Download Human Tonsil-derived Dendritic Cells are Poor

MAJOR ARTICLE
Human Tonsil-derived Dendritic Cells are Poor
Inducers of T cell Immunity to Mucosally
Encountered Pathogens
Claire M. Hallissey,1 Robert S. Heyderman,1,2 and Neil A. Williams1
1
School of Cellular and Molecular Medicine, University of Bristol, Bristol, United Kingdom and 2Malawi-Liverpool-Wellcome Trust Clinical Research
Programme, University of Malawi College of Medicine, Blantyre, Malawi
The mucosal immune system must initiate and regulate protective immunity, while balancing this immunity
with tolerance to harmless antigens and bacterial commensals. We have explored the hypothesis that mucosal
dendritic cells (DC) control the balance between regulation and immunity, by studying the responses of human
tonsil-derived DC to Neisseria meningitidis as a model organism. We show that tonsil DC are able to sample
their antigenic environment, internalizing Nm and expressing high levels of HLA-DR and CD86. However, in
comparison to monocyte-derived DC (moDC), they respond to pathogen encounter with only low level cytokine production, largely dominated by TGFβ. Functionally, tonsil DC also only stimulated low levels of
antigen-specific T cell proliferation and cytokine production when compared to moDC. We therefore propose
that the default role for DC in the nasopharynx is to maintain tolerance/ignorance of the large volume of harmless antigens and bacterial commensals encountered at the nasopharyngeal mucosa.
Keywords.
Dendritic cells; mucosal immunity; Neisseria meningitidis; tolerance; tonsils.
The nasopharyngeal mucosa is the site of colonization
for numerous capsulated bacteria, including Neisseria
meningitidis (Nm), which may invade to cause lifethreatening disease [1, 2]. Typically, potential pathogens are carried harmlessly at the mucosal surface for a
limited time and invasion does not occur or is contained by innate processes. However, under certain
conditions bacteria cross the nasopharyngeal mucosa
and cause systemic disease. The meningococcus, particularly serogroup B (MenB), represents a major health
challenge in Europe and North America. Although
there are MenB vaccines approaching licensure [3],
their ability to provide broad protection and induce
mucosal immunity is uncertain [4].
Received 22 August 2013; accepted 26 November 2013; electronically published
26 December 2013.
Some of this information has previously been presented at the 10th International
Symposium on Dendritic Cells, Kobe, Japan 2008.
Correspondence: Prof. Neil A. Williams, University of Bristol, School of Medical
Science, Cellular and Molecular Medicine, University Walk, Bristol, BS8 1TD, UK
([email protected]).
The Journal of Infectious Diseases 2014;209:1847–56
© The Author 2013. Published by Oxford University Press on behalf of the Infectious
Diseases Society of America. All rights reserved. For Permissions, please e-mail:
[email protected].
DOI: 10.1093/infdis/jit819
Dendritic cells (DC) are professional antigen presenting cells (APC) that internalise and process antigen
for presentation to, and activation of, T lymphocytes.
Various subtypes of DC have been identified and are
believed to play different, although frequently overlapping, roles in controlling immune responses [5]. The
type of T cell response initiated following pathogenic
encounter can be directed via DC surface expression of
various co-stimulatory/regulatory molecules, and secretion of immunomodulatory cytokines and chemokines
[6, 7].
Interestingly, Nm has been shown to stimulate Th1
responses by driving production of IL-12 in monocytederived DC (moDC) [8–10]. Although a useful tool in
cellular immunology, moDCdo not fully represent
tissue-derived DC. This is particularly true for mucosal
DC, which exist in an antigen-rich mucosal environment
and must balance induction of inflammatory Th1 responses against promotion of local antibody production
via a Th2 phenotype, and/or induce regulatory activity
to minimize immunopathological damage [11–13].
We have previously found that naturally-acquired
human mucosal immune responses to MenB emerge with
a Th1 bias, whereas peripheral blood T cell responses are
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more balanced [14]. Our studies of responses from human tonsillar T cells also suggested that the Th1 response was balanced
by the presence of meningococcal-specific regulatory T cells
(Treg). Regulation dominates in teenagers, but T helper responses prevail by adulthood [15]. In this study we examine the
role that nasopharyngeal DC play in either the eventual
skewing of the mucosal immune response towards a Th1 type
and/or the creation of meningococcal specific Treg. We isolated
DC directly from the human nasopharynx and investigated
their responses to pathogen-associated antigens.
from PBMC using anti-CD14 MicroBeads (Miltenyi), according
to manufacturer’s instructions. The monocytes were resuspended in complete RPMI 1640 (containing 100 U/mL penicillin,
0.1 mg/mL streptomycin, 4 mM L-glutamine (Invitrogen), and
10 mM HEPES (Sigma-Aldrich)) with 1000 U/mL rhGM-CSF
and 500 U/mL rhIL-4 (Peprotech EC, London, UK), and incubated in 24-well plates for 6 days at 37°C. On days 2 and 4, cells
were given fresh complete RPMI supplemented with rhGMCSF and rhIL-4.
Isolation of Mononuclear and Dendritic Cells From the Tonsil
METHODS
Ethics Statement
Palatine tonsils (PT) were obtained from consenting patients
undergoing routine tonsillectomy for recurrent tonsillitis or
airway obstruction at St Michael’s Hospital, Southmead Hospital or the Bristol Royal Hospital for Children, in Bristol, UK.
No patient was suffering from tonsillitis at the time of operation. Patients with immunodeficiency or serious infections
were excluded. The collection of tonsils and subsequent research complies with appropriate guidelines and institutional
practices, and the study was approved by University of Bristol
Hospital Trust Local Research Ethics Committee (reference
E4388). Informed consent was obtained from participants or,
for children, their legal guardians.
Bacteria and Other Antigens
Neisseria meningitidis strain H44/76 (B:15:P1.7,7,9) was used
after overnight growth on horse brain heart infusion (HBHI)
plates and fixation in 0.5% paraformaldehyde (PFA) in PBS.
FITC-labelled PFA-fixed bacteria were created by incubation
with 0.5 mg/mL FITC (Sigma Aldrich, Gillingham, UK) in PBS
(30 minutes, 37°C). Dialysed inactive trivalent split virion influenza vaccine (Fluarix 2002/2003) was a control antigen. E. coli
0111:B4 LPS (Sigma-Aldrich) and CpG-ODN (InvivoGen,
Tolouse, France) were positive controls for DC activation.
Antibodies for Flow Cytometry
Fluorochrome-conjugated mouse monoclonal antibodies
(mAb) were used in flow cytometry and for purification of
tDC: CD19-PE, CD3-PE, CD123-PE, CD11c-PE, CD11c-APC
(Miltenyi Biotec, Surrey, UK); CD14-PE, CD56-PE, CD16-PE
(BD Biosciences, Oxford, UK); CD19-FITC, HLA-DR-PECy5.5,
CD86-PE, CD80-PE, CD83-FITC (Invitrogen, Paisley, UK);
CD123-PECy7, (eBioscience, Hatfield, UK); CD86-Pacific Blue
(Cambridge Bioscience Ltd., Cambridge, UK).
Preparation of moDC
Peripheral blood mononuclear cells (PBMC) were isolated
from buffy coat (National Blood Service, UK) via separation over
Histopaque 1077 (Sigma-Aldrich). Monocytes were purified
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Tonsil mononuclear cells (TMNC) were isolated from PT using
a method adapted from Davenport et al [14]. In brief, tonsils
were cut into 2 mm3 pieces using a scalpel and dispersed
through industrial steel mesh (Potter and Son, Bristol, UK.).
After washing with PBS, MNC were recovered from the tonsil
cell suspension by separation on Histopaque 1077.
A DC-enriched TMNC fraction was isolated using a method
adapted from Ruedl et al [16]. Tonsil MNC were resuspended
in HBSS + 5 mM EDTA, then mixed with OptiPrep for a final
iodixanol solution of density ρ = 1.085 g/mL. The sample was
overlayed with 1.065 g/mL OptiPrep/RPMI solution, followed
by HBSS. Following centrifugation at 400 × g for 15 minutes
(20°C), DC-enriched cells were harvested from the top of the
1.065 g/mL iodixanol layer. Cells were then stained with PEconjugated mAb against CD3, CD19, CD14, CD16 and CD56.
For some experiments, antibodies against CD11c or CD123
were also added. PE-stained cells were depleted using anti-PE
magnetic MicroBeads (Miltenyi), according to the manufacturer’s instructions but with the addition of anti-CD19 beads to
enhance B cell removal. Labelled cells were removed from the
cell fraction using an LD column (Miltenyi).
T cell Proliferation Assays
TMNC or PBMC were enriched for T cells by using CD3 MicroBeads (Miltenyi), according to manufacturer’s instructions.
Labelled cells were recovered using an LS Column (Miltenyi).
T cell proliferative responses were monitored via incorporation
of 0.4 µCi [3H]thymidine (Amersham Pharmacia Biotech,
Little Chalfont, U.K.), as previously described [14]. Incorporation of tritiated thymidine was quantified using a Wallac MicroBeta TriLux liquid scintillation counter.
Cytokine Analysis
Levels of the cytokines IL-2, IL-4, IL-6, IL-10, IL-12p40, IL12p70, IL-13, IL-17, IFN-γ, TNF-α, and the chemokine MCP-1,
were measured using Milliplex Human Cytokine Multiplex
Immunoassay Kits (Millipore, UK) according to the manufacturer’s protocol. The assay plate was read on a Luminex 200
Total System (Luminex Corporation, Texas, USA), and data
evaluated using Luminex xPONENT software. Levels of TGF-β
in culture supernatant were measured using a Human TGF-b1
DuoSet ELISA Development System (R&D Systems Europe,
Ltd. Abingdon, UK) according to the manufacturer’s protocol.
The assay plate was read on an ELISA reader measuring absorbance at 450 nm, with reference 570 nm.
Statistical Analysis
Statistical analysis was carried out using GraphPad PRISM
6.03.
RESULTS
Uptake of FITC-labelled Bacteria
Dendritic cells in complete RPMI 1640 were incubated with
FITC-labelled killed N. meningitidis, at a ratio of 1:100. At specified time points, cells were recovered by vigorous washing with
refrigerated PBS, and analysed by flow cytometry or confocal
microscopy. For flow cytometry, association of meningococci
with DC was demonstrated using a method adapted from [17].
Cells were stained for expression of DC-associated surface
markers and fixed in PFA prior to analysis. In some experiments, trypan blue (0.5 mg/mL) was added to quench extracellular FITC-fluorescence. For confocal microscopy, DC were
incubated on Poly-L-lysine (Sigma-Aldrich) coated. Cells were
fixed in 4% PFA, blocked with 5% BSA in PBS, and stained
with non-conjugated anti-human HLA-DR mouse mAb (Invitrogen), followed by Alexa Fluor 594-conjugated goat anti-mouse
IgG (Invitrogen). Stained cells were mounted using a glass cover
slip and VECTASHIELD HardSet Mounting Medium with DAPI
(Vector Laboratories, Peterborough, UK), and imaged using a
Leica AOBS SP2 confocal imaging system.
Characterization of Tonsil DC
The DC population isolated from adult human tonsils was
characterized using their expression of key surface proteins previously identified as markers of DC-subtypes [5, 18, 19]. Purified cells did not have any PE fluorescence and so were negative
for cell markers used in the purification process, which included CD14 for macrophage removal. Expression of HLA-DR was
detected on 87%–98% of this enriched DC population, Approximately 85% of the HLA-DR+ population were CD123+
(Figure 1B) and designated as plasmacytoid DC ( pDC); the
minor CD11c+ population were designated as conventional
DC (cDC). Compared to pDC, cDC had higher surface expression of HLA-DR and activation markers including CD83 and
CD86 (Figure 1C).
Comparison of forward scatter-side scatter plots demonstrated
that tDC were smaller and less granular than moDC (Figure 1A).
Expression of CD11c was similar on cDC and moDC populations
(Figure 1B), but expression of the pDC marker CD123 was low or
Figure 1. Phenotypic analysis of tDC and moDC. A, Density plots show FSC/SSC profiles for the two cell types, with gates around the DC populations.
B–C, Density plots and histograms show expression of indicated surface markers, following staining with fluorochrome-conjugated monoclonal antibodies.
B, Data is shown for freshly isolated tDC and day 6 moDC. C, Data is shown for freshly isolated cells (dashed line) or following an overnight culture period
with no antigenic stimulation (solid line). Shaded histograms are for unstained, fresh, cells. In the pDC and cDC plots the unstained cells are the whole
tDC population, with no gating of CD11c+ or CD123+ populations. Data is representative of results from 3 similar experiments for each set of data, using
cells obtained from 3 different subjects for tDC and 3 additional subjects for moDC.
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absent on moDC. Both tDC subsets upregulated surface expression of markers including HLA-DR, CD83 and CD86 after 18
hours of in vitro culture without exogenous antigen (Figure 1C).
In contrast, freshly generated moDC had little change in surface
marker expression after an additional 18 hours of culture.
Tonsil DC Stimulation of Antigen-specific T cell Proliferation
We examined the ability of tDC to stimulate antigen-specific
T cell proliferation, by culturing both tDC and moDC with purified autologous T cells in the presence or absence of antigen.
Proliferation of cultured cells was measured by tritiated
Figure 2. Comparison of the ability of moDC and tDC to stimulate antigen-specific T cell proliferation. Proliferation of the indicated cell populations was
measured at various time points by pulsing with 3H thymidine for 18 hours. A, Collated data is shown for peak proliferation, measured over a 9 day culture,
for TMNC and PBMC in the presence of H44/76 or influenza vaccine (Flu). Lines link data points from individual experiments. B, Data is shown for proliferation of purified T cells in the presence of PFA-fixed H44/76 (1 × 108 cells/mL) (○) or influenza vaccine (90 ng hemagglutinin/mL) (▵) and for T cells in coculture with DC and H44/76 (•) or influenza (▴). Data is shown for 4 different subjects and isrepresentative of experiments with cells from 4 subjects for
blood T cells with moDC and 14 subjects for tonsil T cells with tDC. Results were similar regardless of the age of subjects. After optimization experiments
to determine the cell concentrations which had the greatest proliferation, blood T cells were used at 0.25 × 106 cells/mL, tonsil T cells were at 0.7 × 106
cells/mL. The DC:T Cell ratio was 1:25 for moDC/blood T cells and 1:70 for tDC/tonsil T cells. Values plotted are means of 3 triplicate wells with error bars
showing standard deviation across the wells. C, Collated data is shown for peak antigen-dependent proliferation, measured on days 3 and 5, for tDC or
moDC-stimulated T cells. Proliferation was measured for tonsil T cells in co-culture with tDC at a DC:T cell ratio of 1:50 (•) and 1:5 (○), and for blood
T cells in co-culture with moDC at a ratio of 1:50 (▪). Stimulatory antigens were as indicated on the x-axis. Horizontal bars represent the median value for
each data set Significance of proliferation was assessed by comparison of thymidine incorporation in the presence and absence of antigen. *P < .05,
**P < .01 (One way ANOVA with Dunnett’s multiple comparisons). D, Data is shown for day 3 and 5 proliferation of tonsil T cells in co-culture with CD123
or CD11c-depleted tDC. Black bars are control cultures, white bars show thymidine incorporation due to proliferation in the presence of H44/76. T cells
were at 5 × 105 cells/mL, CD123- tDC were ata ration of 1:10, and CD11c- tDC were at 1:5. Data is from individual experiments and is representative of
results with cells taken from 4 subjects. Results were similar regardless of the age of subjects. Values plotted are means of 3 triplicate wells with error
bars showing standard deviation across the wells. Data for all figures has been normalised by subtraction of the background level of thymidine incorporation by control cells cultured in medium alone, with no antigen. c.c.p.m – corrected counts per minute.
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thymidine incorporation over a 9 day time course (Figure 2).
As reported previously [14], whole TMNC and PBMC from
most donors proliferated in response to both killed H44/76
MenB and influenza (Figure 2A). In all subjects where proliferative responses to both antigens occurred in whole PBMC
cultures, moDC generated from the same donor stimulated
proliferation of autologous T cells in response to these antigens
(Figure 2B). At a similar DC:T cell ratio, tDC were poor stimulators of T cell proliferation, with responses being seen in only a
minority of subjects. In the majority of donors (9 of 14) there was
no T cell proliferation in response to either H44/76 or influenza.
Although there is increasing evidence that pDC can stimulate antigen-dependent activation and proliferation of T cells
[20, 21], this major DC subset have historically been characterized as poor stimulators of antigen-dependent T cell proliferation in comparison to cDC. We therefore investigated whether
tDC had the capacity to stimulate antigen-dependent T cell
proliferation when at a higher DC:T cell ratio than necessary
for moDC to stimulate proliferation. At a tDC/T cell ratio of
1:5, tDC stimulated T cell proliferation in response to influenza
comparable to moDC at a ratio of 1:50 (Figure 2C). The tDC at
this higher ratio also stimulated a significant level of antigendependent proliferation in response to H44/76.
Additional T cell proliferation assays were carried out using
tDC depleted of either CD11c+ or CD123+ cells. Although individual data were variable, the tDC population enriched for
pDC by removal of CD11c+ consistently stimulated less
antigen-dependent T cell proliferation when compared with
non-depleted tDC (Figure 2D). Similarly, removal of CD123+
DC consistently produced a cell population which stimulated
greater T cell proliferation than untouched tDC but was still
less stimulatory than moDC.
Cytokine Production in Tonsil DC/T cell Cultures
We used a Luminex assay to analyze cytokine production by
DC-stimulated T cells, in order to generate a picture of the
types of T cell response likely to be induced by tDC [22–24].
T cell cultures stimulated with H44/76 in the presence of moDC
produced high levels of the proinflammatory/Th1-associated
cytokines IFNγ and TNF-α (Figure 3A). In contrast, cultures of
T cells with tDC produced much lower levels of cytokines.
These responses were dominated by IL-17 and TGF-β, both of
which were produced at similar levels in cultures without
antigen stimulation. Figure 3B confirms that proliferation occurred in cultures from which supernatants were taken for cytokine analysis.
Figure 3. Analysis of the effects of H44/76 on cytokine secretion from T cells co-cultured with tDC and moDC. Purified T cells were cultured with DC
for 5 days, in the presence of H44/76 (1 × 108 cells/mL). Proliferation was measured on days 3 and 5 via the incorporation of 3H thymidine, and cytokine
levels were measured for supernatant samples taken on day 4. A, Data is shown for cytokine production by tonsil T cells in co-culture with tDC added at a
1:5 ratio, and for blood T cells in co-culture with moDC added at a 1:50 ratio. Black bars are control cells, white bars are cells in the presence of H44/76.
Values plotted are mean concentrations from duplicate wells in the assay plate, with samples taken from a single experimental well in the co-culture
plates. Error bars show standard deviation across the duplicates. B, Data is shown for proliferation of T cells within the same experiment as A. All data is
from individual experiments and is representative of results using cells from 4 subjects. Results were similar regardless of the age of subjects.
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Comparison of moDC and tDC Stimulation of T cell Proliferation
To address whether the poor tonsillar T cell responses were due
to poor DC function, or impairment in the innate capacity of
tonsillar T cells to respond to presented antigen, we collected
blood samples from patients prior to tonsillectomy. This
enabled us to investigate whether blood moDC were able to
stimulate antigen-dependent responses in autologous tonsil
T cells, and whether tDC were able to stimulate responses in autologous blood T cells. In the presence of meningococcal or influenza antigen, moDC stimulated similar levels of proliferation
in T cells from tonsils and blood, indicating no difference in
the capacity for responsiveness of T cell populations in the two
tissues (Figure 4). Similarly, tDC were poor stimulators of both
T cell responses (Figure 4).
Uptake of FITC-labelled Bacteria
To determine whether tDC have the ability to internalize
antigen, we compared uptake of FITC-labelled H44/76 by
moDC and tDC. Flow cytometric analysis of DC after a 20
hour culture with fluorescent H44/76 indicated that both tDC
and moDC had a similar level of association with bacteria
(Figure 5A). Two approaches were taken to verify that vesicular
uptake of the associated bacteria had occurred. Firstly, trypan
blue quenching was used to remove any fluorescence from externally attached bacteria [25]. The presence of a DC-associated
fluorescent signal from bacteria after quenching established
that internalization was taking place (Figure 5B). The levels of
fluorescence were similar between tDC and moDC, suggesting
a comparable endocytic capacity. The internalization of H44/76
was further confirmed by using confocal microscopy to visualize cells incubated with labelled bacteria (Figure 5C).
Staining with fluorochrome-conjugated anti-CD11c and
anti-CD123 antibodies was used to distinguish the cDC and
pDC populations respectively during flow cytometric analysis
(Figure 5D). Association with bacteria occurred more rapidly in
the cDC subset, but both tDC groups had similar levels of association at the end of a 20 hour culture period (Figure 5E).
Comparison of DC Responses to CpG DNA, Influenza and N.
meningitidis
In order to investigate whether the poor capacity for T cell
stimulation by tDC could be due to a failure to upregulate DC
surface molecules involved in the interaction with T cells, we
incubated tDC and moDC with various TLR agonists and used
flow cytometry to measure surface expression of DC activation
markers at different time points (Figure 6). After culture with
H44/76, moDC had upregulated expression of HLA-DR as well
as co-stimulation molecules including CD83 and CD86, but
they did not upregulate expression of surface markers in response to influenza or CpG DNA. Tonsil DC expressed higher
initial levels of HLA-DR and costimulatory markers than
moDC, and stimulation with influenza or H44/76 antigens
caused additional upregulation. In the case of influenza, upregulation of HLA-DR and CD86 was observed on cDC and
HLA-DR on pDC. Exposure to H44/76 increased expression of
CD86 on cDC. Predictably, CpG DNA primarily exerted effects
on pDC, which are known to be high expressors of TLR9.
Comparison of Cytokines Produced by tDC and moDC in
Response to Antigen
Figure 4. Comparison of the ability of moDC and tDC to stimulate
antigen-specific proliferation of tonsil or blood T cells. Purified tonsil
T cells (5 × 105 cells/mL) were cultured with autologous tDC (•) at a 1:5
ratio or moDC (▾) at a 1:50 ratio. Blood T cells were similarly cultured
with tDC (▴) or moDC (▪). Proliferation of T cells in the presence of influenza vaccine (open shapes) or PFA-fixed H44/76 (filled shapes) was measured at day 3 and 5 by pulsing with 3H thymidine for 18 hours. Influenza
vaccine was at 90 ng hemagglutinin/mL, H44/76 was at 1 × 108 cells/mL.
Data shown is the peak proliferation value for T cells in the presence of
the indicated antigens. Values plotted are the means of triplicate wells in
individual experiments, horizontal bars represent the median value for
each data set. Data has been normalised by the subtraction of control
levels of thymidine incorporation by T cells cultured in medium alone. Significance of proliferation was assessed by comparison of thymidine incorporation in the presence and absence of antigen. *P < .05 (One way
ANOVA with Dunnett’s multiple comparisons).
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We compared levels of immunomodulatory cytokines in tDC
and moDC cultures after overnight stimulation with antigen,
using Luminex and ELISA analysis (Figure 7). Monocytederived DC produced a range of cytokines (IL-6, IL-10, IL12p40, IL-12p70 and TNF-α) when cultured with H44/76.
They also produced MCP-1 and TGF-β, both in the presence
and absence of H44/76. Importantly, tDC produced undetectable or low levels of most cytokines examined. Supernatants
from tDC cultures contained only low levels of IL-6, TNF-α
and IL-10; and IL-12p40 and p70 were undetectable. Cultures
of tDC were dominated by constitutively high levels of TGF-β.
DISCUSSION
The functional interaction of upper respiratory tract mucosal DC
with colonizing bacteria has been largely unexplored. We have
Figure 5. Analysis of the uptake of FITC-labelled H44/76 by tDC and moDC. Purified tDC or moDC (5 × 105 cells/mL) were cultured for up to 48 hours
with FITC-labelled H44/76 (1 × 108 cells/mL). The level of association of DC with MenB was assessed by flow cytometry and confocal microscopy. A, Data
is shown for association of tDC and moDC with FITC-labelled bacteria after a 20 hour culture. Shaded histograms show the fluorescence of control cells.
Values shown are the percentage of FITC positive DC. B, Data is shown for FITC fluorescence of tDC before (solid line) and after (dashed line) quenching of
fluorescence due to external bacteria, via the addition of trypan blue (0.5 mg/mL). C, Images show the presence of internalized FITC-labelled H44/76
(green) within tDC and moDC. Cells were stained with a DAPI nuclear stain (blue), and an anti-surface HLA-DR antibody (red). D and E, Gating of the cDC
and pDC populations within the tDC was carried out using staining with fluorochrome conjugated monoclonal antibodies against surface CD11c and
CD123. D, Data is shown for association of CD11c+ and CD123+ tDC with FITC-labelled bacteria after a 20 hour culture. Shaded histograms show the fluorescence of control cells. E, Data is shown for CD11c+ (•) and CD123+ (▴) tDC over a 20 hour time course of incubation with FITC-labelled H44/76. Data
points are median values from 3 experiments, error bars show standard deviation between experiments. **indicates a significant difference in pDC and
cDC proliferation (P < .01, 2-way ANOVA with Sidak multiple comparisons). Data in A–D is from individual experiments and is representative of results with
cells from 3 subjects.
shown here that, in comparison to moDC, tDC are poorly responsive to a variety of antigens. In addition to the limited capacity of tDC to stimulate antigen-specific T cell proliferation,
cytokines released in co-cultures of tDC and tonsil T cells were at
lower levels than cytokines produced in moDC/blood T cell cultures and had a different profile. Whereas moDC/T cell responses
were dominated by high-level production of the Th1 cytokines
TNF-α and IFNγ, tDC/T cell responses were dominated by
TGFβ. Our failure to detect the Th2-associated cytokines IL-4
and IL-13 in tDC/T cell cultures supports previous work in the
group, which detected Th1-dominated memory T cell responses
in TMNC stimulated with meningococcal antigens [15]. Low
level cytokine production and the absence of inflammatory TNFα, along with high levels of constitutive TGFβ, in our tDC/T cell
cultures could indicate that tDC are involved in induction of the
Nm-specific Treg activity also identified in this previous study.
As DC undergo maturation, they lose their ability to take up
and respond to newly encountered antigen [26]. Our data
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Figure 6. Analysis of the effect of exposure to antigen on the surface marker expression of tDC and moDC. Purified tDC or moDC (5 × 105 cells/mL)
were incubated with influenza vaccine (90 ng hemagglutinin/mL), PFA-fixed H44/76 (1 × 108 cells/mL) or CpG DNA (6 µg/mL). Data is shown for the expression of indicated surface markers at 6 hours (black bars), 18 hours (white bars) and 42 hours (grey bars). Values plotted are the median fluorescence intensity following staining with fluorochrome-conjugated antibodies. Data is from a single experiment with individual donors for the tDC and moDC, and is
representative of results from 2–6 similar experiments using cells from different subjects (different total numbers of experiments were carried out for different antigens, time points and cell types). Results were similar regardless of the age of the subjects.
indicated that tDC were capable of internalizing antigen; but
also that they matured rapidly in culture following isolation,
even in the absence of antigen. Tissue DC isolated from other
sites have been shown to mature and upregulate surface marker
expression following isolation, yet retain the ability to stimulate
strong T cell responses [27, 28]. It is therefore unlikely that the
relative inability of tDC to stimulate T cells responses was due
to the isolation procedure.
Studies of DC from mucosal sites including the gut and
lungs have highlighted their role in maintaining immune homeostasis and minimizing inflammation, in addition to initiating protective immune responses. Mucosal DC in the gut
appear particularly adapted to a more immunoregulatory role,
and DC dysregulation is implicated in various diseases [29, 30].
Comparatively little information exists about the role mucosal
DC might play in protection against disease caused by invasive
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pathogens within the nasopharynx. In this study we have explored how tDC might shape the immune response following
encounters with potential pathogens, and help balance tolerance of nasopharyngeal commensals with establishment of
protective immunity to potential pathogens. Although it is
important to note that our studies use tonsils from patients undergoing tonsillectomy for either recurrent inflammation or
obstruction, material from patients with active inflammation at
the time of removal is excluded and over 10 years of using this
material for studies of cellular immunity we have not noted
consistent differences in levels of responses from these two
sources.
Compared with moDC, the most notable deficit in tDC
function was poor ability to produce cytokines that promote
antigen-dependent T cell responses. The tDC constitutively
secreted TGF-β at similar levels to moDC, but without the
Figure 7. Effect of H44/76 on chemokine and cytokine secretion from tDC and moDC. Purified DC (5 × 105 cells/mL) were cultured in the presence of
H44/76 (1 × 108 cells/mL), for 18 hours. Data is shown for cytokine production by control DC (black bars) and for DC in the presence of H44/76 (white bars).
Values plotted are mean concentrations from duplicate wells in the assay plate, with samples taken from a single experimental well. Error bars show standard deviation across duplicates. Data is from individual experiments and is representative of results using cells from 4 subjects for tDC and 3 subjects for
moDC. Results were similar regardless of the age of the subjects.
counteracting effect of the moDC’s inflammatory cytokine
production to stimulate a Th1 immune response to antigen.
The other cytokine secreted constitutively by tDC was IL-17,
which might enhance mucosal immunity by aiding with the
maintenance of mucosal barrier integrity [31]. The low level of
tDC-derived cytokines in our studies suggests characteristics of
‘semi-mature’ DC, a recently described DC subset that have
been associated with induction of Treg and T cell anergy.
The majority of antigen encountered in the nasopharynx is
in the form of harmless environmental antigens or commensal
bacteria. There would therefore be an evolutionary advantage
to a default state of local APC which fails to stimulate T cell reactivity, or even promotes the development of tolerance. In
keeping with this, we have shown that naturally acquired immunity to pneumococcal and meningococcal bacteria develops
slowly over a number of years, and Th1 and Th17 responses are
constrained by the induction of mucosa-associated Treg cells
[15, 32]. It seems likely that the non-responsive tDC phenotype
which we detected in this study might play a role in the development of these mucosal Treg. TGF-β plays a dual role in directing naive T cell differentiation; promoting the generation of
inducible Treg and, in concert with IL-6, driving differentiation
of Th17 cells [33]. In culture, the suppressive effects of tDCderived TGF-β may limit T cell proliferation; while in vivo its
presence may eventually lead to the generation of Treg cells and
a controlled level of Th17 responsiveness.
It seems likely that mechanisms might exist that enable tDC
to acquire a more responsive and stimulatory phenotype to initiate T cell immunity during pathogenic invasion [34]. This
would be in keeping with the phenotype of DC from mucosal
sites such as the lung and intestine, and could be dependent on
a change in function of the overall tDC population or activation
of specific tDC subsets [30, 35–37]. We have found that the
cDC subset of tDC are slightly more stimulatory than the pDC
subset. Given the appropriate environment and stimulation, it
could therefore be possible for at least a subset of tDC to initiate
protective immune responses. Alternatively, immunity may
depend on invasion of N. meningitidis across the nasopharyngeal epithelium and encounter with local lymph node DC. The
fact that Th1 responses can be stimulated in whole TMNC populations in vitro, suggests that once initiated, recall responses
can be supported in the local tissue, potentially as a result of
presentation by cognate memory B cells, which are abundant in
the tonsils of adults.
Knowledge of the mechanisms involved in switching the DC
role between maintenance of mucosal tolerance and initiation
of inflammatory immunity will be important for enhanced
understanding of host-pathogen interactions at the mucosa.
Tonsil DC have the capability to control the balance between the
establishment of commensal colonies and the development of
host immunity. This balance is particularly critical in immunity
to the meningococcus; a species encountered as a commensal
throughout life, which establishes a profile of responses that protects against pro-inflammatory damage following colonization.
These responses may subsequently prove to be inappropriate in
circumstances where the organism invades and establishes infection. The ability to modify normal DC responsiveness and
trigger protective mechanisms then becomes critical, providing
the potential for an alternative route towards development of a
broadly-reactive mucosally-active MenB vaccine.
Notes
Acknowledgments. We thank Mark Jepson and Alan Leard for their assistance with use of the Wolfson Bioimaging Facility Light Microscopy Unit
at the University of Bristol. We thank Mumtaz Virji and Natalie Griffiths
for advice and assistance with microbiology experiments.
Financial support. This work was supported by Medical Research
Council grant number G0600286.
Potential conflicts of interest. The authors report no conflicts of interest.
Tonsil-derived Dendritic Cells and Mucosal Immunity
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JID 2014:209 (1 June)
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All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the
content of the manuscript have been disclosed.
18.
19.
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