3. TCR transgenic OT-I mice were purchased from The Jackson Laboratory.
CD45.1 congenic C57BL/6 (a gift from Pierre Guermonprez, Institut Curie,
Paris, France) were bred to OT-I mice to obtain OT-I/CD45.1. Animal care
and treatment were conducted in conformity with institutional guidelines in
compliance with national and international laws and policies (European
Economic Community (EEC) Council Directive 86/609; OJL 358; Decem-
ber 12, 1987).
Cells
Bone marrow-derived DCs (BM-DCs) were differentiated in vitro from the
bone marrow of wild-type (WT) or WASpϪ
mice as described previously
(22). DCs were used for experiments between days 8 and 10 when expres-
sion of Cd11c was higher than 80%. OT-I and OT-I/CD45.1 cells were
isolated from total LN suspension by negative selection using a MACS
isolation kit. To isolate endogenous DCs, single cell suspensions of LNs
were treated with 400 U/ml collagenase D (Roche) for 30 min followed by
enrichment (depletion of B, T NK) or positive selection by Cd11cϩ
beads
(Miltenyi Biotec).
Abs and reagents
The following Abs for FACS analysis were purchased from BD Pharmin-
gen: FITC and PE-conjugated anti-CD11c, FITC, and PE-conjugated anti
I-Ab
, PE-conjugated antiCD86, PE-conjugated anti-CD11b, PE-Cy5 con-
jugated anti-CD8, PE-conjugated anti-CD45.1, biotinilated anti-CD69, bio-
tinilated anti-CD3. CFSE and orange-fluorescent tetramethylrhodamine
cell tracker orange (CMTMR) were from Molecular Probes. To target DCs
in vivo, we generated a recombinant anti-DEC OVA fusion as described in
Ref. 23. The peptides corresponding to residues of 257–264 of OVA were
synthesized on solid phase (Fmoc/t-Bu chemistry). The peptides were pu-
rified by reverse phase HPLC and purity was verified mass spectrometry.
Time-lapse video microscopy
For the dynamic analysis of DCs trajectories, 3 ϫ 105
immature or LPS-
pulsed DCs (overnight, 10 g/ml) were plated on fibronectin coated cov-
erslips and placed into a chamber on a Zeiss LSM510 META Axiovert
200M reverse microscope at 37°C in a 5% CO2 atmosphere. Transmitted
light images were taken with a ϫ63 objective and a 3CCD camera every
30 s for 40 min. Recording of the trajectories, displacement analysis, and
velocity measurements were made using the Image J software. For analysis
of conjugate formation, mature DCs were incubated for 1 h with 0.1 nM of
the SIINFEKL peptide before plating. One ϫ 105
OT-1 cells were added
to the dish and images were taken starting 5 min after landing on the same
plane of DCs. Each DC was analyzed along the length of the movie and the
number and duration of contacts established with T cells was scored.
In vivo migration assay
WT and WASpϪ
BM-DCs were harvested at day 8 and labeled with 2 M
of CFSE (Molecular Probes) according to the manufacturer’s instructions.
After labeling, 5 ϫ 105
to 2 ϫ 106
cells, depending on the experiments,
were injected s.c. into the footpad of the C57BL/6 host. For cotransfer
experiments, 2 ϫ 105
WT DCs labeled with carboxy-SNARF (Molecular
Probes) were mixed with 6 ϫ 105
WASpϪ
DCs labeled with CFSE and
injected in the footpad of the WT recipient. To quantify the number of
migrating DCs, single-cell suspensions from the draining popliteal LN
were obtained by digestion in collagenase D at days 1, 2, and 3 postinjec-
tion. The absolute number of CFSEϩ
/CD11cϩ
cells was quantified by
FACS by acquiring all cells in each sample.
Immunostaining on LNs section
For localization of DCs within LNs, a mixture of 2 ϫ 105
WT DCs
(SNARF labeled) and 6 ϫ 105
WASpϪ
DCs (CFSE labeled) was injected
into the footpad of a WT recipient. LNs were harvested 24-h postinjection,
fixed in paraformaldehyde (2%), and snap frozen in Tissue-Tek. Frozen
sections (8 M) were fixed in cold acetone and analyzed using the fol-
lowing Abs: biotinylated anti-B220 followed by streptavidin Alexa 647
(Molecular Probes); biotinylated anti-CD3 followed by streptavidin Alexa-
647; rat anti PNAd (MECA 79) followed by anti-rat Alexa 647. Images
were acquired using a LSM 510 Meta using a 40/0.40 NA oil objectives
and MetaView 4.6 software (Molecular Devices).
In vitro T cell activation
To test Ag presentation of the MHC class-I OVA epitope by LN DCs upon
uptake of ␣DEC205-OVA, we performed an in vitro assay of OT-I acti-
vation using conditions that bypasses defects in conjugate formation.
Five ϫ 104
immature DCs isolated from LNs of WT or WASpϪ
mice were
incubated with increasing doses of the Ag for 3 h in the presence of 1
g/ml LPS. After washing, 1 ϫ 105
OT-I cells were added to the wells and
the plate was spun to force DC-T interaction. Cells were harvested after
24 h and up-regulation of CD69 on T cells was measured by FACS. To
correlate defective synapse formation and T cell priming in vitro, we used
a previously established assay (9). DCs were pulsed with 0.1 nM of SI
INFEKL peptide, washed, and transferred into round-bottom or flat-bottom
wells (2 ϫ 104
/wells). Two ϫ 104
OT-I cells were added to the wells and
harvested 24 h later to quantify the percentage of CD8ϩ
cells that up-
regulated CD69ϩ
.
Adoptive transfer and T cell activation
One ϫ 106
OT-I/CD45.1 cells were purified as described above and in-
jected i.v. into the recipient host. For priming with BM-DCs, 2 ϫ 105
WT
or 6 ϫ 105
WASpϪ
DCs were pulsed with a graded dose of the MHC class
I restricted peptide of OVA (SIINFEKL) and injected in the footpad 24 h
after transfer T cell transfer. At day 3 after DCs injection, popliteal drain-
ing LNs were collected, digested in collagenase, and the percentage of
OT-I/CD45.1 cells were evaluated by gating on OT-I/CD45.1. For com-
parison of the priming ability of DCs in LNs, we quantified the number of
CFSE DCs in each sample (by gating on CFSE cells). To analyze the CFSE
dilution profile of transferred OT-I, T cells were labeled with CFSE and the
dilution profile was analyzed by gating on CD8/CD45.1 cells. For priming
with ␣DEC205OVA, mice were adoptively transferred with OT-I, as
above, and injected s.c. with graded dosed of ␣DEC205-OVA plus 10 g
of anti-CD40 in the footpad 24 h later. LNs were collected at day 3 to
quantify the percentage of OT-I/CD45.1 and CFSE dilution by FACS.
Two-photon laser scanning microscopy
For in vivo imaging of T cell trajectories in LNs control or WASpϪ
re-
cipient mice were injected i.v. with 10 ϫ 106
CMTMR-stained (10 M)
OT-I cells. One hour later, mice were injected s.c. in the hind footpad with
0.1 g of ␣DEC205-OVA plus 10 g of anti-CD40. To directly visualize
DC-T cell interactions in LNs, mice were adoptively transferred with
CFSE-labeled OT-I cells. In brief, 0.5 ϫ 106
or 1.5 ϫ 106
WT and WASpϪ
DCs pulsed with 0.1 nM peptide were labeled with CMTMR (10 M) and
injected into the right and left footpad of the same mouse, respectively.
Eighteen hours later, draining inguinal LNs were carefully dissected and
placed into an imaging chamber which was perfused with harmed medium
bubbled with a gas mixture containing 95% O2 and 5% CO2. The temper-
ature close to the sample was regulated at 37°C. Details on the microscope
setting are as described in Ref. 24. The trajectories of each T cell in the
imaging session and the duration of contacts established by each individual
T cells during the imaging session was quantified using the Metamorph
software on at least 20 movies/genotype.
Statistical analysis
Non-normally distributed data were compared with the Wilcoxon rank sum
test. Graphed distributions were analyzed with a Chi-square test on con-
tingency tables.
Results
Ag targeting to endogenous DCs in WASpϪ
mice induce faint
activation of T cells
We set out to study the ability of DCs resident in lymphoid organs
of WASpϪ
null animals to crosspresent exogenous Ags to naive
CD8ϩ
T cells. We first examined DCs frequencies and subtypes
composition in WASpϪ
mice. The frequency of total conventional
CD11chigh
DCs in LNs of WT and WASpϪ
mice was similar and
within the expected range (around 2% of total cells). Within total
CD11cϩ
cells in LNs we found the same percentage of tissue-
derived DCs (CD11cϩ
MHCIIhigh
) in WT and mutant animals in-
dicating normal trafficking from the periphery to the LN at steady
state (Fig. 1a). Blood-derived CD8␣ and CD11b cells were also
present in equal numbers in LNs (Fig. 1b) and spleen (data not
shown) of WT and WASpϪ
animals. Thus, WASp is dispensable
for DCs development in vivo.
To study Ag presentation by lymphoid organ-resident DCs, we
used a previously described system to deliver Ag to DCs in vivo.
Mice were injected with a recombinant anti-DEC205 Ab fused to
OVA (␣DEC205-OVA) in the presence of anti-CD40 Ab
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4. (␣CD40). Immunization with ␣DEC205-OVA leads to cross-pre-
sentation of the MHC class I epitope of OVA mainly by CD8ϩ
LN-resident DCs and the adjuvant ␣CD40 is required to expand
long term memory CD8ϩ
T cells (7, 23, 25). A control irrelevant
Ab coupled to OVA does not induce T cell activation (23). To
exclude interference by defective activation of T cells, we ana-
lyzed responses of OVA-specific TCR transgenic T cells (OT-I)
that are of WT origin. WT and WASpϪ
mice were adoptively
transferred with CFSE-labeled OT-I cells followed by s.c. immu-
nization with different doses of ␣DEC205-OVA plus ␣CD40. The
maturation profile of DCs after anti-CD40 treatment is equivalent
in WT and WASpϪ
mice (data not shown). At first we measured
the up-regulation of the early activation marker CD69 on OT-I
cells 24 h after immunization. As shown in Fig. 2a, delivery of 0.1
g of ␣DEC205-OVA induced up-regulation of CD69 on 49% of
OT-I cells whereas activation was almost undetectable at this dose
in WASpϪ
host. At higher Ag dose, OT-I cells were primed also
in WASpϪ
mice, yet at lower levels that in WT. The mean reduc-
tion in the percentage of OT-I cells that up-regulated CD69 at 24 h
in WASpϪ
mice at 0.1 and 1 g of ␣DEC205-OVA is depicted in
Fig. 2b. By an in vitro assay that measures presentation of the
MHC class-I OVA peptide to T cells (see Materials and Methods),
we excluded that this difference is due to reduced internalization
and processing (Fig. 2c). Therefore, WASp expression in DCs is
required to induce early markers of CD8ϩ
T cell activation down-
stream of Ag processing.
We next analyzed the proliferation of OT-I cells primed in a WT
or WASpϪ
context. Mice were immunized with increasing doses
of ␣DEC205-OVA plus ␣CD40 and adoptively transferred with
CFSE-labeled OT-I/CD45 cells. LNs draining the immunization
site were harvested at day 3 to analyze the cell division profile
(Fig. 3a). At low doses, the amount of Ag required to trigger entry
of T cells in cycle was 4-fold higher in WASpϪ
than in WT mice
(12.5 ng WT vs 50 ng WASpϪ
). Increasing the Ag dose induced
entry in cycle of the majority of the OT-I. However, T cells primed
in WASpϪ
mice remained in large proportions within the seventh
cycle and only few cells beyond the seventh division accumulated
in LNs of WASpϪ
mice (Fig. 3b). Quantification of Ag specific T
cell expansion indicates that accumulation of OT-I cells was dra-
matically decreased in WASpϪ
animals, especially at low Ag
doses (Fig. 3c). Thus, WASp expression in LN-resident DCs is
required to achieve optimal T cell responses at limiting Ag doses.
Correction of defective migration to LNs does not rescue CD8ϩ
T cell priming
The above results indicate that priming of CD8ϩ
naive T cells by
DCs resident in the lymphoid organ of WASp-deficient mice is
inefficient. To clearly dissect migration to and priming in LNs, we
used adoptively transferred BM-DCs. Different doses of BM-de-
rived WT or WASpϪ
DCs labeled with CFSE were injected into
the footpad of the WT recipient. LNs were harvested at different
time points to measure the number of DCs that have reached the
draining LN. An inhibition of at least 2-fold in migration of
WASpϪ
cells was observed along the entire range of doses and
kinetics tested, in agreement with previous reports (21) (data not
shown). To study the intrinsic ability of WASpϪ
DCs to prime
naive T cells within LNs, we set the conditions to bypass defective
homing by increasing the input of WASpϪ
DCs. To achieve com-
parable numbers of cells in LNs we injected three times as much
FIGURE 1. DCs frequencies and subtype composition in WT and
WASpϪ
mice. Total LN cell suspensions were enriched in DCs by mag-
netic depletion of B, T, and NK cells and analyzed by flow cytometry. a,
Phenotypic profile of the DC-enriched fraction labeled with fluorescent
Abs against CD11c/MHC-II. gate A: CD11cϩ
/MHCIIhigh
, Langerhans and
interstitial DCs; Gate B: CD11chigh
/MHCIIint
, blood-derived DCs (left pan-
els). Percentage of total CD11cϩ
cells in LNs of WT and WASpϪ
mice.
The results are the means Ϯ SD of five mice/genotype. b, Density plot of
WT and WASp LN cells (gate B) stained with anti-CD11c and anti-CD8 or
anti-CD11b fluorescent Abs to identify CD8␣ϩ
CD11cϩ
and
CD11bϩ
CD11cϩ
cells. Mean percentage of CD8␣ϩ
CD11cϩ
and
CD11bϩ
CD11cϩ
over total CD11c in LNs of eight WT and eight WASp
mice.
FIGURE 2. Early events of T cell activation induced by Ag targeting to
DCs in vivo. a, WT or WASpϪ
mice were adoptively transferred with 1 ϫ
106
CFSE-labeled OVA specific OT-I cells (expressing the CD45.1 con-
genic marker) followed by immunization with 0.1 or 1 g of ␣DEC205-
OVA plus ␣CD40 (10 g). Profiles shows CD69 expression on OT-I cells
(gated on CFSE cells) in draining LN at day 1. Numbers indicate the
percentage of cells with high expression of CD69. b, Results from a rep-
resented as the percentage of inhibition of the frequency of CD69high
OT-I
cells in LNs of WASpϪ
animals relative to WT. Results are pooled from
three independent experiments with three mice/condition. c, CD11cϩ
cells
were purified from LNs of WT and WASpϪ
mice and pulsed in vitro with
the indicated doses of ␣DEC205-OVA. After pulsing, OT-I cells were
added to the culture to measure the amount of processed Ag presented by
DCs. Numbers indicate the percentage of OT-I cells that up-regulate CD69
after 24 h of coculture with WT () or WASpϪ
() DCs.
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5. WASpϪ
DCs than WT DCs. Under these conditions the total num-
ber of DCs in LNs was the same at 24, 48, and 72 h post injection
(Fig. 4a). The maturation profile of migrated DCs was similar for
WT and WASpϪ
DCs (data not shown). To study the localization
of migrated DCs within LNs, we performed tissue immunofluo-
rescence on LN sections 24 h after injection of labeled DCs. The
total number and the distribution profile of DCs with respect to B
cell follicles, T cell areas, and HEV was similar for WT and
WASpϪ
cells coinjected in the same LN (Fig. 4b). This result
indicates that WASp expression is important for migration to,
rather than localization within, LNs.
To test the priming potential of DCs in LNs, DCs were left
unpulsed or pulsed with two different doses of OVA peptide and
used at 1:3 ratio to immunize WT hosts that had been adoptively
transferred with OT-I cells (CD45.1 congenic). T cell expansion
was evaluated as the ratio between Ag specific OT-I/CD45 cells
and endogenous CD8ϩ
T cells at day 3. Despite equal numbers of
Ag presenting DCs in LNs draining the immunization site, OT-I
cell expansion was significantly inhibited in mice immunized with
WASpϪ
DCs (Fig. 5a). The profile of OT-I CFSE dilution showed
that at the lowest peptide doses immunization with wt DCs induce
up to 40% of T cells to enter division and 30% of the cells to
undergo more than seven cycles of division (fully divided cells).
At the same peptide dose, most OT-I cells primed by WASpϪ
DCs
remained undivided (60%) and a little proportion underwent two to
seven division (13%). At 0.05 nM of peptide WT DCs induced all
OT-I to enter the cell cycle and a large proportion of fully divided
cells to accumulate (56%). Interestingly, priming with WASpϪ
DCs loaded with 0.05 nM of peptide caused up to 70% of OT-I
cells to enter division but the cells remained trapped between two
to seven cycles with very few cells beyond the seventh division
(13%) (Fig. 5, b and c). Thus, WASp-deficient DCs can stimulate
division of Ag-specific CD8ϩ
T cells but do not provide the signals
to achieve full expansion.
We conclude that WASp expression in DCs is important to ini-
tiate CD8ϩ
T cells responses at two levels: by promoting the mi-
gration to draining LN and by supporting efficient T cell activation
in LNs.
Imaging DC-T cell contacts
To understand the mechanism of defective priming in LNs, we
moved to analyze the dynamics of DC-T cell interaction during
priming. We have previously shown that, during the initial phases
of T cell priming, DCs project polarized membrane extensions that
facilitate the formation of DC-T cell conjugates. This activity is
regulated by small GTPases of the Rho familiy (22). Because
WASp drives actin polimerization downstream of Rho GTPases,
we asked whether WASp expression in DCs contribute to facilitate
the interaction with T cells during priming. We first studied con-
jugate formation by time-lapse in vitro. Despite several evidences
on the role of WASp in T cells, its function in DCs during immune
synapse formation has not been investigated. To pin on the role of
WASp expression in DCs, we studied synapse formation using
CD8ϩ
T cells of WT origin. DCs were activated by LPS treatment
and pulsed with the MHC class-I OVA peptide or left unpulsed.
OT-I cells were added to DCs at 1:1 ratio and differential inter-
ference images were collected every 30 s for the first 40 min of the
coculture (Fig. 6a and web movies 1, a and b).5
WT DCs displayed
5
The online version of this article contains supplemental material.
FIGURE 3. Activation of naive WT CD8ϩ
T cells by endogenous DCs
is highly compromised in WASpϪ
mice. Mice were adoptively transferred
with Ag-specific naive CD8ϩ
T cells (CD45.1) followed by immunization
with different doses ␣DEC205-OVA plus ␣CD40. LNs were harvested at
day 3 to evaluate T cell division. a, Histograms plots represent the CFSE
dilution profile of transferred OT-I cells for the indicated Ag doses (gated
on CD45.1/CD8). b, Data in a were expressed as the percentage of OT-I
cells that remain undivided, that divided two to seven times, or that fully
diluted CFSE. c, Percentage of CD45.1/CD8 on total CD8ϩ
cells. Values
are pooled from three independent experiments with three mice per
condition.
FIGURE 4. DC migration to draining LNs. A mix of 2 ϫ 105
WT and
6 ϫ 105
WASpϪ
immature BM-derived DCs labeled, respectively, with
CFSE and SNARF were injected into the footpad of WT recipient mice. a,
Dot plot of WT and WASpϪ
DCs migrated to lymph nodes 24 h after
injection. Bars represent the absolute number of WT and WASpϪ
DCs
recovered in LNs at different time points after injection. Results are the
means Ϯ SD of three injected mice/group/time point. b, LNs sections were
labeled with Abs against B cells (B220), T cells (CD3), and high endo-
thelial venules (HEV) to analyze the relative distribution of WT and
WASpϪ
DCs.
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6. extensive ruffling activity and moved on fibronectin with a mean
velocity of 0.080 Ϯ 0.005 m/sec (n ϭ 36). Ruffling activity was
maintained in WASpϪ
DCs but net translocation was reduced be-
cause the cell body remained anchored to the matrix. The mean
velocity of WASpϪ
DCs was reduced to 0.059 Ϯ 0.004 m/sec
(n ϭ 31; p Ͻ 0.01) (Fig. 6b). DC movements facilitate the scan-
ning and capture of naive T cells in the culture. As shown in Fig.
6a and supplemental movies, WT DCs translate to capture naive T
cells when they come in close proximity. In contrast, a proportion
of WASpϪ
DCs failed to establish tight contacts with T cells de-
spite initial scanning. We quantified the percentage of DCs that
form a contact that last more than 20 min with at least one naive
T cell. Both WT and WASpϪ
DCs establish only few long contacts
in the absence of OVA-specific peptide (WT ϭ 13.7%; WASpϪ
ϭ
14.5%). Addition of 0.1 nM of OVA peptide induced a high pro-
portion of WT DCs to form long-lasting contacts with Ag specific
T cells (47 Ϯ 0.13%). In contrast, peptide loading on WASpϪ
DCs
induced only a modest increase in the percentage of long lasting
interactions (21 Ϯ 0.15%; p Ͻ 0.001) (Fig. 6c). We confirmed
these observations using DCs freshly isolated from the LNs of WT
and WASpϪ
mice. CD11cϩ
cells from LNs were loaded with
OVA MHC class-I peptide and coculture with naive OVA-specific
T cells. The overall duration of Ag-specific DC-T cell interaction
was significantly decreased in freshly isolated WASpϪ
DCs
(WT ϭ 18.58 min Ϯ 1.41; WASpϪ
ϭ 13.05 min Ϯ 1.73, p Ͻ
0.05) (Fig. 6d and web movies 2, a and b). To correlate defective
DC-T interaction with priming in vitro, we used a previously es-
tablished assay (9). DCs were plated with T cells in flat-bottom
wells at a 1:1 ratio to reproduce the conditions of in vitro imaging.
As a control cells were plated in round-bottom wells. The profile
of CD69 expression on T cells 12 h after culture shows that
WASpϪ
DCs are impaired when DCs need to patrol for the pres-
ence of T cells and to capture and stabilize interaction with
FIGURE 5. CD8ϩ
T cell priming induced by WT and WASpϪ
DCs in
LNs. a, Mice were adoptively transferred with CD45.1, CFSE-labeled
OT-I cells. T cell priming was induced by injection of 2 ϫ 105
WT or 6 ϫ
105
WASpϪ
DCs loaded with the indicated doses of MHC class-I OVA
peptide. Values show percentage of OT-I cells over the total CD8ϩ
pop-
ulation at day 3 postimmunization (gated on CD45.1/CD8ϩ
cells) and are
the means Ϯ SD of four mice per group. b, Histogram plots show the CFSE
dilution profile of transferred OT-I cells (gated on CD8ϩ
/CD45.1ϩ
) in
draining LNs at day 3. Values are means Ϯ SD of four mice per group. One
of three independent experiments with similar results is shown. c, Data in
b expressed as the percentage of OT-I cells that remained undivided, that
underwent two to seven division or that fully diluted CSFE (fully divided).
FIGURE 6. DC-T cell interaction in vitro. BM-derived DCs matured by
overnight treatment with LPS were loaded with 0.1 nM of the MHC class-I
OVA peptide and let to adhere to fibronectin. OT-I cells were added to the
culture (1:1 ratio) and time-lapse movies were recorded during the first 30
min of interaction. a, Sequential images from supplemental movies 1, a and
b show an example of a WT (upper panel) and a WASpϪ
(lower panel) DC
cocultured with OT-I cells. In the WT sequence the DC forms a stable
contact few frames after the first dendrite mediate sampling of the T cell
surface. In the WASpϪ
sequence, the T cell bounces repeatedly on the
dendrites but does not stop to form a stable contact. b, Mean velocities
were calculated by manual tracking of 36 and 31 mature WT and WASpϪ
respectively (,ءء p Ͻ 0.01). Each dot represents a single cell. Horizontal
bars indicate mean velocities. c, Percentage of mature BM-derived DCs
that establish a long lasting contact (duration Ͼ20 min) with at least one T
cell in the absence (no pep) or in the presence of 0.1 nM peptide (pep)(49
WT and 42 WASpϪ
cells were analyzed, ,ءءء p Ͻ 0.001). d, CD11cϩ
cells
were isolated from total LNs cells suspensions using magnetic beads and
pulsed with 0.1 nM of MHC class-I peptide. OT-I cells were added to the
culture and time-lapse movies were acquired. Dots represent the duration
of contact for each individual DC. Horizontal bars indicate the mean con-
tact duration for WT or WASpϪ
DCs (56 wt and 91 WASp cells were
analyzed; ,ء p Ͻ 0.05). e, DCs pulsed with 0.1 nM peptide were plated in
round or flat-bottom wells. OT-I cells were added to the plate for 12 h. Bars
show the percentage of T cells that up-regulated CD69 after coculture with
WT or WASpϪ
DCs.
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7. surrounding T cells (flat-bottom) whereas priming is normal when
DC and T cells are forced to interact (round-bottom) (Fig. 6e).
To evaluate the impact of defective DC mobility on the ability
to interact with T cells in the complex LN environment, we used
two-photon microscopy. Several previous studies established that
T cells stop their migration and interact for several hours with
mature Ag-presenting DCs during the initial phase of T cell prim-
ing (6–8). To determine the role of WASp expression in DCs on
T cell dynamics within LNs, WT, and WASpϪ
mice were adop-
tively transferred with labeled OT-I and immunized with
␣DEC205-OVA plus ␣CD40, in the same conditions used to study
priming in Fig. 2 and 3. LNs were harvested 20 h after immuni-
zation, a time point that corresponds to the peak of arrest of naive
T cells on Ag-presenting DCs, to track T cells movements (Fig. 7a,
and web movies 3, a and b). Three parameters were quantified: T
cells’ mean velocities, confinement ratio (maximal distance over
total distance), and arrest coefficient (percent of the time during
which each T cell was arrested). In immunized WT recipient mice,
T cells migrated with a low mean velocity (WT ϭ 3.6 Ϯ 2.2
m/min) and within a restricted area, as reflected by their low
confinement ratio (0.38 Ϯ 0.2). A high proportion of cells were
arrested for long periods of time (arrest coefficient: 46 Ϯ 22%).
Thus, as expected, naive T cells are stopped on Ag-presenting DCs
at 20 h after immunization in the WT recipient. In contrast, in
immunized WASpϪ
hosts OT -I cells migrated with increased
mean velocities (Vmean: 5.2 Ϯ 2.6 m/min, p Ͻ 0.001). Further-
more, their migration paths were less restrained and the percentage
of arrested cells was decreased (confinement ratio: 0.5 Ϯ 0.2; p Ͻ
0.001; arrest coefficient: 37 Ϯ 21%; p Ͻ 0.001) (Fig. 7b). These
results indicate that naive T cells arrest less on Ag-bearing DCs in
LNs of WASp null mice. To confirm that lack of T cell arrest in
WASpϪ
mice is due to reduced interaction with DCs, we trans-
ferred WT or WASpϪ
DCs (1:3 ratio) and a fixed number of OT-I
cells into the WT recipient. Analysis of fixed LNs sections showed
a similar distribution of wt and WASpϪ
DCs and OT-I cells in T
cell areas 20 h after transfer (Fig. 8a). At the same time point, we
imaged individual cell movements (Fig. 8b). CD8ϩ
T cells mi-
grated with lower mean velocities and were less stopped in the
FIGURE 7. T cell trajectories in LNs of WT and
WASpϪ
mice. CMTMR-labeled OT-I cells were
adoptively transferred into WT or WASp mice that
were injected with 0.1 g of ␣DEC205-OVA plus
␣CD40. Draining LNs were collected 20 h later and
T cells were imaged by two-photon microscopy
(supplemental movie 3, a and b). a, Migratory paths
of individual OT-I cells along the duration of the
movie (40 min) are indicated on the images. b, Data
on T cell mean velocity, confinement ratio, and T
cell arrest coefficient were pooled from two inde-
pendent experiments (n Ͼ 300 cells/genotype). Hor-
izontal bars indicate mean values within each group
(,ءءء p Ͻ 0.001).
FIGURE 8. Dynamic DC-T interaction in lymph nodes is regulated by WASp expression in DCs. In brief, 0.5 ϫ 106
WT or 1.5 ϫ 106
WASpϪ
DCs
pulsed with 0.1 nM peptide were labeled with CMTMR and injected respectively into the left and right footpad of WT recipients that received CFSE-labeled
OT-I cells. LNs were collected 20 h later for analysis. a, Overall distribution of WT and WASpϪ
DCs (red) and OT-I cells (green) in LNs relatively to B
cell areas (blue). b, Interactions between CD8ϩ
T cells and DCs were imaged during 30 min. Time-lapse two-photon laser scanning microscopy images
of WT and WASpϪ
DCs (red) and T cells (green) from web movies 4, a and b. Colored tracks represent 30Ј lasting paths of individual T cells. c, Mean
velocities (V mean) of T cells in the LN draining WT or WASpϪ
DCs. Dots represent single T cell velocities. Data are pooled from at least 15 different
movies in four independent experiments (,ءءء p Ͻ 0.001). d, Overall duration of contacts established by individual T cells with WT or WASpϪ
DCs (,ءءء
p Ͻ 0.001).
1140 INEFFICIENT CD8ϩ
T CELL PRIMING BY WASpϪ
DCs
byguestonMay2,2015http://www.jimmunol.org/Downloadedfrom
8. presence of WT as compared with WASpϪ
DCs (Vmean: 3.8 Ϯ
2.1 m/min and 4.7 Ϯ 2.1 m/min, p Ͻ 0.0001), indicating that
DC-T interactions were more stable in the presence of WT DCs
(Fig. 8c and web movies 4, a and b). Indeed, the overall mean
duration of individual DC-T interaction was of 23.3 Ϯ 9.2 min for
WT DCs and of 11 Ϯ 3 min for WASpϪ
DCs ( p Ͻ 0.0001) (Fig.
8d). Collectively, these results show that WASp expression in DCs
is required to optimize the encounter and the stable interaction
with T cells within LNs.
Discussion
We have investigated the role of WASp in DCs during synapse
formation and priming of naive CD8ϩ
T cell responses. To selec-
tively assess the impact of altered DC functions to initiation of
adaptive immune responses, we followed activation of naive
CD8ϩ
T cells of WT origin. Our results show that WASp expres-
sion in DCs is important at two levels: 1) trafficking from the
periphery to secondary lymphoid organs and 2) interaction with
and activation of T cells in LNs.
Alterations in the cytokeletal architecture of immature WASp
null DCs have been described in detail (26). However, the defects
that affect mature cells and the impact of DCs dysfunction on prim-
ing of adaptive immunity have just begun to be addressed. On a
functional point of view, the role of WASp in DCs has been mainly
associated to trafficking of immature DCs to secondary lymphoid
organs. A recent report showed that DCs differentiated from the
bone marrow of WASpϪ
animals and adoptively transferred into
WT recipients are poor stimulators of T cell responses because of
altered trafficking to secondary lymphoid organs (21). In vivo, DCs
are a heterogeneous class that comprises different subtypes with
different location and Ag-presenting properties. In several infec-
tious models, initiation of adaptive immunity has been shown to
depend on capture and presentation of Ags by lymphoid organ-
resident DCs (27, 28). In this study, we set up the experimental
model to study the priming potential of endogenous DCs that re-
side in lymphoid organs of WASpϪ
null animals. The Ab against
DEC-205 coupled to OVA is a powerful tool to study the functions
of untouched DCs in vivo. Its ability to induce cross-presentation
of exogenous Ags by CD11cϩ
CD8␣ϩ
DCs in LNs is well docu-
mented (7, 29). We found that WASp-deficient mice are strongly
impaired in presenting Ag to naive CD8ϩ
T cells following im-
munization with DEC205, an effect that was especially evident at
low Ag doses. In this model, we cannot exclude that impaired
trafficking of Ag-bearing Langerhans cells to LNs contributes to
the observed defect. However, this is unlikely because peripheral
DCs start to present Ag between day 2 and 3 postinfection (4, 30),
when OT-I have already undergone more than seven division cy-
cles. The additional evidence that WASp expression in DCs is
important to prime naive T cells within LNs is given by the adop-
tive transfer experiments. These data (Figs. 4 and 5) demonstrate
that correction of defective migration is not sufficient to rescue T
cell priming. We ruled out that altered T cell priming arises from
mislocalization of DCs within lymph nodes because WT and mu-
tant cells differentially labeled and coinjected were found to home
to the same zones in the draining LN. This result contrasts a recent
report showing that the few WASpϪ
DCs that arrive to LNs lo-
calize inefficiently to T cell areas (21). DCs already in LN affect
the behavior of incoming DCs (31) thus, the discrepancy may de-
pend on the fact that we compared localization of equal absolute
numbers of WT and WASpϪ
DCs. Therefore, we conclude that the
primary intrinsic defect is migration to rather than localization
within lymphoid organs.
This study discloses a second important mechanism to explain
defective priming by WASp null DCs, i.e., the reduced capacity to
stabilize the interaction with T cells. So far, the analysis of the role
of WASp in synapse formation was limited to T cells, whereas we
show in this study that WASp is required also on the other side of
the immune synapse. The importance of the DC cytoskelton during
priming has been highlighted by previous studies (22, 32, 33). DCs
use a pathway that depends on the small Rho GTPases Rac to
extend polarized ruffles that help to actively capture T cells. Ma-
ture WASpϪ
cells extend ruffles similarly to WT cells, indicating
that WASp does not control peripheral actin protrusions in mature
cells. Instead, inspection of time-lapse movies suggests that a pro-
portion of WASp null DCs fail to perform net translocation toward
the T cell, thus reducing the number of T cells that are engaged
with DCs. This does not depend on altered adhesive properties
because levels of ICAM-1 are similar in WT and WASpϪ
cells
(data not shown). We are currently investigating whether those
synapses that still form between WASp null DCs and T cells are
functional in terms of signaling. Imaging of cell movements in
LNs proves that WASp expression in DCs is necessary to stably
interact with T cells in vivo. Indeed OT-I cells are less stopped on
Ag-bearing DCs in LNs of WASpϪ
mice. In addition, quantifica-
tion of DC-T interactions in lymph nodes evidenced a reduced
contact duration with WASpϪ
DCs. The T cell division profiles
induced by WASpϪ
DCs in vivo (both adoptively transferred and
endogenous) indicates that OT-I cells do encounter Ag-bearing
WASpϪ
DCs because they enter division, but they fail to fully
divide and accumulate. These data are in agreement with a recent
report indicating that long-lasting stable interaction are required
for full T cell expansion (24, 34). However, it remains to be es-
tablished the long-term fate of T cells primed by WASp deficient
DCs in terms of memory development.
WASp knockout mice models have proved useful to understand
the cellular basis of the disease pathogenesis. Previous analysis of
different hematopoietic cells ex vivo identified a common inability
to properly reorganize the cytoskeleton in response to environmen-
tal and Ag-specific signals (16, 35, 36). Few studies have assessed
in an integrated fashion the ability of WASp null animals to initiate
T cell immune responses in vivo (16, 37). Our results on naive
CD8ϩ
T cell activation by endogenous DCs in WASp null mice
extend recent data on priming by BM-DCs by providing a mech-
anism for impaired T cell priming. Altogether these findings high-
light the crucial relevance of WASp function in DCs during initi-
ation of adaptive immune response. This has important clinical
implications because current gene therapy protocols have docu-
mented mainly the functional reconstitution of the T cell compart-
ment (38, 39) whereas our data indicate that CD8ϩ
T cells of WT
origin are not properly primed by WASp-deficient DCs.
Acknowledgments
We are grateful to Clotilde Thery, Claire Hivroz, and Stephanie Hugues for
critical reading. We thank Mauro Sturnega for mice genotyping and animal
handling.
Disclosures
The authors have no financial conflict of interest.
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