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Estrogen Selectively Promotes the Differentiation of Dendritic Cells with Characteristics of Langerhans Cells¹

Allen Mao^*, Vladislava Paharkova-Vatchkova^*, John Hardy, Marcia M. Miller and Susan Kovats^2,*

^* Division of Immunology and Division of Molecular Biology, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA 91010

	Abstract

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The steroid hormone estrogen regulates the differentiation, survival, or function of diverse immune cells. Previously, we found that physiological amounts of 17-estradiol act via estrogen receptors (ER) to promote the GM-CSF-mediated differentiation of dendritic cells (DC) from murine bone marrow progenitors in ex vivo cultures. Of the two major subsets of CD11c⁺ DC that develop in these cultures, estrogen is preferentially required for the differentiation of a CD11b^intLy6C^– population, although it also promotes increased numbers of a CD11b^highLy6C⁺ population. Although both DC subsets express ER, only the CD11b^highLy6C⁺ DC express ER, perhaps providing a foundation for the differential regulation of these two DC types by estrogen. The two DC populations exhibit distinct phenotypes in terms of capacity for costimulatory molecule and MHC expression, and Ag internalization, which predict functional differences. The CD11b^intLy6C^– population shows the greatest increase in MHC and CD86 expression after LPS activation. Most notably, the estrogen-dependent CD11b^intLy6C^– DC express langerin (CD207) and contain Birbeck granules characteristic of Langerhans cells. These data show that estrogen promotes a DC population with the unique features of epidermal Langerhans cells and suggest that differentiation of Langerhans cells in vivo will be dependent upon local estrogen levels and ER-mediated signaling events in skin.

	Introduction

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Langerhans cells (LC)³ are a specialized dendritic cell (DC) subset resident in the epidermis and mucosal tissue (1). In the skin, LC act during initial immune responses to pathogens, neoplasia, or cellular damage resulting from exposure to environmental chemicals or UV light. LC are potent APC capable of internalizing and processing Ags, responding to activating signals via TLRs, and, upon migration to lymph nodes (LN), efficient activation of T cells via MHC and costimulatory molecules (2). Skin immunity is regulated by LC and, therefore, indirectly by factors that modulate LC differentiation, survival, or function. The steroid sex hormone estrogen is one such potential regulator of LC differentiation. Estrogen is known to have effects on estrogen receptor (ER)-expressing DC, macrophages, lymphocytes, or their undifferentiated progenitors as well as on epidermal keratinocytes and dermal fibroblasts (3, 4, 5).

Estrogen is a regulator of growth, differentiation, survival, or function in many cell types, including cells of the immune system (6). In addition to estrogens, ER ligands include selective ER modulators, such as tamoxifen and raloxifene; phytoestrogens; or other environmental endocrine disruptors, such as bisphenol A (7, 8, 9). Upon ligand binding, ER modulate gene transcription or mediate rapid nongenomic signaling events. Each structurally distinct ligand imparts a specific conformation to either ER or ER dimers, leading to recruitment of distinct profiles of coactivators or corepressors into multiprotein transcription complexes that bind DNA (10). Thus, ligation of one or both ER or ER may have disparate outcomes in different cell types depending on ligand form and concentration, the relative cellular expression of the two ER, and the availability of coactivators or corepressors (11). Both ER and ER are expressed in lymphoid organs. Although there are some reports of differential ER and ER expression by immune cells, the relative roles of ER and ER in myeloid cell regulation remain poorly understood (12, 13, 14, 15, 16).

LC are characterized by the presence of unusual cytoplasmic rod-like or tennis-racket-shaped structures termed Birbeck granules (BG), which can be visualized by electron microscopy as two apposed membranes separated by a striated zipper-like lamella (17, 18). LC express langerin (CD207), a type II membrane-associated C-type lectin that binds mannose residues via a single carbohydrate recognition domain (19). Langerin-deficient mice lack BG, and introduction of the langerin gene into fibroblasts induces the formation of BG, suggesting that langerin has a formative role in BG structure (19, 20). In the steady state, langerin is retained primarily in endosomal recycling compartments, and BG form apparently where langerin accumulates in pericentriolar endosomes (21). Upon binding mannose or anti-langerin Abs, cell surface langerin rapidly internalizes preferentially into BG-rich, and not MHC class II (MHCII)-rich, compartments (19, 21). Several studies indicate that langerin functions as an endocytic receptor to target mannosylated ligands to BG, leading to Ag presentation by CD1a (reviewed in Ref.22).

Upon arrival to LN, epidermal LC may be distinguished from other CD11c⁺ DC subsets by the CD4^–CD8^lowDEC-205^high phenotype (23). A recent report of a mouse expressing langerin linked to enhanced GFP showed that langerin is expressed at high levels by skin-derived MHCII^highCD8^–CD205^high DC found predominantly in cutaneous LN and at lower levels by MHCII^intCD8⁺CD205^int DC found in spleen, LN, and thymus (24). In bone marrow (BM) DC cultures, langerin mRNA was most highly expressed by immature MHCII^intCD86^– DC; langerin protein expression also was reported in BM DC cultures supplemented with TGF- (25, 26). Of two distinct CD11c⁺ DC populations (CD11b^high and CD11b^int) that appear in GM-CSF-supported BM DC cultures, the CD11b^int DC express the characteristic LC molecules E-cadherin and CD205 and are enhanced by the presence of TGF- (27, 28).

We previously demonstrated that physiological amounts (0.1 nM) of 17-estradiol (E2) promote the GM-CSF-mediated differentiation of DC from BM progenitors in ex vivo cultures derived from either female or male mice (29). Our experiments with the ER antagonist ICI 182,780, selective ER modulators, and ER^–/– BM cells indicated that estrogen acts via the ER to promote DC differentiation (29, 30). The CD11c⁺ DC that differentiate in this culture system may be divided into two major subsets based on relative expression of CD11b and Ly6C and dependence upon estrogen. E2 is preferentially required for the differentiation of a CD11b^intLy6C^– population, although it also promotes increased numbers of a CD11b^highLy6C⁺ population. In this report we have further characterized functional features of these two predominant DC populations. The two DC subsets show distinct capacity for Ag internalization when immature and for MHC and costimulatory molecule expression after LPS activation. The estrogen-dependent CD11b^intLy6C^– DC uniquely express langerin and contain BGs characteristic of Langerhans cells. Although both DC populations express ER, only the CD11b^highLy6C⁺ DC express ER, consistent with the differential regulation of these two DC types by E2. Taken together, these data indicate that differentiation of Langerhans cells in vivo may be regulated by ER-mediated signaling events.

	Materials and Methods

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Cell culture reagents

DC were differentiated in steroid hormone-deficient RPMI 1640 medium containing 10% charcoal-dextran (Dex)-stripped FBS (Omega Scientific) and lacking phenol red. Charcoal-Dex stripping of FBS extracts steroid hormones and reduces their levels below the detection limits of a standard RIA (31). Phenol red was omitted because it can have weak estrogenic activity at the concentration that is present in RPMI 1640 medium (32). The medium was supplemented with 2 mM glutamine, 10 mM HEPES buffer, 1 mM sodium pyruvate, 50 µM 2-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin. Medium conditioned by the GM-CSF-producing cell line, J558L (33), was added to DC culture medium at 5% to promote DC development. J558L cells were provided to us by Dr. R. Steinman (Rockefeller University, New York, NY). E2 (Sigma-Aldrich) was solubilized in 95% ethanol and diluted into cultures at the concentrations indicated from a 1-µM stock solution. An equal amount of ethanol alone was added to cultures as a vehicle control.

DC generation from murine BM

DC were generated from murine BM precursors as previously described (34). BM was isolated from 8- to 12-wk-old female 129S6 mice (Taconic Farms), which were housed at the City of Hope animal resource center in compliance with federal and institutional guidelines. BM was flushed from femurs and tibiae with PBS, resuspended in medium containing GM-CSF at 1.65–2 x 10⁵ cells/ml, and plated at a cell density of 3.3–3.5 x 10⁴ cells/cm². Cells were cultured for 7 days in 100-mm Teflon dishes or six-well plate inserts (Savillex) to reduce cell adherence. E2 was added from the first day of culture (day 0), and cells were fed every 3 days with medium containing GM-CSF and E2. On day 3, cells were fed by adding a volume of fresh medium equivalent to that of the culture. For feeding on day 6, one-half of the culture supernatant was first removed and centrifuged to recover any cells before disposal. The cell pellet was then resuspended in an equal volume of fresh medium and added back to the original culture. For DC maturation, cultures were incubated for 12–14 h, spanning days 6–7, with 0.2–2.0 µg/ml LPS from Escherichia coli serotype 055:B5 (Sigma-Aldrich).

Flow cytometry

DC cultures were analyzed on day 7 by four-color flow cytometry. FACS staining buffer (PBS, 5% newborn calf serum, and 0.1% sodium azide) was used for all washes and incubations. After harvest, 2 x 10⁵ cells were incubated for 5 min with anti-CD16/32 mAb (2.4G2) to block FcR. Then surface staining was performed by incubating samples for 15 min with combinations of optimally titrated, fluorochrome- or biotin-conjugated mAbs specific for CD11c, CD11b, Ly6C, CD86, CD80, CD83, MHCI (K^b), MHCII (I-A^b), CCR5, CCR7, and TLR4. Subsequently, fluorochrome-conjugated streptavidin (SA) was added for 15 min to detect biotin conjugates. Cells were fixed with 1% paraformaldehyde before analysis. Unlabeled anti-CD16/32 and biotin-conjugated anti-MHCII (mAb Y3P) were produced from hybridomas (American Type Culture Collection) and were purified and labeled in our laboratory. SA-allophycocyanin, SA-PE, and the following mAbs: anti-CD80-biotin (16-10A1), -CD86-PE (GL1), -Ly6C-FITC (AL-21), -CD11c-allophycocyanin (HL3), -MHC class I-bio (AF6-88.5), and -CD11b-PerCP-Cy5.5 (M1/70), were obtained from BD Pharmingen. Anti-TLR4-PE (MTS510), -CCR5-PE (HM-CCR5), and -CCR7-PE (4B12) were purchased from BioLegend. Anti-CD83-PE was obtained from eBioscience. Appropriately conjugated, isotype-matched control mAbs were used and did not nonspecifically stain cells in BM cultures. Samples were run on a FACSCalibur instrument (BD Biosciences) capable of four-color detection and were analyzed with FlowJo software (TreeStar).

Cell sorting

CD11c⁺ DC from day 7 cultures were isolated using anti-CD11c (mAb N418) microbeads and magnetic columns according to the manufacturer’s instructions (Miltenyi Biotec). Subsequently, DC were labeled with fluorochrome-conjugated mAbs specific for CD11c (mAb HL3), Ly6C, and CD11b and sorted into two groups on a Mo-Flo cytometer (DakoCytomation) in the City of Hope analytical cytometry core facility. The purity of the sorted cells was >95%.

Ag internalization and processing

Chicken OVA labeled with Alexa 488 (OVA₄₈₈) and Dex (10,000 m.w.)-labeled fluorescein (DexFITC) were obtained from Molecular Probes. Human serum albumin (HSA488) was labeled with Alexa 488 using an mAb labeling kit (Molecular Probes). Cells from day 7 BM DC cultures (2 x 10⁵) were resuspended in fresh medium at a concentration of 1.5 x 10⁶ cells/ml and were incubated with 10 µg/ml OVA488, 50 µg/ml HSA488, or 100 µg/ml DexFITC for 30 min at 37°C. Ag uptake controls were conducted at 0°C with all other conditions being equal. The samples were then washed twice in FACS staining buffer and surface stained for CD11c, CD11b, and Ly6C as described above. No E2 or ethanol was included in the medium during the 30-min Ag uptake.

Detection of ER and langerin mRNA by RT-PCR

Total RNA was extracted from cell pellets using an RNeasy Micro Kit (Qiagen) and was treated with a DNase kit (Promega) before being converted to cDNA using a SuperScript kit (Applied Biosystems) according to the manufacturer’s instructions. To detect langerin cDNA (25) or ER, ER, and -actin cDNA (16), gene-specific primers were used as previously described. PCR products were resolved on agarose gels and visualized with SYBR Green stain and a TYPHOON gel imager (Amersham Biosciences). No bands were observed in control samples of RNA not treated with reverse transcriptase.

Electron microscopy

CD11c⁺ DC were sorted into CD11b^highLy6C⁺ and CD11b^intLy6C^– populations, and pellets containing 1 x 10⁶ cells were prepared for microscopy as described previously (21). Briefly, cells were fixed overnight in 3% glutaraldehyde/2% sucrose in 0.1 M sodium cacodylate buffer, pH 7.3. After washing three times in the buffer, the pellet was postfixed with 1% osmium tetroxide at 4°C for 1 h, washed three times in sodium cacodylate buffer, and dehydrated in sequential 10-min steps through ethanol (60, 70, 80, 95, and 100%). The cell pellet then was incubated sequentially in 100% propylene oxide, propylene oxide/eponate (1/1, v/v), and 100% eponate at room temperature before polymerization at 70°C for 72 h. Thin sections were cut on the Leica Ultracut UCT at 50 nm with a diamond knife, picked up on the shiny side of 300-mesh pore size, uncoated copper grids, and stained with 5% aqueous uranyl acetate for 15 min, followed by Satos’ lead stain for 1 min. Sections were observed and photographed in an FEI Tecnai G2 transmission electron microscope in the City of Hope electron microscope core facility.

	Results

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Total BM cells from female 129S6 mice were cultured in GM-CSF-supplemented, steroid hormone-deficient medium with or without the addition of 0.1 nM E2 for 7 days. DC that had differentiated under these conditions were identified by expression of the integrins CD11b and CD11c using flow cytometry and could be divided into three subsets of CD11c⁺ DC: CD11b^high, CD11b^int, and CD11b^low (Fig. 1). The presence of E2 markedly promoted the differentiation of CD11b^int DC and, to a lesser degree, enhanced the differentiation of the CD11b^high subset (Fig. 1, A and B). To more accurately define these DC subsets, CD11c⁺ DC (Fig. 1, C and D) were assessed for relative expression of CD11b and Ly6C, a GPI-anchored membrane glycoprotein variably expressed on hemopoietic cells. Using this gating scheme, CD11c⁺ DC were divided into three subsets: CD11b^highLy6C⁺, CD11b^intLy6C^–, and CD11b^lowLy6C^– (Fig. 1, E and F). Remarkably, the CD11b^intLy6C^– subset was observed only in the presence of E2. The total number of each CD11c⁺ DC subset (defined in Fig. 1, A and B) that differentiated in a 5-ml culture in the absence or the presence of E2 is graphed in Fig. 1G. Although the number of CD11b^lowLy6C^– DC did not change, the numbers of CD11b^highLy6C⁺ and CD11b^intLy6C^– DC were increased significantly after culture with E2.

View larger version (23K): [in this window] [in a new window]   FIGURE 1. Estrogen most profoundly promotes the differentiation of CD11bintLy6C– DC. Female 129S6 BM cells were cultured in GM-CSF-supplemented, steroid hormone-deficient medium in the absence (left panels) or the presence (right panels) of 0.1 nM E2. After 7 days, cells were assessed for the relative expression of CD11c, CD11b, and Ly6C by flow cytometry. A and B, Analyses of CD11b and CD11c expression show three DC populations differing in the level of CD11b, and that E2 preferentially promotes the CD11bint subset. In an alternate gating scheme, CD11c+ cells were gated (indicated by the horizontal bar; C and D) and those gated CD11c+ cells (E and F) were divided into three subsets based on the relative expression of CD11b and Ly6C: CD11bhighLy6C+, CD11bintLy6C–, and CD11blowLy6C–. The E2-dependent subset is defined as CD11bintLy6C– by this scheme. G, The total number of DC of each indicated phenotype (based on boxed populations in A and B) is shown. Data are representative of >10 independent experiments.

View larger version (45K): [in this window] [in a new window]   FIGURE 2. CD11c+ DC subsets show differential expression of ER and ER. Cells in day 7 BM DC cultures containing E2 were divided into CD11c+ and CD11c– populations using anti-CD11c-conjugated magnetic beads. Subsequently, CD11c+ cells were sorted into CD11bhighLy6C+ and CD11bintLy6C– subsets by FACS. Each population was assessed for the presence of ER, ER, or -actin mRNA using RT-PCR and specific primers. ER mRNA was uniquely present in CD11bhighLy6C+ DC. Data are representative of two independent experiments.

View larger version (25K): [in this window] [in a new window]   FIGURE 3. CD11bintLy6C– DC show more significant increases in cell surface MHC and costimulatory molecules after LPS activation. Cells in E2-supplemented day 7 BM cultures were divided into CD11bhighLy6C+ (left panels) or CD11bintLy6C– (right panels) CD11c+ DC subsets and assessed for the expression of MHCI, MHCII, CD80, CD83, and CD86 by flow cytometry before ( ) or after () activation with bacterial LPS for 14 h. Data are representative of five independent experiments.

View larger version (27K): [in this window] [in a new window]   FIGURE 4. CD11c+ DC subsets show differential ability to internalize native Ags. Cells in E2-supplemented day 7 BM cultures were incubated for 30 min with FITC- or Alexa488-labeled dextran, HSA, or OVA at 4°C (left panels) or 37°C (right panels). Subsequently, cells were stained with anti-CD11c, -CD11b, and -Ly6C to define DC subsets. The relative amounts of Ag internalization by CD11bhighLy6C+ ( ) and CD11bintLy6C– () CD11c+ DC are shown. Data are representative of three independent experiments.

View larger version (124K): [in this window] [in a new window]   FIGURE 5. CD11c+CD11bintLy6C– DC uniquely express langerin mRNA and contain BGs visualized by electron microscopy. CD11c+ DC from E2-supplemented day 7 BM cultures were sorted into CD11bhighLy6C+ and CD11bintLy6C– DC subsets, as defined in Fig. 1F. A, The two DC subsets and CD11c– cells were assessed for the presence of langerin or -actin mRNA using RT- PCR and specific primers. CD11bintLy6C– DC selectively express langerin mRNA. Data are representative of two independent experiments. B and C, Sorted DC subsets were prepared for electron microscopy. At lower magnification, cells representative of the two DC subsets show that CD11bintLy6C– DC generally had more extensive filopodia. D and E, At higher magnification, 31 cells of each DC subset were examined in the pericentriolar region for the presence of BG. Only CD11bintLy6C– DC contained BG. Centrioles are indicated by asterisks, and BG are indicated by arrowheads.

	Discussion

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Our definition of two major DC subsets in BM DC cultures is consistent with previous reports. The differentiation of two phenotypically distinct CD11c⁺ DC populations from lineage^–c-kit⁺ progenitors in the presence of GM-CSF, stem cell factor, and TNF- was observed (28). The CD11b^highLy6C/G^lowc-Fms⁺ population retained pluripotent myeloid progenitor potential, because it was capable of differentiating to macrophages in the presence of M-CSF or to mature DC in the presence of GM-CSF and TNF-. In contrast, the CD11b^–/+Ly6C/G^–c-Fms^– population remained DC under all conditions and exhibited features of LC, with expression of E-cadherin and CD205; these DC were less capable of internalizing DexFITC and displayed greater levels of MHCII and allostimulatory capability. A second study extended these observations and divided immature MHCII^int BM DC into two groups based on the ability to internalize DexFITC (27). DexFITC⁺ DC were CD11b^highLy6C/G^highE-cadherin^–, whereas DexFITC^– DC were CD11b^intLy6C/G^–E-cadherin⁺. As we also report in this study, the latter CD11b^int population matured more readily upon stimulation with LPS. These authors hypothesize that CD11b^high DC correspond to myeloid monocyte-derived DC, whereas CD11b^int DC correspond to LC (although significant langerin expression or BG was not found).

The precise developmental relationship between the CD11b^highLy6C⁺ and CD11b^intLy6C^– DC populations that differentiate in GM-CSF-supported BM cultures remains unclear, although the above-mentioned studies suggest that the CD11b^int DC may arise via multiple pathways (27, 28). We do not yet have strong evidence that one DC population arises from the other, or that the two DC populations arise from distinct undifferentiated myeloid progenitors. BM progenitors of distinct DC populations have been identified in several studies (35, 36, 37), although it is unclear how these previously defined progenitors relate to our E2-responsive populations. The differential E2 requirement of the two DC populations may help us to dissect their origin from myeloid progenitors. Recently, we determined that E2 acts on undifferentiated CD31⁺Ly6C⁺ myeloid blasts present in murine BM (36) to promote differentiation of the CD11b^intLy6C^– subset (V. Paharkova-Vatchkova, A. Mao, and S. Kovats, manuscript in preparation).

It is difficult to directly correlate the BM DC in this ex vivo culture system with DC in secondary lymphoid organs, because we did not observe the expression of CD4 or CD8 by BM DC. However, analyses of splenic DC in mice treated with Flt3 ligand, which expands the naturally occurring DC populations, identified two populations of CD11c⁺ DC that correspond to the subsets defined in BM DC cultures (38). In the spleen, CD11b^high DC are Ly6C^highCD8^–CD205^–, more phagocytic, and located in the marginal zone. In contrast, CD11b^int/lowLy6C^–/+CD8⁺CD205⁺ DC are less phagocytic and located in the T cell areas of the white pulp. Interestingly, a study of the langerin-enhanced GFP-knockin mice showed that, in addition to LC, langerin is expressed in CD8⁺CD205^low DC in spleen and LN (24). These observations suggest that the estrogen-dependent DC in BM DC cultures may correspond to in vivo populations of both LC and CD8⁺ lymphoid DC.

Differentiation of CD1a⁺ LC from human CD34⁺ cells or monocytes requires TGF-1, which has been shown to promote the survival of DC progenitors by protecting them from apoptosis (39, 40). Mice deficient in TGF-1 lack epidermal LC, indicating that TGF- is required for LC differentiation and migration to skin (41, 42). Interestingly, estrogen induces the expression of TGF- in multiple cell types (43, 44, 45, 46, 47). Thus, we currently are testing the hypothesis that E2 promotes the differentiation of LC-like DC by increasing the level of TGF- in BM cultures.

Recent studies have suggested that human and murine LC renew from skin-resident proliferating precursors in the steady state (48, 49). The estrogen necessary to support LC differentiation in skin may be produced remotely in gonads or locally in tissue. Skin fibroblasts and adipose tissue are sites of extragonadal estrogen synthesis by aromatase, the P450 enzyme that catalyzes the conversion of C (19) steroid precursors to estrogens (50). Estrogen synthesized in extragonadal tissue acts via paracrine mechanisms, and it is likely that the concentration of E2 in the skin will be sufficient to mediate cellular ER signaling (51). In addition to potential direct effects of estrogen on gene expression patterns in LC progenitors, estrogen increases GM-CSF production by keratinocytes and TGF-1 production by fibroblasts (52). Taken together, these observations are consistent with the hypothesis that the paracrine action of locally produced estrogen in the skin, leading to enhanced production of TGF- and GM-CSF, will promote LC differentiation in vivo.

	Acknowledgments

 
We thank Lucy Brown and Claudio Spalla in the City of Hope Analytical Cytometry core facility for cell sorting. We thank Drs. Ralph Steinman, Greg Nalbandian, and Jose Alberola-Ila for helpful discussions.

	Disclosures

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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 National Institutes of Health, National Institute of Allergy and Infectious Diseases, Grant AI44922 (to S.K.), National Institutes of Health Cancer Center Core Grant CA33572(to City of Hope Medical Center), and National Science Foundation Grant DBI9970143 and Office of Naval Research Grant N00014-02-1 0958 (to the City of Hope Electron Microscopy core facility).

² Address correspondence and reprint requests to Dr. Susan Kovats, Division of Immunology, City of Hope Medical Center, 1450 East Duarte Road, Duarte, CA 91010. E-mail address: skovats{at}coh.org

³ Abbreviations used in this paper: LC, Langerhans cell; BG, Birbeck granule; BM, bone marrow; DC, dendritic cell; Dex, dextran; DexFITC, FITC-conjugated dextran; E2, 17-estradiol; ER, estrogen receptor; int, intermediate; HSA, human serum albumin; LN, lymph node; MHCII, MHC class II; OVA₄₈₈, Alexa 488-conjugated OVA; SA, streptavidin.

Received for publication July 11, 2005. Accepted for publication August 11, 2005.

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