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The Journal of Immunology, 2007, 178: 2973-2978.
Copyright © 2007 by The American Association of Immunologists, Inc.

CC Chemokine Receptor 7 Contributes to Gi-Dependent T Cell Motility in the Lymph Node1

Takaharu Okada and Jason G. Cyster2

Department of Microbiology and Immunology and Howard Hughes Medical Institute, University of California, San Francisco, CA 94143


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Naive T cells migrate extensively within lymph node (LN) T zones to scan for Ag-bearing dendritic cells. However, the extracellular signals controlling T cell motility in LNs are not well defined. In this study, by real-time imaging of LNs, we show that the inhibition of Gi signaling in T cells severely impairs their migration. The chemokine CCL21, a ligand of CCR7, strongly induces chemokinesis in vitro, and T cell motility in LNs from CCR7 ligand-deficient plt/plt mice was reduced. CCR7-deficient T cells in wild-type LNs showed a similar reduction in motility, and antagonism of CXCR4 function did not further decrease their motility. The effect of CCR7 or CCR7-ligand deficiency could account for ~40% of the Gi-dependent motility. These results reveal a role for CCR7 in promoting T cell migration within lymphoid organ T zones, and they suggest the additional involvement of novel Gi-coupled receptors in promoting T cell motility at these sites.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Migration of T cells within the secondary lymphoid organs contributes to rapid scanning for Ags (1, 2). Recent real-time imaging studies by multiphoton microscopy revealed that T cells migrate at 10–12 µm/min in lymph nodes (LNs),3 and this extensive migration together with vigorous extension of processes by relatively sessile dendritic cells (DCs) enables each DC to contact as many as 500~5000 naive T cells per hour (3, 4).

Chemokines are the key factors orchestrating lymphocyte compartmentalization within lymphoid organs (5). However, their role in controlling lymphocyte motility in lymphoid organ subcompartments is not clearly defined. In the T cell zone of LNs, multiple chemokines are expressed including CCL19 and CCL21, the receptor of which is CCR7, and CXCL12, the ligand for CXCR4 (5, 6). It is known that some chemoattractants enhance leukocyte migration even in the absence of concentration gradients, which is referred to as chemokinesis (7, 8, 9, 10). Because migration of T cells in the T zone does not show apparent directionality (11), it has been speculated that chemokine-induced chemokinesis may be involved.

Chemokine receptors signal to mediate chemotaxis predominantly by activating G{alpha}i-containing heterotrimeric G-proteins (5). B cells deficient in Gnai2, one of two G{alpha}i proteins expressed in B cells, were reported to have 20% reduced motility in LNs (12). However, the impact of more complete inhibition of Gi signaling on lymphocyte motility in LNs has not been reported. Pertussis toxin (PTX) is a multisubunit enzyme that ADP-ribosylates a cysteine in the carboxyl-tail of G{alpha}i and renders the heterotrimeric Gi complex incapable of coupling to receptors (13). Although Gi signaling is essential for lymphocyte entry into LNs, we recently developed a PTX pulse-loading and adoptive transfer procedure in which Gi signaling is inhibited after the treated cells have entered recipient LNs (14). This procedure allowed us to analyze the effect of near complete inhibition of Gi signaling in T cells on their migratory behavior in the LN parenchyma without detectably perturbing Gi signaling in other cells. We found a striking requirement of Gi signaling for T cell motility in LNs. We also found that CCR7 and its ligands are required for optimal migration of T cells in LNs, although CCR7 only partially accounts for the Gi signaling requirement.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice, cells, Abs

C57BL/6 (B6) and B6-CD45.1 mice were obtained from The Jackson Laboratory or National Cancer Institute. CCR7-deficient mice (15) and plt/plt mice (16) were 10 generations backcrossed with B6. T cells were purified from spleen and LN cells by immunomagnetic depletion of B cells, DCs, macrophages, granulocytes, and erythrocytes using autoMACS (Miltenyi Biotec). B cells were purified from splenocytes of B6 mice by depleting CD43-expressing cells. Abs used for depletion and staining were purchased from BD Biosciences, Invitrogen Life Technologies, eBioscience, and Jackson ImmunoResearch Laboratories.

Adoptive transfer

Purified T and B cells were labeled with 10 µM CFSE (Molecular Probes), 20 µM 5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTMR; Molecular Probes), or 50 µM 7-amino-4-chloromethylcoumarin (CMAC; Molecular Probes) at 37°C for 25 min. For analysis of PTX-treated T cell motility, recipients received CFSE- or CMTMR-labeled T cells treated with saline (5 x 106), 200 ng/ml PTX (Sigma-Aldrich and List Biological Laboratories) (9 x 106), or 200 ng/ml B oligomer (List Biological Laboratories) (5 x 106) in RPMI 1640 containing 2% FBS at 37°C for 10 min and washed three times at room temperature. Treated T cells were transferred into recipient mice that had received CMAC-labeled B cells 1 day before. Two hours after T cell transfer, one inguinal LN from each recipient was used for imaging, and cells from the other were subjected to Transwell assays. It was necessary to transfer more PTX-treated than control T cells in these experiments because the treatment causes a gradual reduction in entry efficiency over the 2-h period. We did not observe any adverse effect of the PTX treatment on cell viability. Transfers into plt/plt mice for imaging involved 107 CFSE-labeled T cells (4 x 106 for wild-type controls) and 4 x 106 CMTMR-labeled B cells for 1 day. For analysis of CCR7-deficient T cell motility, 4 x 106 B6 T cells and 15 x 106 CCR7-deficient T cells labeled with CFSE or CMTMR and B cells labeled with CMAC were cotransferred into B6 mice. One day after transfer, mice were either left untreated or implanted s.c. in the back with osmotic pumps (1-day duration, 8 µl/h pumping rate; Model 2001D; Durect) loaded with 40 mg/ml of the CXCR4 antagonist 4F-benzoyl-TN14003 (6, 17) for 1 day. As an analgesic after surgery, 0.05–0.1 mg/kg of buprenorphine (Sigma-Aldrich) was given s.c. For analysis of the surface phenotype of transferred T cells in LNs, 30 or 90 million splenocytes from B6-CD45.1 mice were transferred for 1 day into B6 or plt/plt recipients, respectively, and 26 million B6-CD45.1 splenocytes and 108 million CCR7-deficient splenocytes, both labeled with 1.5 µM CFSE, were cotransferred into B6 mice for 1 day. Protocols were approved by the Institutional Animal Care and Use Committee of the University of California San Francisco.

Two-photon imaging and analysis

Imaging of excised LNs was performed as described previously with some modifications (18). Briefly, inguinal LNs were isolated, maintained in 36°C RPMI 1640 bubbled with 95% O2/5% CO2, and imaged through the capsule in a region distal to the efferent lymphatic by two-photon microscopy. Each x-y plane spanned 240 µm by 288 µm at a resolution of 0.6 µm per pixel, and images of 25–36 x-y planes with 3-µm z spacing were formed by averaging 10 video frames, using emission wavelengths of 500–540 nm (for CFSE-labeled cells), 567–640 nm (for CMTMR-labeled cells), 455–485 nm (for CMAC-labeled cells), and 360–440 nm (to detect second harmonic emission) every 20 s. Image acquisition was performed using Video Savant software (IO Industries). The z-projection videos were made using MetaMorph (Molecular Devices). Three-dimensional cell tracking was performed by using Imaris 5.0.1 x 64 (Bitplane). Motility coefficient was calculated as M = D2/6t, where M is motility coefficient, D displacement, and t time (19) using 10-min tracks.

Transwell migration assay

Transwell migration assays were performed with spleen or LN cells as described previously (6). Diluted chemokine was placed either only in the bottom wells or both in the top and bottom wells at the concentrations indicated. In some experiments, cells were treated with 200 ng/ml PTX for 2 h before assays.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Gi signaling is essential for T cell motility in LNs

To examine the role for Gi signaling in promoting T cell motility within LNs, we pulsed T cells with PTX and cotransferred them with saline-treated control T cells into recipient mice. PTX causes gradual enzymatic inactivation of G{alpha}i and after cell loading Gi signaling is not inhibited for 60–90 min, allowing time for chemokine-mediated entry of transferred cells into LNs (14). Two hours after transfer, both saline- and PTX-treated T cells are localized uniformly in the T cell zone; by 8 h, PTX-treated cells were mostly localized in the peripheral area of the T cell zone (data not shown). Transwell assays with recipient LNs 2.5 h after transfer showed that migration of PTX-treated T cells to 3 µg/ml CCL21 was inhibited (6.8% input migrated vs 2.3% in the absence of chemokine) to a similar extent as cells exposed to PTX continuously for 2 h (4.1% migrated), whereas migration of cotransferred saline-treated T cells was unaffected (82.3% migrated vs 77.5% for T cells in a control LN). Multiphoton imaging at the 2–3 h time point revealed that migration of PTX-treated T cells within LNs was strikingly perturbed compared with saline-treated control T cells (Fig. 1; see also Supplemental Video 1).4 PTX-treated T cells scarcely showed stretches of migration longer than 5 µm within every 20 s (Fig. 1D), and their median velocity was 2-fold less than that of control T cells (Fig. 1E). The majority of PTX-treated cells showed only marginal displacement throughout the imaging time of 30 min to 1 h (Fig. 1C and F), and this corresponded to a 90% reduction in mean motility coefficient (Fig. 1F). The more pronounced suppression in displacement than in velocity was due to the fact that PTX-treated T cells had broader turning angles (mean 72.4 ± 1.1°, where 0° indicates no turns) than saline-treated T cells (44.0 ± 2.4°). We also examined the motility of T cells treated with B oligomer, the non-ADP ribosylating subunit of PTX, and found it virtually identical to that of saline-treated cells (Fig. 1, D–F). These results indicate an essential role of autonomous Gi signaling for T cell motility within LNs.


Figure 1
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  		FIGURE 1. G{alpha}i signaling is essential for T cell motility in LNs. A, A 75-µm z-projection of a two-photon image stack of an inguinal LN from a mouse that received fluorescently labeled, B cells (blue), saline-treated T cells (green), and PTX-treated T cells (red). B and C, Ten-minute tracks of saline-treated T cells (B) and PTX-treated T cells (C) in the 120 µm (x) x 120 µm (y) x 75 µm (z) box indicated in A. Tracks of migration start in blue and end in white. D, Velocity distribution for saline-, PTX-, or B oligomer-treated T cells. The velocity data were obtained from movements in every 20 s, and grouped into bins with 1 µm/min width. The data for each kind of T cell are from three imaging data sets (five imaging data sets from four independent experiments in total). Twenty to 56 cells of each kind were tracked for >10 min from each data set. E, Median velocity was obtained for individual cells, and averaged for each kind of T cell in each imaging experiment. The histogram shows mean and SD of median velocities from three imaging data sets. *, p < 0.05; **, p < 0.01. F, Displacement from starting coordinates of tracks is plotted against square root of time. The data for each kind of T cell are from three imaging data sets. Difference in displacement between saline-treated T cells and PTX-treated T cells was statistically significant (p < 0.05) between 0.58 min1/2 and 3.16 min1/2. Mean motility coefficient 3.8 ± 1.8 µm2/min for PTX-treated T cells and 44.2 ± 16.4 µm2/min for saline-treated T cells.

 
CCL21 induces strong gradient-independent T cell migration in vitro

Because migration of T cells in the T cell zone is seemingly random in directionality (20), chemokinesis is likely to be involved in Gi-dependent T cell migration. Therefore, we tested the chemokines expressed in the T cell zone for their potency to induce T cell chemokinesis by Transwell migration assay. As shown in Fig. 2A, CCL21 induced remarkable T cell chemokinesis at 3 µg/ml but not at 1 µg/ml or lower concentrations. The chemokinesis by CCL21 was abrogated by PTX-treatment (Fig. 2A). In contrast, CXCL12 induced only marginal chemokinesis even at 3 µg/ml (Fig. 2B).


Figure 2
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  		FIGURE 2. CCL21 induces T cell chemokinesis. Plotted is percentage of T cells that migrated in response to the indicated concentration of CCL21 (A) or CXCL12 (B) in the bottom wells only ({circ}, dashed line) or both in the top and bottom wells (•, filled line). The {blacksquare} in A indicates percentage of T cells that migrated in the presence of 3 µg/ml CCL21 both in the top and bottom wells after 2-h treatment with 200 ng/ml PTX. The data are from three independent experiments.

 
CCR7 signaling is partially responsible for T cell motility in LNs

To test the contribution of CCR7 ligands to T cell motility in LNs, we analyzed the migration of wild-type T cells transferred into plt/plt mice that lack expression of CCL21 and CCL19 in lymphoid organs (21, 22, 23). As shown in Fig. 3 (see also Supplemental Video 2), T cell motility in LNs from plt/plt mice was ~20% lower than that in LNs from wild-type mice. In contrast, B cell motility in plt/plt LNs was not significantly different from that in wild-type LNs (Fig. 3B). The displacement of T cells in plt/plt LNs was notably reduced (Fig. 3, B–D), and the mean motility coefficient was decreased from 52.9 ± 5.6 µm2/min for cells in wild-type LNs to 21.7 ± 1.1 µm2/min for cells in plt/plt LNs. The mean turning angle for T cells in plt/plt LNs (50.5 ± 1.9°) was not significantly different from that for T cells in wild-type LNs (46.3 ± 4.0°). Because CCL21 plays an important role in T cell entry into LN, it was important to test the possibility that different subsets of T cells were enriched in plt/plt LNs compared with the wild-type control LNs. Flow cytometric analysis of surface marker expression showed that the majority of transferred T cells in wild-type or plt/plt LNs had the naive (CD69–CD62LhighCD44low and CXCR5–) T cell phenotype (Fig. 2E). The minor increase in CD62Llow and CD44high memory T cell frequencies in plt/plt LNs cannot account for the global reduction in median velocity observed in these LNs (Fig. 2D).


Figure 3
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  		FIGURE 3. T cell motility is reduced in LNs from plt/plt mice. A, Velocity distribution for T cells in LNs from wild-type and plt/plt mice. B, The histogram shows mean and SD of median velocities for T cells and B cells in wild-type or plt/plt LNs from three imaging data sets. Twenty-five to 52 T cells and 18–30 B cells were tracked for >10 min from each data set. *, p < 0.05. C, Displacement of T cells and B cells in wild-type or plt/plt LNs. Difference in displacement between T cells in wild-type LNs and T cells in plt/plt LNs was statistically significant (p < 0.05) between 1.83 min1/2 and 3.16 min1/2. D, Distribution of median velocity of individual T cells in wild-type and plt/plt LNs. E, The surface phenotype of transferred T cells in wild-type and plt/plt LNs. Cells were stained for the indicated markers. Dotted lines show control staining with no primary Abs. Percentage of cells with nonnaive phenotype for each marker in total transferred T cells in wild-type and plt/plt LNs is indicated.

 
Because it is possible that the T zone structure in plt/plt LNs has developmental alterations, which might indirectly affect T cell migration, we also analyzed the motility of CCR7-deficient T cells transferred into wild-type mice. Although most of the transferred CCR7-deficient T cells localized in the peripheral part of the T zone and at the B-T boundary area, we were able to compare motility of CCR7-deficient T cells and cotransferred wild-type T cells in overlapping regions in the same LNs (Fig. 4A; see also Supplemental Video 3). Similar to findings in plt/plt mice, the migration velocity of CCR7-deficient T cells was reduced by ~20% compared with that of wild-type T cells (Fig. 4, C, D, and F), and the displacement of CCR7-deficient T cells was notably reduced (Fig. 4, B and E). The mean motility coefficient was decreased from 41.4 ± 2.7 µm2/min for wild-type T cells to 25.0 ± 7.7 µm2/min for CCR7-deficient T cells. The mean turning angle was not significantly different between CCR7-deficient T cells (48.7 ± 1.9°) and wild-type T cells (46.3 ± 3.2°). The majority of both wild-type and CCR7-deficient T cells in the recipient LNs had a naive T cell phenotype. Again, although there were minor increases in activated (CXCR5 or CD69-positive) and memory T cells (CD62Llow or CD44high) (Fig. 4G), these cells cannot account for the global reduction in median velocities of the whole CCR7-deficient T cell population (Fig. 4F). We also treated mice receiving CCR7-deficient T cells with the CXCR4 antagonist, 4F-benzoyl-TN14003 (6). The antagonist treatment increased CXCR4 on the surface of all T cells including transferred CCR7-deficient T cells, presumably by blocking CXCL12-triggered internalization (Fig. 4H). The antagonism appeared to be maintained throughout the imaging period in excised LNs because the surface CXCR4 level remained increased (Fig. 4H). However, the motility of wild-type or CCR7-deficient T cells in LNs from antagonist-treated animals was not distinguishable from that in untreated animals (Fig. 4D).


Figure 4
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  		FIGURE 4. CCR7-deficient T cells have reduced motility in LNs. A, Twenty to 30-min tracks of CCR7+/+ T cells and CCR7–/– T cells from the same imaging data set. Tracks start in blue and end in white. B, Superimposed 10-min tracks of 40 randomly selected CCR7+/+ T cells and CCR7–/– T cells in the x-y and x-z plane, setting the starting coordinates to the origin. Units are in micrometers. C, Velocity distribution of CCR7+/+ T cells and CCR7–/– T cells. D, The histogram shows mean and SD of median velocities of CCR7+/+ and CCR7–/– T cells from three imaging data sets of LNs from untreated mice (•) and two imaging data sets of LNs from 4F-benzoyl-TN14003-treated mice ({circ}). Eighteen to 62 cells of each kind were tracked for >10 min from each data set. *, p < 0.05. E, Displacement of CCR7+/+ and CCR7–/– T cells. Difference in displacement between CCR7+/+ and CCR7–/– T cells was statistically significant (p < 0.05) between 1.63 min1/2 and 2.71 min1/2 and between 2.83 min1/2 and 3.00 min1/2. F, Distribution of median velocity of individual CCR7+/+ T cells and CCR7–/– T cells. G, The surface phenotype of transferred CCR7+/+ T cells and CCR7–/– T cells. H, The surface CXCR4 level on transferred CCR7–/– T cells in LNs from nontreated or 4F-benzoyl-TN14003(T140)-treated mice. LNs isolated from recipient mice were either kept on ice or subjected to imaging before staining.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The above findings strongly suggest that Gi signaling is necessary for the majority of T cell motility within LNs. CCR7 and CCR7 ligands are shown to make a significant contribution to promoting the T cell motility, providing evidence that chemokines and chemokine receptors can contribute to promoting nondirectional as well as directional lymphocyte migration within lymphoid tissues. Antagonism of CXCR4 function did not identify a major role for this receptor in promoting T cell motility within the LN T zone. These observations also lead us to conclude that one or more still-to-be-defined Gi-coupled ligand-receptor systems make important contributions to T cell motility in lymphoid tissues.

The more complete block in lymphocyte motility observed in our study than in previous experiments with G{alpha}i2-deficient B cells (12), suggests that additional G{alpha}i proteins contribute to lymphocyte motility or that T cell motility is more Gi dependent than B cell motility. Consistent with the possible involvement of additional G{alpha}i family members, like G{alpha}i2, G{alpha}i3 is abundantly expressed in lymphocytes (24, 25). B cell motility was also only partially inhibited in LNs of mice treated systemically with PTX, although in this case it was noted that Gi function was incompletely blocked (12). It is not clear whether the remaining movement we observed in the marginally displaced PTX-treated T cells is their spontaneous behavior or passive fluctuation caused by the migration of surrounding cells. It is also not clear whether the occasional significant displacement of PTX-treated T cells (arrowhead in Fig. 1C) is due to remaining Gi activity or reflects the involvement of Gi-independent mechanisms of cell migration in some cells.

Our results indicate that CCR7 is active in promoting gradient-independent T cell migration in vitro (Fig. 2). CCL21-induced chemokinesis was also recently reported by Stachowiak et al. (26), using videomicroscopy to analyze in vitro migration of CD4+ T cells on ICAM-1-coated two-dimensional surfaces. Immunohistochemical analysis has shown the T zone to be a broad region of high CCL21 expression, and total tissue analysis indicates that CCL21 is present at 2–10 µg per g of tissue in LNs (roughly corresponding to 2–10 µg/ml) (27, 28, 29). Local concentrations of CCL21 within the LN T zone are likely to be higher than these estimates because the CCL21-positive area accounts for only part of the LN volume. These data suggested that CCL21-driven chemokinesis might contribute to the Gi-dependent T cell migration in the T zone. However, local variations in T zone CCL21 concentration can be observed, with the protein being most abundant on stromal cells (22), and a role for local CCL21 concentration differences in promoting T cell motility is not excluded. A very recent study has provided evidence that T cells tend to migrate along the surface of T zone stromal cells (30), and it may be the case that CCL21 is most active in promoting T cell motility when encountered in a surface-bound form (31). Our studies do not rule out involvement of the second CCR7 ligand, CCL19, which can also promote T cell chemokinesis (data not shown and Ref. 32), although this ligand was found in only low concentrations (~0.01 µg/g) in unstimulated LNs (29). The efficacy of CCR7 ligands in promoting chemokinesis (this study and Refs. 26 , 32) suggests that CCR7 may be especially adapted for promoting and maintaining cell motility for prolonged periods. We speculate that CXCR5 is similarly adapted for promoting prolonged B cell motility within lymphoid follicles.

Our studies show that in addition to the established role of CCR7 in cell recruitment to the T zone (15, 16), CCR7 signaling contributes to T cell motility within this zone. CCR7 signaling accounts for possibly 40% of the Gi signaling function in T cell motility. These conclusions pertain to the region of the T zone adjacent to B cell follicles, areas where CCR7-deficient cells are able to gain access and that are within ~200 µm of the capsule and accessible to multiphoton imaging. Although it is possible that the contribution of Gi and CCR7 to T cell motility in the deeper T zone may differ from these findings, the distribution of CCL21 suggests that CCR7 and CCR7 ligands will have a similar role in this region. The CCR7 requirement for T cells to access this zone provides additional support for this view. Because we previously saw an overlapping role for CCR7 and CXCR4 in Gi-dependent lymphocyte entry into LNs (33), it seemed possible that CXCR4 could contribute to the remaining Gi-dependent motility. In preliminary experiments, motility of CXCR4-null T cells in wild-type LNs was found to be unchanged from wild-type T cell motility (data not shown). Because the entry studies showed that the contribution of CXCR4 in T cells was obscured by the larger contribution of CCR7, we focused our efforts on testing the effect of combined deficiency in CXCR4 and CCR7 function through the use of a CXCR4 antagonist and CCR7-deficient T cells. These experiments did not identify a major contribution of CXCR4/CXCL12 to T cell motility in the wild-type T zone. Consistent with this result, our in vitro Transwell experiments showed that CXCL12 was poor at promoting gradient-independent T cell migration. Although we cannot rule out the possibility that inhibition of CXCR4 signaling was incomplete in LNs of the treated animals, treatment with the same dose of the antagonist was able to disrupt cell compartmentalization within LN germinal centers to the same extent as genetic CXCR4 deficiency (6). It remains possible that CXCR4 contributes to lymphocyte motility in some regions of the LN such as the medulla, where CXCL12 is abundant (34). Because the majority of naive T cells show little in vitro response to ligands for the other defined chemokine receptors, these observations suggest that additional, still-to-be-characterized Gi-coupled receptors are involved in promoting T cell motility within lymphoid organs. Sphingosine-1-phosphate receptor 1 is expressed on naive T cells, but the ligand sphingosine-1-phosphate is present in only low amounts in LNs (14, 35), and in studies where sphingosine-1-phosphate receptor function was modulated using agonists or antagonists, there were no effects on T cell motility in the T zone (36, 37).

The analysis of the surface phenotype and median velocity distribution for transferred T cells in the recipient LNs shows that deficiency in CCR7 signaling reduces naive T cell motility. Transferred T cells in the recipient LNs also contain small populations of activated/memory T cells, and it is not clear at this point whether these cells have different motility from naive cells in LNs. Although the reduction in T cell motility was partial, CCR7-deficiency may still impact on the rapid surveillance by naive T cells for specific Ags. In this regard, it is notable that plt/plt mice have slower kinetics of Ag-specific T cell responses following immunization (38). Because plt/plt mice have additional defects such as inefficient DC migration, it will be important in future studies to develop approaches to dissect the role of CCR7-driven T cell motility from the other roles of CCR7 in the initiation of the T cell immune response.


   Acknowledgments
 
We thank Drs. Hirotaka Tamamura and Nobutaka Fujii for providing 4F-benzoyl-TN14003, Drs. Martin Lipp and Reinhold Forster for CCR7-deficient mice, and Chris Allen for help with data processing.


   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 work was supported by National Institutes of Health Grants AI 45073 and AI 40098, by the Howard Hughes Medical Institute, and by a Sandler New Technology Award. T.O. was supported in part by the Japan Society for Promotion of Science. Back

2 Address correspondence and reprint requests to Dr. Jason G. Cyster, Department of Microbiology and Immunology, University of California San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143. E-mail address: jason/cyster{at}ucsf.edu Back

3 Abbreviations used in this paper: LN, lymph node; DC, dendritic cell; PTX, pertussis toxin; CMTMR, 5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine; CMAC, 7-amino-4-chloromethylcoumarin. Back

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

Received for publication October 18, 2006. Accepted for publication December 21, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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