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CD11chigh Dendritic Cells Are Essential for
Activation of CD4 + T Cells and Generation
of Specific Antibodies following Mucosal
Immunization
Linda Fahlén-Yrlid, Tobias Gustafsson, Jessica Westlund,
Anna Holmberg, Anna Strömbeck, Margareta Blomquist,
Gordon G. MacPherson, Jan Holmgren and Ulf Yrlid
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2009 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2009; 183:5032-5041; Prepublished online 28
September 2009;
doi: 10.4049/jimmunol.0803992
http://www.jimmunol.org/content/183/8/5032
The Journal of Immunology
CD11chigh Dendritic Cells Are Essential for Activation of
CD4ⴙ T Cells and Generation of Specific Antibodies following
Mucosal Immunization1
Linda Fahlén-Yrlid,* Tobias Gustafsson,* Jessica Westlund,* Anna Holmberg,*
Anna Strömbeck,* Margareta Blomquist,* Gordon G. MacPherson,† Jan Holmgren,*
and Ulf Yrlid2*
M
ucosal immunization is often necessary to generate
immunity at mucosal surfaces (1). This protection requires generation of Ag-specific T cells and Abs. To
activate Ag-specific T cells, peptides must be displayed on MHC
molecules on the surface of activated APCs, particularly dendritic
cells (DCs)3. Indeed, the superior capacity of DCs to activate naive
T cells ex vivo following nasal and oral immunizations has been
determined in several studies (2–10). Additionally, the strong T
cell priming capacity of Ag-loaded DCs has not only been shown
after i.v. transfer but also after intratracheal injection (11). A transgenic system has been developed where diphtheria toxin (DTx)
administration conditionally ablates CD11chigh conventional DCs
*Department of Microbiology and Immunology, Institute of Biomedicine, The Mucosal Immunobiology and Vaccine Center (MIVAC) and University of Gothenburg
Vaccine Research Institute (GUVAX), The Sahlgrenska Academy at the University of
Gothenburg, Göteborg, Sweden; and †Sir William Dunn School of Pathology, Oxford,
United Kingdom
Received for publication December 1, 2008. Accepted for publication August 7, 2009.
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 study was supported by grants from the Swedish Research Council, Swedish
Foundation for Strategic Research through its support of the Mucosal Immunobiology
and Vaccine Center, Marianne and Marcus Wallenberg Foundation, Jeansson Foundation, Åke Wiberg Foundation, Clas Grochinsky Foundation, Magnus Bergvall
Foundation and the Royal Arts and Society of Arts and Science in Göteborg.
2
Address correspondence and reprint requests to Dr. Ulf Yrlid, Department of Microbiology and Immunology, Institute of Biomedicine, University of Gothenburg,
Box 435, 40530 Göteborg, Sweden. E-mail address: [email protected]
3
Abbreviations used in this paper: DC, dendritic cell; DTx, diphtheria toxin; cDC,
conventional dendritic cell; DTR, diphtheria toxin receptor; VSV, vesicular stomatatis
virus; LN, lymph node; pDC, plasmacytoid dendritic cell; CT, cholera toxin; Tg,
transgenic; BM, bone marrow; WT, wild type; 7AAD, 7-aminoactinomycin D; CTB,
cholera toxin subunit B; MLN, mesenteric LN; PP, Peyer’s patches.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803992
(cDCs) in vivo (12). Using these CD11c-DTx receptor (CD11cDTR) mice, the absolute requirement for cDCs to activate naive
CD8⫹ T cells following infections and parenteral immunizations
has been demonstrated in several reports (12–16).
Despite the requirement for cDCs in naive CD8⫹ T cell activation,
the role of cDCs in CD4⫹ T cell activation is not clear. For example,
although depletion of CD11chigh cells significantly reduces the expansion of adoptively transferred vesicular stomatatis virus (VSV)specific CD4⫹ T cells following i.v. infection (17), it does not affect
the VSV-driven generation of CD4⫹ T cell-derived cytokines (13).
CD4⫹ T cell activation is delayed but not abrogated in Mycobacterium tuberculosis-infected cDC-depleted mice (18). Additionally,
cDCs are not essential for achieving maximal clonal expansion of
CD4⫹ T cells following vaccinia virus infection (19). Finally, a recent
study has shown that CD4⫹ T cells in lymph nodes (LNs), but not
spleen, can be activated in vivo following parenteral administration of
protein, even in the absence of cDCs (16). This suggests that cells
other than cDCs, such as plasmacytoid DCs (pDCs), can prime CD4⫹
T cells in a tissue- or route-dependent fashion. Despite these data from
parenteral immunization systems, the types of APCs required for activation of CD4⫹ T cells in vivo following mucosal immunization
have not been investigated.
A role for DCs in B cell priming has been suggested by both in
vitro studies and investigations using adoptively transferred Agloaded DCs (20 –22). However, a recent study showed that
MHC-II molecules on B cells could be rapidly loaded with peptides despite cDC ablation, illustrating that DCs are not required
for loading Ag-specific B cells (23). Furthermore, other experiments using CD11c-DTR mice have shown that differentiation of
plasma cells in T-independent immune responses is independent of
cDCs (24). T-independent Ab responses to VSV infection do not
require CD11chigh cells, despite the fact that these cells are required for induction of Abs to purified VSV-G protein (17).
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To generate vaccines that protect mucosal surfaces, a better understanding of the cells required in vivo for activation of the
adaptive immune response following mucosal immunization is required. CD11chigh conventional dendritic cells (cDCs) have been
shown to be necessary for activation of naive CD8ⴙ T cells in vivo, but the role of cDCs in CD4ⴙ T cell activation is still unclear,
especially at mucosal surfaces. The activation of naive Ag-specific CD4ⴙ T cells and the generation of Abs following mucosal
administration of Ag with or without the potent mucosal adjuvant cholera toxin were therefore analyzed in mice depleted of
CD11chigh cDCs. Our results show that cDCs are absolutely required for activation of CD4ⴙ T cells after oral and nasal immunization. Ag-specific IgG titers in serum, as well as Ag-specific intestinal IgA, were completely abrogated after feeding mice OVA
and cholera toxin. However, giving a very high dose of Ag, 30-fold more than required to detect T cell proliferation, to cDC-ablated
mice resulted in proliferation of Ag-specific CD4ⴙ T cells. This proliferation was not inhibited by additional depletion of plasmacytoid DCs or in cDC-depleted mice whose B cells were MHC-II deficient. This study therefore demonstrates that cDCs are
required for successful mucosal immunization, unless a very high dose of Ag is administered. The Journal of Immunology, 2009,
183: 5032–5041.
The Journal of Immunology
Materials and Methods
Mice
CD11c-DTR transgenic (B6.FVB-Tg Itgax-DTR/GFP 57Lan/J) mice (12),
OT-II (C57BL/6) TCR transgenic mice harboring OVA-specific CD4⫹ T
cells (26), B cell-deficient mice (C57BL/10-IgH-6tm1Cgn) (␮Mt) (27), and
MHC-II-deficient mice (B6.129S2-H2dlAb1-Ea/J) (MHC-II-KO) (28) were
all bred and maintained under specific pathogen-free conditions at the Experimental Biomedicine Animal Facility, University of Gothenburg,
Göteborg, Sweden. CD11c-DTR Tg:␮Mt mice were obtained by intercrossing the respective lines, and CD45.1⫹CD45.2⫹OT-II mice were obtained from OT-II ⫻ C57BL/6-CD45.1 mating. All experiments were performed using protocols approved by the Swedish government’s Animal
Ethics Committee and followed institutional animal use and care
guidelines.
Bone marrow (BM) chimeras and DC depletions in vivo
Femurs and tibias were taken from donor mice and BM was flushed out.
Cells were passed through a 70-␮m nylon mesh and RBCs were lysed.
CD11c-DTR BM donor cells were used to generate CD11c-DTR3wildtype chimeras (CD11c-DTR/WT) (29). Alternatively, a mix of CD11cDTR Tg:␮Mt and MHC-II⫺/⫺ BM donor cells at a 4:1 ratio were used to
generate mixed CD11c-DTR/MHC-IIB⫺/⫺3 WT chimeras (CD11c-DTR/
MHC-IIB⫺/⫺) (30). C57BL/6 recipient mice were irradiated (1000 rad)
before 2–5 ⫻ 106 BM cells were transferred i.v. Mice were rested for 7 wk
before use. For cDC depletion, mice were injected i.p. with 4 ng/g body
weight DTx (Sigma-Aldrich) 3 days and 18 h before immunization. For
combined cDC and pDC depletion the mice were, in addition to the DTx
injections, injected i.p. with 0.5 mg of Ab 120.G8 (31) (Schering-Plough)
for 3 consecutive days before immunization. Chimerism and DC depletions
were confirmed at the time of immunization by flow cytometric analysis of
splenocytes.
Immunizations
For oral immunizations, food was removed 16 –18 h before the mice receiving OVA (Sigma-Aldrich) in 3% NaHCO3 with or without 10 ␮g of
CT (Sigma-Aldrich). For nasal i.p. and i.v. immunizations, mice received
LPS-low OVA containing 1.1 endotoxin unit/mg of protein. OVA was
given nasally with or without 1 ␮g of CT or 50 ␮g of CpG 1826 (Operon)
in a total volume of 20 ␮l and given i.p. with or without 2 ␮g of CT.
Flow cytometry
Single-cell suspensions were washed in FACS buffer (PBS containing 2%
FCS and 1 mM EDTA), which was used for all stainings. Cells were
stained for 15 min at 4°C with the following Abs conjugated to FITC, PE,
allophycocyanin, biotin, Pacific Blue, Alexa Fluor 700, or allophycocyanin-Alexa Fluor 780: anti-CD3 (clone 17A2), anti-CD4 (clone L3T4), antiCD11b (clone M1/70), anti-CD11c (clones HL3 and N418), anti-CD19
(clone 1D3), anti-CD45.1 (A20), anti-CD69 (H1.2F3), anti-V␣2 (clone
B20.1), anti-MHC-II (clone M5/114), anti-Ly6C (clone AL-21), anti-Ly6G
(clone 1A8), and anti-PDCA1 (clone JF05-1C2.4.1). All mAbs except antiCD11c (clone N418) (BioLegend), anti-MHC-II (eBioscience), and mPDCA-1 (Miltenyi Biotec) were purchased from BD Pharmingen. SAQdot605 (Invitrogen) was used as secondary reagent to detect biotinylated
mAbs. 7-Aminoactinomycin D (7AAD; Sigma-Aldrich) was included in all
stainings to define nonviable cells. Cells were acquired on a FACSCalibur
or LSR II (both BD Bioscience) and analyzed using FlowJo software (Tree
Star).
Histochemistry
For histological studies of nasal-associated lymphoid tissue, the head was
stripped of facial skin and severed along the line between the upper and
lower jaw. Heads were then fixed in 4% paraformaldehyde, decalcified,
and embedded in Tissue-Tek OCT compound. All other tissues were collected in Histocon (Histolab), embedded in Tissue-Tek OCT compound,
and snap-frozen. Sections (7 ␮m) were cut on a cryostat (Leica), picked up
on microslides, and stored at ⫺70°C until used. All slides, except those for
nasal-associated lymphoid tissue studies, were fixed on ice in 50% acetone
for 30 s and then in 100% acetone for 5 min before staining. The sections
were blocked with normal horse serum in PBS for 20 min in a humidifying
chamber. For detection of DCs, sections were incubated with biotinylated
anti-CD11c for 30 – 60 min followed by Alexa Fluor 488-conjugated
streptavidin (Invitrogen) for 30 – 60 min. Sections were double stained with
PE-conjugated anti-B220 for visualization of B cells. The B220 signal was
further enhanced with Cy3-conjugated anti-rat IgG (Jackson ImmunoResearch Laboratories). Sections were examined using a confocal laser
scanning microscope (Zeiss LSM 510 META). Images were further processed using an LSM Image Browser (Zeiss).
Adoptive transfer experiments
Spleens and LNs were taken from OT-II mice, and CD4⫹ T cells were
negatively enriched by magnetic separation using an autoMACS (Miltenyi
Biotec) and CD4⫹ T cell isolation kit (Miltenyi Biotec). Cells were then
labeled with CFSE (Invitrogen) by resuspending them at 1 ⫻ 107/ml in 5
␮M CFSE in serum-free PBS for 5 min at 37°C. The reaction was
quenched with an equal volume of FCS. Cells were washed, resuspended
in PBS, and 3–5 ⫻ 106 were injected i.v. in to mice that had, 70 and 2 h
earlier, either been given DTx or not. Eighteen hours later the mice were
immunized with OVA with or without CT. Three or 5 days later, mice
were sacrificed and then single-cell suspensions from lymphoid tissues
were stained and analyzed by flow cytometry to determine the frequency of
adoptively transferred cells that had entered division.
Measurements of Ag-specific Ab titers in serum and tissues
To determine OVA-specific Ab titers, 96-well plates (Greiner Bioscience)
were coated with OVA (20 ␮g/ml) and then blocked with PBS/BSA. To
determine CT subunit B (CTB)-specific Ab titers, 96-well plates (Nunc)
were coated with GM-1 (0.3 nmol/ml) and, after blocking with PBS/BSA,
CTB (0.5 ␮g/ml) was added for 60 min. Serially diluted serum samples or
perfusion-extraction samples (32) from intestines or lungs were added to
OVA- and CTB-coated plates and then incubated for 90 min. After washing, goat anti-mouse IgA-HRP (SouthernBiotech) or goat anti-mouse IgGHRP (Jackson ImmunoResearch Laboratories) was added and developed
with o-phenylenediamine dihydrochloride before absorbance determination at 450 nm. IgG or IgA titers were defined as the sample dilution giving
an OD value of 0.4 above the background value.
In vitro proliferation assay
Splenocytes from CD11c-DTR mice given DTx i.p. 24, 48, or 72 h previously or PBS-treated controls were seeded 5 ⫻ 105 in 96-well plates.
Splenocytes were pulsed in six identical wells with titrated amounts of
OVA protein or peptide (323–339) for 2 h. Remaining proteins and peptides were washed away and 1 ⫻ 105 MACS-purified CD4⫹ OT-II T cells
were added to each well. DTx was added to half of the wells to give a final
concentration of 200 ng/ml. Cells were cocultured for 64 h, pulsed for 8 h
with [3H]thymidine, and then incorporation into cellular DNA was
measured.
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Ab responses to usually inert protein Ags are very poor unless
the Ag is coadministered with an adjuvant. Mucosal immunization,
particularly by the oral route, is hampered by the paucity of useful
adjuvants. Cholera toxin (CT) is a strong oral adjuvant that, despite
its toxicity to humans, is an important tool for gaining insight into
the mechanisms of oral immunization and the design of novel mucosal vaccines. To function as a mucosal adjuvant, CT binds ubiquitously expressed GM1 ganglioside receptors (25). CT also
causes activation of DCs after mucosal administration (2, 25).
However, the extent to which cDCs are required for CT to function
as a mucosal adjuvant in vivo has not been addressed.
Here we examine the activation of naive CD4⫹ Ag-specific T
cells and the induction of specific Abs following mucosal administration of OVA and CT in mice depleted of cDCs in vivo. Our
results show that cDCs are required for activation of CD4⫹ T cells
after oral and nasal administration of Ag, with or without CT.
Additionally, cDC depletion abrogates the induction of Ag-specific
intestinal IgA responses as well as Ag-specific serum IgG production following oral immunization. When a very high dose of Ag is
given, proliferation of CD4⫹ T cells is detected in cDC-ablated
mice. We demonstrate that this is not due to a compensatory role
of B cells, pDCs, or recruited CD11bhighCD11c⫺/low myeloid
cells. These results therefore show that cDCs are required for mucosally induced adaptive immune responses unless a very high
dose of Ag is administered.
5033
5034
CD11chigh DCs ARE ESSENTIAL FOR MUCOSAL IMMUNIZATION
Results
Efficient depletion of cDCs in mucosal and lymphoid tissues in
CD11c-DTR/WT following DTx administration
⫹
Our aim was to determine the role of cDCs in activating CD4 T
cells in vivo following mucosal Ag administration. We thus took
advantage of CD11c-DTR Tg mice, which allow conditional ablation of cDCs upon administration of DTx (12). However, injection of DTx leads to death of CD11c-DTR mice within a week (29)
in a process mediated by nonhematopoietic cells. The use of
CD11c-DTR3 WT BM chimeras avoids this problem, and chimeric mice can be given multiple injections of DTx without any
adverse effects (29). CD11c-DTR3 WT BM chimeras (CD11cDTR/WT) were generated and used throughout this study unless
otherwise stated.
To determine the chimerism of DCs, flow cytometric analysis
was performed. A small frequency of GFP-negative resident
CD11chigh splenocytes was present in chimeric CD11c-DTR/WT
mice (Fig. 1A, upper left plot). Importantly, an identical population
of CD11chighGFP⫺ DCs was observed in the spleen and mesenteric LN of (nonchimeric) CD11c-DTR mice (Fig. 1A, upper middle and right plots), demonstrating that this is background staining
or that the penetrance of GFP expression in cDCs of CD11c-DTR
mice is not 100%. Furthermore, irradiation experiments using congenic mice confirmed the lack of remaining cDCs of recipient
origin in the chimeras (data not shown). To ensure complete ablation of cDCs, the mice received two injections of DTx. This
treatment depleted 85–98% of donor GFP⫹ cDCs from spleen,
mucosal tissues, and draining lymphoid tissues (Fig. 1).
Histochemical analysis confirmed the depletion of CD11c-expressing cells and showed retention of lymphoid follicles with no
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FIGURE 1. Efficient ablation of cDCs in CD11c-DTR and CD11cDTR/WT following DTx administration. A, Flow cytometric analysis of
GFP vs CD11c expression by 7AAD⫺CD3⫺CD19⫺ splenocytes or cells
from MLN of CD11c-DTR/WT and CD11c-DTR mice treated with or
without DTx. B, Flow cytometric analysis of GFP vs CD11c expression by
7AAD⫺CD3⫺CD19⫺ cells from cervical LN (CLN), nasal-associated lymphoid tissue, PP, or intestinal lamina propria (LP) of CD11c-DTR/WT
mice treated with or without DTx.
FIGURE 2. Ablation of cDCs in CD11c-DTR/WT retains lymphoid follicles and does not effect baseline recruitment, retention, or activation of
adoptively transferred CD4⫹ T cells. A, Histological analysis of tissue
sections from mice given DTx (⫹DTx) or not (⫺DTx) stained with antiCD11c (green) and B220 (red) (⫻20 magnification). B and C, Flow cytometric analysis of CD45.1 and CD4 for detection of adoptively transferred
OT-II cells in CLN cells from mice given DTx (⫹DTx) or not (⫺DTx).
OT-II cells were transferred at the same time as the second DTx treatment
and the tissues were taken 18 (B) or 90 h (C) later. B and C, Left panel, The
number in the dot plot indicates the frequency of gated cells among total
viable cells. C, Right panels, Expression of CD45.1 and CD69 by CD4⫹
cells gated as indicated by the arrow. The numbers in the dot plot indicate
the frequency of CD69 expressing cells among the transferred
CD45.1⫹CD4⫹ OT-II T cells.
The Journal of Immunology
5035
cDCs are required for efficient generation of specific Abs
following mucosal administration of Ag and CT
FIGURE 3. cDCs are required for activation of CD4⫹ T cells following
OVA feeding. Flow cytometric analysis of 7AAD⫺V␣2⫹CD4⫹ cells from
PP (A) or MLN (B) of CD11c-DTR/WT mice adoptively transferred with
CFSE-labeled OT-II cells treated with DTx or not and fed PBS (control) or
OVA as indicated. Cells were analyzed 72 h after OVA feeding. The histograms represent 7AAD⫺V␣2⫹CD4⫹ cells gated as shown. The number
in the histograms indicates the frequency of transferred T cells that have
entered division taking into account the expansion of divided cells (undivided cells are indicated with a gate in the histogram). Plots are of three
pooled mice and are representative of at least three experiments. The statistical analysis (C and D) was performed using Student’s t test where ⴱ,
p ⬍ 0.05 and ⴱⴱ, p ⬍ 0.01. The bars show the SEM.
gross abnormalities in lymphoid tissue structure following DTx
treatment (Fig. 2A). Additionally, adoptive transfer of OVA-specific OT-II CD4⫹ T cells resulted in comparable cell recruitment
and retention in DTx-treated recipients and control mice with no
aberrant cell activation (Fig. 2, B and C). A reduction in lymphoid
tissue cellularity (10 –20%) was sometimes observed following
DTx treatment, but this did not change the frequency of total
CD4⫹ T cells (Fig. 2C and data not shown). Thus, the DTx depletion of cDCs was very effective but not absolute in DTx-treated
chimeras. It also had no detectable effect on baseline recruitment,
retention, or activation of adoptively transferred CD4⫹ T cells.
cDCs are essential for priming naive CD4⫹ T cells following
mucosal immunization
CFSE-labeled OT-II T cells were adoptively transferred into
CD11c-DTR/WT mice 48 h after receiving DTx or PBS. DTxtreated mice received a second injection of DTx shortly after the
transfer. Eighteen hours later both groups of mice were fed different doses of OVA, and cells from Peyer’s patches (PP) and
To investigate the role of cDCs in generating Ag-specific Abs after
oral and nasal immunization, CD11c-DTR/WT mice were fed
OVA and CT. Before feeding, half of the mice were depleted of
cDCs, as described above, and Ag-specific Ab titers were measured. The IgG titers in serum (10 –12 days postadministration)
and IgA in intestinal tissues (3 wk postadministration) were determined by ELISA. Anti-OVA and anti-CTB IgG titers in serum,
following oral (Fig. 5, A and B) and nasal immunization (Fig. 5, D
and E), were abrogated in CD11c-DTR/WT given DTx compared
with controls. Although no significant intestinal anti-OVA IgA
could be detected regardless of the dose of OVA given orally and
irrespective of cDC-depletion (data not shown), anti-CTB IgA was
clearly detected and completely lost in CD11c-DTR/WT mice
given DTx (Fig. 5C). These results show that cDCs are required
for the generation of OVA- and CTB-specific serum IgG after
mucosal administration, and of CTB-specific intestinal IgA after
feeding mice OVA with CT.
Immunization of cDC-depleted mice with very high doses of Ag
results in proliferation of transferred CD4⫹ T cells
A very high dose of protein Ag is sometimes required to initiate
immune responses at mucosal surfaces. The abundance of Ag has
also been suggested to affect activation of the adaptive immune
response, possibly because additional APC populations access the
Ag in peripheral or lymphoid tissues (17). To test this, we mucosally immunized cDC-depleted mice with a very high dose of
OVA, 30-fold more than that required to detect proliferation of
Ag-specific CD4⫹ T cells in WT mice after mucosal immunization
(Fig. 6, A and B). OVA administration at 300 mg orally or 3 mg
nasally in the presence of CT resulted in extensive proliferation
of adoptively transferred OT-II T cells in control mice not given
DTx. However, treatment with DTx had no significant effect on
the frequency of cells that entered division (Fig. 6, A, B, D, and
E). To determine whether this was due to the route of immunization, a very high dose of OVA (0.3 mg) was injected i.v.
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MLNs were analyzed for proliferation of the adoptively transferred
T cells 3 days later (Fig. 3, A and B). Little, if any, OT-II T cell
expansion was detected in either organ of mice receiving DTx
before feeding OVA, while significantly more proliferation was
readily observed in mice not receiving DTx (Fig. 3, C and D).
To determine the impact of a mucosal adjuvant on the expansion
of transferred T cells, CT was given together with OVA. Again, a
significant reduction in the proliferation of labeled CD4⫹ T cells
was found in the draining LNs after oral (Fig. 4, A and C) or nasal
(Fig. 4, B and D) administration to DTx-treated mice compared
with controls not receiving DTx treatment. Expression of CD69 by
the transferred OT-II T cells was detected among undivided OT-II
cells in both DTx-treated mice and untreated controls following
oral immunization with OVA and CT (Fig. 4E). In the absence of
DTx treatment, CD69 expression was down-regulated upon cell
division. The same CD69 expression pattern was detected on
OT-II T cells from cervical LN 5 days after nasal immunization
with OVA (Fig. 4F). Restimulation of OT-II T cells from these
animals resulted in Ag-specific proliferation irrespective of DTx
treatment before immunization (data not shown). This suggests
that the observed lack of OT-II T cell division following
DTx treatment (Figs. 3 and 4) was not due to a delayed response
in the transferred cells and that these cells were not anergic to
subsequent stimulation. These results hence show that cDCs are
required for priming of CD4⫹ T cells following nasal and oral
administration of protein both in the absence and presence of CT.
5036
CD11chigh DCs ARE ESSENTIAL FOR MUCOSAL IMMUNIZATION
FIGURE 4. cDCs are essential for proliferation of CD4⫹ T cells following mucosal administration of OVA and CT. Flow cytometric analysis
of 7AAD⫺V␣2⫹CD4⫹ cells from (A and E) MLN or (B and F) CLN of
CD11c-DTR/WT mice adoptively transferred with CFSE-labeled OT-II
cells treated with or without DTx. Mice were then given OVA plus CT (A
and E) orally, (B) nasally, or (F) OVA nasally. Cells were analyzed (A, B,
and E) 72 h or (F) 96 h after OVA administration. A and B, Histograms
represent 7AAD⫺V␣2⫹CD4⫹ cells gated as shown. The number in the
histograms indicates the frequency of transferred T cells that have entered
division taking in to account the expansion of divided cells (undivided cells
are indicated with a gate in the histogram). E and F, Expression of CD69
and CFSE by CD45.1⫹CD4⫹ OT-II T cells. The numbers in the dot plot
indicate the frequency of CD69-expressing cells among the transferred
CD45.1⫹CD4⫹ OT-II T cells. Plots are of three pooled (A, E, and F) or
individual mice (B). Data are representative of three separate experiments.
Statistical analysis (C and D) was performed using Student’s t test where
ⴱⴱ, p ⬍ 0.01 and ⴱⴱⴱ, p ⬍ 0.001. The bars show the SEM.
(Fig. 6C) and titrated amounts of OVA were given i.p. (Fig. 6G)
and then the proliferation of the transferred cells in the spleen
was measured. As observed with mucosal immunization, increasing the dose of OVA to a very high dose resulted in similar
proliferation of the transferred cells in cDC-depleted mice and
in animals not treated with DTx (Fig. 6, F, G, and H). This is
in contrast to cDC-dependent proliferation where lower doses
of Ag are used (Fig. 6H). These results show that immunizing
mice depleted of cDCs with a very high dose of Ag results in
proliferation of CD4⫹ T cells.
CD11bhighCD11c⫺/low cells cannot compensate for cDC
depletion by activating CD4⫹ T cells
A possible explanation for CD4⫹ T cell activation in DTx-treated
chimeric mice given a very high dose of Ag is that other APCs
accumulate in tissues following DTx-induced cell death of DTR⫹
DCs. The cellular composition of lymphoid organs from CD11cDTR/WT mice treated twice with DTx was therefore analyzed.
After giving one or two injections of DTx, CD11bhighCD11c⫺/low
cells accumulated in lymphoid tissues (Fig. 7A). Multicolor flow
cytometry of the splenic CD11bhighCD11c⫺/low cells revealed a
decrease in frequency of Ly6Chigh6G⫺/low and an increase in
Ly6Chigh6Ghigh cells, representing monocytes and neutrophils, respectively (33) (Fig. 7B). The increase of Ly6Chigh6Ghigh cells was
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FIGURE 5. cDCs are essential for efficient generation of Ag-specific
Abs following mucosal administration of OVA and CT. The indicated
amounts of OVA and CT were administered (A–C) orally or (D and E)
nasally to CD11c-DTR/WT mice. The graphs show log10 titers of antiOVA- (A and D), anti-CTB- (B and E) specific serum IgG or anti-CTBspecific intestinal IgA (C) in CD11c-DTR/WT mice treated with DTx
(open bars) or not (filled bars). DTx was administered 72 and 18 h before
the immunizations. Serum samples were collected 10 –12 days postimmunization and intestines were collected 3 weeks postimmunization. The IgG
or IgA titer was defined as the sample dilution giving an OD value of 0.4
above the background value in ELISA. Unimmunized controls showed a
titer ⬍10 for IgA and IgG. Error bars show the SEM and statistical analysis
was performed using Student’s t test where ⴱ, p ⬍ 0.05, ⴱⴱ, p ⬍ 0.01, and
ⴱⴱⴱ, p ⬍ 0.001.
The Journal of Immunology
detected very rapidly and at all time points when cDCs were efficiently ablated. In contrast, no increase of these cells was observed in DTx-treated WT mice (data not shown). Recruitment of
neutrophils is thus an effect of the DTx-induced ablation in
CD11c-DTR/WT mice.
FIGURE 7. CD11bhighCD11c⫺/low cells with very poor CD4⫹ T cell
activation capacity accumulate in DTx-treated CD11c-DTR mice. A, Flow
cytometric analysis of CD11b vs CD11c expression by 7AAD⫺
CD3⫺CD19⫺ splenocytes or cells from MLN of CD11c-DTR/WT mice
treated with or without DTx. B, Flow cytometric analysis of Ly6C/Ly6G
expression by 7AAD⫺CD3⫺CD19⫺CD11bhighCD11c⫺/low splenocytes. C,
In vitro proliferation of OT-II cells cocultured with CD11c-DTR splenocytes pulsed for 2 h with titrated amounts of OVA protein (left) or peptide
(right) in the presence (F) or absence (E) of DTx. After 72 h the cocultures
were pulsed with [3H]thymidine and incorporation was measured after 6 h.
D, In vitro proliferation of OT-II cells cocultured with splenocytes from
CD11c-DTR mice given PBS (E) or DTx i.p. 24 (F), 48 (⽧), or 72 h (⽧)
earlier and subsequently pulsed for 2 h with titrated amounts of OVA
protein (left) or peptide (right). After 72 h the cocultures were pulsed with
[3H]thymidine and incorporation was measured after 6 h. One representative experiment out of two is shown.
The depleted splenic cDC population in DTx-treated mice is
restored 6 days following treatment, during which time the observed neutrophil accumulation occurs. Therefore, we wondered
whether cells (precursor or inflammatory cells) recruited to the
tissue during this period of cDC absence could contribute to the
activation of CD4⫹ T cells. To address this, depletion of
CD11chigh cells from the splenocytes was conducted in vitro (12)
so that no recruitment of cells was possible. Initial experiments
showed that 0.2 ␮g/ml DTx efficiently depleted CD11c⫹ cells
from splenocyte cultures (0.4% compared with 23% CD11c⫹ cells
among nonlymphocytes with or without DTx, respectively). There
was no increase in CD11bhighCD11c⫺/low cells (data not shown).
Addition of DTx, followed by a pulse with OVA peptide or
protein, abrogated the proliferative response of cocultured
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FIGURE 6. Priming of CD4⫹ T cells after mucosal and parenteral administration of a very high dose of OVA. Flow cytometric analysis of
7AAD⫺V␣2⫹CD4⫹ cells from (A) MLN, (B) cervical LN, or (C and G)
spleen of CD11c-DTR/WT mice adoptively transferred with CFSE-labeled
OT-II cells treated with or without DTx and given OVA plus CT orally (A),
OVA plus CT nasally (B), OVA i.v. (C), or OVA i.p (G). Histograms
represent 7AAD⫺V␣2⫹CD4⫹ cells, gated as shown. The number in the
histograms indicates the frequency of transferred T cells that have entered
division taking into account the expansion of divided cells (undivided cells
are indicated with a gate in the histogram). Plots are of (A) three pooled or
(B, C, and G) individual mice and the data are representative of three or
more separate experiments. Statistical analysis was performed (D–F) using
Student’s t test or (H) one-way-ANOVA where ⴱⴱ, p ⬍ 0.01 and n.s, not
significant. The bars show the SEM.
5037
5038
CD11chigh DCs ARE ESSENTIAL FOR MUCOSAL IMMUNIZATION
OT-II cells unless very high peptide or protein concentrations
were used (Fig. 7C).
Having shown that cDCs were required to induce proliferation
of CD4⫹ T cells in vitro, we next assessed whether the cells recruited to the spleen after DTx treatment could activate CD4⫹ T
cells. CD11c-DTR mice were therefore given DTx before sacrifice. Splenocytes from CD11c-DTR mice given DTx 72, 48, or
24 h earlier were pulsed with OVA protein or peptide and cultured
with OT-II cells. While proliferation was observed in PBS-treated
controls, little proliferation was detected when splenocytes from
CD11c-DTR mice given DTx in vivo were used (Fig. 7D). Taken
together, these results show that CD11chigh cells are efficiently
ablated by DTx treatment in lymphoid tissues of CD11c-DTR/WT
mice. Concomitantly, CD11bhighCD11c⫺/low neutrophils are recruited to tissues, but these cells have a very poor capacity to
present peptides to CD4⫹ T cells in vitro.
Having shown that the recruited CD11bhighCD11c⫺/low cells are
not capable of inducing proliferation of CD4⫹ T cells after Ag
exposure, we next addressed if other APCs could be responsible
for the observed activation of OVA-specific CD4⫹ T cells after
administration of a high dose of OVA to cDC-ablated mice. To
determine whether B cells were capable of priming T cells in the
absence of cDCs, we crossed the DTR Tg mice with B cell-deficient ␮MT mice, generating DTR Tg:␮Mt mice. Similar to ␮MT
mice, these mice display a multitude of architectural defects in the
spleen, including absence of follicular DCs, marginal zone macrophages, and metallophilic macrophages (34), as well as differences in DC function (35). We therefore made mixed BM chimeras
(CD11c-DTR/MHC-IIB⫺/⫺) in which the B cell compartment is
normal in number but MHC-II is deficient, as described by Crawford et al. (30). Seven weeks after engraftment, ⬃80% of splenic
cDCs (CD11c⫹B220⫺ cells) from CD11c-DTR/MHC-IIB⫺/⫺
mice were MHC-II⫹ and expressed DTR (i.e., were GFP⫹) (Fig.
8A). Importantly, the remaining DCs (GFP⫺) and all B cells were
MHC-II⫺. These mice were then adoptively transferred with
CFSE-labeled OT-II cells and 1 day later given OVA at a high
dose nasally or at titrated amounts of OVA i.p. Although expansion of the transferred T cells was cDC-dependent when lower
doses of Ag were administered i.p. (Fig. 8C, left panel), cDCs were
not essential for OT-II T cell division when a high dose of Ag was
given nasally or i.p. (Fig. 8, B and C, middle panel). Because the
DTx-treated mice had a MHC-II-deficient B cells, the observed T
cell proliferation must have been initiated by cells other than B
cells.
Activation of CD4⫹ T cells and induction of serum Ab
responses in mice depleted of both cDCs and pDCs
Our data so far show that neither B cells nor recruited myeloid
cells are responsible for CD4⫹ T cell proliferation in DTx-treated
mice given a very high dose of OVA. We thus determined if pDCs
contribute to this CD4⫹ T cell expansion by administering the
pDC-depleting Ab 120.G8 (31) to DTx-treated CD11c-DTR/WT
mice before administering OVA (Fig. 9). This combined treatment
resulted in a complete loss of cDCs (CD11chighmPDCA-1⫺) and
pDCs (CD11cintmPDCA-1⫹) in the spleen (Fig. 9A). As shown
above, cDC depletion had a relatively minor effect on the proliferation of transferred OT-II CD4⫹ T cells in CD11cDTR/WT mice given 3 mg of OVA nasally (Fig. 9B) or i.p.
(Fig. 9C). Depleting both pDCs and cDCs had little additional
FIGURE 8. Expansion of CD4⫹ T cells following administration of a
very high dose of OVA to mice with MHC-II-deficient B cells and ablated
cDCs. A, Flow cytometric analysis of MHC-II and GFP expression by
CD11c⫹B220⫺ cells (cDCs) and CD11c⫺B220⫹ cells (B cells) from
CD11c-DTR/MHC-IIB⫺/⫺ mixed BM chimeric mice at the time of immunization with OVA. B and C, Flow cytometric analysis of
7AAD⫺V␣2⫹CD4⫹ cells from cervical LN or spleen taken from CD11cDTR/WT mice adoptively transferred with CFSE-labeled OT-II cells
treated with or without DTx and given OVA or PBS nasally (B) or i.p. (C).
Histograms represent 7AAD⫺V␣2⫹CD4⫹ cells, gated as shown. The number in the histograms indicates the frequency of transferred T cells that
have entered division (determined from two separate experiments with a
total of four mice per group) taking in to account the expansion of divided
cells (undivided cells are indicated with a gate in the histogram).
effect, and significant expansion could still be observed in the
pDC/cDC-depleted mice.
We next measured the OVA-specific IgG response in CD11cDTR/WT mice primed with OVA plus CT nasally in the presence
or absence of DCs (cDCs or cDCs/pDCs) and boosted with OVA
i.p. with neither DTx nor 120.G8 treatment (Fig. 9D). This immunization regime led to a high anti-OVA IgG titer in the serum of
CD11c-DTR/WT mice that was significantly reduced by DTx
treatment. However, there was no significant effect of additional
pDC depletion. To confirm that the pDC depletion was functional,
depleted mice were immunized with OVA and CpG, as pDCs are
essential for CpG-induced activation of cDCs (36). Only the combined cDC and pDC ablation led to a significant reduction in serum
anti-OVA IgG titers upon OVA plus CpG immunization (Fig. 9D).
These results show that a high dose of OVA results in CD4⫹ T
cell proliferation even in mice depleted of both cDCs and pDCs.
Finally, depletion of pDCs in addition to cDCs at the time of
priming with OVA and CT via the nasal route did not further
reduce the anti-OVA Ab response compared with cDC depletion alone.
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Expansion of CD4⫹ T cells following administration of high
doses of Ag in mice with MHC-II-deficient B cells
and ablated cDCs
The Journal of Immunology
Discussion
To generate vaccines that protect mucosal surfaces, a better understanding of the cells required in vivo for activation of the adaptive immune response following mucosal immunization is required. To determine the role of DCs in this regard, we analyzed
the activation of CD4⫹ T cells following oral and nasal immunization of mice ablated of cDCs in vivo. Our results show that cDCs
are required for the activation of naive OVA-specific CD4⫹ T cells
in vivo and for the generation of mucosal and systemic Ag-specific
Abs after both oral and nasal administration of OVA and CT,
unless a very high dose of OVA is used.
Our results with high doses of Ag show that an APC population
present in, or recruited to, the tissue of DTx-treated CD11c-DTR
mice possesses the capacity to present peptides, but only when Ag
is abundant. DC depletion resulted in an immediate recruitment of
cells to lymphoid tissues, most of which express a high level of
CD11b, Ly6C, and Ly6G, but no, or very low level of, CD11c.
These cells are likely neutrophils, but the recruited population
could also contain other cells potentially capable of differentiating
into APCs (33). However, this appears unlikely, as splenocytes
from CD11c-DTR mice treated with DTx 3 days earlier (a time
frame that should allow for differentiation) could not activate
CD4⫹ T cells. This result is identical to that observed with splenocytes following DC depletion in vitro where no recruitment was
possible.
The appearance of neutrophils in tissues of CD11c-DTR mice
after DTx-induced ablation of DCs has not previously been reported, even though this Tg mouse has been used in several studies
(12–17, 19, 24, 29, 37–39). Although our results show that the
recruited cells do not have the capacity to efficiently present peptides on MHC-II ex vivo, these results emphasize the need for a
cautious approach when using DTx-treated CD11c-DTR mice to
study the role of inflammatory cells and secretion of soluble factors in the absence of DCs.
Furthermore, we could not detect a significant role for B cells in
the activation of T cells following immunization with a high dose
of Ag in the absence of cDCs. Our results therefore support a
previous report showing that CD4⫹ T cell activation in LNs could
still be observed in a B cell-deficient animal where cDCs had been
ablated (16). Importantly, our results also extend this observation,
as we used mice in which B cells are present but are MHC-II
deficient. Using this system, we ensured that the observed MHC-II
presentation was not due to changes in LN cellularity or altered
structure of peripheral lymphoid tissues, which is a caveat when
using B cell-deficient mice (34, 35). Our results could seem contradictory to a study using mice with MHC-II⫺/⫺ B cells, which
showed that B cells provide extra Ag presentation capacity above
that provided by DCs (30). This could be because different doses
of Ag were used in that study compared with the doses used by us,
and/or that Crawford et al. performed all immunizations in the
presence of alum (30). It remains possible, however, that B cells
may collaborate with cDCs during CD4⫹ T cell activation.
Our study shows that immunization of mice depleted of both
cDCs and pDCs with a very high dose of OVA still resulted in
activation of CD4⫹ T cells, comparable to that seen when only
cDCs were depleted. It has been reported that OVA-specific CD4⫹
T cells can be activated by pDC-specific Abs carrying OVA, and
also in LNs of cDC-depleted mice following s.c immunization
with OVA (16). Sapoznikov et al. (16) found no significant depletion of pDCs after DTx treatment, while we consistently observe a
partial depletion, which has also been reported by others (13, 17).
It is therefore possible that this partial depletion is sufficient to
deplete mucosal pDCs that have a capacity to activate CD4⫹ T
cells. However, only after combining DTx treatment with the
pDC-depleting Ab could we significantly inhibit the generation of
OVA-specific Abs in mice that had been given CpG and OVA
mucosally. This shows that pDCs remaining after DTx treatment
are indeed fully functional, since CpG-induced immune activation
is dependent on IL-15-mediated cross-talk between pDCs and
cDCs (36). Although Sapoznikov et al. (16) showed that specific
targeting of Ag to pDCs resulted in activation of CD4⫹ T cells,
they did not address whether removal of pDCs abrogated the observed activation. As emphasized by our study, the dose of Ag
administered will also have a significant impact. This makes comparison between different routes of immunization difficult unless
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FIGURE 9. Activation of CD4⫹ T cells and induction of serum Ab
responses in mice depleted of both cDCs and pDCs after administration of
a very high dose of Ag. A, Flow cytometric analysis of mPDCA-1 and
CD11c expression by 7AAD⫺CD3⫺CD19⫺ splenocytes from CD11cDTR/WT treated with PBS, DTx, or DTx plus 120.G8. The mAb 120.G8
was given for 3 consecutive days before immunization. The numbers indicate the frequency of cells among gated 7AAD⫺CD3⫺CD19⫺ cells. B,
Flow cytometric analysis of 7AAD⫺V␣2⫹CD4⫹ cells from the cervical
LN or spleen from CD11c-DTR/WT mice adoptively transferred with
CFSE-labeled OT-II cells treated with DTx, DTx plus 120.G8, or untreated
and then given OVA nasally (B) or i.p. (C). Analysis was performed 72 h
after OVA administration. Histograms represent 7AAD⫺V␣2⫹CD4⫹ cells,
gated as shown. The number in the histograms indicates the frequency of
transferred T cells that have entered division (determined from three separate experiments) taking into account the expansion of divided cells (undivided cells are indicated with a gate in the histogram). Plots are of individual mice and are representative of three separate experiments. D,
Log10 titers of anti-OVA-specific IgG in serum from CD11c-DTR/WT
mice treated with DTx, DTx plus 120.G8, or untreated before nasal immunization with OVA plus CT or OVA plus CpG. Ten to 14 days later
these mice and unimmunized controls (filled gray bars) received OVA i.p.
Serum was collected 1 wk later and analyzed for OVA-specific IgG titers
by ELISA. Titers were defined as the sample dilution giving an OD value
of 0.4 above the background value. Error bars show the SEM and statistical
analysis was performed using Student’s t test where ⴱ, p ⬍ 0.05 and n.s.,
not significant.
5039
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CD11chigh DCs ARE ESSENTIAL FOR MUCOSAL IMMUNIZATION
role of cDCs shown in this study suggests that targeting of the
vaccine to DCs would be a useful way to ensure successful mucosal vaccination.
Acknowledgments
We thank medical physicist Elin Haglund and the Department of Radiation
Physics, Sahlgrenska University Hospital, for excellent assistance in the
generation of chimeric mice.
Disclosures
The authors have no financial conflicts of interest.
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the Ag is titrated. Finally, it is also possible that pDCs in skin are
functionally different from those in the spleen and mucosal tissues.
For example, we have not been able to detect pDCs in afferent
lymph from mucosal tissues (40), while this has been reported in
lymph draining the skin (41).
CT holotoxin is the most potent oral adjuvant, but the mechanism behind its adjuvanticity is not fully known. Ab responses to
both OVA and CT following mucosal coadministration have been
shown to be completely dependent on CD4⫹ T cell help (42). The
effect of CT could therefore be to make DCs more potent activators
of CD4⫹ T cells. Alternatively, CT could act on B cells, rendering
them capable of activating naive T cells. Our results show that
coadministering CT and OVA mucosally did not overcome the
essential role of cDCs for activation of CD4⫹ T cells and induction
of intestinal IgA and serum IgG. Detection of anti-OVA IgA responses in mucosal tissues required that the mice be immunized a
second time with OVA and CT. However, ablation of cDCs before
both immunizations creates a problem, as plasma cells in the
spleen express CD11c and thereby become sensitive to DTx treatment (24). Indeed, in preliminary experiments we have found that
the number of CT- and OVA-specific IgA-secreting cells in the
intestine are reduced after DTx treatment (OVA, 120 ⫾ 50, CT,
3515 ⫾ 1889 per million cells (⫺DTx) compared with OVA, 18 ⫾
13, CT, 275 ⫾ 129 per million cells (⫹DTx)). In these experiments DTx was only given 1 wk after the second immunization
with OVA and CT, making it unlikely that the effect of DTx was
on DCs.
DTx treatment of CD11c-DTR mice has also been reported to
ablate marginal zone macrophages and their sinusoidal counterparts in LN when using the same dose of DT used by us in this
study (43). This makes it unlikely that these cells contribute to
activation of CD4⫹ T cells following immunization with a very
high dose of Ag. Additionally, removal of macrophages using
chlodronate-containing liposomes increases, rather than reduces,
the amount of Ab-secreting cells following immunization with a T
cell-dependent Ag (44). The mucosal epithelium has also been
suggested to present Ags on MHC-II (45). However, in preliminary experiments using chimeric mice, we have been unable to find
a role for nonhematopoietic cells (including epithelial cells) for
CD4⫹ T cell activation in the absence of cDCs (data not shown).
It is still possible that a hematopoietic cell type that we have
been unable to define could be responsible for the activation of
CD4⫹ T cells when a very high dose of Ag is administered. During
the revision of this manuscript it has been shown that basophils
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required (46). Another possibility is that, although only few cDCs
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anergized.
Our study therefore shows the crucial and irreplaceable role of
cDCs for nasal and oral immunizations unless a very high dose of
protein is used. Whether mucosally applied Ags are directly taken
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Ag after transport over the epithelium or following transport in
lymph (cell associated or not), or if all of these pathways operate
is not known. Additionally, if the same rules apply for Ag and
different adjuvants after mucosal coadministration also remain to
be addressed. Answering these questions will be crucial to improving the efficacy of mucosal vaccinations. However, the essential
The Journal of Immunology
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