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Related Collections Immunobiology Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 15 July 2002, Vol. 100, No. 2, pp. 569-577 IMMUNOBIOLOGY Identification of CD8+CD11c lineage phenotype-negative cells in the spleen as committed precursor of CD8+ dendritic cells Yong Wang, Yanyun Zhang, Hiroyuki Yoneyama, Nobuyuki Onai, Taku Sato, and Kouji Matsushima From the Department of Molecular Preventive Medicine and CREST, School of Medicine, The University of Tokyo, Tokyo, Japan.     Abstract Top Abstract Introduction Materials and methods Results Discussion References CD8+ dendritic cells (DCs) represent a functionally distinct DC subset in vivo, which plays a critical role in initiating various cellular immune responses. However, the committed precursor of CD8+ DCs remains to be identified. We reported here that murine splenic CD8+CD11c lineage phenotype (Lin) cells could differentiate into CD8+ DCs in vivo after intravenous transplantation. Immunohistochemistry staining showed that donor-derived DCs mainly located in T-cell areas of the spleen. Functionally, these CD8+CD11cLin cell-derived DCs were capable of stimulating allogenic T-cell response, as well as secreting bioactive interleukin 12 p70 and interferon . Freshly isolated CD8+CD11cLin cells expressed CC chemokine receptor (CCR)2, CCR5, and CCR7 messenger RNA, whereas CD8+ DCs derived from CD8+CD11cLin cells further obtained the expression of CCR6 and macrophage-derived chemokine. Flow cytometry analysis showed that CD8+CD11cLin cells were identified in bone marrow and lymph nodes. Moreover, transplanted splenic CD8+CD11cLin cells could also home to thymus and lymph nodes and were capable of developing into CD8+ DCs in these locations. However, CD8+CD11cLin cells failed to differentiate into CD8 DCs, T cells, natural killer cells, or other myeloid lineage cells in irradiated chimeras. Taken together, all these findings suggest that CD8+CD11cLin cells are a committed precursor of CD8+ DCs. (Blood. 2002;100:569-577) © 2002 by The American Society of Hematology.     Introduction Top Abstract Introduction Materials and methods Results Discussion References Dendritic cells (DCs) are professional antigen-presenting cells that play a pivotal role in the control of immunity.1-3 DCs are heterogeneous in populations, and at least 3 DC subpopulations have been identified in the mouse spleen based on the expression of CD4 and CD8, namely CD8CD4, CD8CD4+, and CD8+CD4, respectively.4 It has been demonstrated that these DC subsets are different not only in phenotype and function but also in geographic localization in lymphoid organs.1-3,5 CD8+ DCs reside in the T-cell areas of periarterial lymphoid sheath (PALS), whereas CD8 DCs are present in the marginal zones.5 Functionally, CD8+ but not CD8 DCs secrete high levels of interleukin-12 (IL-12) and interferon- (IFN-) to promote Th1 response.6-8 Süss and Shortman9 and Kronin et al10 previously reported that CD8+ DCs could regulate both CD4 T-cell and CD8 T-cell responses, associated with DC-mediated immune tolerance.9,10 It has been recently shown that CD8+ but not CD8 DCs cross-prime cytotoxic T cells in vivo.11,12 All these data indicate that CD8+ DCs represent a functionally distinct DC subset in vivo that plays a critical role in initiating various cellular immunities.1,13 Accumulating evidence indicates that several committed hematopoietic progenitor cells can differentiate into CD8+ DCs.14-20 Ardavin et al14 reported that CD8+ DCs could be generated in vivo from lymphoid-committed CD4low precursors isolated from the thymus, indicating that CD8+ DCs were of lymphoid lineage origin. The fact that mice deficient for Ikaros,21 a transcription factor that controls the differentiation of lymphoid cells, do not show impairment of CD8+ DC development in vivo suggests that there may be an alternative pathway for the generation of CD8+ lymphoid DCs. In contrast, recent studies indicate that both common myeloid progenitors (CMPs) and common lymphoid progenitors (CLPs) can differentiate into CD8+ DCs with an identical DC function and localization in lymphoid organs.19,20 These data suggest that CD8+ DCs may be generated in vivo from different hematopoietic progenitor cells (HPCs) via a common differentiation pathway that is still ill defined. Lacking a system to exclusively generate CD8+ DCs in vivo and in vitro has hampered the insight into the ontogeny of CD8+ DCs. It was previously reported that CD8+CD11c/dull cells were increased in Flt3 ligand (Flt3L)-treated mice, along with the significantly increased splenic CD8+ DCs.5 However, the relationship between CD8+CD11c/dull cells and CD8+ DCs remains unknown. In the present report, we describe CD8+CD11c lineage phenotype (Lin) cells in the spleen as a committed precursor of CD8+ DCs, which fail to give rise to CD8 DCs, T cells, natural killer (NK) cells, or other myeloid lineage cells in irradiated chimeras.     Materials and methods Top Abstract Introduction Materials and methods Results Discussion References Mice C57BL/6 Ly5.2 and BALB/c mice were obtained from CLEA Experimental Animal Co Ltd (Tokyo, Japan), and congenic C57BL/6 Ly5.1 mice were obtained from Dr H. Ishikawa (Keio University School of Medicine, Tokyo, Japan). They were maintained under specific pathogen-free conditions in the Animal Facility of Department of Molecular Preventive Medicine, School of Medicine, The University of Tokyo (Tokyo, Japan). All animal experiments complied with the standards set out in the Guidelines for Care and Use of Laboratory Animals of the University of Tokyo. Antibodies Antibodies that were used for immunofluorescence staining were obtained from BD PharMingen (San Diego, CA) unless otherwise indicated. DEC-205 (NLDC-145) monoclonal antibody (MoAb) was obtained from BMA Biomedicals (Augst, Switzerland). Fluorescein isothiocyanate (FITC)-conjugated goat F(ab')2 antirat immunoglobulin G (IgG; heavy and light chains (H&L)) was purchased from Leinco Technologies (Ballwin, MO). Phycoerythrin (PE)-conjugated anti-CD40 (3.23), PE-conjugated anti-CD86 (RMMP1), and FITC-conjugated anti-CD86 were purchased from Immunotech (Marseille, France). CD11c (N418) MoAb was obtained from Serotec (Oxford, England). As secondary antibodies, alkaline phosphatase-conjugated goat antirat IgG was bought from Jackson ImmunoResearch Laboratories (West Grove, PA), whereas alkaline phosphatase-conjugated goat antihamster IgG was from Cedarlane (Ontario, Canada). Streptavidin-peroxidase (Histofine) was purchased from Nichirei Corporation (Tokyo, Japan). Purification and transplantation of CD8+CD11cLin cells CD8+CD11cLin cells were isolated from the mouse spleen. In brief, mononuclear cells (MNCs) from the splenocytes were isolated by Lymphoprep (density, 1.077 ± 0.001 g/mL; Nycomed Pharma AS, Oslo, Norway) gradient centrifugation and then were enriched using MACS (Miltenyi Biotec, Germany) by incubation with CD8-microbeads (Miltenyi Biotec). Cells retained in the column were eluted and then 3-color stained with FITC-conjugated anti-CD8 (53-6.7), a cocktail of PE-labeled MoAbs to CD3 (145-2C11), B220 (RA3-6B2), Gr-1 (RB6-8C5), CD11b (M1/70), NK1.1 (PK136), and biotin-conjugated anti-CD11c (HL3), followed by APC-conjugated streptavidin. CD8+CD11cLin cells were isolated by using a cell sorter (EPICS ELITE; Beckman Coulter). The purity was consistently more than 98% as reanalyzed with the cell sorter. Similarly, CD8+CD11cLin cells were isolated from the bone marrow (BM) and lymph nodes (combined axillary, cervical, inguinal, and mesenteric lymph nodes). In some experiments, the sorted CD8+CD11cLin cells were further stained with PE-conjugated anti-Ia (AF6-120.1), anti-CD40, anti-CD86, anti-CD4 (H129.19), or biotin-conjugated anti-CD8 (53-5.8) revealed with APC-streptavidin, respectively. BM-derived Lin c-kit+ HPCs were isolated and purified as previously described.22,23 Before intravenous transfer, the congenic Ly5.1 B6 mice received 10.5 Gy -radiation and then used as recipients. CD8+CD11cLin cells (3-5 × 105) isolated from Ly5.2 B6 mice were intravenously transferred to the lethally irradiated congenic Ly5.1 B6 recipient mice, accompanied by 2 × 105 recipient-type (Ly5.1-type) BM cells to rescue the recipient mice from lethal irradiation. Mice transferred with Ly5.1-type BM cells only were used as negative control, whereas mice transferred with Ly5.2-type BM-derived Linc-kit+ HPCs or BM cells were used as positive control. The spleens of the recipient mice were analyzed 1 to 4 weeks after transfer. Immunofluorescence analysis Immunofluorescence analyses were performed as previously described.22,23 Cells were preincubated with 2.4G2 to prevent binding to FcRII/III to reduce the nonspecific staining unless rat DEC-205 MoAb was used as the primary antibody staining. In 2-color analyses, 4 × 105 indicated cells were incubated with FITC-conjugated anti-Ly5.2 (104) as well as PE-conjugated anti-Ia, anti-CD3, anti-B220, anti-NK1.1, or anti-Gr-1. In tri-color analyses, 4 × 105 cells were incubated with optimal concentration of biotin-conjugated anti-Ly5.2, followed by APC-conjugated streptavidin, as well as stained with PE-conjugated anti-Ia and FITC-conjugated anti-CD11c, anti-CD8, anti-CD40, anti-CD86, or anti-CD11b. In some experiments, cells were stained with purified rat anti-DEC-205, followed by FITC-conjugated goat F(ab')2 antirat IgG (H&L) as the second stage. The instrument compensation was set in each experiment by using single-color and/or 2-color stained samples. Dead cells were excluded with forward scatter, side scatter, and propidium iodide gating. In some experiments, the corresponding cell subpopulations were isolated with the use of a cell sorter. Immunohistochemistry staining Double immunostaining was performed by indirect immunoalkaline phosphatase or immunoperoxidase methods.24 In short, spleen specimens were removed and embedded in Tissue-Tek O.C.T. compound (Miles, Elkhart, IN), frozen in liquid nitrogen, and cut by a cryostat into 7-µm-thick sections, air-dried overnight, and fixed in acetone for 10 minutes at room temperature. The sections were sequentially incubated with optimal dilution of biotin-conjugated anti-Ly5.2 followed by streptavidin-peroxidase. Peroxidase activity was visualized with 3-amino-9-ethylcarbazole (Vector Laboratories, Burlingame, CA) showing red color. The sections were then incubated with hamster antimouse CD11c (N418) followed by alkaline phosphatase-conjugated goat antihamster IgG staining. Sections were also separately stained with rat anti-DEC-205 or anti-CD8 followed by goat antirat IgG complexed to alkaline phosphatase. Alkaline phosphatase activity was developed with the Vector Blue substrate (Vector Laboratories) revealing in blue color. Levamisole (0.024%; Sigma Chemical, St Louis, MO) was added to the reaction mixture to block endogenous alkaline phosphatase activity. In these preparations, peroxidase activity yields a red color reaction product, whereas alkaline phosphatase activity appears blue in color. Isolation of DCs Different DC subsets were isolated from the spleens, thymi, and lymph nodes (combined axillary, cervical, inguinal, and mesenteric lymph nodes) as previously described25 with slight modification. Briefly, spleens, thymi, and lymph nodes were digested with collagenase D (1 mg/mL; Boehringer Mannheim, Roche, Germany), and MNCs were isolated by Lymphoprep gradient centrifugation. After these MNCs were incubated with microbead-conjugated anti-CD11c MoAb (N418; Miltenyi Biotec), CD11c+ cells were sorted by MACS and used for phenotyping analyses by 2-color or 3-color immunofluorescence staining. In some experiments, the corresponding cell subpopulations were further purified with the cell sorter. The purity is more than 98% as revealed by immunofluorescence reanalysis. Cytokine assay Sorted host-type CD8+ DCs from Ly5.1 BM cell-reconstituted Ly5.1 B6 mice and Ly5.2+CD8+Ia+ DCs from Ly5.2+CD8+CD11cLin precursor- reconstituted Ly5.1 B6 mice were cultured in different conditions to stimulate the production of IL-12 and IFN-. To measure IL-12 production, 7.5 × 104 DCs were stimulated in vitro with granulocyte-macrophage colony-stimulating factor (GM-CSF) (20 ng/mL; Kirin Brewery, Tokyo, Japan) + IFN- (20 ng/mL; PeproTech EC, London, England) + Pansorbin (50 µg/mL; Calbiochem, Darmstadt, Germany) for 40 hours. To measure IFN- production, 3.3 × 104 DCs were stimulated in vitro with rmIL-12 (16 ng/mL; kindly provided by Nippon Roche Research Center, Kamakura, Japan) for 48 hours. Supernatants were collected and quantified by enzyme-linked immunosorbent assay (ELISA). Mouse IL-12 p70 ELISA kit was purchased from Genzyme (sensitivity, < 2.5 pg/mL; Minneapolis, MN), whereas mouse IFN- ELISA kit was from Endogen (sensitivity, < 15 pg/mL, Woburn, MA). Mixed leukocyte reaction Allogenic CD4+ T cells from BALB/c mice were used as responders and prepared as previously described.23,26 Briefly, the adherent cells were first removed by incubating splenic MNCs at 37°C for 60 minutes in Iscoves modified Dulbecco medium (GIBCO, Rockville, MD) containing 10% fetal bovine serum. The nonadherent splenic MNCs were incubated with microbead-conjugated antimouse CD4 MoAb (Miltenyi Biotec, Germany), and CD4+ T cells were separated with magnetic cell sorting. BM Linc-kit+ HPCs were obtained and cultured to generate mature DCs as previously described.23 Splenic Ly5.2+CD8+Ia+ cells from naive Ly5.2 B6 mice and reconstituted mice, as well as BM-derived mature DCs, were used as stimulators and treated with mitomycin C (15 µg/mL; Sigma Chemical) as previously described.27 Graded doses of stimulator cells (from 100 to 3 × 104 cells) were added to the T cells (3 × 105) in wells of 96-well round-bottomed microtest tissue-culture plates (Nunc, Roskilde, Denmark), respectively. After incubating at 37°C for 4 days, cell proliferation was determined by using 3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyltetrazolium bromide (Sigma Chemical). The resultant absorbance at 550 nm was read by a microplate immunoreader. Reverse transcription-polymerase chain reaction Total RNAs were extracted from 2 × 105 indicated cells by using RNAzol B (Biotex Laboratories, Houston, TX), according to the manufacturer's instructions. First-strand complementary DNA (cDNA) was synthesized at 37°C for 1 hour from 200 ng total RNA in 25 µL reaction volume with the use of random primers (Promega, Madison, WI). Thereafter, cDNA was amplified for 35 cycles consisting of 94°C for 30 seconds, 55°C for 1 minute, and 72°C for 1 minute, with a pair of oligonucleotide primers corresponding to each chemokine or chemokine receptor.28 Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript was amplified in parallel as control. The corresponding oligonucleotide primers were as follows: 5'-TGTTACCTCAGTTCATCCACGG-3' and 5'-CAGAATGGTAATGTGAGCAGGAAG-3' were designed for murine CC chemokine receptor (CCR)2, 5'-CATCGATTATGGTATGTCAGCACC-3' and 5'-CAGAATGGTAGTGTGAGCAGGAA-3' for murine CCR5, 5'-ACTCTTTGTCCTCAC-CCTACCG-3' and 5'-ATCCTGCAGCTCGTATTTCTTG-3' for murine CCR6, 5'-CATCAGCATTGACCGCTACGT-3' and 5'-GGTACGGATGATAATGAGGTAGCA-3' for murine CCR7, 5'-TCTGATGCAGGTCCCTATGGT-3' and 5'-TTATGGAGTAGCTTCTTCACCCAG-3' for murine macrophage-derived chemokine (MDC), and 5'-CCTTCATTGACCTCAACTAC-3' and 5'-AGTGATGGCATGGACTGTGGT-3' for GAPDH. The polymerase chain reaction (PCR) products were fractionated on 1.5% agarose gel and visualized by ethidium bromide staining. Statistical analysis Significant differences were evaluated by using the Student t test. P < .05 was considered to be statistically significant.     Results Top Abstract Introduction Materials and methods Results Discussion References Isolation of CD8+CD11cLin cells To identify the committed precursor of CD8+ DCs in vivo, CD8+CD11cLin cells were isolated from the spleens. Tricolor-fluorescence analyses showed that these CD8+CD11cLin cells did not express any detectable level of CD3, B220, CD11b, Gr-1, NK1.1, Ia, CD40, CD86, or CD4 (Figure 1A-B). They expressed CD8 (Figure 1B). As shown in Figure 1C, the expression level of CD8 on the spleen-derived CD8+CD11cLin cells was as high as on CD8 T cells. This cell population was scarce and only represented 0.2% to approximately 0.25% of the whole splenocytes in the C57BL/6 mice (Figure 1A). Giemsa staining showed that freshly sorted CD8+CD11cLin cells displayed a round lymphoid cell-like morphology (Figure 1D). In addition, as shown in Figure 2, CD8+CD11cLin cells were also detected in the BM and lymph nodes (combined axillary, cervical, inguinal, and mesenteric lymph nodes). They represented 0.2% of the lymph node cells and 0.06% of BM leukocytes, respectively. View larger version (25K): [in this window] [in a new window]   Figure 1. Isolation of CD8+CD11cLin cells from the spleen. (A) The splenocytes enriched by CD8-microbeads, as described in "Materials and methods," were stained with biotin-conjugated anti-CD11c MoAb, followed by APC-conjugated streptavidin, as well as FITC-conjugated anti-CD8, PE-conjugated anti-Lin markers (CD3, B220, Gr-1, CD11b, and NK1.1). The CD8+Lin cells for sorting were gated on CD11c cells. The purity of the CD8+CD11cLin cells after sorting was presented (> 98%). The quads were set up on the isotype-matched control dot plot. The results are representative of more than 6 independent experiments. (B) CD8+CD11cLin cells were first isolated by using a cell sorter and then further stained with PE-conjugated anti-Ia, anti-CD40, anti-CD86, anti-CD4, as well as biotin-conjugated anti-CD8, revealed with APC-streptavidin. Solid and dotted lines indicated the immunofluorescence intensity of cells stained with a control and the test antibodies, respectively. The results are representative of 4 independent experiments. (C) CD8 expression of spleen-derived CD8+CD11cLin cells. The CD8+CD11cLin cells were first isolated by using a cell sorter as described in (A), and then reanalyzed by FACS (shown as solid line). For CD8CD3 T cells, the CD8-microbead-enriched cells were stained with FITC-conjugated anti-CD8 and PE-conjugated anti-CD3. The CD8CD3 T cells were isolated by using a cell sorter and then reanalyzed by FACS (shown as dotted line). The results are representative of 3 independent experiments. (D) Giemsa staining was performed on CD8+CD11cLin cells after sorting. The results are representative of 4 independent experiments. Original magnification × 400. View larger version (22K): [in this window] [in a new window]   Figure 2. Isolation of CD8+CD11cLin cells from BM and lymph nodes. BM cells (A) and lymph node cells (combined axillary, cervical, inguinal, and mesenteric lymph nodes; B) were enriched by CD8-microbeads and then stained with biotin-conjugated anti-CD11c, revealed with APC-streptavidin, as well as FITC-conjugated anti-CD8, PE-conjugated anti-Lin markers (CD3, B220, Gr-1, CD11b, and NK1.1). The CD8 versus Lin markers staining was gated on CD11c cells. The gate indicated the BM-derived (A) and lymph node-derived (B) CD8+CD11cLin cells, respectively. The results are representative of 3 independent experiments. CD8+CD11cLin cells differentiate into CD8+ DCs in vivo To elucidate whether CD8+CD11cLin cells might be able to differentiate into CD8+ DCs in vivo, 5 × 105 CD8+CD11cLin cells isolated from the spleens of Ly5.2 B6 mice were injected intravenously into lethally irradiated Ly5.1 congenic B6 mice. Recipient type (Ly5.1-type) BM cells (2 × 105) were simultaneously transferred to rescue the mice from lethal irradiation. At the indicated time points after transfer, the recipients were killed, and splenocytes were analyzed by immunofluorescence staining. As shown in Figure 3A and Figure 4, donor-derived Ly5.2+Ia+ cells were readily detected as early as 7 days after transplantation in the spleen and peaked at day 14. Donor-derived DCs from CD8+CD11cLin cells gradually disappeared from the spleen and could not be detected at day 28 (Figure 4), suggesting that the life span of CD8+CD11cLin cell-derived DCs may be limited. These donor-derived cells expressed moderate to high levels of Ia, as well as CD11c, DEC-205, CD40, and CD86 molecules, which are characteristics of DC phenotype (Figure 3C). They also expressed CD11b molecule (Figure 3C). CD11b is expressed at levels ranging from low to high on different lymphoid tissue DC subsets.4,25,29 Most strikingly, all of these donor-derived cells were positive for CD8 molecule with analyses performed 7 to 21 days after transplantation (Figure 3C and not shown). Morphologically, these freshly isolated Ly5.2+Ia+CD8+ cells from CD8+CD11cLin precursor-reconstituted mice displayed round or irregular shape with small membrane projections (Figure 3B). After culture in the presence of GM-CSF overnight, these donor-derived Ly5.2+Ia+CD8+ cells demonstrated a typical DC morphology with irregular membranes, beanlike nuclei, and fine dendritic processes (Figure 3B). Functionally, these donor-derived Ly5.2+CD8+Ia+ DCs stimulated allogenic T-cell proliferation, as wild-type CD8+ DCs did in a mixed leukocyte reaction (MLR) assay (Figure 5). However, it was less potent than mature DCs generated from BM-derived HPCs (Figure 5). As described in Figure 3B-C, donor-derived DCs expressed moderate to high levels of Ia, and freshly isolated donor-derived DCs displayed round to irregular shape, which indicated that donor-derived cells consisted of immature and mature DCs. The heterogeneous immature and mature stages of donor-derived DCs may explain the lower allogenic T-cell response than mature BM-derived DCs. View larger version (54K): [in this window] [in a new window]   Figure 3. Development of donor-derived DCs in the spleens from CD8+CD11cLin cells in vivo. (A) CD8+CD11cLin cells isolated from the spleens of Ly5.2 B6 mice were intravenously transferred to the lethally irradiated congenic Ly5.1 B6 recipient mice, accompanied by Ly5.1-type BM cells to rescue the recipient mice from lethal irradiation. Mice transferred with Ly5.1-type BM cells only were used as negative control, whereas mice transferred with Ly5.2-type BM-derived Linc-kit+ HPCs were used as positive control. At day 14, splenocytes were isolated and enriched with CD11c-conjugated microbeads, as described in "Materials and methods" and then stained with FITC-conjugated anti-Ly5.2 and PE-conjugated anti-Ia. The quads were set up on the isotype-matched control dot plot. The results are representative of 6 independent experiments. (B) Giemsa staining was performed on sorted Ly5.2+CD8+Ia+ cells from CD8+CD11cLin cell-reconstituted mice, which were isolated freshly or cultured overnight with GM-CSF. The results are representative of 3 independent experiments. Original magnification × 400. (C) FACS analyses of CD8+CD11cLin cell-derived DCs by tricolor staining. Cells were collected as described in (A) and then stained with biotin-conjugated anti-Ly5.2, followed by APC-streptavidin, as well as PE-conjugated anti-Ia, FITC-conjugated anti-CD11c, anti-CD8, anti-CD40, anti-CD86, or anti-CD11b. As for the staining of DEC-205, rat DEC-205 was used as first antibody, followed by FITC-conjugated goat F(ab')2 antirat IgG (H&L). Data from mice transferred with Ly5.1-type BM cells only were shown as negative control. The quads were set up on the isotype-matched control dot plot. The results are representative of 6 independent experiments. View larger version (16K): [in this window] [in a new window]   Figure 4. The kinetics of generation of CD8 +CD11cLin cell-derived splenic DCs. Each group of 4 irradiated Ly5.1 B6 mice was reconstituted with 5 × 105 sorted CD8+CD11cLin cells from the spleens of Ly5.2 B6 mice. Donor-derived DCs were identified as Ly5.2+Ia+ cells as described in Figure 3A at various time points (1 to 4 weeks) after transplantation. Each point is the mean ± 1 SD of 3 such reconstitution experiments, each based on a pool of 4 spleens from the reconstituted mice. View larger version (16K): [in this window] [in a new window]   Figure 5. Allogenic MLR. Allogenic MLR was performed by using purified CD4+ T cells (3 × 105 cells/well in 96-round-well plate) as responder cells.  represents Ly5.2+CD8+Ia+ DCs from naive Ly5.2 B6 mice (wild-type CD8+ DCs), whereas  represents Ly5.2+CD8+Ia+ DCs from Ly5.2+CD8+CD11cLin precursor-reconstituted Ly5.1 B6 mice (donor-derived CD8+ DCs).  represents mature DCs generated from BM-derived HPCs (BM-derived DCs). All the indicated cell subpopulations were purified or generated as described in "Materials and methods." Results are expressed as the mean ± 1 SD of the triplicate cultures and are representative of 3 independent experiments. To elucidate whether CD8+CD11cLin cells could also home to other lymphoid organs, thymi and lymph nodes were isolated from the recipient mice. As shown in Figure 6A, donor-derived Ly5.2+Ia+ cells were detected in the thymi at day 14 after transplantation. These donor-derived cells expressed CD11c, CD8, DEC205, CD40, and CD86 molecules, some of which expressed CD11b molecule (Figure 6B). In addition, as shown in Figure 7A, donor-derived Ly5.2+Ia+ cells were also detected in the lymph nodes (combined axillary, cervical, inguinal, and mesenteric lymph nodes) at day 14 after transplantation. These donor-derived cells expressed CD11c, CD8, DEC205, CD40, and CD86 molecules, some of which expressed CD11b molecule (Figure 7B). View larger version (49K): [in this window] [in a new window]   Figure 6. Development of donor-derived DCs in the thymi from CD8+CD11cLin cells in vivo. (A) CD8+CD11cLin cells isolated from the spleens of Ly5.2 B6 mice were intravenously transferred to the lethally irradiated congenic Ly5.1 B6 recipient mice, accompanied by Ly5.1-type BM cells to rescue the recipient mice from lethal irradiation. Mice transferred with Ly5.1-type BM cells were used as negative control, whereas mice transferred with Ly5.2-type BM cells were used as positive control. At day 14 after transplantation, thymi were digested with collagenase D. Collected thymocytes were enriched with CD11c-conjugated microbeads and then stained with FITC-conjugated anti-Ly5.2 and PE-conjugated anti-Ia. The quads were set up on the isotype-matched control dot plot. The results are representative of 3 independent experiments. (B) Phenotype characterization of CD8+CD11cLin cell-derived thymic DCs. Cells were collected as described in (A) and then stained with biotin-conjugated anti-Ly5.2, followed by APC-streptavidin, as well as PE-conjugated anti-Ia, FITC-conjugated anti-CD11c, anti-CD8, anti-CD40, anti-CD86, or anti-CD11b. As for the staining of DEC205, rat DEC205 was used as first antibody, followed by FITC-conjugated goat F(ab')2 antirat IgG (H&L). Solid and dotted lines indicated the histograms of specific stainings and isotope-matched controls gated on the Ly5.2+Ia+ cells, respectively. The results are representative of 3 independent experiments. View larger version (51K): [in this window] [in a new window]   Figure 7. Development of donor-derived DCs in the lymph nodes from CD8+CD11cLin cells in vivo. (A) CD8+CD11cLin cells isolated from the spleens of Ly5.2 B6 mice were intravenously transferred to the lethally irradiated congenic Ly5.1 B6 recipient mice, accompanied by Ly5.1-type BM cells to rescue the recipient mice from lethal irradiation. Mice transferred with Ly5.1-type BM cells were used as negative control, whereas mice transferred with Ly5.2-type BM cells were used as positive control. At day 14 after transplantation, combined axillary, cervical, inguinal, and mesenteric lymph nodes were collected and digested with collagenase D. Cells were enriched with CD11c-conjugated microbeads and then stained with FITC-conjugated anti-Ly5.2 and PE-conjugated anti-Ia. The quads were set up on the isotype-matched control dot plot. The results are representative of 3 independent experiments. (B) Phenotype characterization of CD8+CD11cLin cell-derived DCs in the lymph nodes. Cells were collected as described in (A) and then stained with biotin-conjugated anti-Ly5.2, followed by APC-streptavidin, as well as PE-conjugated anti-Ia, FITC-conjugated anti-CD11c, anti-CD8, anti-CD40, anti-CD86, or anti-CD11b. As for the staining of DEC205, rat DEC205 was used as first antibody, followed by FITC-conjugated goat F(ab')2 antirat IgG (H&L). Solid and dotted lines represent the histograms of specific stainings and isotope-matched controls gated on the Ly5.2+Ia+ cells, respectively. The results are representative of 3 independent experiments. To examine the potential of CD8+CD11cLin cells to generate other lineages in vivo, the expressions of CD3 (T cells), B220 (B cells), NK1.1 (NK cells), and Gr-1 (granulocytes) on Ly5.2-positive cells were also analyzed by immunofluorescence staining at various time points (1, 2, 3, and 4 weeks) after transplantation. None of these cell lineage markers such as for T cells, NK cells, or myeloid cells were detectable on the surface of donor-derived cells. However, there were a small number of scattered donor-derived B220+ cells that could not be detected 4 weeks after transplantation (Figure 8 and not shown). View larger version (17K): [in this window] [in a new window]   Figure 8. CD8+CD11cLin cells fail to differentiate into T cells, NK cells, or other myeloid lineage cells. At day 21, the splenocytes from the CD8+CD11cLin cell-reconstituted mice were collected and stained with FITC-conjugated anti-Ly5.2 and PE-conjugated anti-CD3, anti-B220, anti-NK1.1, or anti-Gr-1, respectively. The quads were set up on the isotype-matched control dot plot. The results are representative of 6 independent experiments. Localization of donor-derived DCs in the spleen sections CD8+ DCs have been identified in the T-cell areas of lymphoid organs,5 whereas CD8 DCs mainly distribute in the areas of marginal zone surrounding T-cell areas.13 To investigate the localization of donor-derived DCs in the spleen, series of frozen splenic sections from the CD8+CD11cLin precursor-reconstituted mice were stained with Ly5.2 marker (visualized in red color) in combination with CD8, CD11c (N418), or DEC-205 (visualized in blue color). CD8 staining represents the T-cell areas of the PALS. As shown in Figure 9A-B, all the donor-derived cells were colocalized in the T-cell areas (Figure 9A), and double-stained donor-derived Ly5.2+CD8+ cells were shown in purple color (Figure 9B). The donor-derived cells also expressed CD11c and DEC-205 antigens (Figure 9C-D). These results suggest that newly generated CD8+ DCs from donor CD8+CD11cLin cells exclusively reside in the T-cell areas. View larger version (147K): [in this window] [in a new window]   Figure 9. Localization of donor-derived DCs in the spleen sections. At day 14, the splenic sections were performed and double stained as described in "Materials and methods." (A) Donor marker Ly5.2 was stained as red color, whereas CD8 marker was stained as blue color. Original magnification, × 100. (B) The same staining was performed as described in (A). Original magnifications, × 200 and × 400 (the insert picture at the right-bottom side). (C-D) Donor marker Ly5.2 was stained as red color, whereas CD11c (C) and DEC205 (D) markers were stained as blue color, respectively. Original magnifications, × 200 and × 400 (the insert pictures at the bottom right sides). The results are representative of 3 independent experiments. Donor-derived CD8+ DCs secrete IL-12 and IFN- on ex vivo culture One of the functional features of CD8+ DCs is that this DC subset exclusively secretes high levels of IL-12 and IFN- in response to appropriate stimulations.6-8 To characterize the cytokine profile of the donor-derived DCs, Ly5.2+CD8+Ia+ DCs from Ly5.2+CD8+CD11cLin precursor-reconstituted Ly5.1 B6 mice and host-type CD8+ DCs from Ly5.1 BM cell- reconstituted Ly5.1 B6 mice were highly purified by cell sorting. They were cultured in the presence of GM-CSF (20 ng/mL) + IFN- (20 ng/mL) + Pansorbin (50 µg/mL) for 40 hours for the secretion of IL-12 p70, or rmIL-12 (16 ng/mL) for 48 hours for the secretion of IFN-. As shown in Figure 10A-B, high levels of IL-12 p70 and IFN- were detected in the supernatant of donor-type DC cultures. View larger version (36K): [in this window] [in a new window]   Figure 10. IL-12 p70 and IFN- detection by ELISA. Ly5.2+CD8+Ia+ DCs from Ly5.2+CD8+CD11cLin precursor-reconstituted Ly5.1 B6 mice (donor-derived CD8+ DCs) and host-type CD8+ DCs from Ly5.1 BM cell-reconstituted Ly5.1 B6 mice (host-type CD8+ DCs) were purified as described in "Materials and methods," respectively. (A) Cells (7.5 × 104) from each population were cultured in the presence of GM-CSF (20 ng/mL) + IFN- (20 ng/mL) + Pansorbin (50 µg/mL) in 200 µL medium for 40 hours. (B) Cells (3.3 × 104) from each population were stimulated in vitro with rmIL-12 (16 ng/mL) in 200 µL medium for 48 hours. Supernatants were assayed for IL-12 p70 and IFN- with ELISA, respectively. Results are expressed as the mean ± 1 SD of the triplicate cultures and are representative of 3 independent experiments. Chemokine and chemokine-receptor expression of donor-derived DCs Finally, the expression of chemokine and chemokine receptor of Ly5.2+CD8+Ia+ DCs from naive Ly5.2 B6 mice (Figure 11, lane 1) and Ly5.2+CD8+CD11cLin precursor-reconstituted Ly5.1 B6 mice (Figure 11, lane 2), as well as CD8+CD11cLin cells from Ly5.2 B6 mice (Figure 11, lane 3), was examined by using reverse transcription (RT)-PCR assay. As shown in Figure 11, Ly5.2+CD8+Ia+ DCs from the reconstituted mice expressed CCRs, such as CCR2, CCR5, CCR6, and CCR7, and a T-cell attracting chemokine, MDC. In contrast, CD8+CD11cLin cells expressed CCR2, CCR5, and CCR7, but barely CCR6. CD8+CD11cLin cells did not express MDC. View larger version (56K): [in this window] [in a new window]   Figure 11. Chemokine and chemokine receptor expression by RT-PCR. Ly5.2+CD8+Ia+ DCs from naive Ly5.2 B6 mice (lane 1), Ly5.2+CD8+Ia+ DCs from Ly5.2+CD8+CD11cLin precursor-reconstituted Ly5.1 B6 mice (lane 2), and CD8+CD11cLin cells from Ly5.2 B6 mice (lane 3) were purified as described in "Materials and methods." Total RNAs were extracted from 2 × 105 indicated cells by using RNAzolB. RT-PCR was assessed as described in "Materials and methods." The results are representative of 3 independent experiments.     Discussion Top Abstract Introduction Materials and methods Results Discussion References DCs in vivo are heterogeneous populations on the basis of their phenotype, function, and tissue distribution.1-13 In mouse lymphoid organs, CD8+ and CD8 DC subsets have been identified and termed "lymphoid DCs" and "myeloid DCs," respectively.1,3,14,15 In this study, we characterized a committed DC precursor, which was identified as CD8+CD11cLin phenotype in the mouse spleen. Moreover, the CD8+CD11cLin cells were also identified in BM and lymph nodes. These splenic CD8+CD11cLin cells could differentiate into immature and mature DCs expressing CD8 antigen. These CD8+ DCs derived from CD8+CD11cLin cells resided in the T-cell areas of the spleen. Functionally, these CD8+CD11cLin cell-derived DCs could stimulate allogenic T-cell proliferation and secrete IL-12 and INF-. Interestingly, CD8+CD11cLin cells failed to differentiate into other cell lineages, including T cells, NK cells, and myeloid cells. However, as shown in Figure 8, there were some scattered donor-derived B220+ cells. We currently could not rule out the possibility that CD8+CD11cLin cells might be able to generate some B220+ cells of unknown type after transplantation. All these data indicate that CD8+CD11cLin cells represent a committed precursor of CD8+ DCs in the mouse spleen. In addition, these CD8+CD11cLin cells could also home to thymus and lymph nodes and were capable of developing into CD8+ DCs in these locations. It is well established that CD8+ DCs are mainly found in the T-cell areas of PALS of the mouse spleen, whereas CD8 DCs are found in the marginal zones.5,13 DEC-205 is considered a marker of interdigitating cells (IDCs), and the expression of DEC-205 correlates with that of CD8.30,31 It has been further clarified that only IDCs are DEC205+ and CD8+, suggesting that IDCs are the most probable candidates for the CD8+ subset of DCs, leaving the marginal zone DCs as the CD8 subset.29 With the use of double-color immunohistochemistry staining, we found that CD8+CD11cLin cell-derived CD8+ DCs in the spleen mainly located in T-cell areas of PALS. These donor-derived DCs expressed CD8, CD11c, and DEC-205 markers, suggesting that donor-derived DCs represent the IDCs in the spleen. An important role for the chemokine and chemokine receptor system in DC migration and maintenance of the microanatomic environment of secondary lymphoid organs has been studied extensively. It has been reported that CCR2-deficient mice display restrictive DC defects in the localization of CD8+ Th1-inducing splenic DCs,32 whereas ligand interactions with CCR5 can trigger IL-12 production by the CD8+ subset of DCs after microbial stimulation.33 Epstein-Barr virus-induced molecule 1 ligand chemokine (ELC) and secondary lymphoid tissue chemokine (SLC) are specifically expressed in the T-cell areas where mature DCs home to become IDCs.34,35 Studies in plt mice36 and CCR7-deficient mice37 provide strong evidence for a determinant role of SLC and ELC/CCR7 axis in directing DC migration to T-cell areas of lymphoid tissues. It has been recently identified that DCs are a major source of MDC in vitro and in vivo.38 The up-regulation of T-cell-attracting chemokine MDC expression may enhance the encounters between DCs and antigen-specific T cells.39 By using RT-PCR, we observed that CD8+CD11cLin cell-derived CD8+ DCs expressed CCRs, such as CCR2, CCR5, CCR6, and CCR7, as well as T-cell-attracting chemokine MDC, which displayed the similar expression pattern as CD8+ DCs from naive B6 mice. Thus, on the basis of the immunohistochemistry study and the expression pattern of chemokine and chemokine receptor, CD8+CD11c Lin precursor-derived CD8+ DCs represent the splenic CD8+ DC subset. Accumulating evidence suggests that CD8+ and CD8 DCs play distinct roles in initiating immune responses. In vivo studies revealed that both CD8+ and CD8 subtypes of DCs could sensitize naive T lymphocytes and direct the development of distinct T-helper populations. Antigen-pulsed CD8 DCs induce a Th2-type response, whereas injection of CD8+ DCs leads to Th1 differentiation.6,7,40 CD8+ DCs have been shown to produce more IL-12 than CD8 DCs.6 It was elucidated that, after stimulation with IL-12, splenic CD8+ DCs produced more IFN- than CD8 DCs did.8 We observed that CD8+CD11cLin cell-derived CD8+ DCs were able to secrete similar levels of bioactive IL-12 p70 as well as INF- after stimulation with GM-CSF + IFN- + Pansorbin and IL-12 in vitro, respectively, as the counterparts from mice receiving autologous BM transplants. These data indicate that donor-derived CD8+ DCs developed from CD8+CD11cLin precursors display similar characteristics to those of splenic CD8+ DC subset from mice receiving autologous BM transplants functionally. Previous studies have demonstrated that CD8+ DCs can be generated from mouse CD4low lymphoid precursor as well as CD48344+25+ precursor, suggesting that CD8+ DCs are of lymphoid origin.14,15,17 However, recent studies have challenged the conception of lymphoid DCs. Martin et al16 have shown that thymic CD4low precursors can generate both CD8 and CD8+ DCs in the spleen of the irradiated recipient. More recently, it has been demonstrated that both CMPs and CLPs can differentiate into CD8+ and CD8 DC subsets in vivo.19,20 Other studies have also demonstrated that the generation of CD8+ DCs does not always correspond to the development of lymphoid cells in vivo.41-43 All these data suggest that CD8+ DCs can be generated from different hematopoietic progenitor cells in vivo. However, it remains unclear whether there is a common differentiation pathway for the generation of CD8+ DCs among these different hematopoietic progenitor cells. Several lines of evidence suggest that CD8+CD11cLin cells represent a committed DC precursor that can develop into CD8+ DCs. First, CD8+CD11cLin cells failed to differentiate into CD8 DCs in vivo. Second, CD8+CD11cLin cells failed to differentiate into CD8 T cells, NK cells, or myeloid cells in vivo. And third, it was reported that the CD8+CD11c/dull cells were increased in Flt3L-treated mice, accompanied by the significantly increased splenic CD8+ DCs.5,44 Although the differentiation capacity of CD8+CD11c/dull cells to CD8+ DCs had not been investigated in that study, a possible pathway for CD8+CD11c/dull cells to develop into CD8+ DC subset was proposed. Taken together, the above lines of evidence support that CD8+CD11cLin cells may be the immediate precursor that has committed to differentiate into CD8+ DCs in vivo. Further experiments will be focused on whether different hematopoietic progenitor cells, such as CMPs and CLPs, may differentiate into CD8+ DC subset in vivo via the immediate precursor termed as CD8+CD11cLin cells, which are undertaken in our laboratory. In summary, the data presented here first characterized a committed DC precursor, which was identified as CD8+CD11cLin cells in the spleen that could differentiate into CD8+ DCs but not CD8 DCs, T cells, NK cells, or other myeloid lineage cells in vivo. Identification of this committed CD8+ DC precursor will be useful for further understanding the ontogeny of CD8+ DCs from different hematopoietic progenitor cells at the cellular and molecular levels.     Acknowledgments We thank Dr H. Ishikawa (School of Medicine, Keio University, Tokyo, Japan) for his generous provision of C57BL/6 Ly5.1 mice and Dr Yi Zhang (Division of Hematology/Oncology, University of Pennsylvania School of Medicine, Philadelphia) and Dr K. Matsuno (Department of Anatomy I, Dokkyo University School of Medicine, Tochigi, Japan) for their critical reviews of the manuscript.     Footnotes Submitted July 17, 2001; accepted March 7, 2002. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Kouji Matsushima, Department of Molecular Preventive Medicine, School of Medicine, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; e-mail: koujim{at}m.u-tokyo.ac.jp.     References Top Abstract Introduction Materials and methods Results Discussion References 1. Bell D, Young JW, Banchereau J. Dendritic cells. Adv Immunol. 1999;72:255-324[Medline] [Order article via Infotrieve]. 2. Hart DN. Dendritic cells: unique leukocyte populations which control the primary immune response. Blood. 1997;90:3245-3287[Free Full Text]. 3. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature. 1998;392:245-252[CrossRef][Medline] [Order article via Infotrieve]. 4. Vremec D, Pooley J, Hochrein H, Wu L, Shortman K. CD4 and CD8 expression by dendritic cell subtypes in mouse thymus and spleen. J Immunol. 2000;164:2978-2986[Abstract/Free Full Text]. 5. Pulendran B, Lingappa J, Kennedy MK, et al. Developmental pathways of dendritic cells in vivo: distinct function, phenotype, and localization of dendritic cell subsets in FLT3 ligand-treated mice. J Immunol. 1997;159:2222-2231[Abstract/Free Full Text]. 6. Maldonado-López R, De Smedt T, Michel P, et al. CD8+ and CD8 subclasses of dendritic cells direct the development of distinct T helper cells in vivo. J Exp Med. 1999;189:587-592[Abstract/Free Full Text]. 7. Maldonado-López R, De Smedt T, Pajak B, et al. Role of CD8+ and CD8 dendritic cells in the induction of primary immune responses in vivo. J Leukoc Biol. 1999;66:242-246[Abstract]. 8. Ohteki T, Fukao T, Suzue K, et al. Interleukin 12-dependent interferon gamma production by CD8+ lymphoid dendritic cells. J Exp Med. 1999;189:1981-1986[Abstract/Free Full Text]. 9. Süss G, Shortman K. A subclass of dendritic cells kills CD4 T cells via Fas/Fas-ligand-induced apoptosis. J Exp Med. 1996;183:1789-1796[Abstract/Free Full Text]. 10. Kronin V, Winkel K, Süss G, et al. A subclass of dendritic cells regulates the response of naive CD8 T cells by limiting their IL-2 production. J Immunol. 1996;157:3819-3827[Abstract]. 11. den Haan JM, Lehar SM, Bevan MJ. CD8+ but not CD8 dendritic cells cross-prime cytotoxic T cells in vivo. J Exp Med. 2000;192:1685-1695[Abstract/Free Full Text]. 12. Pooley JL, Heath WR, Shortman K. Cutting edge: intravenous soluble antigen is presented to CD4 T cells by CD8 dendritic cells, but cross-presented to CD8 T cells by CD8+ dendritic cells. J Immunol. 2001;166:5327-5330[Abstract/Free Full Text]. 13. Moser M, Murphy KM. Dendritic cell regulation of TH1-TH2 development. Nat Immunol. 2000;1:199-205[CrossRef][Medline] [Order article via Infotrieve]. 14. Ardavin C, Wu L, Li CL, Shortman K. Thymic dendritic cells and T cells develop simultaneously in the thymus from a common precursor population. Nature. 1993;362:761-763[CrossRef][Medline] [Order article via Infotrieve]. 15. Wu L, Li CL, Shortman K. Thymic dendritic cell precursors: relationship to the T lymphocyte lineage and phenotype of the dendritic cell progeny. J Exp Med. 1996;184:903-911[Abstract/Free Full Text]. 16. Martin P, del Hoyo GM, Anjuere F, et al. Concept of lymphoid versus myeloid dendritic cell lineages revisited: both CD8 and CD8+ dendritic cells are generated from CD4low lymphoid-committed precursors. Blood. 2000;96:2511-2519[Abstract/Free Full Text]. 17. Saunders D, Lucas K, Ismaili J, et al. Dendritic cell development in culture from thymic precursor cells in the absence of granulocyte/macrophage colony-stimulating factor. J Exp Med. 1996;184:2185-2196[Abstract/Free Full Text]. 18. Galy A, Travis M, Cen D, Chen B. Human T, B, natural killer, and dendritic cells arise from a common bone marrow progenitor cell subset. Immunity. 1995;3:459-473[CrossRef][Medline] [Order article via Infotrieve]. 19. Traver D, Akashi K, Manz M, et al. Development of CD8-positive dendritic cells from a common myeloid progenitor. Science. 2000;290:2152-2154[Abstract/Free Full Text]. 20. Manz MG, Traver D, Miyamoto T, Weissman IL, Akashi K. Dendritic cell potentials of early lymphoid and myeloid progenitors. Blood. 2001;97:3333-3341[Abstract/Free Full Text]. 21. Wu L, Nichogiannopoulou A, Shortman K, Georgopoulos K. Cell-autonomous defects in dendritic cell populations of Ikaros mutant mice point to a developmental relationship with the lymphoid lineage. Immunity. 1997;7:483-492[CrossRef][Medline] [Order article via Infotrieve]. 22. Zhang Y, Mukaida N, Wang J, Harada A, Akiyama M, Matsushima K. Induction of dendritic cell differentiation by granulocyte-macrophage colony-stimulating factor, stem cell factor, and tumor necrosis factor alpha in vitro from lineage phenotypes-negative c-kit+ murine hematopoietic progenitor cells. Blood. 1997;90:4842-4853[Abstract/Free Full Text]. 23. Zhang Y, Harada A, Wang JB, et al. Bifurcated dendritic cell differentiation in vitro from murine lineage phenotype-negative c-kit+ bone marrow hematopoietic progenitor cells. Blood. 1998;92:118-128[Abstract/Free Full Text]. 24. Matsuno K, Ezaki T, Kudo S, Uehara Y. A life stage of particle-laden rat dendritic cells in vivo: their terminal division, active phagocytosis, and translocation from the liver to the draining lymph. J Exp Med. 1996;183:1865-1878[Abstract/Free Full Text]. 25. Vremec D, Zorbas M, Scollay R, et al. The surface phenotype of dendritic cells purified from mouse thymus and spleen: investigation of the CD8 expression by a subpopulation of dendritic cells. J Exp Med. 1992;176:47-58[Abstract/Free Full Text]. 26. Zhang YY, Zhang Y, Wang Y, et al. Development of dendritic cells in vitro from murine fetal liver-derived lineage phenotype-negative c-kit+ hematopoietic progenitor cells. Blood. 2000;95:138-146[Abstract/Free Full Text]. 27. Cohen PJ, Cohen PA, Rosenberg SA, Katz SI, Mulé JJ. Murine epidermal Langerhans cells and splenic dendritic cells present tumor-associated antigens to primed T cells. Eur J Immunol. 1994;24:315-319[Medline] [Order article via Infotrieve]. 28. Ogata M, Zhang Y, Wang Y, et al. Chemotactic response toward chemokines and its regulation by transforming growth factor-1 of murine bone marrow hematopoietic progenitor cell-derived different subset of dendritic cells. Blood. 1999;93:3225-3232[Abstract/Free Full Text]. 29. Leenen PJ, Radosevic K, Voerman JS, et al. Heterogeneity of mouse spleen dendritic cells: in vivo phagocytic activity, expression of macrophage markers, and subpopulation turnover. J Immunol. 1998;160:2166-2173[Abstract/Free Full Text]. 30. Kronin V, Vremec D, Winkel K, et al. Are CD8+ dendritic cells (DC) veto cells? The role of CD8 on DC in DC development and in the regulation of CD4 and CD8 T cell responses. Int Immunol. 1997;9:1061-1064[Abstract/Free Full Text]. 31. Vremec D, Shortman K. Dendritic cell subtypes in mouse lymphoid organs: cross-correlation of surface markers, changes with incubation, and differences among thymus, spleen, and lymph nodes. J Immunol. 1997;159:565-573[Abstract]. 32. Sato N, Ahuja SK, Quinones M, et al. CC chemokine receptor (CCR)2 is required for langerhans cell migration and localization of T helper cell type 1 (Th1)-inducing dendritic cells: absence of CCR2 shifts the Leishmania major-resistant phenotype to a susceptible state dominated by Th2 cytokines, B cell outgrowth, and sustained neutrophilic inflammation. J Exp Med. 2000;192:205-218[Abstract/Free Full Text]. 33. Aliberti J, Reis e Sousa C, Schito M, et al. CCR5 provides a signal for microbial induced production of IL-12 by CD8+ dendritic cells. Nat Immunol. 2000;1:83-87[CrossRef][Medline] [Order article via Infotrieve]. 34. Dieu MC, Vanbervliet B, Vicari A, et al. Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites. J Exp Med. 1998;188:373-386[Abstract/Free Full Text]. 35. Cyster JG. Chemokines and cell migration in secondary lymphoid organs. Science. 1999;286:2098-2102[Abstract/Free Full Text]. 36. Gunn MD, Kyuwa S, Tam C, et al. Mice lacking expression of secondary lymphoid organ chemokine have defects in lymphocyte homing and dendritic cell localization. J Exp Med. 1999;189:451-460[Abstract/Free Full Text]. 37. Förster R, Schubel A, Breitfeld D, et al. CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs. Cell. 1999;99:23-33[Medline] [Order article via Infotrieve]. 38. Vulcano M, Albanesi C, Stoppacciaro A, et al. Dendritic cells as a major source of macrophage-derived chemokine/CCL22 in vitro and in vivo. Eur J Immunol. 2001;31:812-822[CrossRef][Medline] [Order article via Infotrieve]. 39. Tang HL, Cyster JG. Chemokine up-regulation and activated T cell attraction by maturing dendritic cells. Science. 1999;284:819-822[Abstract/Free Full Text]. 40. Pulendran B, Smith JL, Caspary G, et al. Distinct dendritic cell subsets differentially regulate the class of immune response in vivo. Proc Natl Acad Sci U S A. 1999;96:1036-1041[Abstract/Free Full Text]. 41. Radtke F, Ferrero I, Wilson A, Lees R, Aguet M, MacDonald HR. Notch1 deficiency dissociates the intrathymic development of dendritic cells and T cells. J Exp Med. 2000;191:1085-1093[Abstract/Free Full Text]. 42. Rodewald HR, Brocker T, Haller C. Developmental dissociation of thymic dendritic cell and thymocyte lineages revealed in growth factor receptor mutant mice. Proc Natl Acad Sci U S A. 1999;96:15068-15073[Abstract/Free Full Text]. 43. Merad M, Fong L, Bogenberger J, Engleman EG. Differentiation of myeloid dendritic cells into CD8-positive dendritic cells in vivo. Blood. 2000;96:1865-1872[Abstract/Free Full Text]. 44. Maraskovsky E, Brasel K, Teepe M, et al. Dramatic increase in the numbers of functionally mature dendritic cells in Flt3 ligand-treated mice: multiple dendritic cell subpopulations identified. J Exp Med. 1996;184:1953-1962[Abstract/Free Full Text]. © 2002 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: F. Ishikawa, H. Niiro, T. Iino, S. Yoshida, N. Saito, S. Onohara, T. Miyamoto, H. Minagawa, S.-i. Fujii, L. D. Shultz, et al. The developmental program of human dendritic cells is operated independently of conventional myeloid and lymphoid pathways Blood, November 15, 2007; 110(10): 3591 - 3660. [Abstract] [Full Text] [PDF] H. Wang, N. Peters, V. Laza-Stanca, N. Nawroly, S. L. Johnston, and J. 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