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IMMUNOBIOLOGY
From the Department of Molecular Preventive Medicine
and CREST, School of Medicine, The University of Tokyo, Tokyo, Japan.
CD8 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 Accumulating evidence indicates that several committed hematopoietic
progenitor cells can differentiate into CD8 In the present report, we describe
CD8 Mice
Antibodies
Purification and transplantation of
CD8  +CD11c Lin 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 Immunofluorescence analysis Immunofluorescence analyses were performed as previously described.22,23 Cells were preincubated with 2.4G2 to prevent binding to Fc RII/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.
Isolation 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.
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.
To elucidate whether
CD8
To examine the potential of
CD8
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.
Donor-derived 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.
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.
DCs in vivo are heterogeneous populations on the basis of
their phenotype, function, and tissue distribution.1-13 In
mouse lymphoid organs, CD8 It is well established that CD8 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 Accumulating evidence suggests that CD8 Previous studies have demonstrated that CD8 Several lines of evidence suggest that
CD8 In summary, the data presented here first characterized a
committed DC precursor, which was identified as
CD8
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.
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.
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+CD11c
lineage
phenotype-negative cells in the spleen as committed precursor of
CD8
Top
Abstract
Introduction
represents
Ly5.2+CD8
represents Ly5.2+CD8
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.
