The Rockefeller University Press $30.00
J. Exp. Med. Vol. 208 No. 12 2511-2524
DCs and macrophages are highly hetero
geneous cells (Vremec and Shortman, 1997;
Shortman and Liu, 2002; Gordon and Taylor,
2005; ZieglerHeitbrock et al., 2010). Their
myriad subtypes exhibit a blend of shared and
unique functions that tailor their abilities to
regulate innate and adaptive immune responses.
In mouse LNs, conventional DCs include the
CD8+ and CD11b+ subgroups, the latter sub
divided based on CD4 expression (Vremec
et al., 2000; Allan et al., 2003; Belz et al., 2004;
Hildner et al., 2008). Recent DC immigrants
populating skin draining LNs (DLNs) include
Langerhans cells and CD8 CD103+ dermal
DCs (Randolph et al., 2008; Bedoui et al.,
2009). An additional subset, plasmacytoid DCs
(pDCs) are copious producers of type I IFNs
(Swiecki and Colonna, 2010). Microbial infec
tions typically increase DC numbers in the
DLN, and induce the differentiation of inflam
matory DCs (Serbina et al., 2003).
Macrophage subtypes are less clearly estab
lished than DC subtypes, but can be delineated
based on anatomical location (e.g., marginal
zone splenic macrophages) or expression of cell
surface receptors (e.g., CD169, mannose re
ceptor, MARCO, and dectin1; Taylor et al.,
2005). Within the LN, both subcapsular sinus
(SCS) and medullary macrophages express
sialoadhesin (CD169), with the latter distin
guished by coexpression of F4/80 (Phan et al.,
2009). The inflammatory milieu heavily influ
ences the differentiation state of macrophages
(Serbina et al., 2008; Liu et al., 2009).
LN macrophages have recently been the
subject of intense investigation because of their
rapid and efficient uptake of lymphborne par
ticles deposited into the LN SCS. After antigen
capture, SCS macrophages can relay antigen to
follicular B cells (Carrasco and Batista, 2007;
Junt et al., 2007; Phan et al., 2007), a step that
promotes the affinity maturation of antibodies
Jonathan W. Yewdell:
Abbreviations used: Ab, anti
body; CR, cortical ridge;
DLN, draining LN; DTR,
DTx receptor; DTx, diphtheria
toxin; eGFP, enhanced GFP;
eYFP, enhanced yellow fluo
rescent protein; hpi, hours post
infection; MPM, multiphoton
microscopy; MRR, macro
phagerich region; PIR, periph
eral interfollicular region;
SCS, subcapsular sinus; VV,
Chemokines control naive CD8+ T cell
selection of optimal lymph node antigen
Heather D. Hickman,1 Lily Li,1 Glennys V. Reynoso,1 Erica J. Rubin,1
Cara N. Skon,1 Jacqueline W. Mays,1 James Gibbs,1 Owen Schwartz,2
Jack R. Bennink,1 and Jonathan W. Yewdell1
1Cell Biology and Viral Immunology Sections, Laboratory of Viral Diseases, 2Biological Imaging Facility, Research Technologies
Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
Naive antiviral CD8+ T cells are activated in the draining LN (DLN) by dendritic cells
(DCs) presenting viral antigens. However, many viruses infect LN macrophages, which
participate in initiation of innate immunity and B cell activation. To better understand
how and why T cells select infected DCs rather than macrophages, we performed intravi-
tal microscopy and ex vivo analyses after infecting mice with vaccinia virus (V V),
a large DNA virus that infects both LN macrophages and DCs. Although CD8+ T cells
interact with both infected macrophages and DCs in the LN peripheral interfollicular
region (PIR), DCs generate more frequent and stable interactions with T cells. V V infec-
tion induces rapid release of CCR5-binding chemokines in the LN, and administration
of chemokine-neutralizing antibodies diminishes T cell activation by increasing T cell
localization to macrophages in the macrophage-rich region (MRR) at the expense of PIR
DCs. Similarly, DC ablation increases both T cell localization to the MRR and the duration
of T cell–macrophage contacts, resulting in suboptimal T cell activation. Thus, virus-
induced chemokines in DLNs enable antiviral CD8+ T cells to distinguish DCs from macro-
phages to optimize T cell priming.
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The Journal of Experimental Medicine
Chemokine-dependent APC choice | Hickman et al.
To better understand the priming of antiviral CD8+
T cells in vivo, we have combined intravital multiphoton
microscopy (MPM) with other approaches to dissect
mechanisms controlling T cell activation during viral in
fection. We show that CD8+ T cells interact with both vi
rally infected macrophages and DCs in vivo in the DLN.
Macrophage interactions, however, cannot fully prime
antiviral CD8+ T cells. To optimize priming, CD8+ T cells
use chemokines to guide them to DCs in vivo, and neu
tralizing these chemokines diminishes the antiviral CD8+
T cell response.
Definition of a macrophage-rich region (MRR)
of the inguinal LN
Visualizing macrophages and DCs in vivo using MPM ne
cessitates the unambiguous, simultaneous fluorescent detec
tion of both cell populations. Although CD11ceYFP
reporter mice allow DC analysis via MPM (Lindquist et al.,
2004), no red fluorescent counterpart exists for macrophages.
Gretz et al. (2000) showed that s.c. injected dextran ≥70 kD
was initially excluded from cortical areas of the LN but flowed
into the subcapsular and medullary sinuses, where it accumu
lated in unidentified
phagocytic cells. Sub
and Batista (2007)
identified this region
methylrhodamine-labeled dextran (red) 30
min before excision. (A) ER-TR7 staining (grey)
identifies the following nodal regions: MRR,
macrophage-rich region; PIR, peripheral inter-
follicular region; CA, capsule; SCS, subcapsu-
lar sinus; CR, cortical ridge; B, B cell follicle;
T, T cell zone. (B) Mosaic-tiled confocal images
of whole LNs stained with different Abs (grey,
left images), and higher magnification
images of staining of the MRR (right images).
(C) Number of dextran+ cells per node per cell
type as determined by node dissociation fol-
lowed by flow cytometry. Cells were gated on
FITC-dextran positivity and divided into
CD11b+ DCs (CD11c+CD11b+ cells), CD11b
DCs (CD11c+CD11b cells), and macrophages
(CD11cCD11b+ cells). Macrophages were
further gated on F4/80 to identify medullary
macrophages (F4/80+). Dextran+ cells belong-
ing to each population were quantified
using flow cytometric percentages and
total node counts. (D) MPM images
from CD11c-eYFP mice given dextran. Bottom
panels show higher magnification. CD11c+
cells (green), collagen (second harmonic
generation, blue), dextran+ cells (red).
(E) High-magnification confocal image of
whole-mounted CD11c-eYFP LNs after dextran
administration. CD11c+ cells (green), dextran+
cells (red). Scale bars are shown in micrometers.
We made similar observations in at least three
additional experiments per image.
(Abs; Phan et al., 2009). As SCS macrophages express lower
levels of several proteases involved in proteolytic degradation
of phagocytosed antigen, Phan et al. (2007) suggested that
their function may be to maintain a reservoir of antigen for
relay to LN B cells. However, many SCS macrophages are
not located above B cell follicles, but rather above the T cell
zone in the peripheral interfollicular regions (PIRs) of the
node (Hickman et al., 2008), suggesting their participation in
T cell responses.
As with other particulate antigens, virions trafficking to the
LNs via the lymphatics are internalized by SCS macrophages
(Norbury et al., 2002; Junt et al., 2007; Hickman et al., 2008;
Iannacone et al., 2010). Both vaccinia virus (VV) and vesicular
stomatitis virus readily infect nodal macrophages, which ac
count for up to 85% of virusinfected LN cells within a few
hours of infection (Norbury et al., 2002; Hickman et al., 2008).
Functionally, SCS macrophages limit vesicular stomatitis vire
mia and dissemination (Junt et al., 2007), and are a critical
source of type I IFNs (Iannacone et al., 2010). Although macro
phages express the appropriate machinery for T cell activation,
numerous studies have shown through in vivo ablation or
ex vivo isolation that DCs prime CD8+ T cells after viral
infection (Allan et al., 2003; Probst et al., 2005; Ciavarra
et al., 2006; Kassim et al., 2006). It is currently unknown
whether CD8+ T cells interact with infected LN macrophages.
Additionally, because infected macrophages and DCs are inti
mately intermingled in the PIRs, it is unclear how T cells se
lect DCs for priming over the more numerous macrophages.
Figure 1. Histological characterization
of the MRR of the LN. (A and B) Frozen LN
sections from WT mice given 70-kD tetra-
JEM Vol. 208, No. 12
CD8+ T cells interact with V V-infected macrophages
and DCs in vivo
Previously, we showed that CD8+ T cells in virusDLNs
form intimate, enduring antigenspecific interactions with
virusinfected cells, leading to T cell activation (Norbury
et al., 2002; Hickman et al., 2008). Notably, interactions oc
curred in a relatively small subregion of the LN, the PIR, and
as a macrophagerich area. Therefore, to visualize macro
phages, we injected mice s.c. with 70 kD tetramethylrhoda
mine dextran (red fluorescent) and examined excised nodes
at 30–45 min after injection by immunohistochemistry and
flow cytometry (Fig. 1).
Dextran+ cells were clearly visible in frozen sections at the
periphery of the LN, both in the SC and medullary sinuses,
as well as in the interfollicular channels identified via staining
of the LN stroma with ERTR7 (Fig. 1 A). Dextran+ cells
composed a distinct area of the cortical ridge (CR) region of
the LN (Katakai et al., 2004) between the T cell zone and
B cell follicles. Additional staining clearly identified the dex
tran+ cells as SCS and medullary macrophages (Fig. 1, B and C),
indicated by expression of CD11b and CD169 (SCS mac
rophages) and F4/80 on medullary macrophages (Phan
et al., 2009). Although the overwhelming majority of con
ventional (CD11c+) DCs do not endocytose dextran (visual
ized by MPM; Fig. 1, D and E), we detected a minor
population of dextran+ CD11b+ CD11c+ DCs using flow
cytometry (Fig. 1 C). Conventional DCs are positioned on
either side of the dextran+ cells in the CR, clearly distinct
from macrophages using MPM (Fig. 1, D and E; and Videos
1 and 2). This region of dense dextran+ macrophages over
laps but is not identical to either the PIR or CR and repre
sents the MRR.
Together, these data support the use of dextran endocyto
sis for the in vivo labeling of LN macrophages in the sinuses
SCS and medullary macrophages become infected
by lymph-borne virus
We previously reported that SCS macrophages are the pre
dominant infected cell in the DLN after s.c. VV injection
(Norbury et al., 2002; Hickman et al., 2008). Now other
lymphborne viruses have been shown to infect or accumu
late in SCS macrophages (Hsu et al., 2009; Gonzalez et al.,
2010). Although these studies relied exclusively on CD169 to
identify SCS macrophages, it is now clear that medullary
macrophages also express this marker (Phan et al., 2009).
Thus, we reexamined LNs (Fig. 2) using CD169 and
F4/80 in flow cytometry to distinguish macrophage popula
tions 8–10 h after infecting mice s.c. with a recombinant
VV (rVV) expressing a red fluorescent protein (VVNP
SmCherry; Table I). This revealed that >90% of VV
infected LN cells were CD169+ or CD11chigh/intermediate
(Fig. 2, A–C). Approximately 15% of infected cells were
DCs, with the remainder being CD169high/intermediate. As ex
pected, almost all of the CD169+ cells were also CD11b+,
with 56% being F4/80dim SCS macrophages (Fig. 2 B, far
right) and 38% being F4/80+ medullary macrophages.
To confirm this finding, we performed wholemount
F4/80 staining of nodes removed 10 h post infection (hpi;
Fig. 2 D). We detected a substantial number of F4/80+,
dextran+ VVinfected cells in the LN. Thus, both medullary
macrophages and SCS macrophages are robustly infected by
virus draining to the LN.
Figure 2. LN SCS and medullary macrophages are infected by V V.
(A) Flow cytometric analysis of single-cell LN suspensions from saline-
injected mice. CD169+CD11clo macrophages can be further divided into
CD11b+F4/80 SCS or CD11b+F4/80+ medullary macrophages. (B) LNs
from V V-infected mice 10 hpi. Cells were gated based on expression of a
vaccinia-expressed fluorescent protein, into DCs and CD169+ macro-
phages, and finally on F4/80+ or F4/80 cells. (C) Numbers of vaccinia-
infected cells in different nodal populations. Circles indicate individual
LNs, and bars show mean. (D) Whole-mount confocal images of LNs 10
hpi, with V V-NP-S-mCherry (red). Macrophages, green (FITC-
labeled dextran); F4/80, grey. Scale bars are shown in micrometers.
Data are representative of three experiments.
Chemokine-dependent APC choice | Hickman et al.
viral gene products and DCs. By generating rVVs that express
an antigenic protein genetically fused to mCherry (NPS
mCherry) we could simultaneously image viral antigens, eYFP
expressing DCs, and adoptively transferred, fluorescently
labeled, antigenspecific CD8+ T cells (OTI cells, recognizing
KbSIINFEKL; Clarke et al., 2000).
At 10 hpi, we imaged excised inguinal
LNs via confocal microscopy (Fig. 3 A), or
inguinal LNs in living animals using MPM
(Fig. 3 B). In uninfected nodes or nodes
infected with a control rVV lacking SIIN
FEKL, few OTI cells are present in the
PIR (Video 3). After VV infection with
a SIINFEKLcontaining virus (VVNPS
mCherry [red]), we commonly observed
OTI cells contacting infected DCs (Fig. 3,
A and B; and Video 4). We also identified
OTI cells interacting with likely unin
fected DCs (we cannot rigorously exclude
expression of fluorescent protein below
detection limits) and infected eYFP cells
identified as macrophages in other analyses
antigen was required for T cell accumulation in the PIR and
for sustained DC–T cell interactions (Hickman et al., 2008).
However, we were technically unable to simultaneously
identify virusinfected cells and DCs by MPM because of
spectral overlap of the fluorescent proteins used to visualize
Table I. Viruses used in this study
Abbreviation Full nameAntigen Fluorescent
full-length ovalbumin (secreted)
SIINFEKL as a fusion protein
standard construct V V-Influenza A virus nucleoprotein-
SIINFEKL-enhanced green fluorescent
V V-Influenza A virus nucleoprotein-
SIINFEKL-mCherry fluorescent protein
V V-Venus enhanced yellow fluorescent
V V-Venus enhanced yellow fluorescent
V V-NP-S-mCherry SIINFEKL as a fusion protein mCherrystandard construct; red
SIINFEKL as a minigene liberated via
none for OT-I cells
Venus eYFPcontrol fluorescent virus
none; backbone for recombinant V Vs
none control nonfluorescent virus
Figure 3. CD8+ T cells interact with LN DCs and
macrophages after viral infection. (A) LNs from
CD11c-eYFP mouse (green DCs) excised 10 hpi with
V V-NP-S-mCherry (red) were examined as whole
mounts by confocal microscopy. OT-I cells were la-
beled with Cell Tracker Blue before transfer. (B) MPM
images of the inguinal LN, as in A. (C) MPM images
from the top 30 µm of the inguinal node of CD11c-
eYFP mice given fluorescent-dextran (red) to label
macrophages and OT-I cells (blue). Uninfected nodes
(left) 10 hpi with nonfluorescent backbone virus
(V V--gal; control, middle left) or V V-Ova (middle
right). (right) A schematic of T cell localization in
relation to macrophages after V V infection. DCs
(green), MRR (red), OT-I cells (blue; right). Scale bars
are shown in micrometers. We made similar observa-
tions in two additional experiments.
JEM Vol. 208, No. 12
generation of bonemarrow chimeras, our shortterm assays
were performed simply with transgenic animals. Thus, to mea
sure T cell activation in the absence of DCs, we adoptively
transferred OTI cells (because of the potential effects of DTx
on autologous T cells in DTR mice, which can express CD11c
[and DTR] upon activation; Jung et al., 2002), administered
DTx, and examined OTI cell proliferation in rVVinfected
LNs (Fig. 4, A and E). DTx treatment greatly reduced (but
did not eliminate) OTI cell proliferation in response to VV
encoded antigen. We next analyzed several common T cell
activation markers (Fig. 4, C–E). Macrophageactivated T cells
failed to normally upregulate CD25 or downregulate CD62L
(Fig. 4, C and D). Although CD69 was induced in OTI cells,
it was dysregulated in comparison to cells activated in the pres
ence of DCs (at 36 h there was less downregulation compared
with WT; Fig. 4 E), and CD69 was not upregulated at all
under the same circumstances in different TCR transgenic
T cells (unpublished data). Reducing the number of OTI cells
transferred failed to restore their normal activation phenotype
(unpublished data), indicating that this phenomenon is unlikely
to be an artifact of using a high precursor frequency of TCR
transgenic cells (Badovinac et al., 2007).
Such macrophage activatedCD8+ T cells demonstrated
functional defects as well (Fig. 4, F–I). OTI cells were transferred
into CD11cDTReGFP mice that received DTx 12 h. before
infection with VVOva (Table I). In untreated mice, 90% of
(Video 4). We routinely identified multiple examples of
each interaction occurring in individual LNs (Fig. 3 B).
We next labeled macrophages in CD11ceYFP mice via
s.c. dextran injection. Few OTI cells were located at the pe
riphery of the uninfected LNs, or in nodes infected with con
trol virus (Fig. 3 C left and center panels and Video 3). In
contrast, infection with VV expressing cognate antigen in
duced OTI cells to localize to the PIR (Fig. 3 C, Video 3,
and Video 5). Most (but not all) OTI cells were excluded
from the MRR (Fig. 3 C, schematic, far right).
Collectively, these data indicate that although most CD8+
T cells interact with infected and uninfected DCs, they can
and do interact with VVinfected PIR macrophages under
V V-infected macrophages stimulate T cell division
but not activation
What happens when CD8+ T cells interact exclusively with
infected macrophages? We forced T cells to use infected
macrophages as APCs, using CD11cdiphtheria toxin (DTx)
receptor (DTR)eGFP mice (Jung et al., 2002) that were pre
viously used for ex vivo analyses of immune responses to other
viruses (Ciavarra et al., 2005; Probst et al., 2005; Kassim et al.,
2006). In this system, administration of DTx results in DC
ablation in vivo, leaving macrophages as the only infected cell
type in the DLNs. Although longterm DC depletion requires
Figure 4. CD8+ T cell interactions with
macrophages are largely nonproductive.
(A) LN OT-I cells dividing in response to s.c.
delivery of V V-NP-S-eGFP. 5 × 106 OT-I
CFSE-labeled OT-I cells were transferred into
CD11c-DTR-eGFP mice. 12 h later, mice were
treated with DTx or PBS (untreated). After an
additional 12 h, mice were infected with
V V (untreated and DTx) or left uninfected
(naive). Cells were analyzed at 48 hpi for divi-
sion. Far right panel shows overlays. Un-
treated mice, black lines; DTx-treated mice,
red lines; uninfected DTx-treated, filled grey
lines. Numbers = OT-I cells recovered.
(B) Graph showing percentage of dividing OT-I
cells. Dots represent individual nodes.
(C–E) Activation marker profile of T cells. Histo-
grams were only gated on dividing cells.
Untreated mice, black lines; DTx-treated
mice, red lines; uninfected DTx-treated, filled
grey lines. Numbers in top right corner indi-
cate time after infection. (F) IFN- produc-
tion (y axes) versus division (indicated by
CFSE dilution; x axes) at 2 days after infec-
tion with V V-Ova. (G) Graphical representa-
tion of data shown in F. Dots represent
individual nodes. (H) 2 × 106 OT-I cells were
transferred into CD11c-DTR-eGFP mice (ex-
pressing CD45.2) that were treated with DTx
(middle) or PBS (right) before intradermal infection with V V-SIINFEKL in the ear pinnae. 4 days after infection, ears were removed and analyzed for
the percentage of OT-I cells present (CD8+CD45.1+ cells). (I) Graphical representation of data in H. All experiments were performed at least three times
with three to six animals/group yielding similar results.
Chemokine-dependent APC choice | Hickman et al.
which is decreased viral infection/antigen presentation in the
DCdepleted LNs. To analyze this, we infected DTXtreated
CD11cDTReGFP mice or nontreated WT mice with
VVNPSeGFP and visualized infected cells by MPM
(Fig. 5 A). Numerous VVinfected cells were present just
beneath the SCS of the DLN in untreated or DTxtreated
animals (Fig. 5 A). Most infected cells were macrophages
(Video 6), as indicated by in vivo endocytosis of fluorescent
dextran administered s.c., and confirmed by CD11b+ staining
(Fig. 5 B) or F4/80 staining (Fig. 5 C) of wholemount LN
sections. Using flow cytometry, we found that almost all of
the infected LN cells recovered from DTxtreated mice (10
fold fewer than recovered from untreated mice) were CD11b+,
CD169+, F4/80+ medullary macrophages (Fig. 5 D).
To examine the ability of DTxresistant macrophages to
present rVV antigens, we infected mice with a rVV express
ing a highly fluorescent fusion protein (Venusubiquitin
SIINFEKL) that generates large amounts of cytosolic SIINFEKL
because of cotranslational liberation of SIINFEKL by cellu
lar ubiquitin hydrolases (or an irrelevant control virus ex
pressing VenuseYFP but lacking SIINFEKL; Table I; Fruci
et al., 2003; Lev et al., 2010). We removed DLN 6 hpi and mea
sured expression of KbSIINFEKL on dissociated cells by flow
cytometry using a fluorescent conjugate of the TCRlike
OTI cells produced IFN at 2 d after infection when restim
ulated with SIINFEKL peptide (compared with <0.2% when
stimulated with an irrelevant peptide; Fig. 4, F and G). In
CD11cdepleted mice, however, <8% of cells produced IFN
in response to SIINFEKL peptide, and even cells that had di
vided 5 times synthesized less IFN on average. DTx treatment
did not modify IFN production in non–DTR transgenic
(C57BL/6) mice (unpublished data).
A hallmark of effector CD8+ cells is their ability to traffic
from LNs to peripheral infection sites. We transferred OTI
cells into CD11cDTReGFP mice treated with DTx or
untreatedmice and infected intradermally in the ear with
VVSIINFEKL. 4 d later, ears were removed and examined
for the presence of OTI cells (Fig. 4, H and I). Remarkably,
macrophageprimed OTI cells from DTxtreated mice
completely failed to traffic to the infection site.
Together, these data indicate that macrophageprimed
CD8+ T cells exhibit diminished proliferation, altered activa
tion, and greatly reduced functionality, pointing to the im
portance of DCmediated activation in this system.
DC-ablated LNs still present viral antigen
Several possible explanations exist for the altered priming
in DTxtreated CD11cDTReGFP mice, the simplest of
Figure 5. Infected macrophages present antigen in DC-ablated mice. (A) V V-NP-S-eGFP–infected cells (green) just under the collagenous capsule of
the node (second harmonic generation (blue)). Numbers of infected cells in nonablated (untreated) and DC-ablated (DTx-treated) mice (right). (B) Confocal
microscopy of a frozen section of a DC-ablated LN 10 hpi with V V-NP-S-mCherry (red) showing only the red and green channels (left), only the red and
grey channels (middle), or all channels (right). FITC-dextran (green) and anti-CD11b staining (white) identify macrophages. (C) Same as shown in B, except
staining with F4/80 (grey) to identify medullary macrophages. (D) Numbers of infected cells of each cell type in the node. Cells were gated into CD11c+CD169
(DCs) or CD11cdimCD169+ (CD169+ macrophages), then CD169+ macrophages were further gated based on F4/80 expression. Background (blue) is caused by
low cell recovery and macrophage autofluorescence. (E) Flow cytometric analysis of LN single-cell suspensions from untreated or DTx-treated animals 6 hpi with
V V-venus-ubiquitin-SIINFEKL. Cells were stained with 25D-1.16 recognizing Kb-SIINFEKL complexes. Histograms are gated on infected cells (Venus eYFP+).
Infected with virus lacking SIINFEKL (grey shaded histograms), infected without 25D-1.16 stain (black lines), infected untreated mice (blue lines), and in-
fected DTx-treated mice (red lines). Scale bars are shown in micrometers. Results are shown from one experiment of two (A–C) or four (D and E).
JEM Vol. 208, No. 12
infection, forming stable contacts with virusinfected cells in
the presence of cognate antigen (Hickman et al., 2008). No
tably, in the absence of specific antigen, CD8+ T cells in the
periphery move at reduced speeds compared with those in
the deeper T cell zone, yet fail to form immotile, stable con
tacts with non–antigenexpressing APCs. To analyze T cell
behavior in DCablated mice, we transferred 107 OTI cells
into CD11cDTReGFP mice, treated mice with DTx, in
fected with VVSIINFEKL 12 h later, and imaged the DLN
6 hpi after injection of fluorescent dextran to identify macro
phages (Fig. 6, A and B and Video 6). Remarkably, in DTx
treated mice, 82% of OTI cells localized to the MRR
compared with 18% in untreated mice. Complete T cell acti
vation requires stable prolonged contact between T cells and
APCs (Mempel et al., 2004; Henrickson
et al., 2008). MPM revealed that most
OTI cells in the MRR of DTxtreated
mice were relatively immotile throughout
antibody 25D1.16 (Fig. 5 E). Cells from both untreated and
DTxtreated mice were specifically stained with 25D1.16,
demonstrating that APCs in DTxtreated mice are able to
generate class I peptide complexes from endogenous antigens,
although at lower levels than in untreated mice.
Collectively, these data demonstrate that medullary macro
phages are infected in DTXtreated mice and infected cells
still robustly present antigen in the node, albeit at lower
numbers and antigen levels than in untreated mice.
CD8+ T cells establish stable interactions with infected
macrophages in the absence of DCs
How do CD8+ T cells behave in the absence of DCs? In DC
competent mice, T cells rapidly transit to the periphery after
Figure 6. CD8+ T cells stably interact with
V V-infected cells in DC-ablated mice.
(A) MPM images of untreated or DTx-treated
CD11c-DTR-eGFP animals that were given 1.0 × 107
Cell Tracker Red (red) labeled OT-I cells before
DC depletion. Images acquired 6 hpi with V V-
SIINFEKL (nonfluorescent). The macrophage rich-
region (MRR) was identified by in vivo uptake of
FITC-dextran (green). (B) Percentage of OT-I cells
per 63X field (238 × 238 × 85 µm) located in the
MRR in untreated or DTx-treated mice. Results
were analyzed with an unpaired t test. (C) Time-
lapse MPM images of OT-I cells (red) in the LN of
untreated (top panels) or DC-ablated (bottom
panels) mice 6–10 hpi with V V-NP-S-eGFP
(green). White circles indicate stable contacts. For
untreated mice, 106 OT-I cells were transferred;
for DTx-treated, 107 (it was necessary to transfer
more OT-I cells into DTx-treated mice than un-
treated mice due to decreased homing to the LN
following DTx-mediated ablation). (D) Calculation
of contact times between OT-I cells and V V-
infected cells over the course of a 30 min imag-
ing session. (E) Plot of OT-I cells’ movement (step
size) between individual frames of a 30 min
movie in untreated vs. DC-ablated mice. We clas-
sified movement under 0.5 µm between frames
as a pause step. (F) Calculation of the percentage
of T cells arresting in each condition. (G) Color-
mapped plot of T cell tracks during a 30-min
movie. Each box on grid is 20 × 20 µm. Increasing
track speed is colored from purple to red (bottom
bar). (H) Calculation of average OT-I cell speed in
untreated or DTX-treated mice. Results are shown
from one experiment of three to six with similar
results. No differences between untreated and
DTx-treated mice were statistically significant
according to unpaired Student’s t tests. Time
is shown in minutes. Scale bars are shown
Chemokine-dependent APC choice | Hickman et al.
the protein levels of several chemokines on excised nodes.
VV infection increased levels of 5 chemokines examined at
6 hpi, with all except CCL3 being significantly greater at
12 hpi (Fig. 8). The greatest increases were seen with CCL2
and CCL4. We confirmed VV induction of CCL3 expres
sion in BMDCs (unpublished data).
the course of three consecutive 20min imaging sessions (the
first of which is shown in Video 7). To image the inter
action between OTI cells and infected macrophages, we
transferred OTI cells into mice 12 h before DTx treatment,
and then infected them with VVNPSeGFP. 6–10 hpi, we
imaged OTI cells interacting with VVinfected cells in the
DLN (Fig. 6 C and Video 8). In both control and DTx
treated mice, OTI cells formed numerous tight contacts
with infected cells. DTx treatment had no significant effect
on the mean contact time (27 min in each case during a
30min imaging period), arrest coefficients (the fraction of
time OTI cells paused on infected cells), or mean T cell
speeds (Fig. 6, C–H).
Collectively, these data demonstrate that in the absence of
DCs, OTI cells migrate into the MRR and form contacts
with VVinfected macrophages that are grossly indistinguish
able from their contacts with infected DCs.
CD8+ T cells rapidly scan infected non-DCs
Although T cells can form stable contacts with macrophages
when DCs are absent, we reasoned that the presence of DCs
could significantly impact the nature of contacts between
T cells and macrophages. Therefore, we next used MPM to
analyze contacts in CD11ceYFP mice between T cells and
DCs or macrophages 6 h after VV infection. Over a 20min
imaging period, we tracked T cells moving among infected
(directpriming) DCs, uninfected DCs (which likely con
sisted of crosspriming DCs and those expressing amounts of
fluorescent proteins below detectable levels), and infected
nonDCs (macrophages; Fig. 7 A). Although there was no
clear difference in the speed of T cells interacting with in
fected and uninfected DCs (1.4 µm/min, similar to values
for previously unidentified infected cells; Hickman et al.,
2008), T cells interrogated infected nonDCs at a signifi
cantly higher speed (Fig. 7 B). Likewise, T cells closely scanned
DCs in a curved path, but moved in straighter lines (indicated
by higher track linearity) when interacting with infected
nonDCs (Fig. 7 C). Finally, we monitored T cell arrest over
a 30min period for T cells interacting with each APC cate
gory (Fig. 7 D). T cells arrested for similar periods over in
fected and uninfected DCs, but paused for shorter amounts
of time over nonDCs.
Overall, these data indicate that T cells preferentially form
stable contacts with DCs (whether overtly infected or not)
over infected macrophages.
V V induces chemokine production in the DLN
What factors modulate CD8+ T cell interaction with and acti
vation by DCs? The migration of OTI cells into the MRR
in the absence of DCs demonstrates that CD8+ T cells are
fully capable of entering the MRR, suggesting chemotactic
recruitment of CD8+ T cells to PIR DCs. Chemokines at
tract naive CD8+ T cells to antigenbearing LN DCs in non
infectious models (with peptidepulsed DCs or antigen
targeted to DCs via conjugated Abs; Castellino et al., 2006;
Hugues et al., 2007). Thus, we performed bioplex analysis for
Figure 7. CD8+ T cells rapidly scan infected non-DCs. (A) MPM time
series of T cells interacting with infected DCs, uninfected DCs, or infected
non-DCs. CD11c-eYFP mice were given 107 Cell Tracker Blue-labeled
OT-I cells (blue) and were infected s.c. with V V-NP-S-mCherry 12 h. later.
Six hpi, inguinal LNs were imaged over sequential 20–30 min periods
using MPM. The tracks of individual T cells during a 20-min imaging period
are plotted (right). (B) T cell speeds, (C) track straightness (T cell displace-
ment/track length), and (D) T cell arrest calculated for each type of APC
interaction over 30 min. Open circles, infected DCs; closed triangles, unin-
fected DCs; closed circles, macrophages. Mean and SEM is shown. Statis-
tics were performed using an unpaired Student’s t test. Shown is one
experiment of two analyzed with 10–20 nodes per group.
JEM Vol. 208, No. 12
macrophages and DCs in the LN parenchyma (Hickman
et al., 2008). Because viral peptides are generated primarily
from DRiPs (Dolan et al., 2011), the onset of viral protein
synthesis is immediately accompanied by the generation of
class I peptide complexes, which can rapidly initiate CD8+
T cell priming. For VV, the initial phase of direct priming
likely persists for 12–24 h (Hickman et al., 2008), after which
infected DCs and macrophages succumb to viral cytopatho
genesis or NKmediated lysis. Because the first 12 hpi are
critical for activating antiviral T cells after s.c. VV infection
(Hickman et al., 2008), it is imperative that T cells rapidly
contact the appropriate infected APC.
The second wave of antigen presentation likely involves
transfer or acquisition of antigen by LNresident APCs.
CD8+ DCs are particularly adept at crosspriming antigens
acquired from dead or dying cells (den Haan et al., 2000;
Iyoda et al., 2002). For viruses that infect dermal epithelial
cells, like herpes simplex virus1, these cells provide an anti
gen source for crosspriming by migratory dermal DCs
(Bedoui et al., 2009). To date, antiviral crosspriming has
been investigated largely by ex vivo analyses of cells recov
ered from dissociated tissues (Allan et al., 2003; Belz et al.,
2004; Bedoui et al., 2009). It will be important in future
studies to confirm the central role of CD8+ DCs in antiviral
crosspriming using MPM, although it will be challenging
to unambiguously identify APCs actively engaged in cross
priming and not direct priming after synthesis of undetect
able levels of viral antigens.
The LN CR, a reticular stromal structure at the border of
the T/B zones, likely serves as a staging ground for both direct
and crosspriming T cell–APC interactions (Katakai et al., 2004).
Migratory, antigenbearing DCs are thought to accumulate
in the CR, positioning themselves to maximize crosspriming
interactions with nearby T cells (Bajénoff et al., 2003; Lindquist
et al., 2004). We show here that direct priming interactions in
dependent of DC migration also occur in the outermost edges of
the CR as virions drain to the LN and are captured by resident
APCs. After the first wave of virusinfected cells die or are elimi
nated in the LN, it will be interesting to determine the location
of virusspecific CD8+ T cell–APC interactions in relation to
the macrophagerich and dendritic regions of the CR.
Controversy swirls around the definition of macrophages
versus DCs and their putative differences in priming capacity.
Chemokine-neutralizing Abs impair the primary CD8+ T cell
response to V V
Do VVinduced chemokines play an essential role in attract
ing CD8+ T cells to DCs during viral infection? Remarkably,
administering a cocktail of neutralizing Abs against CCL3,
CCL4, and CCL5 dramatically redistributed OTI cells from
DCs into the MRR (Fig. 9), which is visualized clearly using
either twocolor (red T cells, green macrophages; Fig. 9 A)
or threecolor MPM (blue CD8+ T cells, green DCs, and red
macrophages; Fig. 9 C and Video 9). In untreated mice,
30% of OTI cells were associated with MRR macro
phages, but in mice treated with antiCCL3, CCL4, and
CCL5 Abs, >60% of OTI cells localized in the MRR
(Fig. 9 B). s.c. injection of recombinant CCL3 (introducing
artificial gradients of CCL3 in the uninfected node) also in
duced T cell scanning of both DCs and the MRR early after
VV infection (Fig. 9, D and E and Video 10). Additionally,
CCR5KO OTI cells failed to form large clusters with
VVinfected cells at 6 hpi (Fig. 9 F), and many of the cells
were located in the MRR (visualized using the natural
autofluorescence of the region; Fig. 9 G and Video 11).
We examined the participation of these chemokines in
regulating antiviral immune responses via APC selection by
s.c. injecting a mixture of CCL3, CCL4, and CCL5blocking
Abs at the time of infection. Chemokine neutralization re
duced OTI CD69 induction (an early indicator of activation)
15 hpi with VVOva (Fig. 9 H), and decreased IFN synthe
sis 2 d after infection (Fig. 9 I). In contrast, injection of iso
typecontrol Abs did not impact T cell activation (unpublished
data). In untreated, infected LNs, CCR5KO OTI cells (lacking
the CCR5 receptor for these chemokines, but retaining CCR1
which also signals via several CCR5 ligands) upregulated
CD69 to a lesser extent than WT OTI cells (Fig. 9 J).
Together, these findings demonstrate that the chemokines
CCL3, CCL4, and CCL5 play essential roles in enabling
T cells to reach a complete effector differentiation state and
maximizing primary antiviral CD8+ T cell responses by enhanc
ing CD8+ T cell interactions with DCs in the infected node.
After s.c. delivery of infectious virions, the most rapid anti
gen presentation occurs via direct priming. Within seconds,
lymphatics convey virions to LNs, where they can infect
Figure 8. V V infection induces rapid DC chemokine secretion. Mice were infected s.c. with V V-Ova and LN harvested at 6 or 12 hpi Chemokine protein levels
were determined from clarified node homogenates via Bioplex assay. Dots represent individual mice. Data are shown from two of four independent experiments.
Chemokine-dependent APC choice | Hickman et al.
Additionally, LN depletion of CD11c+ cells, which elimi
nates nearly all DCs and SCS macrophages, has profound
effects on antiviral priming, despite our direct observations
that CD8+ T cells now form contacts with macrophages that
are indistinguishable by IVM from their contacts with DCs
under nonablative conditions. This argues strongly that DCs
have a unique role in direct priming that macrophages can
Hume (2008) has eloquently argued that DCs are simply
mononuclear phagocytes that do not comprise a unique cel
lular subset, and are not especially adept at priming. A key
element of Hume’s argument is that the heightened prim
ing ability of DCs relative to macrophages ex vivo is caused
by removing suppressive macrophages during the process
of isolating DCs. Here, we show that in vivo, T cells scan
DCs and macrophages at different rates in infected LNs.
Figure 9. Chemokine-based CD8+ T cell homing to DCs. (A) MPM images of animals that were given 1.0 × 107 Cell Tracker Red (red)–labeled OT-I
cells in the presence or absence (untreated) of chemokine-neutralizing Abs against CCL3, CCL4, and CCL5. Images acquired 6–8 hpi with V V-Ova (non-
fluorescent). MRR delineated by white lines. (B) Percentage of OT-I cells per 63× field (238 × 238 × 85 µm) located in the MRR in untreated or antibody-
treated mice. Results were analyzed with an unpaired Student’s t test. (C) MPM images from the top 30 µm of the inguinal node of CD11c-eYFP mice
given fluorescent-dextran (red) to label macrophages and OT-I cells (blue). Mice were given chemokine neutralizing Abs; nodes imaged at 8 hpi with non-
fluorescent V V-Ova. (right) A schematic of T cell and macrophage localization after V V infection with CCR5-ligand blockade. Scale bars are shown in
micrometers. (D and E) OT-I cells (red) in the MRR (green) after s.c. administration of recombinant CCL3 at 2.25 hpi with V V-SIINFEKL (nonfluorescent; E)
tracks of OT-I cells in the presence (left) or absence (right) of rCCL3 2–3 hpi. Tracks are colored according to mean track speed (slowest [purple) to fastest
[red]). (F) Distribution of CCR5KO OT-I cells (red, left) or WT OT-I cells (red, right) 6 hpi with V V-NP-S-eGFP (green). (G) CCR5KO OT-I cells (red) in the MRR
(visualized using the intrinsic autofluorescence of macrophages) at 6 hpi (virus=green, collagen=blue) (H) CD69 expression by LN OT-I cells 15 hpi with
V V-Ova in the presence or absence of chemokine-neutralizing Abs. Data were averaged from three independent experiments normalized using the high-
est mean fluorescence intensity in an individual experiment as 100. (I) IFN- production by OT-I cells in the node 48 hpi with V V-Ova. Data were com-
piled from two independent experiments and normalized to the highest mean fluorescence intensity per experiment. (J) Same as described in H, but with
CCR5KO OT-I cells. All experiments were performed at least twice with three to six mice/group.
JEM Vol. 208, No. 12
MATERIALS AND METHODS
Mice. Specific pathogen–free CD11cDTReGFP transgenic mice on a
C57BL/6 background were acquired from The Jackson Laboratory (stock #
4509) and bred in house. Nontransgenic WT controls were C57BL/6 mice.
CD11ceYFP mice (Lindquist et al., 2004) were acquired through the
National Institute for Allergy and Infectious Disease (NIAID) Intramural
Research Repository and are now available from The Jackson Laboratory
(stock# 8829). CCR5KO mice (The Jackson Laboratory; stock #5427)
were crossed to OTI TCR transgenic mice (acquired through the NIAID
Intramural Research Repository) and bred for homozygosity. 6–16wkold
adult mice were used in all experiments. All mice were housed under spe
cific pathogen–free conditions (including MNV, MPV, and MHV) and
maintained on standard rodent chow and water supplied ad libitum. All ani
mal procedures were approved by and performed in accordance with the
NIAID Animal Care and Use Committee.
DC depletion and adoptive transfer. DC depletion was performed as
described previously (Jung et al., 2002). Mice were given a single of dose of
DTx at 4 ng/g body weight (SigmaAldrich) i.p. in PBS unless another dose
is noted. CD8+ T cells (TCR transgenic) were transferred 12 h before deple
tion. Virus infections were always performed 12–24 h after DTx administra
tion, allowing complete DC depletion before infection. Mice received only
one dose of DTx.
Dextran and virus injection. Approximately 25 µg of FITC or tetra
methylrhodamineconjugated 70 kD dextran (Invitrogen) was injected s.c.
30–45 min before LN analysis where indicated. VV was injected s.c. (3.5 × 107
PFU) or footpad (104 PFU) in sterile saline. Route of infection is indicated
for each figure. Previously described VVs used in this study include the fol
lowing: VVOva, VVNPSeGFP, VVSIINFEKL, VVVenusubiquitin
SIINFEKL, VVVenusubiquitinNP366374 (Norbury et al., 2002; Norbury
et al., 2004; Lev et al., 2008). VVNPSmCherry was constructed accord
ing to established protocols (Earl et al., 2001).
Adoptive transfers. CD8+ T cells were purified from TCRtransgenic
rag/ mice by negative selection using magneticactivated cell sorting
(MACS) according to the manufacturer’s instruction (Miltenyi Biotec). Cells
were 95–99% pure by flow cytometry. Purified cells were labeled with 2 µM
CFSE (Invitrogen) for 10 min at room temperature in PBS. For IVM, cells
were labeled with 2 µm CMPTX or CMF2HC (Invitrogen). The amount of
T cells transferred is indicated in specific figure legends.
Confocal whole-mount microscopy. Whole LNs were removed at in
dicated time points after infection and placed in 3.2% paraformaldehyde for
2 h at 4°C. For CD11ceYFP experiments, mice were given 107 CMF2HC
labeled (blue) OTI cells 12 h before infection. Where indicated, mice were
given lysinefixable fluorescent dextran s.c. (Invitrogen) 30–45 min before
removal. Nodes were washed extensively in PBS, and then stained overnight
with antiCD11b (clone M1/70; eBioscience) conjugated to Alexa Fluor
647, or for 2 h with antiF4/80 (clone BM8; eBioscience). Nodes were
washed in PBS before slicing into smaller pieces, which were mounted on
slides in Fluoromount G (Electron Microscopy Sciences). Slides were im
aged on an SP5 confocal microscope (Leica).
Ex vivo analyses of dissociated LNs. For flow cytometric determination
of the nature and number of infected cells per node, untreated or CD11cDTR
eGFP mice treated with DTx were given 3.5 × 107 PFU VVNPSmCherry
8–12 h before removal of inguinal LNs. Singlecell suspensions were gener
ated via digestion in collagenase (Worthington Biochemical Corporation) +
dispase (Roche) before staining for CD11c, CD11b, CD169, F4/80, and
GR1. Cells were gated on mCherry+ vacciniainfected cells before addi
tional analyses of individual cell populations.
For determination of the nature and number of dextran+ cells, mice
were given 0.25 µg FITCconjugated 70 kD dextran 30–45 min before node
removal and digestion with collagenase + dispase. Singlecell suspensions
What is the role for the large numbers of infected LN
macrophages if not for priming naive CD8+ T cells? Re
cently, Asano et al. (2011) showed that these macrophages
serve as facile APCs via crosspresentation of tumor associated
antigens, making their failure to drive antiviral T cell responses
even more enigmatic. Several imaging studies have now
shown antigen acquisition by SCS sinus macrophages and
subsequent antigen donation to LN B cells (Batista and
Harwood, 2009). Perhaps virally infected nodal macrophages
specialize in presentation to B cells and are largely ignored by
T cells (much like CD8+ DCs preferentially present to CD8+
and not CD4+ T cells; Dudziak et al., 2007). Additionally,
these macrophages express the molecule sialoadhesin, a newly
identified participant in regulatory T cell function and expan
sion, raising the possibility that macrophage infection activates
suppressive rather than effector T cells (Wu et al., 2009).
We show that macrophages prime CD8+ T cells that are
suboptimally activated by the standard criteria. It remains to
be determined, however, whether there is method to this
madness and the “partially” activated cells are fully activated
for a specific alternative function in primary or memory re
sponses. Alternatively, macrophages may serve to dampen
initial CD8+ T cell responses generated via direct priming,
favoring instead crosspriming at later time points. In any
event, it is a safe assumption that macrophages perform mul
tiple functions in the infected LNs.
In clearly demonstrating the essential role DCs play in di
rect antiviral priming, our findings emphasize the importance
of understanding the basis for CD8+ T cell attraction to in
fected PIR DCs. This knowledge is likely to be useful in maxi
mizing the ability of vaccines to elicit effective CD8+ T cell
responses. We provide the initial evidence that CCR5 and R1
signaling chemokines, known to guide CD8+ T cells to cross
priming DCs in noninfectious models (Castellino et al., 2006;
Hugues et al., 2007), also play a critical role in direct priming
during viral infection. We show, first, that viruses rapidly in
duce chemokines in the LN, and second, that an Ab cocktail
that neutralizes several of the known CCR5 ligands inhibits
both DC attraction of CD8+ T cells and CD8+ T cell IFN
responses. Additionally, these experiments likely underestimate
the importance of CCR5 agonist chemokines in direct anti
viral CD8+ T cell priming, since it is unlikely that the Ab cock
tail we used covers all CCR5 agonists or completely neutralizes
the agonists covered. Likewise, experiments using CCR5KO
OTI cells will similarly undervalue the effect of these che
mokines on CD8+ T cell responses as the cells retain signaling
through CCR1–CCL3 and –CCL5 interactions.
Because of its relevance for rational vaccine design, there
is tremendous interest in identifying APC types that most
effectively prime functional CD8+ T cell responses. We have
identified a factor that optimizes CD8+ T cell targeting to
appropriate APCs during viral infection. This raises the excit
ing possibility of engineering vaccines to express optimal an
tigens as well as a guidance system to attract CD8+ T cells to
DCs for maximal priming of effector cells with optimal effec
Chemokine-dependent APC choice | Hickman et al.
Bioplex assay. LN chemokines were analyzed during infection using
a beadbased assay (Bioplex; BioRad Laboratories) and results were read
out on a Bioplex Suspension Array System (BioRad Laboratories). Experi
ments were basically performed according to the manufacturer’s instruc
tions. To gain more reproducible virus drainage to the LN, we analyzed
cytokines within the popliteal LN instead of the inguinal. Mice were
infected for various time points with VVNPSeGFP (104 PFU footpad),
and the skindraining popliteal LN was removed and placed 250 µl buffered
saline solution with 0.1% BSA. Two nodes were pooled for a single sam
ple. LNs were homogenized in solution, and samples were centrifuged
at 14,000 g for 6 min to remove cellular debris. Exactly 100 µl of clarified
supernatant was used per sample, and samples performed in duplicate.
Results were analyzed using GraphPad Prism Software Version 5.0 (Graph
Pad). Error bars represent SEM, and groups compared using unpaired
Student’s t tests.
Kb-SIINFEKL complex staining. CD11cDTReGFP mice received
4 ng/g DTx i.p. or PBS i.p. 12 h before s.c. infection with VVVenusubiquitin
SIINFEKL or VVVenusUbNP366374. 6 hpi with VV, draining inguinal
LNs were removed and digested with collagenase type II (Worthington Bio
chemical Corporation). Singlecell suspensions were stained with 25D1.16
Alexa Fluor 647 (produced inhouse but comparable product available from
eBioscience) and propidium iodide and analyzed on a LSRII flow cytometer
(BD). Plots were gated on live cells and Venus (eYFP)+ cells before analysis
of 25D1.16 staining.
Chemokine-neutralizing Abs and recombinant chemokine injec-
tion. 50 µg each of antiCCL3 (MAB450), antiCCL4 (MAB451), and anti
CCL5 (MAB478; all from RND Systems) were given i.v. in sterile saline at
the same time as infection. Alternatively, 150 µg rat IgG2a (BioXcell) was
given in the same manner. 1 µg of rCCL3 (R&D Systems) in sterile saline
was given s.c. 45 min to 1 h before imaging.
CFSE proliferation. Spleens, inguinal, brachial, cervical, and mesenteric
LNs were removed and homogenized to produce singlecell suspensions.
Red blood cells were lysed, and samples were filtered through a 70µm nylon
filter. Cells were labeled for 10 min at room temperature in 2 µM CFSE
(Invitrogen). 5 × 106 OTI cells were transferred i.v. into CD11cDTReGFP
mice, which were treated with DTx or PBS i.p. 12 h later, mice were infected
s.c. with 3.5 × 107 PFU VVNPSeGFP. 48 hpi, inguinal nodes were re
moved and analyzed by flow cytometry. OTI cells were stained for CD45.1
(clone A20) and CD8 (clone 53–6.7) from eBioscience as well as propidium
iodide, and analyzed for CFSE fluorescence on a BD LSRII flow cytometer
(BD). Proliferation was then examined using FlowJo (Tree Star).
OT-I cellular activation assays. 12 h before infection s.c. with 3.5 × 107
PFU VVovalbumin, 5 × 106 OTI cells were transferred i.v. into WT mice.
For activation marker analysis, OTI cells were removed from the draining
ILN at indicated time points after infection using homogenization. Red
bloods cells were lysed, and samples were filtered through a 70 µm nylon fil
ter. Singlecell suspensions were stained for CD69 (clone H1.2F3), CD25
(clone PC61), CD62L (clone Mel14), CD45.1 (clone A20), and CD8
(clone 53–6.7; eBioscience). Results were compiled from four independent
experiments by normalizing the maximum CD69 MFI per experiment
to 100. Where indicated, mice received a single injection of chemokine
neutralizing Abs at the same time as infection.
For analysis of IFN production, WT or CD11cDTReGFP mice
(indicated in figure legends) were infected s.c. with 2.5 × 107 PFU VV
ovalbumin or VVNPSeGFP. Where indicated, mice received a single in
jection of chemokineneutralizing Abs i.v. concomitant with infection. Inguinal
LNs were harvested 48 hpi and homogenized, and cells were resuspended in
RPMI10 + 10 mM Hepes buffer and plated at 2 × 106 cells/well in Ubottom,
96well plates along with SIINFEKL or an irrelevant control peptide (SSIE
FARL) at a final concentration of 100 nM. Cells and peptide were incubated
for 3 h. at 37°C in the presence of 10 µg/ml brefeldin A (SigmaAldrich) to
were stained for CD11c, CD11b, F4/80, and GR1. Cells were gated on the
FITC+ population before analyzing individual cell types.
Histochemistry on frozen LN sections. LNs were removed and embed
ded in OCT medium (Electron Microscopy Sciences) and frozen in dryice
cooled isopentane. 20 µm sections were cut on a Leica cryostat (Leica). Sec
tions were fixed in ice coldacetone for 5 min before blocking with 5% goat
or donkey serum, then staining with the following Abs: ERTR7 (clone
ERTR7; Abcam), CD11b (clone M1/70; eBioscience), F4/80 (clone
BM8; eBioscience), CD11c (clone N418; eBioscience), B220 (clone RA3
6B2; eBioscience), pNAd (clone Meca79; BD), CD8 (clone 53–6.7; eBio
science), and GR1 (clone RB68C5; eBioscience).
MPM. CD8+ T cells labeled with CFSE, CMTPX (C42H40ClN3O4), or
CMF2HC (4chloromethyl6,8difluoro7hydroxycoumarin; all from In
vitrogen) were injected intravenously 12–24 h before injection with virus,
unless otherwise specified. When indicated, mice were given 25 µg FITC
or rhodamineconjugated 70kD dextran (Invitrogen) s.c. for drainage to the
inguinal LN. Twophoton imaging used an inverted TCSSP2 MP confocal
microscope (Leica) equipped with a 20× objective (numerical aperture, 0.7)
or 63× objective (numerical aperture, 1.30) and with 80% glycerol as the
immersion medium for each objective. Twophoton excitation was pro
vided by a Mai Tai Ti:Sapphire laser (Spectra Physics) with a 10Watt pump,
tuned to 800 nm for imaging of cells labeled with CMTPX, FITC, or
CMF2HC; 850 nm for imaging of cells labeled with CMTPX in combina
tion with eGFP; or 900 nm for imaging of eGFP alone or for imaging of
secondharmonic generation. Emitted fluorescence was collected with a
twochannel nondescanned detector. Wavelength separation was accom
plished with a dichroic mirror at 560 nm, followed by emission filters of
525/50 nm bandpass and 610/75 nm bandpass. For imaging of cellular inter
actions, the 63× objective was used, and stacks of 25 sections obtained with
a 2.5µm zstep for a total depth of 40–60 µm were obtained every 30 s.
For imaging of CD11ceYFP LNs after infection, a TCSSP5 MP con
focal microscope (Leica) equipped with dual MP lasers was used. One laser
was set at 800 nm for imaging dyelabeled cells and CMF2HClabeled OTI
cells, and the other was set at 900–930 nm for imaging VVNPSmCherry
and eYFP. Images were obtained in sequential mode using the same 63×
objective as in the previous paragraph. Sections were taken at 2.5µm inter
vals for a total depth of 40 µm. Series were acquired every minute.
Data analyses. Data were analyzed with Imaris 64 version 6.1.2 (Bit
plane). Images were processed using the Gaussian filter algorithm. Con
tact times between VVinfected cells and OTI cells were calculated
manually and compiled from 3–5 separate movies for each condition
(DTxtreated or WT). Contacts were defined as being in close proximity
(<1 µm) from a nuclearexpressed fluorescent protein. Arrest coefficients
were calculated using the Imaris XT function “plot length of selected
track,” and the percentage of pause steps for all of the T cells in a given
image were plotted using GraphPad Prism 5. Overlays of tracks were gen
erated using Imaris XT function “translate tracks,” and then pseudocol
ored according to the mean speed of the cell creating the track. Average
speeds were calculated using the spotdetection function and the follow
ing parameters: autoregressive motion, gapclose 3, 7.5µm object diame
ter, 40 µm maximum distance.
The number of infected cells under DTxtreated or untreated condi
tions was calculated from 3–6 fields 63× fields (238 × 238 × 85 µms) chosen
randomly in 3 separate VVinfected LNs. The percentage of cells in the
MRR was calculated by determining the number of OTI cells associated
with the MRR compared with the total number of OTI cells in the 63×
field. 3–6 fields per LN were selected for the MRR and the zone just outside
it (but otherwise randomly); data from at least four individual LNs were
compiled. Results were analyzed using GraphPad Prism 5 software. Means
are indicated with a solid line and standard error of the mean is shown.
Groups were analyzed using an unpaired Student’s t test.
JEM Vol. 208, No. 12
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Online supplemental material. Video 1 shows the MRR CD11cYFP
mice. Video 2 shows macrophages and DCs in the inguinal LN. Video 3
shows OTI cells interacting with DCs outside the MRR. Video 4 shows
OTI cells interacting with different DCs. Video 5 shows T cells in relation
to the MRR after infection. Video 6 shows infected macrophages in the
LN. Video 7 shows T cells in the MRR in the absence of DCs. Video 8
shows T cell contacts in DC ablated nodes. Video 9 shows T cell location
in the presence of chemokine neutralizing Abs. Video 10 shows T cell
behavior in the presence of rCCL3. Video 11 shows CCR5 KO OTI
T cell location after VV infection. Online supplemental material is available
We thank the NIAID Bldg. 33 Comparative Medicine Branch for their support in care
for the mice used in this study.
This work was funded by the Division of Intramural Research, NIAID, National
Institutes of Health
The authors have no conflicting financial interests.
Author contributions: H.D. Hickman, J.W. Yewdell, and J.R. Bennink designed
experiments and wrote the manuscript. H.D. Hickman, L. Li, G.V. Reynoso,
E.J. Rubin, C.N. Skon, and J.W. Mays performed experiments. H.D. Hickman and
O. Schwartz devised MPM methods. H.D. Hickman analyzed the data. J. Gibbs
Submitted: 8 December 2010
Accepted: 27 September 2011
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