SP-OVA, or control 3?NTs. Ear tissue homogenates obtained
as previously described (4) were analyzed by flow cytometry.
Immunolabeling and flow cytometry. Antibodies used were from BD Pharm-
ingen. Before being stained, all the cells were incubated with an anti-Fc III/II
receptor monoclonal antibody in PBS containing 0.1% bovine serum albumin.
T-cell proliferation was measured by fluorescence-activated cell sorter at the
single-cell level as expressed by the intensity of CFSE labeling. OT-I CD8?T
cells and OT-II CD4?T cells were identified by characteristic size and granu-
larity, in combination with anti-CD45.2 (fluorescein isothiocyanate- or PerCP-
conjugated), TCR ?-chain (phycoerythrin-conjugated), and anti-CD8 CD4 (Cy-
Chrome- or APC-conjugated) surface staining. For each sample, between 20,000
and 400,000 cells were analyzed with CellQuest software and a FACSCalibur
flow cytometer (BD Biosciences, San Jose, CA).
ELISA. IFN-? was quantitated by enzyme-linked immunosorbent assay
(ELISA) in 24- to 72-h cell culture supernatants by using mouse IFN-? antibody
matching pairs (Pierce Endogen, Rockford, IL) and following the manufacturer’s
VOL. 73, 2005 PRESENTATION OF L. MAJOR OVA ANTIGENS TO CD8?T CELLS6621
Expression of nonsecreted and secreted OVA chimeric pro-
teins by L. major transfectants. L. major promastigotes were
transfected with an expression plasmid to express the Leish-
mania donovani 3?NT/NU::OVA chimeric proteins (Fig. 1A)
which contain most of the Ld3?NT/NU protein (Ld3?NT) (10),
amino acids 1 to 333 including an N-terminal signal peptide but
excluding the C-terminal anchoring region (3?NTs) fused with
a 57-amino-acid fragment of the OVA protein (232 to 288)
containing the MHC class I H-2Kb-restricted SIINFEKL
epitope (NT-OVA). Two additional plasmids encoding trun-
cated NT-OVA proteins, lacking either its N-terminal signal
peptide (iNT-OVA17) or its first 156 amino acid N-terminal
domain (iNT-OVA4), were also generated. In addition, a plas-
mid encoding a fusion protein made of the first 52 amino acids
of Ld3?NT/NU, containing its signal peptide (1–26), fused to a
portion of OVA protein (139 to 386) containing both MHC
class I OVA257-264- and class II OVA323-339-restricted
epitopes, was also made (SP-OVA).
The expression of the various 3?NT::OVA fusion proteins by
transfected parasites was assessed by SDS-PAGE and immu-
noblotting of parasite cell lysates with either the anti-
Ld3?NT/NU or an anti-OVA antibody (Fig. 1B). Promastig-
otes transfected with the pKS NEO NT::OVA plasmid
expressed an ?43-kDa protein (Fig. 1B, lane 3), consistent
with the addition of the OVA sequence to the 3?NTs. Western
blots also showed that promastigotes transfected with the pKS
NEO iNT::OVA4, pKS NEO iNT::OVA17, and pKS NEO
SP::OVA plasmids expressed ?28-, 38-, and 32-kDa proteins,
respectively (lanes 4, 5, and 7). Cell lysates from wild-type
parasites (V1) did not react with anti-Ld3?NT/NU (lane 1) or
anti-OVA (lane 6) antibody. In addition, the different
3?NT::OVA fusion proteins were still detected by the anti-
Ld3?NT/NU or anti-OVA antibody by Western blot analysis of
tissue-derived amastigotes 5 weeks postinfection (Fig. 1B) or
in amastigotes isolated from macrophages 72 h after infection
(data not shown). Both the 3?NTs and the NT-OVA proteins
were detected by the anti-Ld3?NT/NU antibody in promasti-
gote culture supernatants after 6 h (Fig. 1 C, lanes 2 to 3) and
24 h of culture (lanes 7 and 8). In contrast, the antibody
showed no reactivity with supernatants of wild-type parasites
(lane 1), and L. major expressing either the iNT-OVA4or the
iNT-OVA17proteins after 6 h (lanes 4 to 5), 24 h (lanes 9 to
10), or 48 h of culture (lanes 11 to 12), indicating that the lack
of an N-terminal signal peptide prevented their secretion.35S-
Met metabolic labeling of parasites transfected with pKS NEO
SP::OVA, followed by immunoprecipitation with anti-OVA
antibody, showed the presence of a labeled, ?32-kDa SP-OVA
protein in parasite lysates (Fig. 1D, lane 1). The protein was
not detected when a control rabbit serum was used for the
immunoprecipitation (lane 2). Labeled SP-OVA protein levels
in parasite lysates decreased after 4 h (lane 3) and were almost
undetectable after 18 h of chase (lane 4). The labeled protein
was clearly detected in culture supernatants after 18 h (lane 6).
These results demonstrate that the SP-OVA protein was
readily secreted by the transfected parasites. However, the
SP-OVA protein was not detected in 18-h culture supernatants
by Western blot analysis (data not shown), suggesting that it is
secreted less efficiently than other 3?NT::OVA fusion proteins
by transfected promastigotes. Compared to wild-type L. major,
all transfected parasites showed similar growth as promastig-
otes in vitro and similar pathogenicity in C57BL/6 mice in vivo,
as measured by lesion scores and parasite burden at different
times postinfection (data not shown).
L. major NT-OVA-infected dendritic cells prime naı ¨ve OT-I
CD8?T cells in vitro. In contrast to fetal skin-derived DC,
which poorly internalized L. major metacyclic promastigotes
(43), this parasite stage was efficiently taken up by bone mar-
row-derived DC. Under the conditions of infection used, 50 to
80% of DCs were infected with three to five parasites per cell,
with comparable infection levels obtained for each of the con-
The ability of DC to present L. major NT-OVA antigen and
activate CFSE-labeled naı ¨ve OT-I CD8?T cells was evaluated
for proliferation and release of IFN-?. DC infected with L.
major secreting NT-OVA (Fig. 2Aa) induced the strongest
proliferation, with 81% of the OT-I T cells showing dilutions in
CFSE content after 72 h in culture. In contrast and despite
comparable levels of infection (NT-OVA, 77%; iNT-OVA4,
83%; iNT-OVA17, 82%; and 5 ? 1 parasites per DC), DC
infected with L. major iNT-OVA4(Ab) or iNT-OVA17(Ac),
which expresses nonsecreted NT-OVA, induced moderate lev-
els of CD8?OT-I T-cell proliferation (44%) or only low levels
(10%), respectively. Noninfected DC (Ad) or 3?NTs-infected
DC (Ae) did not induce OT-I T cells to proliferate (1%),
whereas SIINFEKL-pulsed DC (Af) induced the maximal re-
sponse (99%). The release of IFN-? by activated OT-I T cells
followed a similar pattern, with the highest concentrations
observed for secreted NT-OVA and peptide, followed by non-
secreted NT-OVA4, NT-OVA17, and background levels for
3?NTs and uninfected DC (Fig. 2B).
To address the importance of active secretion, the immuno-
genicity of secreted versus nonsecreted NT-OVA was com-
pared (Fig. 2C), as well as the delivery by live or heat-killed
transgenic parasites (Fig. 2D). DC infected with L. major NT-
OVA were highly effective in priming naı ¨ve OT-I T cells, ac-
tivating cells to secrete high levels of IFN-? (200 ng/ml) (Fig.
2C). DC required a 10-fold-higher multiplicity of infection
(MOI) with L. major iNT-OVA17than with NT-OVA to
achieve a comparable level of OT-I T-cell activation. The
greater immunogenicity of secreted NT-OVA could not be
explained by differences in infectivity or intracellular parasite
growth between L. major NT-OVA and iNT-OVA17(data not
shown). DC infected with live L. major NT-OVA, which se-
cretes NT-OVA, were significantly more potent than heat-
killed parasites in priming OT-I CD8?T cells (Fig. 2D). In
contrast, similar levels of T-cell proliferation were observed
with DC infected with live or heat-killed L. major NT-OVA17.
At an MOI of 5:1, heat-killed iNT-OVA17induced increased
OT-I proliferation compared to heat-killed NT-OVA para-
sites, which might be attributed to differences in levels of pro-
tein expression by the transgenic parasites. DC infected with
live or heat-killed 3?NTs induced only background levels of
T-cell proliferation, with ?0.5% of the OT-I T cells showing
reduced CFSE levels (data not shown).
To evaluate whether soluble antigen released by extracellu-
lar promastigotes or by infected DC could be a source of MHC
class I-binding peptides generated by serum proteases and
presented by noninfected bystander cells, a transwell culture
6622 BERTHOLET ET AL.INFECT. IMMUN.
FIG. 1. Expression of the 3?NT::OVA chimeric proteins by L. major. (A) Map of the L. donovani 3?NT/NU protein (Ld 3?NT). Its N-terminal
signal peptide (SP), putative transmembrane domain (TM), and epitope recognized by the anti-Ld3?NT/NU antibody (dotted box) are indicated.
The truncated Ld3?NT/NU (Ld3?NTs) and the four 3?NT::OVA fusion proteins (NT-OVA, iNT-OVA17, iNT-OVA4, and SP-OVA) containing a
C-terminal OVA257-264 (I) and OVA323-339 (II) peptides are shown. (B to D) Western blot analysis performed on wild-type (V1) and transgenic
L. major parasites, with the anti-Ld3?NT/NU (V1, 3?NTs, NT-OVA, iNT-OVA4, or iNT-OVA17) or an anti-OVA (V1 or SP-OVA) antibody.
(B) Cell lysates obtained from promastigote and tissue-derived amastigotes. (C) Culture supernatants of promastigotes after 6 h, 24 h, and 48 h
of culture in vitro. (D)35S-Met labeling of L. major SP-OVA promastigotes, followed by immunoprecipitation of promastigote lysates and culture
supernatant after 0 h, 4 h, and 18 h with anti-OVA (lanes 1 and 3 to 6) or control rabbit serum (lane 2).
VOL. 73, 2005PRESENTATION OF L. MAJOR OVA ANTIGENS TO CD8?T CELLS6623
system was employed in which infected DC were separated by
a semipermeable membrane from purified CFSE-labeled
CD8?T lymphocytes and noninfected DC (Fig. 3, a to c). A
normal coculture experiment was run in parallel (d to f). NT-
OVA-infected DC induced OT-I proliferation only when di-
rect contact with T cells was allowed (e), while background
levels of T-cell proliferation were detected when infected DC
were physically separated from the OT-I cells and noninfected
DC (b). In contrast, soluble SIINFEKL peptide added to the
upper well crossed the semipermeable membrane to the lower
well where it was presented by DC and primed OT-I T cells as
efficiently as when added directly to the coculture (compare
Fig. 3c and f).
L. major NT-OVA-infected DC but not macrophages prime
OT-I CD8?T cells in vitro. To address which APC can present
L. major antigens to CD8?T cells, bone marrow-derived DC
or macrophages were infected for 24 h with L. major 3?NTs or
NT-OVA or pulsed for 1 h with SIINFEKL peptide and cul-
tured for an additional 72 h with CFSE-labeled OT-I cells. L.
major NT-OVA-infected DC induced a dose-dependent OT-I
proliferation (Fig. 4Aa). In contrast, L. major NT-OVA-in-
fected macrophages failed to prime naı ¨ve OT-I T-cells to pro-
liferate (1%) at all the MOIs tested, despite higher infection
rates (70 to 100% infected Mø compared to 44 to 65% infected
DC) and higher numbers of parasites per cell at 24 h postin-
fection. Noninfected or 3?NTs-infected APC induced only
background OT-IT-cell proliferation
SIINFEKL-pulsed DC or macrophages induced 90 or 91%
T-cell proliferation, respectively (data not shown). IFN-? lev-
els detected in the different coculture conditions reflected the
proliferation results (Fig. 4Ab). L. major NT-OVA-infected
Mø could activate in vitro-primed OT-I T cells to secrete
IFN-? in a 24-h coculture assay (Fig. 4B) but required a 20-
fold-higher MOI than DC to achieve the same level of T-cell
cytokine secretion. The percentage of infected Mø and number
of parasites per cell were greater than DC at all MOIs tested
(data not shown).
L. major SP-OVA-infected DC prime both OT-II CD4?and
OT-I CD8?T cells in vitro. L. major SP-OVA transfectants,
which secrete a longer OVA protein fragment that can poten-
FIG. 2. DC present L. major NT-OVA antigens to OT-I CD8?T
lymphocytes. CFSE-labeled OT-I cells were incubated for 72 h with
DC; DC infected with L. major 3?NTs, NT-OVA, iNT-OVA4, or iNT-
OVA17; or DC pulsed with 50 pM SIINFEKL peptide. (A) Intensity of
CFSE fluorescence of CD8?TCR??cells as an indicator of T-cell
proliferation. Numbers represent percent TCR??CD8?cells showing
a reduced CFSE content. (B and C) IFN-? levels measured by ELISA
in 72-h culture supernatants are the mean ? standard deviation of
three replicates, and the experiment is representative of three separate
experiments. (D) OT-I T-cell proliferation, given as the percentage of
TCR??CD8?T cells with a reduced CFSE content, in response to DC
infected with different MOIs of live or heat-killed parasites.
FIG. 3. L. major NT-OVA antigen presentation to OT-I CD8?T
cells requires contact between T cells and infected DC. CFSE-labeled
purified OT-I cells were incubated for 72 h with DC infected with L.
major 3?NTs or NT-OVA. OT-I T cells either were cultured with
infected DC in the same well (coculture) or were cocultured with
normal DC in the bottom wells while infected DC were placed in the
upper wells (transwell). Soluble SIINFEKL peptide was added in the
upper well as a control for membrane permeability and exchange
between wells. Numbers represent the percentages of TCR??CD8?
cells showing a reduced CFSE content. The experiment is representa-
tive of two separate experiments.
6624 BERTHOLET ET AL.INFECT. IMMUN.
tially be processed to generate both MHC class II and class I
binding peptides, were used to infect bone marrow-derived DC
for coculture with CFSE-labeled OT-II CD4?or OT-I CD8?
T cells (Fig. 5). High levels of infection were observed with L.
major 3?NTs (76%), NT-OVA (86%), and SP-OVA (73%). L.
major SP-OVA infected-DC activated OT-I cells for prolifer-
ation (7%), although less efficiently than L. major NT-OVA-
infected DC (50%). L. major SP-OVA-infected DC also acti-
vated OT-II cells (15%), whereas L. major NT-OVA-infected
DC had no effect. L. major NT-infected DC induced back-
ground levels in each case. DC incubated with OVA-coated
latex beads were used as positive controls and induced prolif-
eration in 59% of OT-I T cells and 76% of OT-II CD4?T cells,
L. major NT-OVA and SP-OVA are presented in vivo to OT-I
CD8?T lymphocytes. To evaluate the potential of L. major
NT-OVA transgenic parasites to prime OT-I cells in vivo,
congenic B6.SJL mice received 2 ? 106to 5 ? 106CFSE-
labeled naı ¨ve OT-I CD8?T cells i.v. and were subsequently
inoculated intradermally with either 5 ? 105NT-OVA or
3?NTs metacyclics, 5 ?g of SIINFEKL peptide, or saline.
Draining lymph nodes were isolated 5 days later, and the
intensity of CFSE fluorescence on CD45.2?TCR??CD8?
OT-I T cells was determined (Fig. 6A). A relatively high-dose
L. major challenge was used in these experiments to favor the
early activation and monitoring of the transferred CFSE-la-
beled cells. In response to injection of L. major NT-OVA, 28%
of the gated cells showed a reduced CFSE fluorescence, com-
pared to 4% and 1% in response to 3?NTs parasites and saline
and 100% in response to SIINFEKL. These results indicate
that L. major NT-OVA-secreted protein can be efficiently pro-
cessed by APC in vivo and presented to CD8?T cells. In a
second set of experiments, adoptively transferred OT-I T-cell
recruitment to the ear dermis in response to saline, 104meta-
cyclic L. major 3?NTs, NT-OVA, or SP-OVA was assessed 4
weeks postinfection (Fig. 6B). Infections with L. major NT-
OVA or SP-OVA induced the recruitment of OT-I T cells
(CD45.2?CD8?TCR??) to the ear dermis, while 3?NTs and
saline had no effect. Infection with either of the transgenic
parasites induced an enhanced influx of CD8?T cells (CD45?
CD8?TCR??) to the ear compared to saline alone. OT-I T
cells recruited to the ear expressed high levels of Ly6C com-
pared to naı ¨ve cells, indicating an activated-memory phenotype
To evaluate the ability of L. major-derived OVA to prime
both CD4?and CD8?T cells for subsequent recruitment to
the inflammatory site following a low-dose infection in the
skin, B6.SJL mice that received 2 ? 106to 5 ? 106OT-I CD8?
and OT-II CD4?T cells i.v. were challenged in the ear dermis
with 104metacyclic L. major SP-OVA. The transferred OT-II
and OT-I cells were detectable in each case in lymph nodes
draining the challenge site throughout the first 3 weeks of
infection, although the OT-I cells appear to have homed to the
draining lymph nodes in greater numbers than the OT-II cells
(data not shown). The OT-I CD8?T cells were also found
within the inoculation site, detectable in low numbers by 2
weeks postinfection and present in substantial numbers (11%
of TCR??cells) by 3 weeks (Fig. 6D). No OT-II cells were
detectable within the infected ear dermis during this time (data
FIG. 4. L. major NT-OVA-infected macrophages are unable to
prime OT-I CD8?T lymphocytes in vitro. Bone marrow-derived mac-
rophages or dendritic cells were compared for their ability to present
L. major NT-OVA to OT-I T cells. (A) Naı ¨ve CFSE-labeled OT-I T
cells were incubated for 72 h with APC, APC infected with L. major
3?NTs, or NT-OVA or with APC pulsed with 50 pM SIINFEKL pep-
tide. The percentages of TCR??CD8?cells showing a reduced CFSE
content as an indicator of T-cell proliferation (a) and IFN-? levels
measured by ELISA in 72-h culture supernatants are shown. These
results are representative of two separate experiments. (B) Primed
OT-I T cells were cultured with L. major NT-OVA infected Mø or DC.
IFN-? levels in 24-h culture supernatants are the means ? standard
deviation of three replicates.
FIG. 5. DC present L. major SP-OVA antigens to OT-I CD8?and
OT-II CD4?T cells. CFSE-labeled OT-I or OT-II cells were incubated
for 72 h with DC infected with L. major 3?NTs, NT-OVA, SP-OVA, or
with OVA-coated latex beads. The intensity of the CFSE fluorescence
of CD8?TCR??cells or CD4?TCR??was measured as an indicator
of T-cell proliferation. Numbers represent the percentages of cells
showing a reduced CFSE content.
VOL. 73, 2005 PRESENTATION OF L. MAJOR OVA ANTIGENS TO CD8?T CELLS6625
not shown), but it was not determined whether this was due to
a lack of antigen presentation to OT-II CD4?T cells in vivo.
Thus, the OVA-producing parasites can be used to drive the
expansion, differentiation, and recruitment of Leishmania-spe-
cific CD8?T cells that can be followed at the clonal level.
Despite their importance to acquired immunity, the antigens
that activate Leishmania-specific CD8?T cells have not
been identified. The absence of a defined, immunodominant
epitope and a corresponding clonally restricted CD8?T cell
has made it difficult to follow the evolution of antigen-
specific CD8?T-cell responses in vivo or to understand the
cell biology of the processing and presentation of Leishma-
nia antigens in vitro. In the present studies, transgenic L.
major promastigotes were generated to express an intracel-
lular or a secreted form of the NT-OVA fusion protein
bearing the SIINFEKL epitope recognized by OT-I TCR
transgenic CD8?T cells or to secrete a longer OVA protein
fragment bearing epitopes recognized by either OT-II CD4?
or OT-I CD8?TCR transgenic T cells (SP-OVA). The ex-
pression of NT-OVA by transgenic parasites was associated
with the ability of infected DC, but not macrophages, to
prime naı ¨ve OVA-specific OT-I TCR transgenic CD8?T
cells to proliferate and release IFN-? in vitro. Secreted
NT-OVA was significantly more immunogenic than nonse-
creted or heat-killed NT-OVA, suggesting that antigens ac-
tively released into the host cell phagosome might prefer-
entially drive the CD8?T-cell response. DC infected with
transgenic parasites secreting SP-OVA also primed OT-I
CD8?T cells in vitro and in addition were able to prime
OT-II CD4?T cells. In vivo infections with L. major NT-
OVA or SP-OVA resulted in the proliferation of adoptively
transferred naı ¨ve OT-I CD8?T cells and in the recruitment
of primed OT-I cells to the inoculation site between 2 to 3
weeks postinfection. The studies are the first to monitor the
effector phase of a clonotypic CD8?T-cell response to a
Leishmania-derived epitope as it is produced during the
FIG. 6. Proliferation and recruitment of adoptively transferred OT-I cells following intradermal inoculation of L. major OVA transgenic
parasites. (A) CFSE-labeled CD45.2?OT-I splenocytes were transferred i.v. into B6.SJL congenic recipient mice, which were subsequently
challenged in the dermis with 5 ? 105L. major 3?NTs or NT-OVA or 5 ?g of SIINFEKL peptide or saline. Five days after infection, levels of CFSE
fluorescence of CD45.2?TCR??CD8?cells from the draining lymph nodes were analyzed. Numbers represent the percentages of CD45.2?
TCR??CD8?cells showing a reduced CFSE content. (B) Recruitment at the site of challenge of OT-I T cells 4 weeks postinfection with 104L.
major 3?NTs, NT-OVA, SP-OVA, or saline. Absolute numbers of CD45.2?and CD45.2?TCR??CD8?T cells per ear are given. (C) Ly6C
expression by OT-I CD8?T cells in ear (a) and draining lymph nodes (DLN) (b) from adoptively transferred mice infected with L. major SP-OVA
or from total lymph nodes (LN) of naı ¨ve OT-I transgenic mice (c). Numbers represent the percentage of CD45.2?TCR??cells in each quadrant.
(D) Recruitment at the site of challenge of OT-I T cells 9, 15, 19, and 22 days postinfection with 104L. major SP-OVA. Absolute numbers of
CD45.2?TCR??CD8?cells per ear are shown, with a percentage of the total TCR??CD8?cells given in parentheses.
6626BERTHOLET ET AL.INFECT. IMMUN.
course of infection in the skin and reinforce prior observa-
tions regarding the delayed appearance of these cells in the
inflammatory site (3, 4).
The finding that nonsecreted NT-OVA is significantly less
immunogenic than NT-OVA secreted by the parasites can be
attributed to a difference in NT-OVA concentration available
to the DC for processing and presentation to OT-I CD8?T
cells. The concentration of the nonsecreted form is likely lim-
ited by the requirement for release following parasite death
within the phagosome, as demonstrated by the similar immu-
nogenicity of nonsecreted NT-OVA delivered by live or heat-
killed parasites. In this case, the absence of accumulation of
continuously synthesized, nonsecreted NT-OVA may have
been compensated for by the more efficient release of NT-
OVA from rapidly degraded, prekilled promastigotes, such
that similar amounts of antigen were available to the DC. In
each case, however, the concentration of NT-OVA available
for processing following uptake of heat-killed parasites or par-
asites expressing nonsecreted NT-OVA will be low compared
to NT-OVA actively secreted by live parasites that can accu-
mulate within the vacuole and enter the phagosome-associated
MHC class I-restricted presentation pathway (1, 18, 19). Pre-
vious work using poorly defined antigens (e.g., live parasites or
whole cellular extracts) suggested that secreted and cell sur-
face-associated Leishmania antigen, but not nonsecreted anti-
gens, were presented by APC to CD8?T cells (7, 16, 20, 23, 40)
and CD4?T cells (reviewed in reference 29). Similar differ-
ences in the ability of APC to present secreted versus nonse-
creted antigens to CD8?T cells were reported in other patho-
genic infections, including Trypanosoma (17), Toxoplasma (22),
Listeria (37), and Salmonella (37). The L. major NT-OVA
transgenic parasites more clearly establish the importance of
antigen compartmentalization in driving the CD8?T-cell re-
The transwell experiment effectively ruled out the possibility
that NT-OVA secreted by the promastigotes or by infected
cells was a source of MHC class I binding peptides generated
by serum proteases. The results also indicate that peptide re-
gurgitation following antigen processing by infected DC did
not significantly contribute to OT-I priming in vitro. The re-
sults do not, however, rule out the possibility that noninfected
DC might take up released NT-OVA for processing and pre-
sentation or that uptake of infected cells, including macro-
phages, by noninfected DC might provide an important clas-
sical cross-presentation pathway in vivo.
Comparing L. major NT-OVA-infected DC versus macro-
phages revealed that only infected DC could prime naı ¨ve OT-I
CD8?T cells to release IFN-? and proliferate in vitro. This
observation is in agreement with the specialized capacity of DC
to present exogenous antigens to CD8?T cells (13, 34). Prior
studies using infected macrophages to activate CD8?T cells in
each case used primed CD8?T cells (7, 20, 40), suggesting that
whereas DC may be necessary to prime naı ¨ve CD8?T cells,
infected macrophages can present MHC class I-restricted
epitopes to trigger cytokine release from CD8?effector T cells
and thus be activated for killing. We confirmed these observa-
tions by showing that at a high MOI, infected Mø can present
NT-OVA to primed OT-I T cells, though 20-fold-less effi-
ciently than DC. Together, these studies point out the impor-
tance of parasite antigen compartmentalization and the type of
APC used to present Leishmania antigens to CD8?T cells and
might explain why Garcia et al. failed to detect OVA-specific
CD8?T-cell hybridoma activation by macrophages infected
with a Leishmania construct expressing nonsecreted forms of
OVA (16). These authors postulated that Mø were destroying
the OVA epitope. Recently, Delamarre et al. reported a strik-
ing difference between Mø and DC in lysosomal enzyme con-
tent and activity, suggesting that their phagolysosomes may
differ in their capacity to generate MHC class I ligands (12).
The utility of the transgenic parasites to track the recruit-
ment of antigen-specific, effector CD8?T cells to the site of a
low-dose challenge in the skin was demonstrated in the present
study. It is interesting that the OT-I cells were not detectable
within the inoculation site until approximately 3 weeks postin-
fection, reflecting a similar delay reported for the polyclonal
population of CD8?T cells recruited in response to L. major
infection in the skin (3, 4). This delay might be explained by the
duration of parasite replication in macrophages required be-
fore a sufficient number of released amastigotes are available
for targeting to DC and/or by the relative paucity and low
concentration of secreted antigens that become available to
the MHC class I processing machinery. The fact that OT-I
recruitment to the challenge site was not observed until long
after the injected metacyclic promastigotes had been trans-
formed or cleared provides strong evidence that the tissue
amastigotes remained capable of producing OVA and activat-
ing OT-I cells in vivo.
In conclusion, transgenic L. major parasites expressing the
model antigen OVA are a powerful tool for addressing multi-
ple aspects of the CD8?T-cell response to Leishmania infec-
tion, including the hierarchy of antigens involved in cross-
presentation, and the fate of clonotypic, L. major-driven CD8?
T cells as they are activated during chronic infection in the
We thank Steve Beverley and Ron Germain for the OVA plasmids,
Roger Kurlander for advice on OT-I adoptive transfers, and Nancy
Lee and Sandra Cooper for their technical expertise.
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Editor: J. F. Urban, Jr.
6628BERTHOLET ET AL.INFECT. IMMUN.