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Mouse lung dendritic cells (LDCs) have been recently shown to contain two major .... Taken together, this accumulating evidence confirms that LDCs comprise two ma- ...... Sung SS, Fu SM, Rose CE Jr, Gaskin F, Ju ST, Beaty SR. A major lung.
Mouse Lung CD1031 and CD11bhigh Dendritic Cells Preferentially Induce Distinct CD41 T-Cell Responses Kazuki Furuhashi1, Takafumi Suda1, Hirotsugu Hasegawa1, Yuzo Suzuki1, Dai Hashimoto1, Noriyuki Enomoto1, Tomoyuki Fujisawa1, Yutaro Nakamura1, Naoki Inui1, Kiyoshi Shibata2, Hirotoshi Nakamura1, and Kingo Chida1 1 Second Division, Department of Internal Medicine, and 2Research Equipment Center, Hamamatsu University School of Medicine, Hamamatsu, Japan

Mouse lung dendritic cells (LDCs) have been recently shown to contain two major subpopulations: CD1031 CD11blow or negative (CD1031 LDCs) and CD1032 CD11bhigh LDCs (CD11bhigh LDCs). Although several studies have demonstrated functional differences between them, it is unclear whether the subpopulations induce distinct T helper (Th) cell responses. The present study was conducted to examine whether CD1031 and CD11bhigh LDCs preferentially generate different Th responses. Naive DO11.10 CD41 T cells were primed with CD1031 or CD11bhigh LDCs obtained from normal BALB/c mice. The primed CD41 T cells were restimulated, and their cytokine secretions were assessed. The expression of intracellular cytokines and the mRNA levels of chemokine receptors were also measured. We found that the CD41 T cells primed with CD1031 LDCs secreted significantly larger amounts of IFN-g and IL-17A, whereas those primed with CD11bhigh LDCs released significantly higher levels of IL-4, IL-6, and IL-10. Intracellular cytokine assay showed that CD1031 LDCs induced greater frequencies of CD41 T cells producing IFN-g and IL-17A, whereas CD11bhigh LDCs were more efficient at inducing CD41 T cells producing IL-4 and IL-10. The mRNA levels of CXCR3 and CCR5, which are expressed preferentially in Th1 cells, were significantly higher in CD41 T cells primed with CD1031 LDCs. The mRNA levels of CXCR4 and CCR4, which are expressed primarily in Th2 cells, were significantly greater in those primed with CD11bhigh LDCs. These data suggest that mouse CD1031 LDCs predominantly elicit Th1 and Th17 responses, whereas CD11bhigh LDCs primarily provoke a Th2 response under the steady state. Keywords: dendritic cells; Th1; Th2; Th17

In the lung, dendritic cells (DCs) are the most potent antigenpresenting cells (APCs), playing a central role in initiating the primary immune response (1). Lung DCs (LDCs) are localized within and beneath the epithelium and in the alveolar septa (2–4) and display an excellent capability for the uptake and processing of inhaled antigens (5). LDCs migrate through lymphatic vessels to the mediastinal lymph node (MLN), where they present antigen to T cells and induce an immune response (6). Previous studies have shown that LDCs are involved in a variety of immunologic processes through their potent antigen-presenting capacity (6–10). For example, we previously reported the increased numbers of LDCs in the airways of diffuse panbronchiolitis, a possible involvement of LDCs in pulmonary granuloma formation, and a potent capability of LDCs to induce mucosal IgA production (7–10). DCs are a heterogeneous population of professional APCs and consist of several distinct subsets discernible by surface

(Received in original form March 4, 2011 and in final form August 10, 2011) This work was supported by grant-in-aid for scientific research grant 21,590,985 (T.S.) from Japan Society for the Promotion of Science. Correspondence and requests for reprints should be addressed to Takafumi Suda, M.D., Ph.D., 1-20-1 Handayama, Hamamatsu 431-3192, Japan. E-mail: [email protected] Am J Respir Cell Mol Biol Vol 46, Iss. 2, pp 165–172, Feb 2012 Copyright ª 2012 by the American Thoracic Society Originally Published in Press as DOI: 10.1165/rcmb.2011-0070OC on August 25, 2011 Internet address: www.atsjournals.org

and intracellular phenotypic markers, immunological function, and anatomic distribution. These distinct DC subsets were shown to produce discrete cytokine profiles and to induce different T-cell responses, indicating that each DC subset has a distinct role in the immune response against pathogens and in the maintenance of self-tolerance (11–14). As a phenotypic marker discriminating DC subsets, integrin aE (CD103) has recently attracted special attention (15–17). In the skin and gut, CD1031 DCs, which express langerin (CD207), are primarily involved in cross-presentation of self or foreign antigens to CD81 T cells, the generation of gut-tropic effector T cells, and the induction of regulatory T cells (Treg) (18–21). In contrast, CD103– DCs are predominantly responsible for priming CD41 T cells and the production of proinflammatory cytokines and chemokines (19). In the mouse lung, several recent studies have demonstrated the existence of CD1031 DCs, which comprise a major LDC population and have distinct functional properties from other LDCs (22–26). First, Sung and colleagues reported that LDCs contain the two major DC subsets, identified as CD11c1 MHC class II1 CD11blow or negative CD1031 lung DCs (CD1031 LDCs) and CD11c1 MHC class II1 CD11bhigh CD1032 lung DCs (CD11bhigh LDCs) in the mouse lung (24). CD1031 LDCs express tight junction proteins, such as claudin-1, claudin-2, and ZO-2, and are the main DC population that migrates from the lung to the MLN, where they cross-present inhaled or self antigens (22–25). By contrast, CD11bhigh LDCs, which are superior to CD1031 LDCs in secreting chemokines, are major producers of chemokines during homeostatic and inflammatory conditions of the lung (22, 26). Anatomically, CD1031 LDCs are located along the mucosal lining and vascular wall, whereas CD11bhigh LDCs are located mainly within the perivascular regions (22, 24). More recently, CD1031 LDCs and CD11bhigh LDCs were shown to stimulate CD81 T and CD41 T cells. For example, in the late phase of influenza infection, CD1031 and CD11bhigh LDCs efficiently migrate from the lungs to the MLN, where CD11bhigh LDCs become the primary DC subset for cross-presenting acquired exogenous antigens to CD81 T cells (27). Taken together, this accumulating evidence confirms that LDCs comprise two major phenotypically and functionally distinct subsets, CD1031 and CD11bhigh LDCs. During the primary immune response, the T helper (Th) cell subtype preferentially induced by the distinct DC subset is one of the critical determinants in T-cell–mediated immunity. However, there have been few studies to examine this capability of CD1031 and CD11bhigh LDCs, and the results of such studies have been controversial. Initially, Beaty and colleagues reported no substantial difference in the capability to direct Th responses between CD1031 and CD11bhigh LDCs in vitro (26). In contrast, Raymond and colleagues recently demonstrated that CD11bhigh LDCs, but not CD1031 LDCs, migrating to the MLN are essential for inducing a Th2 response in the mouse model of allergic airway inflammation (28). Thus, the present study was conducted to clarify whether the two major LDC populations preferentially induce

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distinct Th responses. We obtained highly purified CD1031 and CD11bhigh LDCs from the normal mouse lung using magnetic and flow cytometric cell sorting and examined their ability to induce Th-cell differentiation from naive T cells under the steady state.

Statistical Analysis

MATERIALS AND METHODS

RESULTS

Further details are provided in the online supplement.

CD1031 LDCs and CD11bhigh LDCs Are the Major Populations among CD11c1 MHC Class II1 LDC Subsets

Mice

The majority (. 75%) of CD11c1 cell preparations obtained from lung digests using MACS with anti-CD11c mAb-conjugated magnetic beads were macrophages with high autofluorescence (data not shown). CD11c1 MHC class II1 LDCs with low autofluorescence comprised 20 to 25% of the CD11c1 cell preparations (Figure 1B). When gating on CD11c1 MHC class II1 cells, two distinct subpopulations of LDCs were present in terms of their expression of CD103 and CD11b (Figure 1C). CD1031 CD11b– and CD103– CD11bhigh LDCs comprised 34.9 6 0.9% and 32.8 6 0.9% of CD11c1 MHC class II1 LDCs, respectively. These two LDC populations were purified using a FACSAria flow cytometer, and the yields of CD11c1 MHC class II1 CD1031 CD11b– (CD1031 LDCs) and CD11c1 MHC class II1 CD1032 CD11bhigh DCs (CD11bhigh LDCs) were 2.1 to 3.2 3 104 cells and 2.0 to 3.1 3 104 cells per mouse, respectively (Figure 1D).

Experiments were performed on 8- to 12-week-old male BALB/c mice (Nippon-SLC, Shizuoka, Japan) and ovalbumin (OVA)-specific T-cell receptor transgenic mice (DO11.10) (Jackson Laboratory, Bar Harbor, ME).

Preparation of CD1031 and CD11bhigh LDCs LDCs were isolated as described in our previous studies (9, 10), with some modifications. Total CD11c1 cells were obtained from digested lung using magnetic cell sorting (MACS; Miltenyi Biotec, Auburn, CA) with anti-CD11c mAb-conjugated magnetic microbeads (Miltenyi Biotec). CD11c1 MHC class II1 CD1031 CD11b2 LDCs (CD1031 LDCs) and CD11c1 MHC class II1 CD1032 CD11bhigh LDCs (CD11bhigh LDCs) were then isolated using a FACSAria flow cytometer (Becton Dickinson Systems, San Jose, CA). The surface and intracellular expression of various molecules in LDCs were examined by the FACSAria.

Preparation of Naive CD41 T Cells Naive CD41 CD62Lhigh T cells were obtained from DO11.10 murine spleen, using a CD41 CD62L1 T Cell Isolation Kit II (Miltenyi Biotec).

Data were expressed as the mean 6 SEM. Statistical evaluation of differences between the means for experimental groups was performed by the Wilcoxon/Kruskal-Wallis test. P values , 0.05 were considered significant.

Expression of Surface and Intracellular Molecules by CD1031 LDCs and CD11bhigh LDCs

Measurements of Cytokine Production

CD1031 and CD11bhigh LDCs were analyzed for their expression of surface and intracellular markers by flow cytometry (Table 1; Figure 2). CD1031 and CD11bhigh LDCs expressed MHC class II and CD40 at comparably high levels. Expression of CD4 or CD8a was not detected in either of these LDC subpopulations. CD1031 and CD11bhigh LDCs showed moderate expression of B7–1 (CD80) and B7–2 (CD86) and weak or no expression of B7-H1 (PDL-1), B7-DC (PDL-2), DEC205, or DC-SIGN. Langerin (CD207) was moderately expressed in CD1031 LDCs but not in CD11bhigh LDCs, whereas F4/80 was present in CD11bhigh LDCs. Consistent with the results of flow cytometry, RT-PCR analysis showed that mRNA levels of langerin were significantly higher in CD1031 LDCs than in CD11bhigh LDCs (Figure 3).

The levels of IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-g, and TNF in the supernatants were measured using cytometric bead array kits (BD Biosciences, San Jose, CA). The levels of IL-12p70, IL-23, and TGF-b were determined with ELISA kits (R&D Systems, Minneapolis, MN).

Cytokine Production by CD41 T Cells Primed with CD1031 LDCs or CD11bhigh LDCs

Priming CD41 T Cells with CD1031 or CD11bhigh LDCs and Restimulating Primed CD41 T Cells Naive CD41 CD62Lhigh T cells were primed with CD1031 or CD11bhigh LDCs in the presence of OVA323–339 peptide (Invitrogen, Carlsbad, CA) for 5 to 7 days in RPMI 1640 medium including 10% heat-inactivated FCS (RPMI complete medium) (GIBCO, Carlsbad, CA). CD41 T cells were then purified using the FACSAria and restimulated with anti-CD3/CD28 mAbs-conjugated beads (DYNAL, Carlsbad, CA) (29).

Intracellular Cytokine Staining The primed CD41 T cells were restimulated with phorbol myristate acetate and ionomycin in the presence of brefeldin A (all from Sigma-Aldrich, St. Louis, MO). After fixation and permeabilization, cells were stained with various anticytokine mAbs and analyzed using the FACSAria.

Real-time Quantitative RT-PCR Assay for Langerin and Chemokine Receptors Total RNA was obtained from freshly isolated LDCs or CD41 T cells that were primed with CD1031 or CD11bhigh LDCs. cDNAs were generated from total RNA, and real-time quantitative PCR was performed for assessing the expressions of langerin and chemokine receptors.

Treatment of CD1031 and CD11bhigh LDCs with Toll-like Receptor Ligands Purified CD1031 or CD11bhigh LDCs were stimulated with LPS (Sigma-Aldrich), PAM3CSK4 (IMGENEX, San Diego, CA), or CpGmotif oligodeoxynucleotides (CpG-ODN) (IMGENEX) in RPMI complete medium.

To clarify whether the two major LDC subpopulations, CD1031 and CD11bhigh LDCs, induced distinct subtypes of Th cells, we first examined the cytokine profiles produced by T cells primed with CD1031 or CD11bhigh LDCs. Naive CD41 CD62Lhigh T cells from DO11.10 mice were primed with CD1031 or CD11bhigh LDCs in the presence of OVA323–339 peptide for 7 days. These primed T cells were isolated from the cultures and restimulated with anti-CD3/CD28 mAb-conjugated microbeads. Cytokine levels were measured in the culture supernatants of restimulated T cells. The levels of IL-2 and TNF were significantly higher in the cultures of CD41 T cells primed with CD1031 LDCs than in those primed with CD11bhigh LDCs (IL-2: 1,581 6 341 pg/ml versus 628 6 201 pg/ml, P ¼ 0.035; TNF: 4,896 6 978 pg/ml versus 2,363 6 333 pg/ml, P ¼ 0.0181) (Figure 4). In addition, CD41 T cells primed with CD1031 LDCs produced significantly greater levels of IFN-g and IL-17A than those primed with CD11bhigh LDCs (IFN-g: 4,591 6 787 pg/ml versus 1,595 6 381 pg/ml, P ¼ 0.0088; IL-17A: 268 6 48 pg/ml versus 132 6 26 pg/ml, P ¼ 0.0181). By contrast, CD41 T cells primed with CD11bhigh LDCs released significantly larger amounts of IL-4, IL-6, and IL-10 than those primed with CD1031 LDCs (IL-4: 693 6 330 pg/ml

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Figure 1. Isolation of CD1031 and CD11bhigh lung dendritic cells (LDCs). Each LDC population was purified from the enzymatically digested lungs of BALB/c mice using magnetic and flow cytometric cell sorting, as described in MATERIALS AND METHODS. Cells in the gated area in (A) were analyzed for CD11c and MHC class II (B). CD1031 and CD11bhigh LDC populations were sorted from CD11c1 MHC class II1 cells using a FACSAria flow cytometer. (C) Presorted CD11c1 MHC class II1 cells in the gated regions. (D) The sorted LDC populations showed . 98% purity.

versus 25 6 9 pg/ml, P ¼ 0.0350; IL-6: 73.6 6 10.8 pg/ml versus 4.8 6 1.0 pg/ml, P ¼ 0.0017; IL-10: 441 6 191 pg/ml versus 55 6 17 pg/ml, P ¼ 0.0297). Intracellular Cytokine Staining of Primed CD41 T Cells with CD1031 LDCs or CD11bhigh LDCs

We next performed an intracellular cytokine staining assay in CD41 T cells that had been primed with CD1031or CD11bhigh LDCs. CD1031 LDCs induced greater frequencies of CD41 T cells producing IFN-g and IL-17A (Figure 5), whereas CD11bhigh LDCs were more efficient at inducing CD41 T cells producing IL-4 (Figure 5) and IL-10 (see Figure E1 in the online supplement). Regarding IL-6, a few CD41 T cells producing this cytokine were found when primed with CD1031 LDCs or CD11bhigh LDCs, and there was no significant difference in the proportions of CD41 T cells producing IL-6 (Figure E1). These results were mostly consistent with those of measurements of cytokine production from restimulated CD41 T cells that had been primed with CD1031or CD11bhigh LDCs, except for IL-6.

Expression of Chemokine Receptor mRNA in Primed T Cells with CD1031 LDCs or CD11bhigh LDCs

We also examined the expression of chemokine receptor mRNA in T cells primed with CD1031or CD11bhigh LDCs using real-time PCR. Messenger RNA levels of CXCR3 and CCR5, which are expressed preferentially in Th1 cells, were significantly higher in CD41 T cells primed with CD1031 LDCs than in those primed with CD11bhigh LDCs (Figure 6). By contrast, mRNA expression levels of CXCR4 and CCR4, which are present primarily in Th2 cells, were significantly greater in CD41 T cells primed with CD11bhigh LDCs than in those primed with CD1031 LDCs. No significant differences were found in the mRNA levels of CCR1, CCR2, CCR7, or CCR8 between the T cells primed with the two subpopulations of LDCs. Cytokine and Chemokine Production from CD1031 LDCs and CD11bhigh LDCs

To examine whether the two types of LDCs displayed different cytokine profiles that would skew Th responses, we compared

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TABLE 1. SURFACE AND INTRACELLULAR EXPRESSION LEVELS OF VARIOUS MOLECULES ON CD1031 LUNG DENDRITIC CELLS AND CD11BHIGH LUNG DENDRITIC CELLS Mean Fluorescence Intensity Surface and Intracellular Markers

CD1031 LDCs

MHC class II CD40 CD4 CD8a F4/80 Langerin (CD207) B7-1 (CD80) B7-2 (CD86) B7-H1 (PDL-1) B7-DC (PD-L2) DEC205 DC-SIGN

141.2 332.0 51.1 64.1 175.0 103.6 125.2 93.8 85.6 68.1 63.4 69.45

6 6 6 6 6 6 6 6 6 6 6 6

2.1* 57.0 2.3 2.8 21.9 8.8 20.7 3.4 8.0 8.2 8.2 5.1

CD11bhigh LDCs 184.0 409.5 63.2 85.8 286.5 101.6 151.7 112.8 100.0 71.3 67.8 80.7

6 6 6 6 6 6 6 6 6 6 6 6

3.6 82.4 7.1 8.0 11.7 9.5 11.7 3.3 9.9 8.0 7.7 8.8

Definition of abbreviations: LDCs ¼ lung dendritic cells. * Surface and intracellular expression levels of various molecules on CD1031 LDCs and CD11bhigh LDCs were assessed by five-color flow cytometry using the indicated monoclonal antibodies. Data were expressed as mean 6 SEM from five independent experiments.

the capabilities of CD1031 and CD11bhigh LDCs to release cytokines in response to various toll-like receptor (TLR) ligands. CD11bhigh LDCs secreted significantly higher amounts of IL-10

and MCP-1 than CD1031 LDCs, regardless of the TLR ligands (Figure 7). When stimulated with PAM3CSK4 (specific for TLR1 and TLR2) or CPG-ODN (specific for TLR9), CD11bhigh LDCs also produced significantly greater levels of IL-6 and TNF. By contrast, CD1031 LDCs released significantly higher amounts of IL-12p70 in response to PAM3CSK4 than CD11bhigh LDCs. No significant difference was found in the levels of TGF-b between CD1031and CD11bhigh LDCs. The levels of IL-23 were below the detection limit (detectable dose . 2.28 pg/ml) in both of the culture supernatants.

DISCUSSION In the present study, we examined whether the two distinct subsets of LDCs, CD1031 and CD11bhigh LDCs, elicited different Th responses in the resting conditions. The measurements of cytokine production, intracellular cytokine assay, and assessment of chemokine receptor mRNA suggested that CD1031 LDCs preferentially generated Th1 and Th17 responses, whereas CD11bhigh LDCs primarily induced a Th2 response under the steady state. Recent advances in the field of LDC biology have highlighted a unique subset of LDCs, CD11c1 MHC classII1 CD11blow or negative CD1031 LDCs (CD1031 LDCs). Several studies have shown that CD1031 LDCs are phenotypically, functionally, and anatomically distinct from other LDC populations, such as CD11c1 MHC classII1 CD11bhigh CD1032 LDCs

Figure 2. Flow cytometric analysis of CD1031 and CD11bhigh LDCs. Surface and intracellular expression levels of various molecules on CD1031 LDCs (top) and CD11bhigh LDCs (bottom) were examined by five-color flow cytometry using the indicated monoclonal antibodies. Histograms show the expression levels of the cell markers on two LDC subsets (solid line) and isotype-matched control antibodies (shaded area). These results are representative of more than five independent experiments.

Furuhashi, Suda, Hasegawa, et al.: Lung Dendritic Cell Subsets Induce Distinct Th Responses

Figure 3. Langerin mRNA expression in CD1031 and CD11bhigh LDCs. Langerin mRNA expression levels in freshly isolated CD1031 and CD11bhigh LDCs were analyzed by a quantitative real-time PCR assay using TaqMan probes according to the MATERIALS AND METHODS. Bar graphs present the relative expression of langerin mRNA, and values were normalized to GAPDH. Values are expressed as the mean 6 SEM of four independent experiments.

(CD11bhigh LDCs) (22–26). In agreement with previous studies, we confirmed that the CD1031 LDC subset constitutes a major DC population in the mouse lung, comprising 30 to 40% of CD11c1 MHC classII1 LDCs. Compared with CD11bhigh LDCs, CD1031 LDCs exhibited comparable expression levels of MHC class II, CD40, and costimulatory molecules (CD80,

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CD86, PDL-1, and PDL-2). However, langerin (CD207) was exclusively present in CD1031 LDCs but not in CD11bhigh LDCs. Accumulating evidence suggests that CD1031 and CD11bhigh LDCs play different roles in the immunity of the lung, but it has not been determined whether these two types of LDCs preferentially direct distinct Th responses. The findings from the present study clearly demonstrated the functional difference in inducing subtypes of Th cells between CD1031 and CD11bhigh LDCs. Our data suggested that CD1031 LDCs are more potent at eliciting Th1 and Th17 responses than CD11bhigh LDCs, whereas CD11bhigh LDCs are more efficient at evoking a Th2 response under the steady state. Consistent with our results, Raymond and colleagues recently indicated that CD11bhigh LDCs migrating from the lung to the MLN are indispensable for mounting a Th2 response in the lung (28). They showed that impairments in local and systemic Th2 responses observed in CD47–/– mice were associated with a loss of CD11bhigh DCs in the MLN and that the adoptive transfer of CD11bhigh CD471/1 DCs into the lung restored a Th2 response in OVA-sensitized CD47–/– mice. In contrast to our observations, Beaty and colleagues reported no difference in the capabilities of CD1031 and CD11bhigh LDCs to induce subtypes of Th cells, based on the results of an intracellular cytokine assay using CD41 CD25– DO11.10 T cells that had been stimulated with each LDC subset (26). Indeed, in the present study an intracellular cytokine assay was also conducted using naive CD41 CD62Lhigh DO11.10 T cells that had been primed with CD1031 or CD11bhigh LDCs, showing that CD1031 LDCs induced higher frequencies of CD41 T cells producing IFN-g and IL-17A and lower percentages of those producing IL-4 and IL-10 than CD11bhigh LDCs. Regarding IL-6, we detected very few CD41 T cells producing this cytokine. Compared with other cytokines, the levels of IL-6 in the supernatants of the primed CD41 T cells were very low (Figure 4). Thus, such low amounts of IL-6, presumably inside the cells, may be responsible for this, and it appeared to be difficult to assess IL-6 producing capacity of the primed CD41 T cells using our intracellular cytokine assay.

Figure 4. Cytokine production from restimulated CD41 T cells that had been primed with CD1031 or CD11bhigh LDCs. Isolated CD1031 or CD11bhigh LDCs were cocultured with naive DO11.10 CD41 CD62Lhigh T cells in the presence of OVA peptide (OVA323–339) for 7 days. CD41 T cells were then sorted from the cocultures using a FACSAria flow cytometer. The sorted CD41 T cells were restimulated with anti-CD3 and anti-CD28 mAb-conjugated beads. Two days after restimulation, the cytokine levels in the supernatants were measured by a cytometric bead array. Values are expressed as the mean 6 SEM of seven independent experiments.

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Figure 5. Intracellular cytokine staining of primed T cells stimulated with CD103 1 or CD11b high LDCs. (A) Cytokine production by CD41 T cells primed with CD103 1 (left panels; a–d ) or CD11b high LDCs (right panels; e–h). Naive DO11.10 CD4 1 CD62Lhigh T cells were cocultured with CD103 1 or CD11b high LDCs in the presence of OVA 323 –339 peptide for 5 days. CD4 1 T cells were then sorted from the cocultures using a FACSAria flow cytometer. The sorted CD4 1 T cells were stimulated with phorbol myristate acetate and ionomycin for an additional 6 hours in the presence of brefeldin A. After fixation and permeabilization, the cells were stained with allophycocyanin-conjugated anti-CD4 mAb and phycoerythrin-conjugated anti–IFN-g mAb, anti–IL-4 mAb, anti–IL-17A mAb, or rat IgG1k as an isotype control. The stained cells were analyzed using a FACSAria flow cytometer. Numbers indicate the percentage of cytokine-positive cells. One representative experiment is shown from five replicates.

The precise reason for the discrepancy between Beaty’s study and ours remains unclear, but it may be partly attributable to differences in the assay conditions, such as the T-cell populations prepared and the duration of stimulation. For example, we performed the intracellular cytokine assay in naive CD41 CD62Lhigh

DO11.10 T cells, instead of CD41 CD252 DO11.10 T cells as used in Beaty’s study. In assessing the Th-directing capabilities of the LDCs, Beaty and colleagues merely showed the data of their intracellular cytokine assay. However, we further confirmed our observations by analyzing the cytokine profiles of CD41 T cells

Figure 6. Real-time RT-PCR analysis of chemokine receptors in primed CD41 T cells with CD1031 or CD11bhigh LDCs. Isolated CD1031 LDCs or CD11bhigh LDCs were cocultured with naive DO11.10 CD41 CD62Lhigh T cells in the presence of OVA323–339 peptide for 7 days. CD41 T cells were then sorted from the cocultures using a FACSAria flow cytometer. mRNA expression levels of chemokine receptors in the sorted CD41 T cells were analyzed by a quantitative realtime PCR assay using TaqMan probes. Values are expressed as the mean 6 SEM of four independent experiments.

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Figure 7. Cytokine production from CD1031 or CD11bhigh LDCs in response to toll-like receptor ligands. CD1031 or CD11bhigh LDCs were stimulated with 1 mg/mL of LPS, 100 ng/mL of PAM3CSK4, or 5 mg/mL of CpG-ODN for 48 hours. The cytokine levels in the supernatants were measured by a cytometric bead array and an ELISA. Values are expressed as the mean 6 SEM of five independent experiments. *P values , 0.05.

that had been primed with each LDC subset and the expression of chemokine receptors in the primed CD41 T cells. We showed that CD41 T cells primed with CD1031 LDCs released significantly greater levels of IFN-g and IL-17A, whereas those primed with CD11bhigh LDCs produced significantly larger amounts of IL-4, IL-6, and IL-10. In particular, the production of Th2 cytokines, including IL-4 and IL-10, was found almost exclusively in CD41 T cells primed with CD11bhigh LDCs but not in those primed with CD1031 LDCs. In addition, mRNA levels of CXCR3 and CCR5, which are expressed preferentially in Th1 cells, were significantly higher in CD41 T cells primed with CD1031 LDCs. By contrast, mRNA levels of CXCR4 and CCR4, which are present primarily in Th2 cells, were significantly higher in those primed with CD11bhigh LDCs. Taken together, these data support the notion that the two major LDC populations preferentially elicit distinct Th responses. The mechanism by which CD1031 and CD11bhigh LDCs preferentially polarize Th responses toward the distinct types remains unclear. With regard to surface molecule expression, such as that of MHC class II, CD40, and other costimulatory molecules, no significant differences were found between the two LDCs. In terms of their capacities to produce cytokines in response to TLR ligands, although the amounts of released cytokines differed with the type of stimulant, CD11bhigh LDCs commonly secreted greater amounts of IL-6 and IL-10 than CD1031 LDCs. By contrast, CD1031 LDCs generally produced higher levels of IL-12p70 in response to PAM3CSK4 (specific for TLR1 and TLR2). In accordance with our results, a previous study showed that CD1031 LDCs have a more potent ability to release IL-12 than CD11bhigh LDCs when stimulated with multiple TLR ligands (24). Collectively, these data suggest that the capability of CD1031 LDCs to preferentially drive a Th1 response is associated, in part, with their potent IL-12 secreting ability. The development of fully functional mature Th17 cells from naive T cells requires IL-6 and TGF-b, with the coordination of IL-23. In the present study, we found that CD41 T cells primed with CD1031 LDCs produced significantly greater amounts of

IL-17A than those primed with CD11bhigh LDCs. Upon stimulation with TLR ligands, however, CD1031 LDCs secreted lower levels of IL-6 than CD11bhigh LDCs, with no difference in TGF-b production. In addition, we were unable to detect IL-23 in the culture supernatants of CD1031 or CD11bhigh LDCs, irrespective of the stimulant. Thus, the precise reason for the more potent capability of CD1031 LDCs to induce a Th17 response remains unclear. In general, the nature of DC activation does not appear to be the same between the simple stimulation of TLR ligands and the cocultures with naive T cells. Further study is required to elucidate the precise mechanism governing the preferential polarization of Th responses by each LDC subset. In other tissues, a recent study demonstrated that dermal langerin1 CD1031 CD11blow or negative DCs (CD1031 DCs) primarily induced Th1 and Th17 responses in the skin. This study showed that dermal-derived CD1031 DCs efficiently stimulated naive myelin-specific T cells to propagate and secrete IFN-g and IL-17, indicating that these dermal CD1031 DCs play a central and nonredundant role in Th1 and Th17 cell differentiation after subcutaneous immunization (30). In the gut, previous studies have shown that CD1031 DCs promoted the induction of gut-forming receptors such as CCR9 and a4b7 integrin (19, 20) and contributed to the control of inflammatory responses or intestinal homeostasis through the differentiation of Foxp31 Treg (21). In addition, CD1031 DCs in the mesenteric lymph nodes were shown to display a poor capacity to generate IFN-g–secreting Th cells under the steady state. These CD1031 DCs in colitic mice became very efficient at inducing IFN-g– and IL-17–secreting Th cells (31). Thus, the capabilities of CD1031 DCs to direct Th responses may vary between tissues and between different conditions, such as inflammation, infection, or allergic diseases. Further studies will address these issues. In conclusion, the present study demonstrated that the two major LDC populations preferentially induced distinct subtypes of Th cells. CD1031 LDCs were more potent at eliciting Th1 and Th17 responses, whereas CD11bhigh LDCs primarily provoked a Th2 response under the steady state. Our findings contribute

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toward a greater understanding of the functional differences between LDC subpopulations and raise the possibility that, for the therapeutic intervention of immunological lung disorders such as asthma, a particular LDC subpopulation may be a therapeutic target depending on the immunologic process involved in each disorder. Author disclosures are available with the text of this article at www.atsjournals.org.

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