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The Journal of Immunology, 2008, 181, 1135 -1142
Copyright © 2008 by The American Association of Immunologists, Inc.
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Expression of Wiskott-Aldrich Syndrome Protein in Dendritic Cells Regulates Synapse Formation and Activation of Naive CD8+ T Cells1

Julian Pulecio2,*, Elisa Tagliani2,*, Alix Scholer{ddagger}, Francesca Prete*, Luc Fetler{dagger}, Oscar R. Burrone* and Federica Benvenuti3,*

* International Centre for Genetic Engineering and Biotechnology, Trieste, Italy; {dagger} Centre National de la Recherche Scientifique UMR 168, Laboratoire Physico-Chimie Curie, Institut Curie, Paris, France; and {ddagger} Institut National de la Santé et de la Recherche Medicale U653, Immunité et Cancer, Pavillon Pasteur, Institut Curie, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The Wiskott-Aldrich syndrome protein (WASp) is a key regulator of actin polimerization in hematopoietic cells. Mutations in WASp cause a severe immunodeficiency characterized by defective initiation of primary immune response and autoimmunity. The contribution of altered dendritic cells (DCs) functions to the disease pathogenesis has not been fully elucidated. In this study, we show that conventional DCs develop normally in WASp-deficient mice. However, Ag targeting to lymphoid organ-resident DCs via anti-DEC205 results in impaired naive CD8+ T cell activation, especially at low Ag doses. Altered trafficking of Ag-bearing DCs to lymph nodes (LNs) accounts only partially for defective priming because correction of DCs migration does not rescue T cell activation. In vitro and in vivo imaging of DC-T cell interactions in LNs showed that cytoskeletal alterations in WASp null DCs causes a reduction in the ability to form and stabilize conjugates with naive CD8+ T lymphocytes both in vitro and in vivo. These data indicate that WASp expression in DCs regulates both the ability to traffic to secondary lymphoid organs and to activate naive T cells in LNs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cells (DCs)4 are potent inducers of naive CD8+ T cell responses. Most cytotoxic responses against infected cells and intracellular pathogens require the cross-presentation of exogenous Ags on MHC class I products. Cross-presentation in vivo is mainly accomplished by the CD8{alpha} subset of blood-derived lymph node (LN)-resident DCs, which possess a specialized machinery to preferentially drive the formation of MHC class-I complexes from internalized Ags (1, 2, 3). Two pathways contribute to Ag acquisition by LN-resident DCs: direct uptake of soluble Ags drained from the periphery and Ag transfer from skin emigrant and blood circulating DCs that have captured the Ag in the periphery (4, 5). Thus, efficient priming of cytotoxic responses depends on the ability of DCs to carry Ags from the periphery to the draining LN and to present it to naive T cells once in the LN. Both of these two functions are tightly regulated by cytoskeletal dynamics. Actin polimerization provides the mechanical force to exit tissues and to migrate along chemotactic gradients toward the LN. In LNs, DCs scan the T cell repertoire in search of rare T cells presenting a matching TCR. Upon Ag recognition, T cells stop migrating and engage in long-lasting stable contact with the DCs body, with a kinetic that varies depending on the activation state of the DCs (6, 7). In vitro and in vivo imaging of single-cell dynamics showed that an intact DCs cytoskeleton is important to establish and maintain interaction with T cells (8, 9, 10).

The Wiskott-Aldrich syndrome is a primary immunodeficiency caused by mutations in Wiskott-Aldrich syndrome protein (WASp), a key regulator of actin polimerization in hematopoietic cells (11). The syndrome is characterized by a failure to correctly initiate and control adaptive immune responses, resulting in increased susceptibility to infections and high incidence of autoimmune phenomena. Extensive investigations demonstrated that WASp deficiency in T cells affects signal transduction upon TCR engagement and suppression in regulatory T cells (12, 13, 14, 15). Several evidences suggest that altered Ag presentation may also affect immune responses in WAS patients (16, 17, 18, 19). However, the mechanism and the extent of such contribution are just beginning to be elucidated. It was previously established that WASp-null DCs fail to assemble specialized substratum contacts points called podosomes and fail to respond to chemotactic gradients. In vivo, DCs migrate inefficiently to the secondary lymphoid and this has been correlated to reduced priming of T cells responses (20, 21).

In this study, we studied priming of naive CD8+ T cells by adoptively transferred or lymphoid-resident WASp– DCs. By in vitro and in vivo imaging, we show that inefficient T cell priming by WASp null DCs depends on a dual mechanism of altered trafficking from periphery to secondary lymphoid organs and defective priming within LNs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

WASp– mice on a C57BL/6 (CD45.2) genetic background were a gift from S. Snapper (Massachusetts General Hospital, Boston, MA). Mice were bred and maintained in sterile isolators. In vivo experiments were performed using 6- to 8-wk-old WASp– females and control C57BL/6 females purchased from Harlan. OVA specific, MHC class I restricted, 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; December 12, 1987).

Cells

Bone marrow-derived DCs (BM-DCs) were differentiated in vitro from the bone marrow of wild-type (WT) or WASp– mice as described previously (22). DCs were used for experiments between days 8 and 10 when expression 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 Cd11c+ beads (Miltenyi Biotec).

Abs and reagents

The following Abs for FACS analysis were purchased from BD Pharmingen: FITC and PE-conjugated anti-CD11c, FITC, and PE-conjugated anti I-Ab, PE-conjugated antiCD86, PE-conjugated anti-CD11b, PE-Cy5 conjugated anti-CD8, PE-conjugated anti-CD45.1, biotinilated anti-CD69, biotinilated 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 purified by reverse phase HPLC and purity was verified mass spectrometry.

Time-lapse video microscopy

For the dynamic analysis of DCs trajectories, 3 x 105 immature or LPS-pulsed DCs (overnight, 10 µg/ml) were plated on fibronectin coated coverslips 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 x63 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 x 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 WASp– BM-DCs were harvested at day 8 and labeled with 2 µM of CFSE (Molecular Probes) according to the manufacturer’s instructions. After labeling, 5 x 105 to 2 x 106 cells, depending on the experiments, were injected s.c. into the footpad of the C57BL/6 host. For cotransfer experiments, 2 x 105 WT DCs labeled with carboxy-SNARF (Molecular Probes) were mixed with 6 x 105 WASp– 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 postinjection. The absolute number of CFSE+/CD11c+ 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 x 105 WT DCs (SNARF labeled) and 6 x 105 WASp– 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 µM) were fixed in cold acetone and analyzed using the following 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 {alpha}DEC205-OVA, we performed an in vitro assay of OT-I activation using conditions that bypasses defects in conjugate formation. Five x 104 immature DCs isolated from LNs of WT or WASp– mice were incubated with increasing doses of the Ag for 3 h in the presence of 1 µg/ml LPS. After washing, 1 x 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 SIINFEKL peptide, washed, and transferred into round-bottom or flat-bottom wells (2 x 104/wells). Two x 104 OT-I cells were added to the wells and harvested 24 h later to quantify the percentage of CD8+ cells that up-regulated CD69+.

Adoptive transfer and T cell activation

One x 106 OT-I/CD45.1 cells were purified as described above and injected i.v. into the recipient host. For priming with BM-DCs, 2 x 105 WT or 6 x 105 WASp– 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 draining 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 comparison 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 {alpha}DEC205OVA, mice were adoptively transferred with OT-I, as above, and injected s.c. with graded dosed of {alpha}DEC205-OVA plus 10 µg 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 WASp– recipient mice were injected i.v. with 10 x 106 CMTMR-stained (10 µM) OT-I cells. One hour later, mice were injected s.c. in the hind footpad with 0.1 µg of {alpha}DEC205-OVA plus 10 µg 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 x 106 or 1.5 x 106 WT and WASp– DCs pulsed with 0.1 nM peptide were labeled with CMTMR (10 µM) 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 temperature 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 contingency tables.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Ag targeting to endogenous DCs in WASp– mice induce faint activation of T cells

We set out to study the ability of DCs resident in lymphoid organs of WASp– null animals to crosspresent exogenous Ags to naive CD8+ T cells. We first examined DCs frequencies and subtypes composition in WASp– mice. The frequency of total conventional CD11chigh DCs in LNs of WT and WASp– mice was similar and within the expected range (around 2% of total cells). Within total CD11c+ cells in LNs we found the same percentage of tissue-derived DCs (CD11c+ MHCIIhigh) in WT and mutant animals indicating normal trafficking from the periphery to the LN at steady state (Fig. 1a). Blood-derived CD8{alpha} and CD11b cells were also present in equal numbers in LNs (Fig. 1b) and spleen (data not shown) of WT and WASp– animals. Thus, WASp is dispensable for DCs development in vivo.


Figure 1
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  		FIGURE 1. DCs frequencies and subtype composition in WT and WASp– mice. Total LN cell suspensions were enriched in DCs by magnetic 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: CD11c+/MHCIIhigh, Langerhans and interstitial DCs; Gate B: CD11chigh/MHCIIint, blood-derived DCs (left panels). Percentage of total CD11c+ cells in LNs of WT and WASp– mice. The results are the means ± 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{alpha}+CD11c+ and CD11b+CD11c+ cells. Mean percentage of CD8{alpha}+CD11c+ and CD11b+CD11c+ over total CD11c in LNs of eight WT and eight WASp mice.

 
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 ({alpha}DEC205-OVA) in the presence of anti-CD40 Ab ({alpha}CD40). Immunization with {alpha}DEC205-OVA leads to cross-presentation of the MHC class I epitope of OVA mainly by CD8+ LN-resident DCs and the adjuvant {alpha}CD40 is required to expand long term memory CD8+ 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 analyzed responses of OVA-specific TCR transgenic T cells (OT-I) that are of WT origin. WT and WASp– mice were adoptively transferred with CFSE-labeled OT-I cells followed by s.c. immunization with different doses of {alpha}DEC205-OVA plus {alpha}CD40. The maturation profile of DCs after anti-CD40 treatment is equivalent in WT and WASp– 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 µg of {alpha}DEC205-OVA induced up-regulation of CD69 on 49% of OT-I cells whereas activation was almost undetectable at this dose in WASp– host. At higher Ag dose, OT-I cells were primed also in WASp– mice, yet at lower levels that in WT. The mean reduction in the percentage of OT-I cells that up-regulated CD69 at 24 h in WASp– mice at 0.1 and 1 µg of {alpha}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 CD8+ T cell activation downstream of Ag processing.


Figure 2
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  		FIGURE 2. Early events of T cell activation induced by Ag targeting to DCs in vivo. a, WT or WASp– mice were adoptively transferred with 1 x 106 CFSE-labeled OVA specific OT-I cells (expressing the CD45.1 congenic marker) followed by immunization with 0.1 or 1 µg of {alpha}DEC205-OVA plus {alpha}CD40 (10 µg). 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 represented as the percentage of inhibition of the frequency of CD69high OT-I cells in LNs of WASp– animals relative to WT. Results are pooled from three independent experiments with three mice/condition. c, CD11c+ cells were purified from LNs of WT and WASp– mice and pulsed in vitro with the indicated doses of {alpha}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 ({nu}) or WASp– (µ) DCs.

 
We next analyzed the proliferation of OT-I cells primed in a WT or WASp– context. Mice were immunized with increasing doses of {alpha}DEC205-OVA plus {alpha}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 WASp– than in WT mice (12.5 ng WT vs 50 ng WASp–). Increasing the Ag dose induced entry in cycle of the majority of the OT-I. However, T cells primed in WASp– mice remained in large proportions within the seventh cycle and only few cells beyond the seventh division accumulated in LNs of WASp– mice (Fig. 3b). Quantification of Ag specific T cell expansion indicates that accumulation of OT-I cells was dramatically decreased in WASp– 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.


Figure 3
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  		FIGURE 3. Activation of naive WT CD8+ T cells by endogenous DCs is highly compromised in WASp– mice. Mice were adoptively transferred with Ag-specific naive CD8+ T cells (CD45.1) followed by immunization with different doses {alpha}DEC205-OVA plus {alpha}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 CD8+ cells. Values are pooled from three independent experiments with three mice per condition.

 
Correction of defective migration to LNs does not rescue CD8+ T cell priming

The above results indicate that priming of CD8+ 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-derived WT or WASp– 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 WASp– 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 WASp– DCs to prime naive T cells within LNs, we set the conditions to bypass defective homing by increasing the input of WASp– DCs. To achieve comparable numbers of cells in LNs we injected three times as much WASp– DCs than WT DCs. Under these conditions the total number 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 WASp– DCs (data not shown). To study the localization of migrated DCs within LNs, we performed tissue immunofluorescence 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 WASp– cells coinjected in the same LN (Fig. 4b). This result indicates that WASp expression is important for migration to, rather than localization within, LNs.


Figure 4
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  		FIGURE 4. DC migration to draining LNs. A mix of 2 x 105 WT and 6 x 105 WASp– 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 WASp– DCs migrated to lymph nodes 24 h after injection. Bars represent the absolute number of WT and WASp– DCs recovered in LNs at different time points after injection. Results are the means ± SD of three injected mice/group/time point. b, LNs sections were labeled with Abs against B cells (B220), T cells (CD3), and high endothelial venules (HEV) to analyze the relative distribution of WT and WASp– DCs.

 
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 CD8+ 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 WASp– 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 WASp– 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 WASp– 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 CD8+ T cells but do not provide the signals to achieve full expansion.


Figure 5
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  		FIGURE 5. CD8+ T cell priming induced by WT and WASp– 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 x 105 WT or 6 x 105 WASp– DCs loaded with the indicated doses of MHC class-I OVA peptide. Values show percentage of OT-I cells over the total CD8+ population at day 3 postimmunization (gated on CD45.1/CD8+ cells) and are the means ± SD of four mice per group. b, Histogram plots show the CFSE dilution profile of transferred OT-I cells (gated on CD8+/CD45.1+) in draining LNs at day 3. Values are means ± 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).

 
We conclude that WASp expression in DCs is important to initiate CD8+ T cells responses at two levels: by promoting the migration 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 conjugate 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 CD8+ 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 interference 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 extensive ruffling activity and moved on fibronectin with a mean velocity of 0.080 ± 0.005 µm/sec (n = 36). Ruffling activity was maintained in WASp– DCs but net translocation was reduced because the cell body remained anchored to the matrix. The mean velocity of WASp– DCs was reduced to 0.059 ± 0.004 µm/sec (n = 31; p < 0.01) (Fig. 6b). DC movements facilitate the scanning 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 WASp– DCs failed to establish tight contacts with T cells despite 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 WASp– DCs establish only few long contacts in the absence of OVA-specific peptide (WT = 13.7%; WASp– = 14.5%). Addition of 0.1 nM of OVA peptide induced a high proportion of WT DCs to form long-lasting contacts with Ag specific T cells (47 ± 0.13%). In contrast, peptide loading on WASp– DCs induced only a modest increase in the percentage of long lasting interactions (21 ± 0.15%; p < 0.001) (Fig. 6c). We confirmed these observations using DCs freshly isolated from the LNs of WT and WASp– mice. CD11c+ 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 WASp– DCs (WT = 18.58 min ± 1.41; WASp– = 13.05 min ± 1.73, p < 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 established 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 WASp– DCs are impaired when DCs need to patrol for the presence of T cells and to capture and stabilize interaction with surrounding T cells (flat-bottom) whereas priming is normal when DC and T cells are forced to interact (round-bottom) (Fig. 6e).


Figure 6
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  		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 WASp– (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 WASp– 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 WASp– respectively (**, p < 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 >20 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 WASp–cells were analyzed, ***, p < 0.001). d, CD11c+ 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 contact duration for WT or WASp– DCs (56 wt and 91 WASp cells were analyzed; *, p < 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 WASp– DCs.

 
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 priming (6, 7, 8). To determine the role of WASp expression in DCs on T cell dynamics within LNs, WT, and WASp– mice were adoptively transferred with labeled OT-I and immunized with {alpha}DEC205-OVA plus {alpha}CD40, in the same conditions used to study priming in Fig. 2 and 3. LNs were harvested 20 h after immunization, 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 = 3.6 ± 2.2 µm/min) and within a restricted area, as reflected by their low confinement ratio (0.38 ± 0.2). A high proportion of cells were arrested for long periods of time (arrest coefficient: 46 ± 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 WASp– hosts OT -I cells migrated with increased mean velocities (Vmean: 5.2 ± 2.6 µm/min, p < 0.001). Furthermore, their migration paths were less restrained and the percentage of arrested cells was decreased (confinement ratio: 0.5 ± 0.2; p < 0.001; arrest coefficient: 37 ± 21%; p < 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 WASp– mice is due to reduced interaction with DCs, we transferred WT or WASp– 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 WASp– 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). CD8+ T cells migrated with lower mean velocities and were less stopped in the presence of WT as compared with WASp– DCs (Vmean: 3.8 ± 2.1 µm/min and 4.7 ± 2.1 µm/min, p < 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 ± 9.2 min for WT DCs and of 11 ± 3 min for WASp– DCs (p < 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.


Figure 7
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  		FIGURE 7. T cell trajectories in LNs of WT and WASp– mice. CMTMR-labeled OT-I cells were adoptively transferred into WT or WASp mice that were injected with 0.1 µg of {alpha}DEC205-OVA plus {alpha}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 independent experiments (n > 300 cells/genotype). Horizontal bars indicate mean values within each group (***, p < 0.001).

 

Figure 8
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  		FIGURE 8. Dynamic DC-T interaction in lymph nodes is regulated by WASp expression in DCs. In brief, 0.5 x 106 WT or 1.5 x 106 WASp– 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 WASp–DCs (red) and OT-I cells (green) in LNs relatively to B cell areas (blue). b, Interactions between CD8+ T cells and DCs were imaged during 30 min. Time-lapse two-photon laser scanning microscopy images of WT and WASp– DCs (red) and T cells (green) from web movies 4, a and b. Colored tracks represent 30' lasting paths of individual T cells. c, Mean velocities (V mean) of T cells in the LN draining WT or WASp– DCs. Dots represent single T cell velocities. Data are pooled from at least 15 different movies in four independent experiments (***, p < 0.001). d, Overall duration of contacts established by individual T cells with WT or WASp– DCs (***, p < 0.001).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have investigated the role of WASp in DCs during synapse formation and priming of naive CD8+ T cell responses. To selectively assess the impact of altered DC functions to initiation of adaptive immune responses, we followed activation of naive CD8+ T cells of WT origin. Our results show that WASp expression 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 priming 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 WASp– 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 infectious 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 reside in lymphoid organs of WASp– 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 CD11c+CD8{alpha}+ DCs in LNs is well documented (7, 29). We found that WASp-deficient mice are strongly impaired in presenting Ag to naive CD8+ T cells following immunization 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 cycles. The additional evidence that WASp expression in DCs is important to prime naive T cells within LNs is given by the adoptive 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 mutant 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 WASp– DCs that arrive to LNs localize inefficiently to T cell areas (21). DCs already in LN affect the behavior of incoming DCs (31) thus, the discrepancy may depend on the fact that we compared localization of equal absolute numbers of WT and WASp– 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. Mature WASp– 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 proportion 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 WASp– 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 WASp– mice. In addition, quantification of DC-T interactions in lymph nodes evidenced a reduced contact duration with WASp– DCs. The T cell division profiles induced by WASp– DCs in vivo (both adoptively transferred and endogenous) indicates that OT-I cells do encounter Ag-bearing WASp– 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 established 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 environmental 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 CD8+ T cell activation by endogenous DCs in WASp null mice extend recent data on priming by BM-DCs by providing a mechanism for impaired T cell priming. Altogether these findings highlight the crucial relevance of WASp function in DCs during initiation of adaptive immune response. This has important clinical implications because current gene therapy protocols have documented mainly the functional reconstitution of the T cell compartment (38, 39) whereas our data indicate that CD8+ 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This research was supported by Telethon Grant GGP06267. Back

2 J.P. and E.T. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Federica Benvenuti, Molecular Immunology, International Centre for Genetic Engineering and Biotechnology, Padriciano 99, Trieste, Italy. E-mail address: benvenut{at}icgeb.org Back

4 Abbreviations used in this paper: DC, dendritic cell; WASp, Wiskott-Aldrich syndrome protein; LN, lymph nodes; BM, bone marrow derived; WT, wild type; OT-I cells, OVA-specific CD8 T cell; CMTMR, orange fluorescent tetramethylrhodamine cell tracker orange. Back

5 The online version of this article contains supplemental material. Back

Received for publication October 9, 2007. Accepted for publication May 8, 2008.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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