Spatiotemporally separated antigen uptake by alveolar dendritic cells and airway presentation to T cells in the lung.

Abstract

Asthma pathogenesis is focused around conducting airways. The reasons for this focus have been unclear because it has not been possible to track the sites and timing of antigen uptake or subsequent antigen presentation to effector T cells. In this study, we use two-photon microscopy of the lung parenchyma and note accumulation of CD11b(+) dendritic cells (DCs) around the airway after allergen challenge but very limited access of these airway-adjacent DCs to the contents of the airspace. In contrast, we observed prevalent transepithelial uptake of particulate antigens by alveolar DCs. These distinct sites are temporally linked, as early antigen uptake in alveoli gives rise to DC and antigen retention in the airway-adjacent region. Antigen-specific T cells also accumulate in the airway-adjacent region after allergen challenge and are activated by the accumulated DCs. Thus, we propose that later airway hyperreactivity results from selective retention of allergen-presenting DCs and antigen-specific T cells in airway-adjacent interaction zones, not from variation in the abilities of individual DCs to survey the lung.

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The tissue of the lung is a complex filigree,
supporting gas exchange and presenting a
large surface for antigen surveillance and up­
take. This interface provides a classical example
of the challenges of mucosal immunity; res­
ponses to environmental antigens need to be
minimized, whereas the exposure to patho­
gens requires rapid local responses. The domi­
nant symptoms of asthma, airway constriction
and mucus accumulation, are the results of im­
mune responses near airways. A rich body of
literature has identified lung DCs and alveolar
macrophages (AMs) as predominant phago­
cytic populations, both in the steady­state and
in disease (Robinson et al., 1992; McWilliam
et al., 1996; Belz et al., 2004; Holt, 2005;
van Rijt et al., 2005; von Garnier et al., 2005;
Sung et al., 2006; Grayson et al., 2007; Hammad
et al., 2010). Although it is clear by depletion
protocols that lung DCs play a major role in
airway pathogenesis (Lambrecht et al., 1998;
van Rijt et al., 2005), the spatial dynamics that
define how and where they sample material
and when and where they present antigen has
not been assessed.
DCs in the lung, in contrast to AMs, are
very effective at generating T cell responses
(Belz et al., 2004) and are also instrumental in
initiating and perpetuating T cell hyperrespon­
siveness associated with asthma (van Rijt et al.,
2005; Hammad et al., 2010). CD103+ and
CD11b+ subsets have been proposed to acti­
vate CD8 and CD4 responses, respectively
(Jakubzick et al., 2008b). CD103+ DCs have
been shown to play important roles in viral res­
ponses (Ballesteros­Tato et al., 2010) and apop­
totic cell uptake (Desch et al., 2011), but the
localization and uptake capacity of these cells
has not been addressed in the lung. Increased
numbers of DCs are found in bronchoalveolar
lavage (BAL) fluid of asthmatic patients after
allergen challenge (Robinson et al., 1992; van
Rijt et al., 2002), suggesting that their increase
is associated with disease. In mice, upon OVA
challenge in an OVA/alum mouse model of
CORRESPONDENCE
Matthew F. Krummel:
matthew.krummel@ucsf.edu
Abbreviations used: AM, alveo­
lar macrophage; BAL, bron­
choalveolar lavage; i.n.,
intranasal(ly); TLR, Toll­
like receptor.
Spatiotemporally separated antigen uptake
by alveolar dendritic cells and airway
presentation to T cells in the lung
Emily E. Thornton,1 Mark R. Looney,2,4 Oishee Bose,1 Debasish Sen,1
Dean Sheppard,3,4 Richard Locksley,5 Xiaozhu Huang,3,4
and Matthew F. Krummel1
1Department of Pathology, 2Department of Laboratory Medicine, 3Lung Biology Center, 4Department of Medicine,
and 5Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94143
Asthma pathogenesis is focused around conducting airways. The reasons for this focus have
been unclear because it has not been possible to track the sites and timing of antigen
uptake or subsequent antigen presentation to effector T cells. In this study, we use two-
photon microscopy of the lung parenchyma and note accumulation of CD11b+ dendritic
cells (DCs) around the airway after allergen challenge but very limited access of these
airway-adjacent DCs to the contents of the airspace. In contrast, we observed prevalent
transepithelial uptake of particulate antigens by alveolar DCs. These distinct sites are
temporally linked, as early antigen uptake in alveoli gives rise to DC and antigen retention
in the airway-adjacent region. Antigen-specific T cells also accumulate in the airway-
adjacent region after allergen challenge and are activated by the accumulated DCs. Thus,
we propose that later airway hyperreactivity results from selective retention of allergen-
presenting DCs and antigen-specific T cells in airway-adjacent interaction zones, not from
variation in the abilities of individual DCs to survey the lung.
© 2012 Thornton et al. This article is distributed under the terms of an Attribution–
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The Journal of Experimental Medicine

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Antigen uptake and presentation in the lung | Thornton et al.
Two­photon imaging has previously been used to track
the dynamic behavior of lymphocytes in LNs (Miller et al.,
2002) and provides the possibility to assess cells deep within
their native and complex tissues (Germain et al., 2006; Cahalan
and Parker, 2008). For LNs, both intravital as well as organ
explant models have been described, and the comparisons of
these two has provided insight into when each is applicable.
The use of two­photon microscopy for lung has previously
been demonstrated with viable lung slices for the study of
smooth muscle functions (Bergner and Sanderson, 2002), but
the behavior of the immune system during allergy in these
preparations has not been formally assessed. Recently, stabi­
lized and ventilated intravital lung imaging has also become
possible. Using this method, airflow and blood flow remain
intact, and alveolar imaging is possible (Kreisel et al., 2010;
Looney et al., 2011). Under such a preparation, tissue remains
demonstrably viable and without evidence of immune infil­
trate induced by the preparation (Looney et al., 2011).
In this study, we have applied two­photon live imaging
of viable lung slices and intravital stabilized lung to discover
the antigen surveillance and presentation dynamics in lung.
We tested the hypothesis that surveillance changes at the cel­
lular level after allergen challenge. Using CD11c­EYFP mice
(Lindquist et al., 2004), which, by two­photon imaging, mark
DCs and mark AMs only after allergen challenge, we were
able to differentially track the behavior, antigen uptake, and
cell–cell interactions of CD11b+ DCs in viable lung tissue. We
compared these activities on a per­cell basis, both in the steady­
state and in the widely studied OVA model of allergic lung
disease. Although other mucosa has demonstrated evidence of
changes in motile behavior of DCs during insult, neither acute
allergy induction nor treatment with bacterial LPS significantly
changed their surveillance behaviors within the lung tissue.
We also found that alveoli are unexpectedly the sites of the
most robust DC air surveillance in the steady­state with highly
dynamic dendrites that project across epithelial barriers. We
captured examples of DCs ingesting particles directly from an
alveolar atrium and found that DCs are extremely effective at
phagocytosis of particulate antigens from this location. Airway
DCs, which exhibit greater center of mass motility than paren­
chymal DCs, rarely send processes across the airway epithe­
lium. We have been unable to detect either breakdown of
the epithelium leading to DC access or increased extension
after allergen challenge. However, antigen­bearing DCs accu­
mulate near airways after allergen challenge. Concurrently,
activated T cells accumulate in the same regions. Thus, accu­
mulation of antigen­bearing DCs near airways, rather than
changes in alveolar APC phagocytic function during surveil­
lance, generates a local presentation mechanism. This repre­
sents the dominant variation in antigen handling and a likely
driver of pathogenesis in allergic airways disease and asthma.
RESULTS
CD11b+ DCs accumulate near allergic airways
To understand the nature of antigen processing for presenta­
tion to T cells as it occurs on and in the lung mucosa during
asthma, lung tissue–associated DCs also increase in number
and increase expression of co­stimulatory molecules (van Rijt
et al., 2005). After depletion of DCs and macrophages with
the CD11c­driven diphtheria toxin receptor, OVA­treated
mice lose the hallmarks of asthma (van Rijt et al., 2005).
Despite the large amount of data indicating the importance
of DCs in allergic responses in the lung, their functions
within the tissue have remained unclear.
Despite the wealth of studies on the trafficking of DCs
from the lung taken from endpoint analyses, less is under­
stood about the initial competition for antigens with the
more populous AMs, specifically, how in situ movement and
surveillance by DCs and AMs influences the fate of inhaled
materials. The specific handling of antigens by DCs in the
entire lung has largely been inferred from tracheal prepara­
tions. These have revealed DCs that project dendrites into
but not through the tight junctions of epithelial cells (Jahnsen
et al., 2006). A similar study highlighted tracheal DCs as
interlinked sheets lining the mucosal surface (Lambrecht et al.,
1998). The uptake of fluorescent particulate antigens and
macromolecules that are too large to cross the epithelial bor­
der by lung DCs suggested that these cells have mechanisms
to reach across into the airspace (Byersdorfer and Chaplin,
2001; Vermaelen et al., 2001). A prominent proposal is that
breakdown in the epithelium, perhaps in response to Toll­like
receptor (TLR) ligands, underlies pathogenesis (Lambrecht
and Hammad, 2009). Increased DC motility has been ob­
served in tracheal sections in response to epithelial recog­
nition of TLR4 ligands (Hammad et al., 2009), and occasional
dendritic extensions in tracheal DC preparations have been
reported, although it is not clear that these are present under
normal conditions (Hammad and Lambrecht, 2008). These
extensions mimic similar dendrites that protrude (Rescigno
et al., 2001), increasingly after injury or TLR signaling, into
the lumen of the gut (Chieppa et al., 2006). In vitro studies
using lung airway epithelium cultures have suggested that,
though their cell bodies are often beneath the epithelium, DCs
may protrude through epithelial tight junctions (Vermaelen
et al., 2001; Blank et al., 2007), although the relevance of this
to the whole lung remains undescribed.
It is well established that the majority of particulate anti­
gen transport to the draining LNs is performed by DCs and
not by passive carriage of antigen via the flow of afferent
lymph. Diphtheria toxin–mediated depletion of lung DCs
reduces antigen traffic to the LN (Vermaelen et al., 2001),
and DC homing depends on CC­chemokine receptor 7
(Jakubzick et al., 2006; Hammad and Lambrecht, 2007;
Wikstrom and Stumbles, 2007). Particle­bearing DCs that traf­
fic to the LN can subsequently elicit T cell responses to proteins
borne on the particulate (Byersdorfer and Chaplin, 2001).
The late (adaptive) phase of asthma exacerbations begins only
8 h after antigen inhalation, which would provide little time
for antigen traffic to the LN and T cell traffic back to the lung
(Wenzel et al., 2007). How and where these allergens are pre­
sented to T cells to generate airway­localized inflammatory
responses are not clear.

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The AMs were distinguishable because of their very round
shape, which is associated with a sphericity measure of >0.75,
shown as the second peak of sphericity in the histogram rep­
resenting the CD11c­EYFP population (Fig. 1 F). Side by
side analysis of Siglec­F, a marker closely linked to AMs but
not DCs, confirmed that this sphericity corresponded well to
AM/DC distinction (Fig. 1 G). This morphological criterion
in turn allowed us to separate these two cell types within
images (Fig. 1 H) and ultimately to confirm that the majority
of the DC accumulation observed by FACS was accounted
for in the airway­adjacent DC populations with much less
variation in the alveolar populations (Fig. 1 I).
Because the lung contains subpopulations of DCs, we also
wanted to determine which subpopulations we were exam­
ining in our imaging system. By staining live lung sections
from CD11c­EYFP mice with CD11b APC antibody, we
were able to visualize the CD11b+ and CD11b DC popula­
tions (Fig. 1 J). Quantification of the resulting images shows
that the DCs that accumulated near the airway were CD11b+;
however, CD11b+ and CD11b DCs were not otherwise
strictly segregated by location within the lung (Fig. 1 K). In
sum, this analysis indicated that inflammation during allergen
challenge was largely associated with increased numbers of
CD11b+ DCs retained in the airway­adjacent region, where
the pathology of asthma is most evident.
DCs in lung subregions behave differently
Although this imaging system provided much needed clarity
about phagocyte accumulation after allergen challenge, the
method also provided a route to directly analyze the behav­
iors of those cells in situ. By acquiring time­lapse videos of
these viable lung slices, we were able to track these potential
APCs. Using both the EYFP marking and the sphericity
measures of Fig. 1, we found that DCs exhibited significant
center of mass motility near airways, moving nondirection­
ally just below the surface of the epithelium. In contrast (with
the exception of the rare CD11c­EYFP cell that moved
within the lung parenchyma and was perhaps targeted toward
lymphatics), there was generally very little motility in alveolar
regions (Fig. 2 A and Video 2).
Quantification of the motility of many cells (as defined by
the movement of the weighted center of their cell bodies)
revealed speeds of 2.5 µm/min near the airway surface,
which was not appreciably altered under allergen challenge.
In contrast to the more motile airway­adjacent DCs, DCs
located near the alveoli were typically more stationary (mean
velocity of 1–1.5 µm/min) under both conditions, possibly
indicating different functions for DCs at the two locations.
AMs, in contrast, only moved at very slow speeds after aller­
gen challenge (<1 µm/min; Fig. 2 B). This slow motility of
AMs was also similar in control animals, as assessed using the
c-fms–EGFP mouse (Video 3); however, this data does not
preclude the possibility of different behavior in the case of
infection. Speed analysis is sometimes confounded by the
possible jitter of cells accounting for an apparent center of
mass movement, and so we performed analysis of the tracks
asthma, it was necessary to firmly establish the details of AM
and DC populations as well as a system for marking these
cells for imaging. We opted to perform all of these experi­
ments in the context of the highly studied OVA challenge
model of allergic lung disease, representing a subset of the
features of some forms of human asthma (Blyth et al., 1996).
The system is primarily based on a standard immunization
strategy with OVA in alum (Fig. 1 A) followed by inhalation
of OVA in the recall phase, which routinely results in robust
responses including eosinophil infiltration (not depicted),
airway hyperresponsiveness (Voehringer et al., 2006), and
goblet cell hyperplasia (Blyth et al., 1996). In contrast, PBS
inhalation does not result in significant changes in the lung.
To track AM and DC populations in real time, we took
advantage of transgenic mice in which EYFP expression is
driven under the control of the CD11c locus (CD11c­EYFP;
Lindquist et al., 2004). FACS analysis showed that CD11b+
DC numbers increased in the lungs of allergic mice (Fig. 1,
B and C; gating strategy in Fig. S1, A and B) but showed also
that CD11c­EYFP was expressed on all DC populations and
AMs after allergen challenge (Fig. S1 C). Whereas CD11b+
DCs, CD103+ DCs, and AMs expressed similar levels of
class II before allergen challenge, both DC populations up­
regulated class II expression after allergen challenge (not
depicted). This is consistent with the ability to prime or reac­
tivate T cells within the lung. With understanding of the
CD11c­EYFP marker expression profile and this in situ matu­
ration, we turned toward imaging to segregate DCs spatially
and to use image analysis to deconvolve these populations.
We developed a technique for live cell imaging in the
lung adapted from the previous work of Bergner and Sanderson
(2002). In brief, lungs were filled with agarose, removed
from the mouse, and sectioned into 300­µm sections using a
vibratome (Fig. S2 A). Lung sections are maintained at 36°C
in oxygenated media for imaging. Under these conditions,
propidium iodide staining 4 h after imaging showed only
modest cell death, confined to the cut face and distant from
the sites of our imaging, which always began at least 50 µm
beneath the cut site and proceeded downward (Fig. S2 B).
Cell­autonomous behaviors and viability were evident from
ciliary beating in the large airways (Video 1), which has been
documented to be maintained under these conditions for 10 d
(Bergner and Sanderson, 2002).
Tiling of large three­dimensional volumes obtained in
this way confirms and highlights the dramatic expansion of
CD11c­EYFPhi cells that we observed using flow cytometry
and allowed us to spatially assign this to regions of the alveo­
lar spaces and airways (Fig. 1 D). Quantification of cells
showed accumulation of EYFP+ cells near the airway after
OVA challenge (Fig. 1 E). However, as would be predicted
from the CD11chi gate in the flow analysis, DCs were the
dominant cells imaged under control conditions, whereas
both DCs and AMs were visualized with this marker in the
allergen challenge condition (Fig. S1). To resolve this, we
took advantage of the distinct morphologies of these two
cell types to separate them using automated image analysis.

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Antigen uptake and presentation in the lung | Thornton et al.
Figure 1. Specific accumulation of CD11b+ DCs near allergic airways. (A) Method for inducing allergic airway inflammation involving three i.p.
sensitizations with OVA/alum followed by three i.n. administrations of either OVA or PBS. (B) Quantification of CD11b+ DCs, CD103+ DCs, and AMs as a
percentage of live leukocytes based on CD11c surface antibody staining in lungs of PBS- and OVA-challenged mice. (C) Quantification of CD11b+ DC and
CD103+ DC populations as a percentage of live leukocytes gated on CD11c-EYFPhi cells in PBS- and OVA-challenged mice. (D) Large-scale three-dimensional
surveys of >300-µm slices from CD11c-EYFP mice stained with Hoechst shows DC and AM distribution in PBS- and OVA-challenged mice obtained by

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Airway DCs are motile and send few processes
through the airway
Airway DCs were motile in the airway­adjacent
regions (Fig. 3 A and Video 4). However,
these aggregate descriptions do not give an
accurate view of individual cells. For example,
we occasionally observed slow DC circling,
potentially providing surveillance of a fixed
area of epithelium or representing a cell under
the influence of a local chemoattractant (Fig. 3 B
and Video 5). By using mice expressing CFP
under the actin promoter to mark all cells
(Actin­CFP; Hadjantonakis et al., 2002) crossed
to CD11c­EYFP, we also sought to determine
whether airway DCs send processes across the
epithelium, as has been shown for tracheal
DCs (Hammad et al., 2009). A small fraction
of DCs observed were clearly situated just
below the airway epithelium while sending
projections toward and possibly between the
epithelial cells (Fig. 3 C and Video 6). However,
only 3% of airway DCs generated processes in
this way when observed over 30­min periods
in PBS­treated or allergen­challenged mice (Fig. 3 D), and
comparing the Actin­CFP marking, we were unable to ever
observe a dendrite extend past the outermost epithelium and
into the lumen.
Because previous data suggested DC sampling and/or
motility was augmented by TLR stimulation in the trachea
of airway­ and alveolar­associated DCs. This confirmed that
airway DCs displace over time, whereas alveolar DCs largely
do not (Fig. 2 C). However, further analysis of a large num­
ber of DCs observed over a long period of time revealed
that a trafficking fraction of alveolar DCs displaced >5 µm
in 30 min (Fig. 2 D).
two-photon microscopy with 910-nm excitation. Insets provide a zoomed in view of the two distinct morphologies. AW, airway. (E) Quantification of
CD11c-EYFP+ cells near the airways in PBS- or OVA-challenged mice. (F) Measurement of sphericity to separate DCs from AMs. AMs were defined as
having a sphericity >0.75. (G) Imaris was used to make surfaces of Siglec-F+ CD11c-EYFP+ cells from OVA-challenged mice. The sphericity of these cells is
graphed, showing that 80% of Siglec-F–stained AMs are captured by the sphericity measurement. (H) Color coding of localization based on sphericity
with bulk CD11c-EYFP data shown on the left and subdivided data (DCs, purple; AMs, green; airway, white) on the right from an OVA-challenged mouse.
(I) Images were subdivided by sphericity to determine whether DCs or AMs account for the accumulation within 75 µm of the airway after OVA challenge.
(J) Example images from CD11c-EYFP sections (gated on EYFP+ sphericity <0.75) stained with CD11b APC. Bars: (D [top] and H) 100 µm; (D, bottom)
10 µm; (J) 150 µm. (K) Quantification of CD11b+ and CD11b DC distance from the nearest airway for PBS- and OVA-challenged mice. (B, C, and K) Error bars
represent SEM. Graphs represent at least five mice per group from at least three independent experiments. Images are representative data from at least
three independent experiments.

Figure 2. Airway and alveolar DCs exhibit different
behaviors. (A) Detailed image with overlaid tracks of cell
motility at airway surfaces and alveolar spaces over the
course of 2 h. The top half of the image is mostly alveoli,
whereas the bottom is mostly airway. Arrows indicate
alveolar DCs. Bar, 100 µm. (B) Track speed means calcu-
lated using Imaris for CD11c-EYFP DCs and AMs sepa-
rated by airway and alveolar location in PBS and OVA
challenge conditions. Each dot represents one cell. Error
bars represent SEM. (C) Mean squared displacements ±
SE as a function of time for alveolar or airway-adjacent
DCs from untreated mice. (D) 30-min displacement for
alveolar DCs from OVA-challenged mice with a 5-µm
cutoff, which is the diameter of one cell. Each dot
represents one cell. Data are from at least three indepen-
dent experiments.