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Infect Immun. 2005 October; 73(10): 6620–6628.
PMCID: PMC1230980

Antigen Requirements for Efficient Priming of CD8+ T Cells by Leishmania major-Infected Dendritic Cells


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 parasite 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 predominant T-helper type 1 immune response. Acquired resistance in this model relies on activation of CD4+ T cells, resulting in secretion of high levels of gamma interferon (IFN-γ) that induce NO-dependent parasite killing by infected macrophages (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 resistance to secondary challenge (6, 14, 25-27, 41). These observations 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, bacterial, and some parasitic infections have benefited from the description of immunodominant epitopes that have made it possible to study the expansion, differentiation, and recruitment 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 responses more difficult. The model antigen ovalbumin (OVA) has been successfully used with viruses, bacteria, and parasites to provide important information ranging from antigen processing 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.



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 Diseases 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 isolated from stationary cultures (4- to 5-days old) by density centrifugation on a Ficoll gradient (39). Metacyclic promastigotes (5 × 103) were inoculated intradermally into the ear dermis using a 27.5-gauge needle in a volume of ~5 μl. The evolution of the lesion was monitored by measuring the diameter of the induration of the ear lesion with a direct-reading Vernier caliper (Thomas Scientific, Swedesboro, NJ). Parasite titrations were performed with ear tissue homogenates 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 L. major.


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.


The nucleotide sequences encoding the OVA chimeric protein expressed by this plasmid were amplified by PCR using pCR2.1 NT::OVA as a template and the forward 5′-TAATAAACTAGTATGAGCAAGGGCCACATGTCCGTG-3′ and reverse 5′-TAATAAACTAGTAGCGCTAGCCTATTACTCCAT-3′ primers. The PCR product was cloned into pCR2.1, and the SpeI insert was subsequently cloned into pKS NEO.


This expression plasmid was constructed as the pKS NEO iNT::OVA17 above, with the exception of the forward primer OVA-5 (5′-TAATAAACTAGTATGAACGTCAACCTCTTCTCTAAC-3′).


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′-TGGTGGGCTGAGCCGGCAGATCAAGCCAGAGAGCTC-3′ and reverse 5′-CCACCAAGCGCTCTATTAAGGGGAAACACATCTGCC-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 × 108 cells/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 107 cells/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 previously described (9).

Metabolic labeling and immunoprecipitation.

Log-phase promastigotes (1 × 107 to 3 × 107 cells/ml) were harvested from parasite cultures by centrifugation, washed three times at ambient temperature in RPMI medium lacking methionine (Invitrogen), and resuspended in that medium to 2 × 108 cells/ml. 35S-methionine (NEN Life Science Product) was added to a final concentration of 100 μCi/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 × 108 cells/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 108 cells/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 μg/ml leupeptin, 4 μg/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 fluorography 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 succinimidyl ester (CFSE; Molecular Probes, Eugene, OR). The cells were incubated at 2.5 × 107 to 5 × 107 cells/ml in PBS with 0.5 μM 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 106 cells/well in 24-well plates (Costar; Corning, Inc., Corning, NY) in RPMI 10% FCS and 3 × 105 uninfected, infected, or antigen-pulsed APCs were added for 72 h, at which time the cells were fixed in 4% paraformaldehyde. 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 106 CFSE-labeled CD8+ OT-I T cells and 3 × 105 DC were plated in the lower chamber of a 24-well transwell plate (Costar), and 3 × 105 L. 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-γ 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 poststimulation. 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 intravenously (i.v.) 2 × 106 to 5 × 106 CFSE-labeled OT-I total splenocytes. The mice were challenged the same day in the pinea of the ear with 105 metacyclic promastigotes or 5 μg 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 × 106 to 5 × 106 OT-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 Pharmingen. 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 granularity, in combination with anti-CD45.2 (fluorescein isothiocyanate- or PerCP-conjugated), TCR β-chain (phycoerythrin-conjugated), and anti-CD8 CD4 (CyChrome- 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).


IFN-γ was quantitated by enzyme-linked immunosorbent assay (ELISA) in 24- to 72-h cell culture supernatants by using mouse IFN-γ antibody matching pairs (Pierce Endogen, Rockford, IL) and following the manufacturer's protocol.


Expression of nonsecreted and secreted OVA chimeric proteins by L. major transfectants.

L. major promastigotes were transfected with an expression plasmid to express the Leishmania donovani 3′NT/NU::OVA chimeric proteins (Fig. (Fig.1A)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 truncated 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 plasmid 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).

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 ...

The expression of the various 3′NT::OVA fusion proteins by transfected parasites was assessed by SDS-PAGE and immunoblotting of parasite cell lysates with either the anti-Ld3′NT/NU or an anti-OVA antibody (Fig. (Fig.1B).1B). Promastigotes transfected with the pKS NEO NT::OVA plasmid expressed an ~43-kDa protein (Fig. (Fig.1B,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. (Fig.1B)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 promastigote culture supernatants after 6 h (Fig. (Fig.11 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-OVA4 or the iNT-OVA17 proteins 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. (Fig.1D,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 promastigotes 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 naïve 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 marrow-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 constructs used.

The ability of DC to present L. major NT-OVA antigen and activate CFSE-labeled naïve OT-I CD8+ T cells was evaluated for proliferation and release of IFN-γ. 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 levels 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 response (99%). The release of IFN-γ by activated OT-I T cells followed a similar pattern, with the highest concentrations observed for secreted NT-OVA and peptide, followed by nonsecreted NT-OVA4, NT-OVA17, and background levels for 3′NTs and uninfected DC (Fig. (Fig.2B2B).

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 ...

To address the importance of active secretion, the immunogenicity of secreted versus nonsecreted NT-OVA was compared (Fig. (Fig.2C),2C), as well as the delivery by live or heat-killed transgenic parasites (Fig. (Fig.2D).2D). DC infected with L. major NT-OVA were highly effective in priming naïve OT-I T cells, activating cells to secrete high levels of IFN-γ (200 ng/ml) (Fig. (Fig.2C).2C). DC required a 10-fold-higher multiplicity of infection (MOI) with L. major iNT-OVA17 than 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 secretes NT-OVA, were significantly more potent than heat-killed parasites in priming OT-I CD8+ T cells (Fig. (Fig.2D).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-OVA17 induced increased OT-I proliferation compared to heat-killed NT-OVA parasites, which might be attributed to differences in levels of protein 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 extracellular 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 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 direct 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).

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 ...

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 cultured 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-infected macrophages failed to prime naïve OT-I T-cells to proliferate (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 postinfection. Noninfected or 3′NTs-infected APC induced only background OT-I T-cell proliferation (1%), whereas SIINFEKL-pulsed DC or macrophages induced 90 or 91% T-cell proliferation, respectively (data not shown). IFN-γ levels 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-γ in a 24-h coculture assay (Fig. (Fig.4B)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).

FIG. 4.
L. major NT-OVA-infected macrophages are unable to prime OT-I CD8+ T lymphocytes in vitro. Bone marrow-derived macrophages or dendritic cells were compared for their ability to present L. major NT-OVA to OT-I T cells. (A) Naïve CFSE-labeled ...

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 potentially 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. (Fig.5).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 proliferation (7%), although less efficiently than L. major NT-OVA-infected DC (50%). L. major SP-OVA-infected DC also activated OT-II cells (15%), whereas L. major NT-OVA-infected DC had no effect. L. major NT-infected DC induced background levels in each case. DC incubated with OVA-coated latex beads were used as positive controls and induced proliferation in 59% of OT-I T cells and 76% of OT-II CD4+ T cells, respectively.

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 ...

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 × 106 to 5 × 106 CFSE-labeled naïve OT-I CD8+ T cells i.v. and were subsequently inoculated intradermally with either 5 × 105 NT-OVA or 3′NTs metacyclics, 5 μg of SIINFEKL peptide, or saline. Draining lymph nodes were isolated 5 days later, and the intensity of CFSE fluorescence on CD45.2+ TCRβ+ CD8+ OT-I T cells was determined (Fig. (Fig.6A).6A). A relatively high-dose L. major challenge was used in these experiments to favor the early activation and monitoring of the transferred CFSE-labeled cells. In response to injection of L. major NT-OVA, 28% of the gated cells showed a reduced CFSE fluorescence, compared 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 processed 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, 104 metacyclic L. major 3′NTs, NT-OVA, or SP-OVA was assessed 4 weeks postinfection (Fig. (Fig.6B).6B). Infections with L. major NT-OVA or SP-OVA induced the recruitment of OT-I T cells (CD45.2+ CD8+ TCRβ+) 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+ TCRβ+) to the ear compared to saline alone. OT-I T cells recruited to the ear expressed high levels of Ly6C compared to naïve cells, indicating an activated-memory phenotype (Fig. (Fig.6C6C).

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 ...

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 × 106 to 5 × 106 OT-I CD8+ and OT-II CD4+ T cells i.v. were challenged in the ear dermis with 104 metacyclic 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 TCRβ+ cells) by 3 weeks (Fig. (Fig.6D).6D). No OT-II cells were detectable within the infected ear dermis during this time (data 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-specific 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 Leishmania antigens in vitro. In the present studies, transgenic L. major promastigotes were generated to express an intracellular 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 expression of NT-OVA by transgenic parasites was associated with the ability of infected DC, but not macrophages, to prime naïve OVA-specific OT-I TCR transgenic CD8+ T cells to proliferate and release IFN-γ in vitro. Secreted NT-OVA was significantly more immunogenic than nonsecreted or heat-killed NT-OVA, suggesting that antigens actively released into the host cell phagosome might preferentially 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 naïve 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 course of infection in the skin and reinforce prior observations 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 limited by the requirement for release following parasite death within the phagosome, as demonstrated by the similar immunogenicity 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 parasites expressing nonsecreted NT-OVA will be low compared to NT-OVA actively secreted by live parasites that can accumulate within the vacuole and enter the phagosome-associated MHC class I-restricted presentation pathway (1, 18, 19). Previous work using poorly defined antigens (e.g., live parasites or whole cellular extracts) suggested that secreted and cell surface-associated Leishmania antigen, but not nonsecreted antigens, were presented by APC to CD8+ T cells (7, 16, 20, 23, 40) and CD4+ T cells (reviewed in reference 29). Similar differences in the ability of APC to present secreted versus nonsecreted antigens to CD8+ T cells were reported in other pathogenic 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 response.

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 regurgitation following antigen processing by infected DC did not significantly contribute to OT-I priming in vitro. The results do not, however, rule out the possibility that noninfected DC might take up released NT-OVA for processing and presentation or that uptake of infected cells, including macrophages, by noninfected DC might provide an important classical cross-presentation pathway in vivo.

Comparing L. major NT-OVA-infected DC versus macrophages revealed that only infected DC could prime naïve OT-I CD8+ T cells to release IFN-γ 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 naïve 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 observations by showing that at a high MOI, infected Mø can present NT-OVA to primed OT-I T cells, though 20-fold-less efficiently than DC. Together, these studies point out the importance 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 striking difference between Mø and DC in lysosomal enzyme content 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 recruitment 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 postinfection, 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 before 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 transformed or cleared provides strong evidence that the tissue amastigotes remained capable of producing OVA and activating OT-I cells in vivo.

In conclusion, transgenic L. major parasites expressing the model antigen OVA are a powerful tool for addressing multiple aspects of the CD8+ T-cell response to Leishmania infection, 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 skin.


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.


Editor: J. F. Urban, Jr.


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