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Departments of
* Pediatrics and
Microbiology and Center for Immunology, University of Minnesota, Minneapolis, MN 55455
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently it has become clear that at least two phenotypically distinct
types of dendritic cells exist within lymphoid tissues
(4, 5, 6, 7). Although both subsets express CD11c, one type
expresses the macrophage marker CD11b and does not express the
-chain of CD8 or DEC-205, whereas another type expresses the
-chain of CD8 and DEC-205 but lacks CD11b. In this study, we will
refer to the former cells as CD11b+ dendritic
cells and the latter as CD8+ dendritic cells.
Both subsets of dendritic cells are capable of presenting Ag and
activating T cells in vitro (8). In addition, several
recent studies that relied on in vitro stimulation of specific T cells
indicate that different dendritic cell subsets produce peptide-MHC
complexes in vivo after different regimens of Ag administration. For
example, CD8+ dendritic cells from mice injected
i.v. with soluble Ag or Ag-containing splenocytes stimulated specific T
cells more efficiently than CD11b+ dendritic
cells (9, 10). In contrast, CD11b+
dendritic cells were reported to be better T cell stimulators than
CD8+ dendritic cells after intratracheal
administration of Ag (11). Although these studies show
that dendritic cells produce peptide-MHC complexes in vivo, they do not
formally demonstrate that the dendritic cells actually presented these
complexes to specific T cells in situ. We addressed these issues by
identifying the in situ relationship between
CD11b+ and CD8+ dendritic
cells and adoptively transferred TCR-transgenic naive CD4 T cells in
lymph nodes after s.c. injection of the relevant Ag.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to 8-wk-old BALB/c and BALB/c SCID mice were obtained from
the National Cancer Institute, National Institutes of Health
(Frederick, Maryland). DO11.10 BALB/c TCR-transgenic mice expressing an
-TCR specific for aa 332–339 from chicken OVA presented by
I-Ad (12) were bred in our
facilities. DO11.10 BALB/c SCID were produced by backcrossing DO11.10
BALB/c mice to BALB/c SCID mice. DO11.10 BALB/c
Rag2-/- mice were the kind gift of Dr.
A. Khoruts (University of Minnesota, Minneapolis, MN). All DO11.10 mice
were housed in a specific pathogen-free facility. All experiments
involving the use of mice were performed in accordance with protocols
approved by the Institutional Animal Care and Use Committee of the
University of Minnesota.
Cell transfer
Single cell suspensions of spleen and lymph nodes were prepared from naive DO11.10 or DO11.10 SCID mice. A small sample was stained with biotin-labeled KJ1-26 mAb (an anti-clonotypic mAb that uniquely recognizes the DO11.10 TCR; Ref. 13), streptavidin-FITC (Caltag Laboratories, South San Francisco, CA), and PE-labeled anti-CD4 mAb (BD PharMingen, San Diego, CA), and analyzed on a FACScan flow cytometer (BD Biosciences, Mountain View, CA) using CellQuest software. A portion of the remaining unlabeled cell suspension containing 2.5–5.0 x 106 CD4+KJ1-26+ cells was then injected i.v. into each unirradiated BALB/c recipient. In some cases, lymph node cells from naive BALB/c mice were labeled with CFSE (Molecular Probes, Eugene, OR) using a modification (14) of a previously described technique (15), and then injected i.v. into unirradiated BALB/c recipients. Mice were injected s.c. on the back with chicken OVA (Sigma-Aldrich, St. Louis, MO), from which contaminating LPS was removed as described (16), or with FITC- or Texas Red (TR)3-labeled OVA (Molecular Probes, Eugene, OR).
IL-2 production by DO11.10 T cells
Single-cell suspensions were prepared in PBS containing 10 mM EDTA (to ensure the release of activated T cells) from the draining (brachial, axillary, and inguinal) and nondraining (mesenteric) lymph nodes of recipients of DO11.10 T cells at various times after s.c. injection of OVA. The cells were fixed in 2% formaldehyde, permeabilized with 0.5% saponin, as previously described (17), and stained (1–5 x 106/tube) with CyChrome-labeled anti-CD4 mAb RM4-5 (BD PharMingen), FITC-labeled KJ1-26 (Caltag Laboratories), and PE-labeled anti-IL-2 mAb S4B6 (BD PharMingen) or isotype control mAb R35-95 (BD PharMingen). CD4+KJ1-26+ events were collected on a FACScan (BD Biosciences) and analyzed using CellQuest software (BD Biosciences).
OVA uptake in vivo
Single-cell suspensions were prepared by treating the draining
lymph nodes of OVA-injected mice with 400 U/ml of collagenase D
(Boehringer Mannheim, Indianapolis, IN) and 10 mM EDTA to release
dendritic cells from the tissue. In some experiments, dendritic cells
were enriched using biotin-labeled anti-CD11c mAb and
streptavidin-coated microbeads or anti-CD11c mAb-coated microbeads
according to the manufacturer’s protocol (MACS; Miltenyi Biotec,
Auburn, CA). Cells were incubated on ice with anti-FcR mAb (2.4G2;
American Type Culture Collection, Rockville, MD) to block Fc binding
sites. In experiments where unlabeled OVA was injected, cells were
stained with PE-labeled anti-CD11c mAb, FITC-labeled
anti-CD8 mAb, allophycocyanin-labeled anti-CD11b mAb (in
some, but not all experiments), CyChrome-labeled anti-B220 mAb, and
CyChrome-labeled anti-CD3 mAb (all from BD PharMingen). In
experiments where FITC-labeled OVA was injected, cells were stained
with PE-labeled anti-CD11c mAb, allophycocyanin-labeled
anti-CD8 mAb, CyChrome-labeled anti-B220 mAb, and
CyChrome-labeled anti-CD3 mAb. In other experiments designed to
assess CD4 expression on the CD8- dendritic
cells that took up FITC-labeled OVA, cells were stained with
allophycocyanin-labeled anti-CD11c mAb, PE-labeled anti-CD4 mAb
or an isotype control mAb, and CyChrome-labeled anti-CD8 mAb.
CD11b+ dendritic cells were identified as cells
that expressed CD11c and CD11b, or cells that expressed CD11c and
lacked CD8. CD8+ dendritic cells were identified
as cells that expressed CD11c and CD8. Sorting was performed on a
FACSVantage flow cytometer (BD Biosciences).
Proliferation assay
Naive DO11.10 BALB/c Rag2-/- T cells (3 x 104) were placed in triplicate in 96-well U-bottom plates. Sorted CD11c+CD8+ or CD11c+CD8- dendritic cells were added to each well with or without exogenous OVA and with or without anti-MHC class II (MHC II) mAb (M5/114). Forty-eight or 72 h later, tritiated thymidine (1 µCi/well; NEN, Boston, MA) was added to each well. Cells were harvested 18–24 h later with a Wallac cell harvester (Turku, Finland). Tritiated thymidine incorporation into DNA was measured on a Wallac beta counter.
Immunofluorescent microscopy
Draining lymph nodes were harvested from mice sacrificed at various times after OVA injection. The lymph nodes were frozen in precooled isopentane and sectioned using a refrigerated microtome to a thickness of 10 µm. Sections were dehydrated in acetone, hydrated in PBS, incubated at room temperature with anti-FcR mAb (2.4G2; American Type Culture Collection), avidin and biotin solutions (Vector Laboratories, Burlingame, CA) and stained sequentially with biotin-labeled anti-CD11c mAb (N418; American Type Culture Collection) or biotin-labeled anti-DEC-205 mAb (NLDC145; American Type Culture Collection), or biotin-labeled anti-CD11b mAb, digoxigenin-labeled KJ1-26 mAb, streptavidin-labeled HRP (NEN), sheep anti-digoxigenin IgG (Jackson ImmunoResearch Laboratories, West Grove, PA), tyramide-labeled tetramethylrhodamine (NEN), and Cy5-labeled donkey anti-sheep IgG (Jackson ImmunoResearch Laboratories). Dendritic cells of the CD11b+ subset were identified as anti-CD11b-stained cells present in the paracortex, whereas dendritic cells of the CD8+ subset were identified as DEC-205-stained cells.
Confocal microscopy and image analysis was performed as previously described (18). Briefly, lymph node sections were analyzed using a Bio-Rad MRC-1000 confocal microscope equipped with a krypton/argon laser (Hercules, CA). Separate images were collected for each mAb in each area scanned and overlaid to produce a composite image containing all colors. The entire surface of each lymph node section was scanned to account for differences in cell distribution throughout the lymph node. Final image processing was performed using the Confocal Assistant program (University of Minnesota) and Adobe Photoshop (Mountain View, CA) as previously described (18). Interactions between CD11b+ dendritic cells, DEC-205+ dendritic cells, or TR-OVA-containing cells (colored red) and KJ1-26+ cells or CFSE-labeled polyclonal BALB/c cells (colored green) were quantified by measuring the overlap (yellow) between red and green. To account for differences in staining intensity on different sections, the number of yellow pixels was divided by the product of green and red pixels.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Before searching for physical evidence of Ag uptake, it was
necessary to establish the relevant time frame and location of T cell
activation. Naive DO11.10 T cells were transferred into BALB/c
recipients that were then injected s.c. with OVA. IL-2 production was
first detected in the DO11.10 T cells 6 h after OVA injection,
peaked at 12 h, and was primarily restricted to the draining lymph
nodes (Fig. 1
). These results demonstrate
that the initial presentation of the OVA occurs in the draining lymph
nodes within 24 h after s.c. OVA injection.
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), 4 h
after Ag injection. The only fluorochrome-containing cells that could
be detected by immunofluorescent microscopic examination of tissue
sections at this time were present in the subcapsular sinus (Fig. 3). These cells were probably macrophages
because macrophages are the major occupant of the subcapsular sinus
(19) and because macrophages contained the largest amount
of labeled OVA as revealed by flow cytometry (Fig. 2). No
fluorochrome-labeled OVA was detected by microscopy at 4 h in the
follicles or in the paracortex of the lymph node where naive T cells
reside (Fig. 3B). Thus, the level of fluorochrome detected
by flow cytometry in a subset of B cells, which should reside in the
follicles, and in all CD11b+ dendritic cells and
a subset of CD8+ dendritic cells, both of which
reside in the paracortex, was below the limit of detection by
microscopy at this early time after OVA injection.
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). However, a subset of
CD11b+ dendritic cells bearing very large amounts
of fluorochrome-labeled OVA appeared at this time (Fig. 2). Further
characterization revealed that these cells do not express CD4 (data not
shown) and were located within the paracortex, usually near the B
cell-rich follicles (Fig. 3, C and D). CD11b+ dendritic cells produce OVA peptide-MHC complexes
The capacity of dendritic cells purified from draining lymph nodes
to produce OVA peptide-MHC complexes in vivo was tested by assessing
their ability to directly stimulate DO11.10 T cells in vitro without
the addition of exogenous OVA. As shown in Fig. 4A, neither subset of
dendritic cells from the lymph nodes of mice injected with OVA 4 h
earlier stimulated DO11.10 T cells in vitro. Similar results were
obtained at 2 h (data not shown). In contrast, 18 h after OVA
injection, CD11b+, but not
CD8+, dendritic cells stimulated the DO11.10 T
cells, and the CD11b+ dendritic cells that took
up the largest amounts of OVA in vivo were the most effective
stimulators (Fig. 4A). Similar results were obtained at
12 h, the peak of IL-2 production (data not shown). Addition of an
anti-MHC II mAb completely blocked the Ag-driven proliferation of
the DO11.10 T cells (17,937 ± 1,280 cpm for cultures without mAb,
111 ± 39 for cultures with anti-MHC II mAb).
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B,
CD8+ dendritic cells stimulated DO11.10 T cells
at least as efficiently as the CD11b+ dendritic
cells proving that they are capable of producing OVA peptide-MHC
complexes from intact OVA. Thus, the failure of
CD8+ dendritic cells to produce peptide-MHC
complexes from OVA in vivo could not be explained by an inherent defect
in Ag processing. It is formally possible that Ag is sequestered and
not substantially processed by dendritic cells in vivo. To
address this, we aldehyde-fixed dendritic cells before in vitro
culture. This did not change the finding that the
CD8-, and not the CD8+,
dendritic cells stimulated DO11.10 T cells (25,437 ± 709 cpm for
cultures with CD8- dendritic cells, 679 ±
234 for cultures with CD8+ dendritic cells). CD11b+ dendritic cells undergo OVA-dependent interactions with specific CD4 T cells in situ
We next searched for physical interactions between dendritic cell
subsets and Ag-specific CD4 T cells during the time frame when OVA
peptide-MHC complexes and IL-2 were produced in the draining lymph
nodes. This was assessed by measuring changes in the proximity between
dendritic cells and DO11.10 T cells by two-color immunofluorescence
before and after OVA injection. Initial experiments were performed to
define the anatomic location of the dendritic cell subsets. Lymph node
tissue sections were stained with anti-CD11c mAb to detect all
dendritic cells and anti-CD11b or anti-DEC-205 mAbs to detect
the different dendritic cell subsets. Anti-DEC-205 Ab was used instead
of anti-CD8 Ab because of the confounding effect of the many
CD8+ T cells within the paracortex. As expected
(2, 18), anti-CD11c mAb stained dendritic cells
throughout the paracortex but minimally stained cells in the
subcapsular sinus (data not shown). Anti-CD11b mAb intensely stained
macrophages in the subcapsular sinus, and dendritic cells in the
peripheral region of the paracortex near the B cell-rich follicles
(parafollicular region) (Fig. 5, B and D). This is the area where
CD11b+ dendritic cells containing large amounts
of OVA appeared by 18 h after injection of fluorochrome-labeled
OVA (Fig. 3, C and D). In contrast,
CD8+ dendritic cells (identified by DEC-205
expression) were uniformly distributed throughout the paracortex (Fig. 5, A and C).
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A).
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, A and
B). Some interactions between DO11.10 T cells and dendritic
cells were detected in the absence of OVA, with a greater degree of
interaction with CD8+ dendritic cells observed
(Figs. 5A and 6B). The degree of interaction
between DO11.10 T cells and CD8+ dendritic cells
did not increase at any time after OVA injection, and actually
decreased below the starting level (Figs. 5C and
6B). In contrast, the degree of interaction between DO11.10
T cells and CD11b+ dendritic cells increased as
early as 6 h after OVA injection, with a maximum at 18 h
(Fig. 6). The increased degree of interaction was accompanied by
enrichment of the DO11.10 T cells in the CD11b+
dendritic cell-rich parafollicular region (compare Fig. 5, B
and D) where fluorochrome-containing
CD11b+ dendritic cells appeared in the experiment
shown in Fig. 3. However, it was possible that the
CD11b+ dendritic cells that interacted with
DO11.10 T cells (Figs. 5 and 6) and those that contained large amounts
of OVA (Fig. 3D) were not the same cells. To address this
issue, we measured in situ interactions between fluorochrome-containing
cells and DO11.10 T cells after injection of fluorochrome-labeled OVA.
As shown in Fig. 7, interactions between
DO11.10 T cells (shown green) and fluorochrome-containing cells (shown
red) were easily detected in the parafollicular region, whereas
interactions between polyclonal BALB/c T cells (shown purple) and
OVA-containing cells were rarely observed.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Four hours after s.c. injection, most CD11b+ dendritic cells and about one-third of the CD8+ dendritic cells contained a low level of fluorochrome-labeled OVA that could be detected by flow cytometry, but not immunofluorescent microscopy, in the T cell area. Thus, although it is formally possible that both dendritic cell types could have presented Ag to naive T cells in vivo, we could not document this because neither dendritic cell subset isolated from the draining lymph nodes at this time stimulated specific T cells ex vivo. In contrast, during the period of IL-2 production (6–18 h after Ag injection), CD11b+ dendritic cells containing large amounts of OVA appeared in the T cell areas and, when isolated from the draining lymph nodes, stimulated specific T cells ex vivo. Furthermore, the DO11.10 T cells redistributed to the CD11b+ dendritic cell-rich parafollicular region and interacted with CD11b+, but not CD8+, dendritic cells in vivo. These results are strong evidence that CD11b+, but not CD8+, dendritic cells present peptide-MHC II complexes to Ag-specific CD4 T cells in the draining lymph nodes after s.c. injection of soluble Ag.
In summary, our results together with those in the literature suggest several explanations for the finding that CD11b+ dendritic cells are the dominant APC for CD4 T cells in situations where a pathogen enters the body through the skin. These dendritic cells are situated in the outer T cell area and thus are well-positioned to take up bacterial debris that leaks through the subcapsular sinus where bacterial Ags would first appear (21, 22). Alternatively, the CD11b+ cells that we observed could be tissue monocytes or perhaps dermal dendritic cells that engulf pathogens in the skin and carry particulate Ag from the s.c. injection site to the lymph node, and in the process differentiate into CD11b+ dendritic cells as suggested by Randolph et al. (23). Finally, CD11b+ dendritic cells may be the most efficient producers of peptide-MHC II complexes from soluble Ag as suggested by Shortman and colleagues (24). In contrast, CD8+ dendritic cells may be more important as APC for cross-priming CD8 T cells because these dendritic cells are uniquely capable of producing peptide-MHC class I complexes from exogenous Ag (10, 24).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Elizabeth Ingulli, Department of Pediatrics and Center for Immunology, University of Minnesota, MMC 491, 420 Delaware Street SE, Minneapolis, MN 55455. E-mail address: ingul001{at}umn.edu
3 Abbreviations used in this paper: TR, Texas Red; MHC II, MHC class II.
Received for publication June 4, 2002. Accepted for publication June 18, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and nondraining lymph nodes (
) was
measured by flow cytometry at the indicated times after OVA injection.
The values shown are representative of four independent
experiments.
) or FITClow (
). B, Dendritic cells
were enriched from uninjected BALB/c mice and sorted for cells that
were CD11chigh, B220-, CD3-, and
either CD8- (
