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The Journal of Immunology, 2008, 180, 4371 -4381
Copyright © 2008 by The American Association of Immunologists, Inc.
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Dendritic Cell Differentiation State and Their Interaction with NKT Cells Determine Th1/Th2 Differentiation in the Murine Model of Leishmania major Infection1

Carsten Wiethe2,*, Andrea Debus{dagger}, Markus Mohrs§, Alexander Steinkasserer*, Manfred Lutz3,{ddagger} and André Gessner2,3,{dagger}

* Department of Dermatology and {dagger} Institute for Clinical Microbiology, Immunology and Hygiene, University Hospital Erlangen, Erlangen, Germany; {ddagger} Institute of Virology and Immunobiology, University of Würzburg, Würzburg, Germany; and § Trudeau Institute, Saranac Lake, NY 12983


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Recent reports demonstrated that dendritic cells (DC) sense inflammatory and microbial signals differently, redefining their classical subdivision into an immature endocytic and a mature Ag-presenting differentiation stage. Although both signals induce DC maturation by up-regulating MHC class II and costimulatory molecules, only TLR signals such as LPS are able to trigger proinflammatory cytokine secretion by DCs, including Th1-polarizing IL-12. Here, we explored the murine Leishmania major infection model to examine the CD4+ T cell response induced by differentially matured DCs. When partially matured TNF-DCs were injected into BALB/c mice before infection, the mice failed to control L. major infection and developed a Th2 response which was dependent on IL-4R{alpha} signaling. In contrast, injections of fully matured LPS+CD40-DCs induced a Th1 response controlling the infection. Pulsing DCs with a lysate of L. major did not affect DC maturation with TNF-{alpha} or LPS+anti-CD40. When the expression of different Notch ligands on DCs was analyzed, we found increased expression of Th2-promoting Jagged2 in TNF-DCs, whereas LPS+CD40-DCs up-regulated the Th1-inducing Delta4 and Jagged1 molecules. The Th2 polarization induced by TNF-DCs required interaction with CD1d-restricted NKT cells. However, NKT cell activation by L. major lysate-pulsed DCs was not affected by blockade of the endogenous glycolipid, suggesting exchange with exogenous parasite-derived CD1 glycolipid Ag. In sum, the differentiation stage of DCs as well as their interaction with NKT cells determines Th1/Th2 differentiation. These results have generic implications for the understanding of DC-driven Th cell responses and the development of improved DC vaccines against leishmaniasis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Today, the central role of dendritic cells (DC)4 for the induction of immunity and peripheral tolerance is well documented (1, 2). Based on these properties, DCs are in the focus of immunotherapeutic applications to treat cancer, infections and autoimmune diseases. The mechanisms of how DCs initiate and direct the desired immune response efficient to eradicate tumor cells, to eliminate pathogens, or to suppress immune pathogenic responses against self are not well understood. Of particular interest in this context is the differentiation of naive Th cells into various effector lineages orchestrating different immune responses. Naive Th cells can differentiate into IFN-{gamma}-producing Th1 cells; into Th2 cell secreting IL-4, IL-5, IL-13, and IL-10; or into the recently described IL-17-producing Th17 cells (3). In addition, for treatment of autoimmune diseases, DCs directing the differentiation of naive CD4+ T cells into IL-10-secreting regulatory T cells like regulatory type 1 T cells, IL-10, and TGF-β-producing Th3 cells or into Foxp3-expressing regulatory T cells would be of benefit.

In the past, it has been suggested that different DC subsets have intrinsic capacities to drive either Th1 or Th2 responses (4). However, recent findings indicate that a particular DC subset is capable of instructing different Th cell responses depending on the maturation stimuli (5, 6). Thus, the type of DC stimulus may determine the DC-mediated polarization of Th cell responses. We and others have reported that DCs sense inflammatory and microbial signals differently and while both signals result in DC maturation demonstrated by up-regulation of surface markers like MHC class II (MHC II) and costimulatory molecules, only TLR signals induce cytokine secretion by DCs (7, 8).

Although these TLR-stimulated DCs promote Th1 differentiation, DCs stimulated with agents known to induce Th2 responses in vivo, like fungal products, parasitic nematodes, or cholera toxin, instruct DCs to induce Th2 responses (8, 9). IL-12 is considered as a major DC factor for the induction of Th1 differentiation (4). However, IL-12-deficient mice or DCs deficient for IL-12 still mount Th1 responses (10, 11). For the differentiation of Th2 cells, IL-4 is an important factor, but initially other signals might instruct Th2 differentiation as demonstrated in STAT6–/– mice (12, 13, 14). So far little is known about DC signals that induce Th2 responses. Interestingly, the differential expression of Notch ligands on DCs has recently been reported to instruct Th1 or Th2 differentiation and their expression is regulated differently by Th1- or Th2-promoting stimuli (15).

Previously, we have described a partially matured DC phenotype that was characterized by the up-regulation of MHC I/II, CD80, CD86, and CD40 when bone marrow (BM)-derived DCs were treated with TNF-{alpha} (TNF-DC). In contrast to fully matured DCs stimulated with LPS plus anti-CD40 (LPS+CD40-DC), these cells did not secrete cytokines (7). In the experimental autoimmune encephalomyelitis (EAE) model, repetitive injections of TNF-DCs suppress EAE in C57BL/6 mice by the induction of Ag-specific IL-10+ CD4+ T cells (7). Interestingly, the development of this protective immune response is dependent on CD1d-restricted NKT cells, which recognize a self-glycolipid presented by CD1d on TNF-DCs (16). NKT cell activation by TNF-matured DCs induced high serum levels of IL-4 and IL-13, whereas LPS+CD40-DCs induce secretion of IFN-{gamma}. Furthermore, concomitant and interdependent presentation of MHC II/self-peptide and CD1d/self-glycolipid to T and NKT cells by the same TNF-DCs was required for EAE protection and early serum Th2 deviation (16). To date the tolerogenic capacity of TNF-DCs has also been shown in other autoimmune diseases like thyroiditis and rheumatoid arthritis (17, 18). However, TNF-DCs pulsed simultaneously with MHC II- and MHC I-restricted peptides efficiently initiate CD8 T cell responses leading to autoimmune diabetes, despite the simultaneous induction of a protective IL-10-secreting CD4+ T cell response (19). Thus, more detailed analyses regarding the immune response induced by TNF-DCs are needed.

The course of many infections is controlled by different CD4+ Th effector (Th1 and Th2) populations which secrete different cytokines. In murine cutaneous leishmaniasis caused by Leishmania major, susceptible BALB/c mice mount a dominant Th2 response leading to uncontrolled parasite replication, whereas resistant strains (e.g., C57BL/6) develop a protective Th1 response (20, 21). Therefore, this mouse model has been frequently used to determine components of the immune system participating in Th1/Th2 differentiation and control of the intracellular pathogen. In the present study, using the mouse model of cutaneous leishmaniasis, we found that repetitive injections of partially matured TNF-DCs pulsed with L. major lysate biased the immune system toward a non-protective Th2 response, whereas LPS+CD40-DCs induced a protective Th1 response. In addition, the induction of IL-4-producing Th2 cells by TNF-DCs was also shown in vitro. In vivo, the interaction of TNF-DCs with CD1d-restricted NKT cells was crucial for the development of the non-protective Th2 response. Furthermore, we demonstrate the differential expression of Th1/Th2-instructing Notch ligands on TNF-DCs vs LPS+CD40-DCs, suggesting that these ligands might represent DC signals that are involved in the observed differential regulation of Th1/Th2 responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice, parasite, and parasite lysate

BALB/c, C57BL/6, and DO11.10 mice were maintained at the Department of Dermatology, University of Erlangen (Erlangen, Germany). The 4get reporter mice (kindly provided by M. Mohrs) and 4get IL-4R–/– mice were bred and housed at the Department of Clinical Microbiology, Immunology and Hygiene (University of Erlangen). All animal experiments were performed in accordance with institutional guidelines with age- and sex-matched animals. The L. major strain BNI (MHOM/IL/81/FE/BNI) was maintained by passage in BALB/c mice, and promastigotes were propagated in vitro on blood agar cultures. The parasite lysate was prepared by three cycles of freezing and thawing of stationary-phase promastigotes.

Generation and maturation of DCs

DCs were generated as described previously (7). Briefly, DCs were obtained by culturing BM cells from BALB/c mice with GM-CSF and DC maturation was induced with TNF-{alpha} (500 U/ml; Peprotech) or with LPS (1 µg/ml; Sigma-Aldrich; Escherichia coli 0127:B8) and anti-CD40 (clone FGK 45; hybridoma supernatant) together with L. major lysate (8 parasites/cell). To block isoglobotrihexosylceramide (iGb3) recognition by NKT cells, DCs were differentially matured with or without 10 µg/ml Griffonia simplicifolia isolectin B4 (IB4; Vector Laboratories).

Treatment of mice with DCs, L. major, and anti-CD1d

DC injections were performed essentially as described (7). Briefly, 2.5 x 106 DCs were injected i.v. at days –7, –5, and –3 before infection with L. major. Mice were infected s.c. with 2.5 x 106 stationary-phase promastigotes into the left hind footpad. Swelling of the footpad was measured with a metric caliber, and the ratio was calculated. For blockade of NKT cell activation, mice were injected twice (days –7 and –3) i.p. with 400 µg of anti-CD1d Ab (1B1; purified from hybridoma supernatants).

Measurement of cytokine response

For the determination of cytokine responses, 4 x 105 splenocytes depleted of erythrocytes or lesion draining lymph node (LN) cells were cultured in vitro in serum-free HL-1 medium (Lonza) with penicillin, streptomycin, 2-ME, and L-glutamine in the presence of parasite lysate (8 parasites/cell). Supernatants were collected after 72 h, and secreted cytokines were detected using ELISA kits for IL-4, IL-10, IFN-{gamma} (all from BD Pharmingen), and IL-13 (R&D Systems). Sera collected at the indicated time points or 2 h after the third immunization were analyzed for cytokine production using the above-mentioned ELISA kits. In addition, IL-17 secretion was determined by ELISA (R&D Systems).

DC culture supernatants were collected after 20 h and analyzed for IL-10, IL-12p40, and IL-12p70 by ELISA kits (all from BD Pharmingen).

Detection of Leishmania-specific Ab responses

To detect Leishmania-specific IgG1 and IgG2a Ab, 96-well plates (Costar) were coated overnight with L. major lysate (109 Leishmania organisms/ml, repeated freeze-thaw cycles). Washed plates were incubated overnight with diluted serum samples from mice, and Ab binding was detected by biotinylated anti-IgG1 and biotinylated anti-IgG2a (BD Pharmingen) followed by a StrepAB-complex coupled to HRP (Dako Cytomation) and TMB (Sigma=Aldrich) as substrate.

Flow cytometric analysis

To avoid unspecific binding of Abs to Fc{gamma}R, cells were preincubated with the Ab 2.4G2 (CD16/32; hybridoma supernatant) or with medium containing 10% serum before cells were stained with the following mAbs: anti-CD4-PerCP (145-2C11); anti-CD86-FITC (GL1); anti-MHC II-PE (M5/114); anti-CD40-FITC (3/23); anti-CD80 (16-10A1); anti-ICOS-L-PE (HK5.3); and anti-ICOS-PE (7E.17G9). All Abs were purchased from BD Pharmingen, except for ICOS-L and ICOS (eBiosciences). Isotype control mAbs (BD Pharmingen) were used at the same concentration.

In vitro stimulation of naive CD4+ T cells

Naive CD62L+ CD25– CD4+ T cells were isolated from spleen and peripheral LN from OVA-specific TCR-transgenic DO11.10 mice using Dynal mouse negative CD4+ T cell isolation kit (Invitrogen Life Technologies) followed by positive selection with anti-CD62L coupled beads and columns (Miltenyi Biotech). T cells (5 x 105/ml) were cultured in RPMI 1640 (Lonza; with 10% FCS, penicillin, streptomycin, 2-ME, and L-glutamine) with TNF-DCs or with LPS+CD40-DCs (0.5 x 105/ml) for 7 days. DCs were matured for 4 h as described above and pulsed with OVA327–339 peptide (10 µM). After 7 days, stimulated cells were harvested and restimulated for two more rounds with different DCs for 7 days with rIL-2 (50 U/ml). After three rounds of stimulation, cells were used for intracellular cytokine staining.

Detection of intracellular cytokines by flow cytometric analysis

For intracellular cytokine staining, cells (4 x 106/ml) were stimulated with PMA (50 ng/ml; Sigma-Aldrich) and ionomycin (500 µg/ml; Sigma-Aldrich) in RPMI 1640 (Lonza; with 10% FCS, penicillin, streptomycin, 2-ME, and L-glutamine) for 5 h. Brefeldin A (Sigma-Aldrich) was added for the final 3 h. Then, cells were washed in PBS, 2% FCS and stained with anti-DO11.10-TriColor (KJ1-26; Invitrogen Life Technologies) or isotype control. Unspecific binding was blocked by the preincubation with anti-Fc{gamma}RII/III (2.4G2, hybridoma supernatant). For intracellular cytokine detection, cells were fixed with 2% formaldehyde, permeabilized with Perm/Wash (BD Pharmingen), and stained with anti-IL-4-PE (11B11) and anti-IL-10-FITC (JES5-16E3: all from BD Pharmingen). Isotype control mAbs (BD Pharmingen) were used at the same concentration.

Expression of Notch ligands by DCs

DNase I-treated total RNA from unstimulated, TNF-{alpha}-stimulated, or LPS+anti-CD40-matured DCs was isolated using the RNeasy Mini Kit (Qiagen) and cDNAs synthesized with Superscript II (Invitrogen) using oligo(dT) primers. Quantitation of mRNA expression of the Notch ligands Jagged1, Jagged2 and Delta4 was conducted by RT-PCR with the LightCycler PCR system (Roche) using previously published primers (15). In each PCR run, an external standard curve was generated using a 4-log spanning serial dilution of the vector plasmid pGEM-T Easy (Promega) containing one copy of the respective target sequence per plasmid. The standard curves were created by the LightCycler 5.32 software and applied for calculation of amplification efficiency and mRNA expression levels. The content of the housekeeping gene HPRT was determined in a separate run on the basis of an external standard as well and used for normalization of the acquired values. In addition, porphobilinogen deaminase (synonym: hydroxymethylbilane synthase) levels in the samples were assessed to validate the results of HPRT normalization (not shown).

PCR was performed in a final volume of 20 µl containing 1 U of Platinum Taq polymerase, 2 µl of 10x reaction buffer, 4 mM MgCl2 (all from Invitrogen Life Technologies), 0.4 µM concenrtations of each primer, 0.25 mM dNTP mix (Amersham Biosciences), 0.5 x SYBR Green I dye (Roche), 0.5 mg/ml BSA (New England Biolabs), 5% DMSO (Sigma-Aldrich), and 2 µl of cDNA in a dilution of 1/10 in H2O or external standard plasmid template. PCR was conducted as described above.

Statistical analysis

An unpaired Student t test was used for statistical analysis, and significance was accepted if p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Previously, we have reported that stimulation of BM-derived DCs with TNF-{alpha} (TNF-DC) results in partial maturation of the cells characterized by the up-regulation of surface markers including MHC I/II, CD80, CD86, CD40, but these cells do not secrete cytokines like fully matured DCs stimulated with LPS plus anti-CD40 (LPS+CD40-DC) (7). However, only partially matured TNF-DCs suppressed EAE in an Ag-specific manner as well as other autoimmune diseases (7, 17, 18). In the EAE model, we described the induction of a transient Th2 shift early after TNF-DC injections (16), which suggest that TNF-DCs may promote a Th2 differentiation. To gain more information about the type of immune response induced by TNF-DCs, we explored the murine model of Leishmania major infection.

DC maturation is unaffected by the pulsing with L. major lysate

We first had to assess whether pulsing with L. major lysate, which could contain potential TLR ligands, alters DC maturation, because TNF-DCs can be further matured by TLR ligands resulting in fully matured DCs unable to suppress EAE (22). Therefore, unstimulated DCs or DCs matured with TNF-{alpha} or LPS+anti-CD40 in absence or presence of L. major lysate were subsequently analyzed for the expression of surface markers and secretion of cytokines. As described earlier, both stimuli, TNF-{alpha} and LPS+anti-CD40, induce up-regulation of MHC II, CD86, CD40, and ICOS-L compared with unstimulated DCs (Fig. 1A, left panel). Simultaneous pulsing of DCs during maturation with parasite lysate did not significantly modify the expression of these surface molecules. Furthermore, secretion of the cytokines IL-10, IL-12p40, and IL-12p70 were also unaffected by the parasite lysates, and only fully matured LPS+CD40-DCs secrete substantial amounts of these cytokines in contrast to unstimulated or TNF-DCs (Fig. 1B, right panel).


Figure 1
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  		FIGURE 1. L. major lysate does not contain components that interfere with DC maturation induced by TNF-{alpha} or LPS + anti-CD40. DCs generated from BALB/c mice were harvested on day 8 and stimulated for 4 h with TNF-{alpha} or LPS+anti-CD40, respectively, or left untreated (Ø). During this 4 h, L. major lysate (8 parasites/cell) was added to the DC cultures. Cells were stained for the indicated surface Ags and analyzed by FACS (A). The inserted numbers represent percentage of cells in a particular quadrant. Harvested supernatants were analyzed by ELISA for their cytokine contents (B). Results are representative of three independent experiments.

 
Taken together, this indicates that L. major lysate does not contain components that interfere with the induced DC maturation with TNF-{alpha} or LPS+anti-CD40.

TNF-DCs are unable to protect against leishmaniasis

We then examined the immune response induced by repetitive injection of these differentially matured DCs in the L. major infection model in susceptible BALB/c mice. BALB/c mice were treated with L. major lysate-pulsed TNF-DCs (L.m./TNF-DC) or LPS+CD40-DCs (L.m./LPS+CD40-DC), respectively. Three days later, mice were challenged with L. major, and the course of disease was monitored. Our results indicate that repetitive i.v. injections of L.m./LPS+CD40-DCs induced protection against leishmaniasis, whereas L.m./TNF-DCs were not protective (Fig. 2A). Protective immunity induced by LPS+CD40-DCs was dependent on pulsing DCs with L. major Ags and resulted in a long lasting protection able to control also rechallenge with L. major (data not shown). Upon injections of L.m./TNF-DCs, we observed an accelerated development of footpad swelling in approximately one-half of the conducted experiments compared with control mice (data not shown). However, this difference was only transient, i.e., mainly during the early course of the infection (Fig. 2A).


Figure 2
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  		FIGURE 2. Differentially matured DCs show different Th1/Th2-promoting capacities. A, Groups of BALB/c mice were injected three times at 2-day intervals with L. major-pulsed TNF-DCs or LPS+CD40-DCs, respectively. Control mice were left untreated. Three days after the last DC injection, mice were infected with L. major. Increase in size of infected compared with non-infected footpads was measured. The ratio of infected to noninfected footpads is shown as mean ± SEM (n = 3–5). B, Sera from BALB/c mice were collected 2 h after the third DC injection. Serum cytokines were determined by ELISA and compared with naive BALB/c. Data are shown as mean ± SEM of pooled sera from three to four mice. C, Serum cytokines of C57BL/6 mice 2 h after the third i.v. injection of L. major-pulsed TNF-DCs or LPS+CD40-DCs, respectively. Naive C57BL/6 mice were used as controls. D, 4get and 4get IL-4R–/– mice were treated with DCs and L. major as described above. On day 33 after infection, lesion draining LN cells from individual mice were isolated and analyzed by FACS. The inserted numbers represent percentage of cells in the particular quadrant. E, Lesion-draining LN cells were restimulated in vitro with L. major lysate. Supernatants were collected after 72 h, and cytokine contents were determined by ELISA. Data are expressed as mean ± SEM of triplicate cultures from three mice per group. F, At the end of the infection course, sera were collected from the animals and analyzed individually for L. major-specific IgG1 and IgG2a Abs by ELISA. Results represent means ± SEM from three mice per group. Data are representative of three independent experiments. *, Significant differences (p < 0.05).

 
These data demonstrate that partially matured TNF-DCs are unable to induce protection against Th2-biased leishmaniasis in susceptible BALB/c mice. In contrast, fully matured Th1-polarizing LPS+CD40-DCs were able to protect.

Non-protective TNF-DCs induce a transient Th2 cytokine environment in the serum

Recently, we showed that secretion of Th1 and Th2 cytokines in the serum early after DC injections depends on DC maturation and their interaction with NKT cells via CD1d (16). This early cytokine release was also examined after injections of L. major lysate-pulsed DCs into BALB/c mice. Two hours after the third DC injections we detected the secretion of cytokines (Fig. 2B). Upon injections of protective L.m./LPS+CD40-DCs, high levels of IFN-{gamma} and IL-17 were detected, whereas IL-4 and IL-13 were at low concentrations. In contrast, L.m./TNF-DCs induced high levels of Th2 cytokines IL-4 and IL-13 and low levels of IFN-{gamma} and IL-17, demonstrating that these DCs biased toward a Th2 response before L. major infection. IL-10 secretion was never detected in sera of mice injected either with L.m./TNF-DCs or with L.m./LPS+CD40-DCs (data not shown). Similar cytokine profiles were found in the sera of genetically resistant C57BL/6 mice treated with L.m./TNF-DCs or L.m./LPS+CD40-DCs (Fig. 2C). Despite this early Th2 response, inoculation of TNF-DCs into C57BL/6 mice only resulted in the development of transiently enhanced lesions following L. major infection without redirecting immunity in this resistant mouse strain permanently (data not shown).

These findings indicate that before the L. major infection, injections of TNF-DCs result in a Th2 shift demonstrated by the secretion of IL-4 and IL-13 but without increasing the disease severity. In contrast, protective LPS+CD40-DCs led to the release of IFN-{gamma} and IL-17.

Mice injected with TNF-DCs develop a Th2 immune response after L. major infection

To further prove that the non-protective immune response induced by L.m./TNF-DCs is accompanied by IL-4 producing CD4+ T cells, we used 4get reporter mice that express enhanced GFP (eGFP) as part of a bicistronic IL-4-IRES-eGFP mRNA to identify IL-4-expressing cells (23). Upon injections of the differential matured DCs in 4get mice, no difference from wild-type BALB/c mice in the course of the L. major infection and serum cytokines was observed (data not shown). Between 30 and 35 days after L. major infection, cells from lesion-draining LNs were isolated to determine the induction of IL-4/eGFP+CD4+ T cells. Mice treated with the non-protective L.m./TNF-DCs showed a slightly higher frequency of CD4+IL-4/eGFP+ cells compared with control mice (Fig. 2D, top row). Contrary to this, the lowest frequency of CD4+IL-4/eGFP+ cells was determined in protected mice injected with L.m./LPS+CD40-DCs. Furthermore, the high frequency of CD4+IL-4/eGFP+ cells in L.m./TNF-DC-treated mice and control mice was diminished to levels found with protective L.m./LPS+CD40-DCs, when 4get IL-4R–/– mice were used, indicating that IL-4/eGFP expression requires IL-4R{alpha} signaling (Fig. 2D, top row). This reduction of CD4+IL-4/eGFP+ cells in 4get IL-4R–/– mice was of clinical relevance, because these mice developed no progressive leishmaniasis lesions (data not shown).

Others have reported that the costimulatory molecule ICOS may enhance the IL-4R-mediated signaling and that blockade of ICOS signaling suppresses leishmaniasis in susceptible BALB/c (24, 25). Similar to the frequencies of CD4+IL-4/eGFP+ cells, ICOS expression levels were found to be low in protected mice treated with L.m./LPS+CD40-DCs, whereas L.m./TNF-DCs induced a high ICOS expression (Fig. 2D, bottom row). Furthermore, similar to the expression of IL-4/eGFP, ICOS induction was also dependent on IL-4R signaling, because cells from 4get IL-4R–/– mice showed decreased ICOS expression levels (Fig. 2D, bottom row).

To test whether eGFP expression correlates with concomitant IL-4 production, cells from the lesion-draining LNs were restimulated in vitro with parasite lysate and the secretion of IL-4, IFN-{gamma}, and IL-10 were determined (Fig. 2E). LN cells from protected mice injected with L.m./LPS+CD40-DCs secreted the highest levels of IFN-{gamma} and only very low levels IL-4 and IL-10, indicating that these DCs induce a strong Th1 response resulting in protection against leishmaniasis. In contrast, a Th2 response was found in unprotected mice (Fig. 2E). Here, injections of L.m./TNF-DCs resulted in the highest secretion of IL-4 and IL-10 but only slightly increased levels IFN-{gamma}.

In addition to the cytokine secretion, sera of L. major-infected mice were analyzed for L. major-specific IgG1 and IgG2a Abs as surrogate markers for Th1 or Th2 differentiation. The highest IgG1:IgG2a ratio was detected in L.m./TNF-DC-treated mice (Fig. 2F), which was significantly above the ratio in control mice. Similar to the cytokine response, L.m./LPS+CD40-DCs induced a significantly lower IgG1:IgG2a ratio than in the control animals (p > 0.05).

Together, the detection of IL-4/eGFP-producing CD4+ T cells as well as the L. major-specific cytokine and Ab profiles revealed that non-protective TNF-DCs induce a Th2-biased immune response whereas fully matured LPS+CD40-DCs shift toward a Th1 response resulting in protection against L. major.

Differential expression of Th1/Th2-instructing Notch ligands by DCs

Amsen et al. (15) demonstrated that the Notch pathway contributes to T cell differentiation and, whereas Delta polarizes toward Th1, Jagged induces Th2 differentiation. Thus, the expression of these Notch ligands was examined on the differentially matured DCs. Similar to the findings from Amsen et al., LPS+CD40-DCs, which induce a Th1 bias and protection against L. major, expressed the highest concentrations of the Th1-instructing Delta4 and of Jagged1 (Fig. 3A). In contrast, Th2-driving non-protective TNF-DCs expressed the highest levels of Th2-instructing Jagged2 but only low levels of Jagged1 and Delta4.


Figure 3
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  		FIGURE 3. Expression of Notch ligands on differentially matured DCs, and Th2 differentiation by TNF-DCs in vitro. A, Real-time quantitative RT-PCR for Jagged1, Delta4, and Jagged2 using RNA from BM-derived DCs generated from BALB/c mice. DCs were harvested on day (d) 8 and stimulated for 4 h with TNF-{alpha} or LPS+anti-CD40, respectively, before RNA isolation. Untreated DCs (Ø) served as control. cDNA contents were normalized on predetermined levels of HRPT. Data from one representative experiment (of six experiments performed) are shown. B, In vitro Th2 differentiation induced by TNF-DCs. Naive CD4+ T cells were isolated from OVA TCR-transgenic DO11.10 mice and cultured in vitro with OVA327–339 peptide-pulsed TNF-DCs or LPS+CD40-DCs. After three rounds of stimulation, cells were treated with PMA and ionomycin, and intracellular levels of IL-4 and IL-10 in OVA-specific CD4+ T cells were determined by FACS by gating on KJ1-26+ cells. The inserted numbers represent the percentage of cells in the particular quadrant. One representative experiment of two independent experiments is shown. C, TNF-DCs induce a Th2 response in vivo in the absence of L. major infection. 4get mice were injected three times at 2-day intervals i.v. with L.m./TNF-DCs or left untreated. Ten days after the last DC splenocytes from individual mice were isolated and analyzed by FACS. The inserted numbers represent the percentage of cells in the particular quadrant. Mean ± SEM from five mice per group analyzed separately are displayed. *, Significant differences (p < 0.05).

 
These findings suggest that the Th1/Th2 differentiation induced by the injection of differentially matured DCs may be in part due to the expression of Th1/Th2-instructing Notch ligands.

Induction of Th2 differentiation by TNF-DCs in vitro and in vivo

Next, the ability of TNF-DCs to induce Th2 differentiation was studied in vitro. Naive (CD62L+CD25–) CD4+ T cells from DO11.10 mice were stimulated in vitro three times with OVA peptide-pulsed TNF-DCs (OVA/TNF-DC) or LPS+CD40-DCs (OVA/LPS+CD40-DC) and subsequently IL-4 induction was determined by intracellular cytokine staining. When gated on OVA-specific TCR-transgenic T cells, IL-4-producing cells were induced by OVA/TNF-DCs but not by OVA/LPS+CD40-DCs (Fig. 3B). In contrast to the in vivo differentiation, CD4+ T cells stimulated with TNF-DCs did not secrete IL-10 under these in vitro conditions (Fig. 1D).

Infection of susceptible BALB/c mice with L. major results per se in an excessive Th2 response. Despite the secretion of Th2 cytokines upon TNF-DC injections, we further wanted to investigate Th2 priming induced by TNF-DCs in the absence of L. major. Thus, 4get reporter mice were repetitively treated with L.m./TNF-DCs, and induction of IL-4/eGFP-producing CD4+ T cells was determined. In mice injected with TNF-DCs, the frequency of CD4+IL-4/eGFP+ cells significantly increased from 0.75 ± 0.28% (naive) to 2.88 ± 0.44% (n = 5; p < 0.0001; Fig. 3C).

These data demonstrate that TNF-DCs induce the polarization of naive T cells into Th2 cells in vitro and in vivo.

CD1d-restricted NKT cells contribute to the Th2 differentiation induced by TNF-DCs

Recently, we have reported that simultaneous activation of MOG/MHC II-specific CD4+ T cells and CD1d-restricted NKT is crucial for EAE suppression upon TNF-DC injections (16). To investigate whether NKT cells also contribute to the L. major-specific Th2 response induced by TNF-DCs, the interaction of the injected DCs with CD1d-restricted NKT cells was blocked by administration of anti-CD1d (26, 27). Administration of anti-CD1d did not alter the course of L. major infection in mice treated with protective L.m./LPS+CD40-DCs or control mice (Fig. 4A). However, when L.m./TNF-DCs were injected into anti-CD1d-treated BALB/c mice, leishmaniasis was significantly ameliorated compared with mice receiving L.m./TNF-DCs only, similar to protective LPS+CD40-DCs, suggesting that NKT cells are essential for the Th2 induction by TNF-DCs.


Figure 4
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  		FIGURE 4. TNF-DCs required CD1d-restricted NKT cells for Th2 differentiation. A, BALB/c mice were injected three times (days –7, –5, and –3) with L.m./TNF-DCs or L.m./LPS+CD40-DCs, respectively. To block NKT cell activation, mice were injected twice (days –7 and –3) i.p. with 400 µg of anti-CD1d Ab. Control mice were left untreated. On day 0, mice were infected with L. major and increase in footpad swelling of infected compared with noninfected footpads was measured. Ratio of infected to noninfected footpad is shown as mean ± SEM (n = 4). B, Two hours after the third DC injection, sera from mice were collected on d –3. Serum cytokines were determined by ELISA and compared with naive BALB/c. Data are shown as mean ± SEM of pooled sera from two to four mice. C, On day 37 after infection, lesion spleen cells from individual mice were isolated and restimulated in vitro with L. major lysate. Supernatant were collected after 72 h, and cytokine contents were determined by ELISA. Data are expressed as mean ± SEM from four mice per group analyzed separately. D, L. major lysate does not change DC maturation and/or CD1d surface expression. On day 8 of culture, DCs generated from BALB/c mice were stimulated for 4 h with TNF-{alpha} or LPS + anti-CD40, respectively, or left untreated (Ø). L. major lysate (eight parasites/cell) was added to the DC cultures during maturation. Cells were stained for the indicated surface Ags and analyzed by FACS and plotted as percentages of MHC II+ CD86+ and MHC II+-CD1d+ cells. Pooled data are shown as mean ± SEM from two independent experiments.

 
In addition, the effect of NKT cell blockade was also examined on the early serum cytokine profiles, which have been shown to be regulated by CD1d-restricted NKT cells in the EAE model (16). Two hours after the third DC injection, high serum levels of IFN-{gamma} and low levels of IL-4 and IL-13 upon L.m./LPS+CD40-DC injections were detected independent of anti-CD1d (Fig. 4B). In contrast, L.m./TNF-DCs induced higher concentrations of IL-4 and IL-13 and concentrations levels of IFN-{gamma}. Interestingly, L.m./TNF-DCs + anti-CD1d treatment resulted in significantly diminished levels of IL-4, whereas IFN-{gamma} and IL-13 remained unaltered (Fig. 4B), indicating that CD1d-restricted NKT cells contribute to the early IL-4 response following TNF-DC injections. We identified a small fraction of CD1d-restricted NKT cells ({alpha}-galactosylceramide; {alpha}-GalCer-CD1d tetramer+) as IL-4 secreting by intracellular cytokine staining (data not shown).

Subsequently, we analyzed the cytokine secretion of spleen cells upon in vitro restimulation with L. major lysate. As expected from the course of L. major infection, cells from protected animals treated with L.m./LPS+CD40-DCs or L.m./LPS+CD40-DCs + anti-CD1d showed the highest levels of IFN-{gamma} and the lowest levels of IL-4 and IL-10 (Fig. 4C). In contrast, non-protective L.m./TNF-DCs induced the highest concentrations of IL-4 and IL-10 and intermediate levels of IFN-{gamma}. However, cells from mice treated with L.m./TNF-DCs in combination with anti-CD1d secreted significantly more IFN-{gamma} and less IL-4 and IL-10 when compared with L.m./TNF-DCs (Fig. 4C). To test whether pulsing of DCs with L. major lysate alters CD1d surface expression, CD1d expression was examined by flow cytometry. Although both stimuli induced DC maturation as shown by increased frequencies of MHC II+CD86+ cells, CD1d was only slightly up-regulated on LPS+CD40-DCs compared with TNF-DCs and unstimulated DCs (Fig. 4D). Similar to the data shown in Fig. 1A, pulsing with L. major lysate had no effect surface expression of CD1d, MHC II, and CD86 compared with unpulsed DCs for all the different DC types.

Thus, interaction of the injected L.m./TNF-DCs with NKT cells via CD1d is crucial for the Th2 induction and their inability to protect from L. major infection.

Presentation of a different CD1d ligand on L. major lysate-pulsed DCs

Because no exogenous CD1d ligand such as {alpha}-GalCer has been used in this study, we finally examined the glycolipid presented by CD1d on the injected DCs. Recently, iGb3 has been described as endogenous CD1d ligand recognized by human and mouse NKT cells (28). Recognition of exogenous and endogenous iGb3 can be blocked by IB4 which specifically binds to terminal Gal{alpha}1,3Gal (28, 29, 30). Using IB4, we demonstrated that NKT cells recognize a self-glycolipid on the injected DCs susceptible for IB4 blockade (16). Therefore, the impact of IB4 blockade experiments on the early cytokine secretion upon injections of L. major lysate-pulsed DCs has been investigated.

As previously described, serum levels of the Th2 cytokines IL-4 and IL-13 were significantly decreased upon repetitive injections of unpulsed IB4-treated TNF-DCs compared with TNF-DCs, whereas IFN-{gamma} was unaltered (Fig. 5A). In contrast, injections with IB4-treated LPS+CD40-DCs led to a significant reduction of IFN-{gamma} production. Interestingly, these effects of IB4 were not observed with L. major lysate-pulsed DCs. In this case, IB4 treatment did not alter the secretion of the Th2 cytokines IL-4 and IL-13 after injections of L.m./TNF-DCs (Fig. 5B). Also, the IFN-{gamma} levels remained unmodified when mice were injected with L.m./LPS+CD40-DCs treated with IB4. In addition, IB4 treatment of L. major-pulsed DCs had no effect on the cytokine profile compared with unpulsed DCs and did not influence the maturation state of the DCs (depicted as frequencies of MHC II+CD86+ cells) or CD1d expression (Fig. 5C). This suggests that the endogenous self-glycolipid, which is recognized by NKT cells and inhibited by IB4, is replaced when DCs were pulsed with L. major lysate.


Figure 5
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  		FIGURE 5. Endogenous self-glycolipid presented by CD1d is replaced when DCs were pulsed with L. major lysate. BALB/c mice were treated with unpulsed (A) or L. major-pulsed DCs (B) three times i.v. IB4 was added to the DC cultures during maturation and L. major pulsing. Sera were collected 2 h after the third DC injection, and cytokine contents were measured by ELISA. *, Significant differences (p < 0.05). Data are representative of one of three independent experiments. C, DC maturation and/or CD1d surface expression is not affected by IB4 treatment and pulsing with L. major lysate. DCs (at day 8 of culture) generated from BALB/c mice were stimulated for 4 h with TNF-{alpha} or LPS+anti-CD40, respectively, or left untreated (Ø). IB4 and L. major lysate were added to the DC cultures during maturation. Cells were stained for the indicated surface Ags and analyzed by FACS and plotted as percentages of MHC II+ CD86+ and MHC II+-CD1d+ cells. Pooled data are shown as mean ± SEM from two independent experiments.

 
Overall, these data demonstrate that differentially matured DCs show different Th1/Th2-biasing capacities. Although fully matured LPS+CD40-DCs lead to a Th1-mediated protection against leishmaniasis, a non-protective Th2 response is induced by TNF-DCs. These different Th1/Th2-instructing capacities of the DC may be due to their differential expression of Th1/Th2-directing Notch-L. In addition, TNF-DCs require interaction with CD1d-restricted NKT cells for the Th2-biased non-protective L. major response. Interestingly, the self-glycolipid presented by CD1d on both types of DCs and the recognition of which is inhibited by IB4 is replaced after pulsing of DCs with L. major lysate.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the past, it has been suggested that Th1 or Th2 immune responses are initiated by different DC subsets with different intrinsic capacities (4). This hypothesis has been challenged recently by the findings that a given DC subset is able to drive different Th cell responses depending on the maturation stimuli (5, 6), supporting a view that DC-mediated Th cell polarization relies on the DC stimuli. Here we reveal in the murine L. major infection model that TNF-DCs induce a non-protective Th2-biased immune response whereas fully matured LPS+CD40-DCs shift toward a protective Th1 response (Figs. 2A and 4A). This protective immune response is dependent on pulsing of LPS+CD40-DCs with L. major Ags and results in long-lasting protective immunity against rechallenge with L. major (data not shown). These Th1/Th2-instructing capacities of the differentially matured DC may be due to their different ability to produce IL-12 (Fig. 1B) and their different expression profiles of Th1/Th2-directing Notch ligands (Fig. 3A). In addition, CD1d-restricted NKT cells are also critical for the non-protective L. major response induced by TNF-DCs (Fig. 4). Interestingly, after L. major lysate pulsing, NKT cell activation by the injected DCs is not inhibited by IB4 (Fig. 5, A and B), suggesting that the IB4-sensitive self-glycolipid presented by CD1d on both types of DCs is replaced upon L. major lysate pulsing and is not due to altered DC maturation or CD1d surface expression (Fig. 5C).

Recently, we and others have reported that DCs sense inflammatory and microbial TLR signals differently, resulting in up-regulation of surface markers by both stimuli, but only TLR signals induced secretion of cytokines by DCs (2, 7, 8). For the induction of Th1 responses, IL-12 is considered a major factor (4). In addition, numerous reports have demonstrated the critical role of IL-12 in the establishment and maintenance of immunity against L. major (21, 31, 32, 33). Previously, we have shown that DCs stimulated with TNF-{alpha} are only semi-mature compared with LPS+CD40-DCs due to the lack of cytokine secretion (7). In addition, subsequent stimulation of partially matured TNF-DCs with TLR ligands leads to their further maturation into fully matured DCs that secrete cytokines including IL-12 (22). Here we report that pulsing of partially matured TNF-DCs with L. major lysate does not alter their maturation state including the cytokine secretion (Fig. 1), suggesting that promastigotes of L. major do not contain component leading to complete DC maturation like TLR ligands. This is in agreement with a report showing that, with regard to DC activation, infection with L. major is a silent process (34). Thus, the absence of IL-12 secretion by TNF-DCs pulsed with L. major lysate might support the development of a non-protective Th2 response (Figs. 2A and 4A). Other studies suggested that secretion of IL-12 by recipient cells and not by injected DCs might control DC-mediated Th1 development (11, 35). Thus, despite the knowledge regarding the essential role of IL-12 in the induction of a protective Th1 immune response against L. major, the source of IL-12 upon DC vaccination requires further examinations.

Previously, we reported that repetitive injections of TNF-DCs protect against Th1-mediated EAE by the induction of Ag-specific CD4+ IL-10+ T cells (7). In addition, TNF-DCs also suppress other autoimmune diseases like collagen-induced arthritis and thyroiditis (17, 18). In the EAE model, NKT cells recognizing an endogenous glycolipid on injected TNF-DCs are critical for this protective immune response by regulating the early, transient secretion of Th2 cytokines IL-4 and IL-13 (16). In the L. major infection model in susceptible BALB/c mice, similar serum cytokine profiles were detected upon DC injections as observed already in the EAE model. L.m./TNF-DCs induced high levels of Th2 cytokines IL-4 and IL-13 (Figs. 2B, 4B, and 5B), indicating a shift toward Th2 even before the L. major infection. This early Th2 shift is dependent on IL-4R{alpha} signaling, because IL-4 and IL-13 were not detected when IL-4R{alpha}-deficient mice were used as recipients (data not shown). In contrast to TNF-DCs, protective L.m./LPS+CD40-DCs elicit high levels of IFN-{gamma} and IL-17, but only low levels of Th2 cytokines (Figs. 2B, 4B, and 5B). These different serum cytokine profiles correlate with the course of leishmaniasis: induction of immunity with LPS+CD40-DCs and no protection with TNF-DCs (Figs. 2A and 4A). However, despite the Th2-biased cytokine profile induced by TNF-DCs before L. major challenge, the course of infection was accelerated during the early phase in only approximately one-half of the conducted experiments. This may be due to the excessive Th2 response induced per se by the administered dose of L. major. In contrast to the protective immunity induced by LPS+CD40-DCs, the induction of the non-protective immune response induced by TNF-DCs was not dependent on pulsing of the DCs with L. major Ags (data not shown). Nevertheless, TNF-DCs clearly induced a Th2 response in vivo without L. major infection (Fig. 3C) as well as in vitro (Fig. 3B). Mice treated with L.m./TNF-DCs showed the highest levels of IL-4 and IL-10 upon in vitro restimulation of spleen and lesion-draining LN cells (Fig. 2E) and the highest IgG1:IgG2a ratio compared with control animals (Fig. 2F). In addition, we detected an increased IL-4R{alpha}-dependent induction of IL-4+ CD4+ T cells by L.m./TNF-DCs upon L. major challenge using 4get reporter mice in vivo (Fig. 2D). Thus, our data indicate that semi-mature TNF-DCs induce an Ag-specific Th2-differentiation independent of a subsequent L. major infection.

Several recent reports indicate that DCs are able to polarize different Th cell developments depending on the nature of stimulus and environmental factors, whereas the intrinsic capacity of different DC subsets to instruct Th cell polarization is of minor importance (5, 6, 36, 37). It has been demonstrated that DCs matured via TLR ligands drive Th1 development (8, 9). Conversely, induction of Th2 differentiation is shown for DCs stimulated with cholera toxin, fungal product, helminth products, or PGE2 (9, 37). Amsen et al. (15) demonstrated that expression of Notch ligands contribute to this Th1/Th2 regulation by DCs. The question remains what kind of DC maturation and Th instruction is induced by proinflammatory cytokines alone? In vivo maturation of DCs by endogenous inflammatory mediators has little effect on DC maturation and may lead only to partial maturation of DCs, with subsequent CD4+ T cell activation but without further development into Th1 or Th2 cells, as demonstrated by the absence of IgG switching (8, 38). In contrast, here we find that BM-DCs matured with TNF-{alpha} in vitro will induce a Th2 profile and an IgG1 bias after injection. This may indicate that either in vitro-generated BM-DCs are differentially responsive to TNF-{alpha} as compared with their in vivo counterparts or the doses of TNF-{alpha} added in vitro and produced after LPS injection in vivo are different because additional injections of TNF-{alpha} could further increase DC maturation (38). The type of immune reaction after this TNF-{alpha} injection was not further investigated.

Here we show that semi-mature TNF-DCs express high levels of Th2-instructing Notch ligand Jagged2 and only low levels of Jagged1 and Th1-biasing Delta4 (Fig. 3A). This Notch ligand expression profile on TNF-DCs is identical with that of Th2-promoting DCs stimulated with either cholera toxin or PGE2 (15), suggesting that expression of these Th2-directing Notch ligands might have a role in the Th2-shifted immune response upon TNF-DC injections. In contrast, LPS+CD40-DCs express high levels of Th1-inducing Delta4 and Jagged1 but low levels of Jagged2 (Fig. 3A). This is similar to the previously described LPS-DC profile (15). Therefore, LPS+CD40-DCs might regulate the induction of the L. major protective Th1 immune response by the expression of these Notch ligands.

In the murine L. major model, it has been reported that CD1d-restricted NKT cells play only a minor role in the control of L. major and clinical resolution of disease (39). In accordance with this, we also found no obvious effect of NKT cells in control mice or animals treated with LPS+CD40-DCs in the course of leishmaniasis and secretion of L. major-specific cytokines, when their activation was blocked by anti-CD1d treatment (Fig. 4, A and C). However, anti-CD1d treatment of mice receiving TNF-DCs leads to the conversion of the non-protective Th2 response, induced by TNF-DCs alone, into a protective response with higher IFN-{gamma} levels and lower IL-4 and IL-10 levels after an in vitro restimulation with L. major lysate (Fig. 4 A and C). In addition, this NKT cell blockade resulted in a diminished Th2 bias before challenge with L. major (Fig. 4B). Campos-Martin et al. (40) have shown that infection of human monocyte-derived DCs with Leishmania infantum results in up-regulation of surface CD1d and efficient recognition and killing of these infected DCs by NKT cells. However, we found no obvious effect of L. major lysate on DC maturation (Fig. 1) and CD1d expression (Fig. 4D), which is in agreement with another previous report (34). These controversial results might be explained by the different DCs (BM-derived mouse DCs vs monocyte-derived human DCs), different Leishmania species (L. major causing cutaneous leishmaniasis vs L. infantum causing visceral leishmaniasis), and different types of Ag loading (pulsing with Leishmania lysate vs infection with live parasites) used in these studies.

In the EAE model, we recently described the dependency of TNF-DCs to interact with CD1d-restricted NKT cells to suppress EAE by regulating the early secretion of Th2 cytokines necessary for the development of protective IL-10-secreting CD4+ T cells (16). This interaction of injected TNF-DCs with CD1d-restricted NKT cells is also important for the Th2-biased immune response in the L. major infection model, indicating that activation of CD1d-restricted NKT cells may be an additional mechanism of TNF-DCs to drive Th2 differentiation, in addition to the expression of Th2-promoting Notch ligands. The early serum cytokines are produced by CD4+ T cells and CD1d-restricted NKT cells (16), suggesting that early secretion of IL-4 and IL-13 creates an environment required for the subsequent Th2 polarization of CD4+ T cells and development of a non-protective L. major response. In line with this, we detected by a small fraction of CD1d-restricted NKT cells and CD4+ T cells secreting IL-4 and IL-13 in L. major-infected mice (data not shown). Our findings that induction of IL-4-secreting CD4+ T cells and early Th2 serum cytokines are dependent on IL-4R{alpha} signaling (Fig. 2D and data not shown) support the role of early NKT cell-regulated Th2 deviation for subsequent CD4+ Th2 differentiation. Several studies have revealed the immunogenic adjuvant capacity of CD1d-restricted NKT cells for induction of CD4+ as well as CD8+ T cell responses (41, 42, 43, 44). In addition, NKT cells induce DC maturation in vivo following {alpha}-GalCer injections via CD40-CD40L interaction, resulting in IL-12 secretion by DCs and more efficient T cell stimulation (41, 42, 43, 44). Thus, the regulatory effect of NKT cells on Th2 differentiation after TNF-DC injections might rely on direct modification of CD4+ T cells via IL-4R signaling by the early Th2 environment. Alternatively, feedback signaling toward DCs, a mechanism described as DC education or DC licensing (45, 46), might be responsible for this NKT cell effect. DC feedback signaling is supported by our previous finding that concomitant activation of CD4+ T cells and NKT cells by the same DCs expressing MHC II and CD1d is required for early intermediate Th2 cytokine secretion followed by CD4+ T cell differentiation and suppression of autoimmune disease (16). However, the exact mechanism by which CD1d-restricted NKT cells regulate this Th2 differentiation remains to be elucidated in further studies.

Simultaneous activation of CD4+ T cells and NKT cells by TNF-DCs is essential for the protection in the EAE model, and the release of the Th2 cytokines is regulated by the CD1d-dependent NKT cells recognizing a self-glycolipid presented by CD1d (16). Blocking experiments with IB4, which binds to terminal Gal({alpha}1,3)Gal and inhibits CD1d-restricted recognition of iGb3 by NKT cells (28, 29, 30), suggest that iGb3 might be the CD1d ligand presented by the injected DCs in the EAE model (16). Interestingly, when IB4 blocking experiments were performed with L. major-pulsed DCs, no effect of IB4 on the secretion of serum cytokines was observed when compared with control DCs (Fig. 5, A and B), and this is not due to altered CD1d expression or DC maturation (Fig. 5C). This indicates that pulsing DCs with L. major interferes with the presentation of the IB4-sensitive self-glycolipid on CD1d, suggesting that pulsing with L. major lysate may lead to an exchange of the presented CD1d ligand. Such an exchange of endogenous IB4-sensitive iGb3 by exogenous glycolipids has been described following pulsing of DCs with heat-killed bacteria like Sphingomonas capsulate or Ehrlichia muris (47). In addition, recognition of both of these bacterial glycolipid Ags by NKT cells was not inhibited by the lectin IB4 (47). Furthermore, also pathogenic Borrelia burgdorferi contains glycolipid Ags recognized by human and mouse NKT cells (48). The Leishmania-derived lipophosphoglycan, which is the major component of the surface glycocalyx, has been identified as potential CD1d Ag resulting in NKT cell activation (49). Similar to the study with Sphingomonas capsulate and Ehrlichia muris, IB4 blocking experiments with L. major lysate-pulsed DCs suggest that the self-glycolipid may be exchanged by an L. major-derived glycolipid like lipophosphoglycan following DC pulsing with parasite lysate. Because the thus far undefined CD1d ligand presented on DCs pulsed with parasite lysate was not blocked by IB4 (Fig. 5, A and B) and IB4 had no long-lasting effect in the EAE model (16), we consider it very unlikely that IB4 would change the course of L. major infection upon DC injections.

Initially, the costimulatory molecule ICOS has been associated with a Th2 differentiation (50, 51, 52), whereas other studies identified a role of ICOS in Th1 responses as well as for regulatory T cells (53, 54). Consistent with the initial association with Th2 cells is the finding that ICOS regulates GATA-3 induction and IL-4R-mediated signaling (24). For the L. major infection model, it has been reported that ICOS is crucial for Th2-biased T cell differentiation (25). In line with these publications, we found that CD4+ T cells from mice treated with L.m./TNF-DCs expressed the highest levels of ICOS despite the induction of ICOS-L on both types of matured DCs (Figs. 1A and 2D). Furthermore, ICOS expression on CD4+ T cells depends on IL-4R signaling similar to the induction of IL-4/eGFP expression (Fig. 2D). The results that ICOS-ICOS-L interaction might play a special role in the regulation of DC-mediated Th2 cell expansion (55), suggest that ICOS-ICOS-L might also be involved in the induction of the non-protective Th2 response induced by TNF-DCs.

Inoculation of TNF-DCs into resistant C57BL/6 mice only resulted in transiently enhanced lesion development following L. major infection (data not shown). Despite the clear early Th2 differentiation by TNF-DCs as demonstrated by the early Th2 cytokine secretion (Fig. 2C) and increased frequency of CD4+IL-4/eGFP+ cells without L. major infection, these data suggested that the Th2 response induced by TNF-DCs may not be strong enough to redirect immunity in C57BL/6 mice. However, this was not unexpected because it has been reported previously, that administration of exogenous IL-4 is not sufficient to establish a Th2 response in genetically resistant mice that were infected with L. major. Exogenous IL-4 increased the lesion development only transiently, and all mice eventually healed and controlled parasite replication (56, 57). In addition, L. major infection of mice deficient for B cells and Fc{gamma}–/– mice (on the resistant C57BL/6 background) showed only transiently increased disease development despite strongly enhanced IL-4 production and reduced IFN-{gamma} production (58).

In conclusion, we provide evidence that differentially matured DCs show different Th1/Th2-biasing capacities. Injections of semi-mature TNF-DCs induce a non-protective Th2 immune response whereas fully matured LPS+CD40-DCs drive a protective Th1 response in the murine L. major infection model. Based on our results, there are at least three mechanisms by which TNF-DCs might regulate the polarization of CD4+ T cells into Th2 cells. These different Th1/Th2-instructing capacities may be based on their differential IL-12 expression and Th1/Th2-directing Notch ligands. In addition, TNF-DCs require interactions with CD1d-restricted NKT cells for Th2 polarization. Interestingly, pulsing DCs with L. major lysate leads to the exchange of the self-glycolipid presented by CD1d on DCs, the recognition of which is inhibited by IB4.


   Acknowledgments
 
We thank Robert Tindle for critical reading of the manuscript, Mitchell Kronenberg (La Jolla Institute for Allergy and Immunology) for the provision of the 1B1 hybridoma, and Ulrich Schaible (University of London, London, U.K.) for purified 1B1 Ab. We acknowledge the technical assistance of Astrid Mainka, Andrea Hesse, and Rimma Junker.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Deutsche Forschungsgemeinschaft (DFG) Grant 643 from the Society for Biomaterials (to M.L. and A.G.), and the ELAN Fonds of the University of Erlangen (to C.W.). A.G. and A.S. were supported by the 1ZKF, University Hospital Erlangen. C.W. was further supported by the DFG grant STEB2/5.1. Back

2 Address correspondence and reprint requests to Dr. Carsten Wiethe, Department of Dermatology, University Hospital Erlangen, Hartmannstrasse 14, 91052 Erlangen, Germany. E-mail address: carsten.wiethe{at}derma.imed.uni-erlangen.de or Dr. André Gessner, Institute for Clinical Microbiology, Immunology and Hygiene, Wasserturmstrasse 3, 91054 Erlangen, Germany. E-mail address: gessner{at}mikrobio.med.uni-erlangen.de Back

3 M.L. and A.G. contributed equally to this work. Back

4 Abbreviations used in this paper: DC, dendritic cell; {alpha}-GalCer, {alpha}-galactosylceramide; BM, bone marrow; EAE, experimental autoimmune encephalomyelitis; eGFP, enhanced GFP; MHC I/II, MHC classes I and/or II; iGb3, isoglobotrihexosylceramide; 1B1, anti-CD1d Ab; IB4, Griffonia simplicifolia-derived isolectin B4; L.m., Leishmania major; LN, lymph node. Back

Received for publication July 23, 2007. Accepted for publication December 31, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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