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Differential Chemokine Responses and Homing Patterns of Murine TCR NKT Cell Subsets ¹

Brent Johnston^2,*, Chang H. Kim^3,*, Dulce Soler, Masashi Emoto and Eugene C. Butcher^*

^* Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, and Center for Molecular Biology and Medicine, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304; Millennium Pharmaceuticals, Cambridge, MA 02142; and Department of Immunology, Max Planck Institute for Infection Biology, Berlin, Germany

	Abstract

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NKT cells play important roles in the regulation of diverse immune responses. Therefore, chemokine receptor expression and chemotactic responses of murine TCR NKT cells were examined to define their homing potential. Most NKT cells stained for the chemokine receptor CXCR3, while >90% of V14i-positive and 50% of V14i-negative NKT cells expressed CXCR6 via an enhanced green fluorescent protein reporter construct. CXCR4 expression was higher on V14i-negative than V14i-positive NKT cells. In spleen only, subsets of V14i-positive and -negative NKT cells also expressed CXCR5. NKT cell subsets migrated in response to ligands for the inflammatory chemokine receptors CXCR3 (monokine induced by IFN-/CXC ligand (CXCL)9) and CXCR6 (CXCL16), and regulatory chemokine receptors CCR7 (secondary lymphoid-tissue chemokine (SLC)/CC ligand (CCL)21), CXCR4 (stromal cell-derived factor-1/CXCL12), and CXCR5 (B cell-attracting chemokine-1/CXCL13); but not to ligands for other chemokine receptors. Two NKT cell subsets migrated in response to the lymphoid homing chemokine SLC/CCL21: CD4^- V14i-negative NKT cells that were L-selectin^high and enriched for expression of Ly49G2 (consistent with the phenotype of most NKT cells found in peripheral lymph nodes); and immature V14i-positive cells lacking NK1.1 and L-selectin. Mature NK1.1⁺ V14i-positive NKT cells did not migrate to SLC/CCL21. BCA-1/CXCL13, which mediates homing to B cell zones, elicited migration of V14i-positive and -negative NKT cells in the spleen. These cells were primarily CD4⁺ or CD4^-CD8^- and were enriched for Ly49C/I, but not Ly49G2. Low levels of chemotaxis to CXCL16 were only detected in V14i-positive NKT cell subsets. Our results identify subsets of NKT cells with distinct homing and localization patterns, suggesting that these populations play specialized roles in immunological processes in vivo.

	Introduction

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Natural killer T cells constitute a lineage of hemopoietic cells that share both phenotypic and functional characteristics with NK cells and effector T lymphocytes. Mouse NKT cells are often defined by their expression of TCR and NK markers such as NK1.1 (CD161), DX5 (₂ integrin), CD122, or Ly49 family members (reviewed in Refs.1 and 2). Many NKT cells express a highly restricted TCR repertoire consisting of an invariant TCR chain, V14J18, paired preferentially with the TCR chains V8.2, V7, or V2. These invariant NKT cells, recently designated V14i T cells, recognize glycolipids associated with CD1d and ₂-microglobulin (₂m)⁴ rather than classical MHCs, and can be identified using CD1d tetramers loaded with -galactosylceramide (-GalCer) (3, 4). Interestingly, an immature subset of V14i NKT cells lacks expression of NK1.1 and constitutes the major population of NKT cells proliferating in the thymus of mice (5, 6). These cells are exported from the thymus and subsequently acquire NK1.1 expression in peripheral tissues (5, 6), suggesting that NK1.1 up-regulation is associated with a thymus-independent NKT cell maturation step. Most cells in the tetramer-negative population have diverse TCR usage and are not CD1d-restricted. However, this population also includes small subsets of CD1d-restricted NKT cells that do not recognize -GalCer (2, 7).

NKT cells have been shown to play important roles in diverse immune responses including the control of microbial infections, tumor killing, tolerance induction, and suppression of autoimmunity (reviewed in Refs.1 ,2 , 8 and 9). In vivo, NKT cells are infrequent in the blood and lymph nodes, but accumulate at higher levels in the liver, bone marrow, and spleen. After inoculation with mycobacterial glycolipids, or infection with other microbial agents, NKT cells have been found to accumulate in affected tissues (10, 11, 12, 13, 14). Similarly, NKT cells accumulate in the spleen during tolerance induction to Ags introduced via the ocular route (15), and bone marrow NKT cells repopulate the liver after depletion with low dose anti-CD3 Ab treatment (16). However, little is known about the homing mechanisms that NKT cells use to reglate their distribution in the periphery, or migrate to inflamed tissues.

Chemokines mediate leukocyte adhesion and migration, allowing the homing of specific leukocyte subsets to normal and inflamed tissue sites based on differential expression of chemokines and their receptors (reviewed in Refs.17 and 18). The chemokine secondary lymphoid-tissue chemokine (SLC)/CC ligand (CCL)21 and its receptor, CCR7, regulate the basal homing of T lymphocytes into lymph nodes, while B cell attracting chemokine (BCA)-1/CXC ligand (CXCL)13 mediates the localization of B lymphocytes and specialized T lymphocyte subsets to follicular areas of the lymph node. Differential chemokine receptor expression on memory/effector lymphocyte subsets allows for their recruitment into specific tissue sites. For example, skin homing lymphocytes express CCR4, while CCR9 is preferentially expressed on lymphocytes that home to intestinal tissues. To investigate whether NKT cell homing and tissue segregation could be regulated by chemokine receptor expression and chemokine responsiveness, we examined the chemotactic responses of murine TCR NKT cells in the spleen, liver, bone marrow, and blood. The chemotactic profiles of these cells differ significantly from those of conventional NK cells and T lymphocytes. Additionally, subsets of cells within the NKT cell population exhibit differential chemotactic responses and tissue localization. The identification of NKT cell subsets with unique chemotactic responses suggests specialization of these subsets in terms of tissue homing and function.

	Materials and Methods

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Mice

Male and female C57BL/6J mice were obtained from The Jackson Laboratory (Davis, CA). CXCR5-deficient mice were obtained from Dr. M. Lipp (Max-Delbruck-Center for Molecular Medicine, Berlin, Germany) (19). CXCR6-green fluorescent protein (GFP) knockin mice, generated by replacing the coding exon for CXCR6 with eGFP (enhanced GFP) (20), were obtained from Dr. D. R. Littman (Skirball Institute of Biomolecular Medicine and Howard Hughes Medical Institute, New York University School of Medicine, New York, NY). CXCR6-GFP mice were backcrossed against C57BL/6J mice for eight generations and used as heterozygotes. All mice were housed under conventional conditions in the animal facility at the Veterans Administration Hospital (Palo Alto, CA). Experiments were conducted using mice 8–16 wk of age.

Cell isolation

Mice were anesthetized with a ketamine/xylazine mixture and sacrificed by cervical dislocation. Blood was drawn into a heparinized syringe by cardiac puncture. Leukocytes were isolated from spleen, bone marrow, and peripheral lymph nodes (inguinal, axillary, brachial, and superficial cervical nodes were pooled) by mechanical dispersion through a wire mesh followed by hypotonic erythrocyte lysis. Liver lymphocytes were obtained by mincing the tissue, mechanically dispersing it through a wire mesh, and isolating the lymphocytes on a 33% Percoll (Amersham Pharmacia Biotech, Piscataway, NJ) gradient. All lymphocytes were resuspended in RPMI 1640 and allowed to recover in a CO₂ incubator for >1 h before chemotaxis. In some experiments, cells were treated with 200 µg/ml pertussis toxin (List Biochemical Laboratories, Campbell, CA) or control vehicle for 3 h before chemotaxis.

Chemotaxis assays

Chemotactic migration assays were performed as previously described (21). Briefly, 0.5–1.0 x 10⁶ lymphocytes from different tissues (depending on the NKT cell frequency) were placed in the upper chamber of Transwell inserts (5-µm pore size; Corning Costar, Corning, NY). Inserts were placed in wells containing medium alone (basal) or medium plus chemokine. The chemokines macrophage inflammatory protein (MIP)-1/CCL3, JE/CCL2, Eotaxin/CCL11, monocyte-derived chemokine (MDC)/CCL22, SLC/CCL21, KC/CXCL1, MIP-2, monokine induced by IFN- (MIG)/CXCL9, IFN--inducible protein-10 (IP-10)/CXCL10, and stromal cell-derived factor (SDF)-1/CXCL12 were purchased from PeproTech (Rocky Hill, NJ). MIP-1/CCL4, MIP-3/CCL20, T cell activation protein (TCA)-3/CCL1, thymus-expressed chemokine (TECK)/CCL25, cutaneous T cell-attracting chemokine (CTACK)/CCL27, BCA-1/CXCL13, CXCL16, fractalkine/CX3CL1, and lymphotactin/XC ligand (XCL)1 were purchased from R&D Systems (Minneapolis, MN). Chemokines were used at optimal concentrations determined by titration. After 2 h of migration, inserts were removed and polystyrene beads (Polysciences, Warrington, PA) were added to each well as an internal standard. Three wells were pooled for each condition. Migrated leukocyte populations were stained with Abs or CD1d tetramer and quantified by flow cytometry. Chemotaxis was determined by comparing the bead to cell ratios in the migrated and input populations (21). In some experiments, chemokine was added to both the top and bottom wells (checkerboard analysis) to distinguish chemotaxis from chemokinesis.

Flow cytometry

The following conjugated mAbs were used in various staining protocols: FITC, PE, or allophycocyanin-labeled NK1.1 (clone PK136); FITC, PE, or CyChrome-labeled TCR (clone H57-597); FITC, PE, PerCP, or allophycocyanin-labeled CD4 (clone RM4-5); allophycocyanin-CD8 (clone 53-6.7); PE-CD19 (clone 1D3); allophycocyanin-CD11b (clone M1/70); allophycocyanin-L-selectin (clone Mel-14); allophycocyanin-Ly49G2 (clone 4D11); PE-Ly49C/I (clone SW5E6); biotin-CXCR4 (clone 2B11/CXCR4); and biotin-CXCR5 (clone 2G8) were all from BD PharMingen (San Diego, CA). Monoclonal rat anti-mouse CXCR3 (clone 5B4) and CCR6 (clone 1C12) Abs were obtained from Millennium Pharmaceuticals (Cambridge, MA). Biotin-labeled donkey anti-rat IgG H+L was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Streptavidin-allophycocyanin was purchased from BD PharMingen. PE-labeled CD1d tetramers loaded with -GalCer were generated at the Max Planck Institute for Infection Biology (Berlin, Germany). Unloaded tetramers were used as a control. Four-color flow cytometry was performed on a two laser FACSCalibur (BD Biosciences, San Jose, CA) using CellQuest software, version 3.3 (BD Biosciences). Isotype-matched control Abs were used to establish placement of gates and quadrants.

Mouse CD1d/₂m tetramer

Mouse CD1d/₂m tetramer was prepared in a baculovirus expression system using a CD1d-construct with a BirA biotinylation site followed by a His-6 tag as described previously (3). The Sf9 insect cell line (BD Biosciences, Heidelberg, Germany) was infected with virus expressing mouse CD1d/₂m (kindly provided by Dr. M. Kronenberg (La Jolla Institute for Allergy and Immunology, San Diego, CA) at a multiplicity of infection of 0.1 to expand the viral stock. Culture supernatants were harvested on day 4 postinfection and used at a high multiplicity of infection, 1–5, for large scale production of soluble protein. Large scale preparations were performed in Sf-900 II serum-free medium (Life Technologies/Invitrogen, Karlsruhe, Germany), harvested on day 3 or 4 by centrifugation, and concentrated by passing through a hollow fiber tangential flow module (MiniKros 1100 cm², Spectrum; MembraPure, Boddenheim, Germany). CD1d molecules were purified by metal chelating chromatography (Chelating Sepharose Fast Flow; Amersham Pharmacia Biotech, Uppsala, Sweden) on a nitrilotriacetic acid-Sepharose column (Amersham Pharmacia Biotech) charged with cobalt chloride (Roth, Karlsruhe, Germany). Protein was eluted with 200 mM imidazol (Merck, Darmstadt, Germany) and pooled fractions were concentrated to 0.5 ml by ultrafiltration (Ultrafree Units; Millipore, Bedford, MA). Purity and protein concentration were assessed by SDS-PAGE and Bradford assay (Bio-Rad, München, Germany), respectively. Purified CD1d protein was subsequently biotinylated with BirA enzyme (Avidity, Denver, CO) according to the manufacturer’s instructions. Biotinylated CD1d proteins were purified by gel-filtration (Superdex 200 HR10/30; Amersham Pharmacia Biotech). Soluble biotinylated CD1d/₂m protein was loaded with or without -GalCer (Kirin Brewery, Tokyo, Japan) which was dissolved in PBS containing 0.5% Tween 20 at 220 µg/ml at a molar ratio of 1:3 (protein:lipid) overnight at room temperature. For tetramerization, streptavidin-PE (MobiTec, Göttingen, Germany) was added to -GalCer/CD1d/₂m monomers in 1:4 (monomer:streptavidin-PE) molar ratio. The purification of PE-labeled CD1d/₂m tetramers loaded with or without -GalCer was performed by gel-filtration (Superdex 200 HR10/30; Amersham Pharmacia Biotech).

Statistical analysis

Values are reported as means ± SEM. Data points within groups were compared using paired Student’s t tests, with Bonferroni corrections for multiple comparisons where appropriate. An unpaired Student’s t test was used to compare between groups. Statistical significance was set at p < 0.05.

	Results

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Chemotaxis of murine NK1.1⁺ TCR⁺ NKT cells

In the periphery, NKT cells exhibit preferential localization to the liver, spleen, and bone marrow, with a lower frequency of NKT cells present in the blood and peripheral lymph nodes (1, 8). As chemokines play important roles in the homing of leukocytes to specific tissue sites, we examined migration of murine NKT cells to a panel of chemokines that includes ligands for each of the known chemokine receptors (Table I). NK1.1⁺TCR⁺NKT cells from the spleen, which include V14i-positive and -negative NKT cell subsets, exhibited typical bell-shaped migration curves in response to ligands for the chemokine receptors CCR7 (SLC/CCL21), CXCR3 (MIG/CXCL9), CXCR4 (SDF-1/CXCL12), and CXCR5 (BCA-1/CXCL13) (Fig. 1), but failed to migrate in response to ligands for other chemokine receptors. Migration was specifically inhibited by pertussis toxin treatment, and checkerboard analysis revealed migration was due to chemotaxis rather than chemokinesis (data not shown). CXCR3 and its ligands (MIG/CXCL9, IP-10/CXCL10, and IFN-inducible T cell chemoattractant/CXCL11) are primarily associated with homing of activated memory/effector T cells to inflamed nonlymphoid tissues (22, 23, 24, 25). NKT cells exhibited robust migration (35–45% of input cells) to the inflammatory chemokines MIG/CXCL9 (Fig. 1) and IP-10/CXCL10 (data not shown), suggesting that these chemokines could act to recruit NKT cells to sites of infection and inflammation. The chemokine SLC/CCL21 is expressed at the surface of high endothelial venules and mediates lymphocyte recruitment into lymphoid tissues via CCR7 (26, 27, 28), while BCA-1/CXCL13 is expressed in B cell areas of lymph nodes and spleen and mediates localization of B cells and specialized CXCR5⁺ Th cell subsets to these areas (19, 29, 30, 31). Compared with the maximal T lymphocyte response to SLC/CCL21 and B lymphocyte response to BCA-1/CXCL13, NKT cell chemotactic responses to SLC/CCL21 and BCA-1/CXCL13 were relatively low (10–15% over background), suggesting that only small subsets of NKT cells express CCR7 and/or CXCR5. All leukocyte subsets, including NKT cells, migrated at low levels in response to the ubiquitous CXCR4 ligand SDF-1/CXCL12.

View larger version (43K): [in this window] [in a new window]   FIGURE 1. Dose-response curves for splenic leukocyte populations in chemotactic migration assays. Fresh unfractionated splenocytes were allowed to migrate through Transwell inserts (5-µm pores) for 2 h. Cells were stained with fluorochrome-conjugated Abs against TCR, NK1.1, CD19, and CD11b to distinguish individual leukocyte populations: NKT cells (NK1.1+TCR+), NK cells (NK1.1+TCR-), T lymphocytes (NK1.1-TCR+), B lymphocytes (CD19+), and neutrophils and eosinophils (CD11b+). Neutrophils and eosinophils were differentiated based on their distinctive forward and side scatter characteristics. The proportion of migrated cells in each population was calculated as a fraction of the input population (21 ). The background migration in the absence of chemokine was subtracted from each population to yield specific migration. Each plot shows the mean ± SEM of six individual experiments.

Chemotaxis of NKT cells in liver, bone marrow, and blood

As different subsets of NKT cells have been reported to localize preferentially to liver, spleen, or bone marrow (35, 36, 37, 38), it is possible that they may be recruited to specific tissue sites in response to distinct chemotactic signals. Therefore, in addition to spleen, we examined the chemotactic responses of NK1.1⁺TCR⁺NKT cells in liver, bone marrow, and blood (Fig. 2). There were no additional responses in these tissues to any of the chemokines that failed to elicit chemotaxis of spleen NKT cells. MIG/CXCL9 induced significant chemotaxis of NKT cells from all tissues examined. Consistent with lower mean fluorescence intensity of CXCR4 staining (data not shown), chemotaxis to SDF-1/CXCL12 was lower in the liver and spleen compared with bone marrow and blood. Interestingly, BCA-1/CXCL13 responsive NKT cells were only present in the spleen, while very few SLC/CCL21-responsive cells were present in the liver. This suggests preferential localization of NKT cell subsets within these tissue sites.

View larger version (41K): [in this window] [in a new window]   FIGURE 2. Chemotaxis of NKT cells in spleen, liver, bone marrow, and peripheral blood. Leukocytes were migrated to chemokines as described in Fig. 1. Bars represent the migration of NK1.1+TCR+NKT cells at the optimal concentration of chemokine (Table I). Full dose-response assays were conducted in each tissue for chemokines that did not elicit migration at the optimal dose (data not shown). Each bar shows the mean ± SEM of four to six separate experiments. *, p < 0.05 compared with basal migration.

Currently, there are few reagents available to examine the expression of chemokine receptors on the surface of mouse leukocytes. However, we were able to examine the expression of a limited panel of receptors (Fig. 3). Consistent with the functional chemotaxis data, NKT cells did not stain with a mAb against CCR6. In contrast, most V14i-positive (tetramer binding) and -negative (tetramer-negative, NK1.1⁺) NKT cells stained brightly with a mAb against the CXCR3 chemokine receptor, consistent with their robust chemotactic response to MIG/CXCL9. The chemokine receptor CXCR4 was expressed at higher levels on V14i-negative NKT cells than the V14i-positive subsets, suggesting the potential for differential recruitment of these subsets. A subset of spleen NK1.1⁺TCR⁺NKT cells (15–25%) stained with a mAb against CXCR5 (Fig. 3). Interestingly, CXCR5 was expressed on a larger proportion of V14i-positive NKT cells, compared with the V14i-negative NKT cell population. Consistent with the lack of significant NKT cell chemotaxis to BCA-1/CXCL13 in the liver, bone marrow, and blood (Fig. 2), CXCR5 was not detected on NKT cells from these tissues (data not shown). CXCR6 expression was detected on >90% of V14i-positive NKT cells and 50% of V14i-negative NKT cells via an eGFP reporter in heterozygous mice with a targeted replacement of CXCR6. CXCR6/eGFP was reported previously on CD8⁺ T lymphocytes and a subset of memory CD4⁺ T lymphocytes (20). Receptor expression inferred by eGFP expression is consistent with the observation that NKT cells and CD8⁺ T lymphocytes from normal animals bind to a chimeric CXCR6 ligand (Fc-CXCL16) (34). The expression of this receptor in the absence of significant chemotactic activity in normal resting spleen cells (Fig. 1), or eGFP⁺ splenocytes from CXCR6/eGFP heterozygotes (data not shown), suggests that CXCR6 and CXCL16 may be more important in the homing of activated lymphocytes during inflammation or immune responses. It is interesting to note that B cells exhibit a similar phenomenon with the chemokine receptor CCR6. Even though many B cells express the CCR6 receptor (Fig. 3), few of these cells migrated in response to MIP-3/CCL20 (Fig. 1). It has been shown previously that chemokine responsiveness can be controlled independently of receptor expression in dendritic cells, T cells, and germinal center B cells (39, 40, 41, 42), suggesting that regulation of downstream elements can control responsiveness to chemotactic ligands without altering levels of receptor expression. Therefore, it is critical to examine chemotactic responses in addition to receptor expression to implicate a functional role for a particular receptor.

View larger version (78K): [in this window] [in a new window]   FIGURE 3. Expression of chemokine receptors on splenic leukocyte populations. Fresh unfractionated splenocytes were stained with fluorochrome-conjugated Abs against TCR, NK1.1, and CD19 or -GalCer-loaded CD1d tetramers to distinguish individual leukocyte populations: classical NKT cells (NK1.1+TCR+), mature V14i+ NKT cells (NK1.1+tetramer+TCR+), immature V14i+ NKT cells (NK1.1-tetramer+TCR+), V14i- NKT cells (NK1.1+tetramer-TCR+), NK cells (NK1.1+TCR-), T lymphocytes (NK1.1-TCR+), and B lymphocytes (CD19+). Biotin-conjugated Abs against CXCR4, and CXCR5 were visualized after staining with streptavidin-allophycocyanin. CCR6 and CXCR3 Abs were detected with biotin-labeled donkey anti-rat IgG H+L followed by streptavidin-allophycocyanin. CXCR6 expression was detected using a reporter eGFP construct in heterozygous mice with a targeted replacement of CXCR6. Histograms show chemokine receptor staining (solid line) overlaid with the appropriate isotype control or wild-type mouse in the case of eGFP (dotted line). The number in each plot is the percent of cells with fluorescence exceeding the isotype control. Histograms are representative of at least three individual experiments.

Chemotaxis of V14i-positive and -negative NKT cell subsets

The majority of NKT cells in the thymus and liver are CD1d-restricted V14i T cells, while the spleen and bone marrow contain heterogeneous populations of both CD1d-restricted and CD1d-unrestricted NKT cells (3, 36). As the spleen also contains a unique population of BCA-1/CXCL13-responsive NKT cells, we used CD1d tetramers loaded with -GalCer to examine the chemotactic responses of V14i-positive and V14i-negative NK1.1⁺NKT cells in this tissue (Fig. 4). Tetramer-binding V14i-positive NKT cells (NK1.1⁺ and NK1.1^-) accounted for 40–50% of the total NKT cells present in the spleen (Fig. 4A). Seventy to 75% were CD4⁺, and 25–30% were double-negative (DN), while virtually no CD8⁺ cells were observed in this population. In contrast, 60% of the V14i-negative NK1.1⁺NKT cells were DN, with CD4⁺ and CD8⁺ subsets each making up 20% of the total. Significant differences in the migration of NKT cell subsets within these populations were observed in chemotaxis assays (Fig. 4B).

View larger version (49K): [in this window] [in a new window]   FIGURE 4. Phenotype and chemotaxis of V14i-positive and -negative NKT cells in spleen. A, V14i-positive and -negative NKT cell populations were identified in the TCR+ lymphocyte population using NK1.1 and CD1d tetramers loaded with -GalCer. These cells were also stained for expression of CD4 or CD8. Plots and histograms are representative of three to four experiments. B, Chemotaxis of NKT cell subsets to SLC/CCL21 (250 nM), MIG/CXCL9 (250 nM), SDF-1/CXCL12 (100 nM), BCA-1/CXCL13 (250 nM), or CXCL16 (5 nM). Cells were stained with -GalCer-loaded CD1d tetramers, and Abs against TCR, NK1.1, CD4 and/or CD8 to identify subsets of V14i-positive and -negative NKT cells. The basal migration in the absence of chemokine was subtracted from each population to yield specific migration. Each bar shows the mean ± SEM of five separate experiments.

View larger version (45K): [in this window] [in a new window]   FIGURE 5. Chemotaxis of spleen NKT cell subsets expressing L-selectin, Ly49C/I, and Ly49G2. A, Migration of NK1.1+TCR+NKT cell subsets to SLC/CCL21 (250 nM), MIG/CXCL9 (250 nM), SDF-1/CXCL12 (100 nM), and BCA-1/CXCL13 (250 nM). Cells were stained with Abs against Ly49C/I and Ly49G2 or L-selectin. The basal migration in the absence of chemokine was subtracted from each population to yield specific migration. Each chemotaxis assay shows the mean ± SEM of five separate experiments. B, Expression of Ly49C/I, Ly49G2, and L-selectin on V14i-positive and -negative NKT cells. Histograms are representative of three to four experiments.

View larger version (54K): [in this window] [in a new window]   FIGURE 6. Phenotype of NKT cells in peripheral lymph nodes. A, NKT cell populations were identified using TCR, NK1.1, and CD1d-tetramers loaded with -GalCer. The majority (75–85%) of NKT cells in the peripheral lymph nodes were V14i-negative NK1.1+NKT cells. NKT cells in the peripheral lymph nodes were stained with Abs against CD4 and CD8. B, NKT cells from the peripheral lymph nodes were stained for the markers L-selectin, Ly49C/I, and Ly49G2. Plots and histograms are representative of three to four experiments.

Chemotaxis to SDF-1/CXCL12 was highest in the CD8⁺ and DN subsets of V14i-negative NK1.1⁺ NKT cells (Fig. 4B), consistent with enrichment for L-selectin^high and Ly49G2⁺ cells in migrating populations (Fig. 5A). Migration was consistently lower in the CD4⁺ fraction of both V14i-positive and -negative subsets compared with DN (or CD8⁺) NKT cells.

BCA-1/CXCL13-responsive CXCR5⁺ T lymphocytes include cells that can provide B cell help (19, 29, 30, 31). The migration of CD4⁺ and DN V14i-positive and CD4⁺ V14i-negative NKT cells to BCA-1/CXCL13 (Fig. 4B) suggests that these cells could play roles in mediating B cell help, or modulating the follicular B cell response. This is supported by observations that NKT cell overexpression leads to increases in serum IgE and IgG1 (45), and that -GalCer treatment elicits NKT cell-dependent B cell activation in mice (46, 47). BCA-1/CXCL13-responsive NKT cells were enriched for cells expressing ly49C/I (Fig. 5A), which was present on a similar proportion of both V14i-positive and -negative cells (Fig. 5B). In contrast, Ly49G2⁺ NKT cells were excluded from the BCA-1/CXCL13-responsive population (Fig. 5A).

Although chemotaxis to CXCL16 could not be detected in the bulk population of NK1.1⁺TCR⁺NKT cells (Fig. 1), a low level of migration was consistently observed in the V14i-positive subsets when experiments were gated specifically on tetramer-positive cells (Fig. 4B). It is interesting that V14i-negative NKT cells, and other lymphocyte populations, did not respond to CXCL16 even though many cells in these populations also expressed CXCR6. The gating conditions used in these experiments did not reveal migration of V14i-positive or -negative NKT cell subsets in response to other chemokines in our panel that failed to elicit migration of the bulk NKT cell population (data not shown).

BCA-1/CXCL13-induced NKT cell chemotaxis is dependent on CXCR5

It has been reported in human systems that BCA-1/CXCL13 can mediate chemotaxis through CXCR3 in addition to CXCR5 (48). As NKT cells express high levels of CXCR3 (Fig. 3), we examined CXCR5-deficient animals to address the possibility that CXCR3 mediates BCA-1/CXCL13-induced chemotaxis in mouse NKT cells. As shown in Fig. 7, NKT cells from CXCR5-deficient mice responded normally to SLC/CCL21, MIG/CXCL9, and SDF-1/CXCL12, while chemotaxis to BCA-1/CXCL13 was completely abrogated. We conclude that BCA-1/CXCL13-induced chemotaxis acts exclusively through the CXCR5 receptor in mice.

View larger version (18K): [in this window] [in a new window]   FIGURE 7. Chemotaxis of NKT cells in CXCR5-deficient mice. V14i-positive NKT cells from the spleens of CXCR5-deficient or wild-type control mice were analyzed for their migration in response to SLC/CCL21 (250 nM), SDF-1/CXCL12 (100 nM), MIG/CXCL9 (250 nM), and BCA-1/CXCL13 (250 nM). Each bar shows the mean ± SEM of three separate experiments. *, p < 0.05 compared with basal migration. , p < 0.05 compared with migration in wild type.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The expression of CXCR3 on NKT cells suggests that they could migrate to sites of infection and inflammation. As the CXCR3 ligands MIG/CXCL9, IP-10/CXCL10, and IFN-inducible T cell chemoattractant/CXCL11 are highly inducible by IFN- (49, 50), it suggests that NKT cells would be recruited to inflammatory conditions that up-regulate these cytokines. The lack of chemotactic responsiveness to other inflammatory chemokines was somewhat surprising. However, it is likely that other inflammatory mediators, including TNF-, also contribute to the recruitment of NKT cells to inflamed tissue sites (11). Previously, Faunce et al. (51) reported that MIP-2 elicited NKT cell migration in 8–12 h Boyden chamber assays, and that CXCR2 was required for the induction of tolerance to Ags introduced via the ocular route. Using 2 h Transwell migration assays, we did not observe NKT cell migration to the CXCR2 ligands KC (Figs. 1 and 2) or MIP-2 (not shown) in any of the tissues we examined, despite significant chemotaxis of neutrophils in response to these chemokines (Fig. 1 and data not shown). As spleen NKT cells also failed to migrate to KC or MIP-2 after ocular immunization (our unpublished data), it does not appear that CXCR2 is up-regulated on activated NKT cells in this site. Therefore, we find it likely that that CXCR2 and MIP-2 are required for some other aspect of the ocular tolerance model upstream of NKT cell recruitment to the spleen.

In humans, the chemokine receptor CXCR6 is expressed preferentially on Th1 and Tc1 polarized memory CD4⁺ and CD8⁺ lymphocytes, and has been detected on large proportions of tissue infiltrating lymphocytes from patients with inflammatory disorders (52). However, it is not known whether these cells use CXCR6 to migrate into these tissues. The CXCR6 ligand, CXCL16, does not mediate significant migration of most receptor positive cells without prior activation (34, 53), suggesting regulation of receptor signaling independently from its level of expression. As CXCL16 is up-regulated as a surface bound transmembrane protein on activated APCs (34, 53), it is possible that CXCR6/CXCL16 interactions could also play a role in mediating cell-cell contact and/or signaling interactions between NKT cells and APCs. It is unclear at this stage whether CXCL16 functions primarily as a chemoattractant or plays a more important role in facilitating cell-cell interactions. However, it is interesting that a small percentage of V14i-positive NKT cells exhibited chemotaxis to CXCL16 in the absence of an in vitro activation stimulus, suggesting that some of these cells may have been activated in vivo, or that CXCR6 function is regulated differently in V14i-positive NKT cells compared with other leukocyte populations. As CXCL16 is expressed in the thymus and liver (34, 53), it is possible that CXCR6 could play roles in NKT cell development or homing of these cells to the liver.

Single-positive thymocytes have been shown to up-regulate responsiveness to CCR7 ligands (SLC/CCL21 and EBV-induced molecule 1 ligand chemokine/CCL19) during development (32, 54), and some evidence suggests that this may facilitate exit from the thymus (55). As NKT cells also up-regulate SLC/CCL21 responsiveness in the thymus (our unpublished data), CCR7 may play a role in enhancing NKT cell exit from the thymus. In contrast to single-positive T lymphocytes, however, NKT cells exported from the thymus complete their maturation in the periphery, associated with the up-regulation of NK1.1 expression (5, 6). Immature NK1.1^- V14i-positive NKT cells migrated in response to SLC/CCL21, while mature NK1.1⁺ V14i-positive NKT cells did not. This suggests that down-regulation of CCR7 expression and/or SLC/CCL21 responsiveness are also associated with NKT cell maturation in the periphery, and could be used as an additional developmental marker for V14i-positive NKT cells.

The recruitment of T lymphocytes into lymph nodes from the blood requires L-selectin-mediated rolling and lymphocyte activation via the interaction of endothelial-bound SLC/CCL21 (or EBV-induced molecule 1 ligand chemokine/CCL19) with CCR7 (17, 27, 43). Low levels of L-selectin expression on V14i-positive NKT cells would largely exclude them from entering the peripheral lymph nodes, even though the NK1.1^- subset responds to SLC/CCL21. A similar observation would be expected in humans as few V24⁺V11⁺ NKT cells in human blood coexpress L-selectin and CCR7 (56). In contrast, the high level of L-selectin expression and responsiveness to SLC/CCL21 in the V14i-negative population of NKT cells explains their presence in the peripheral lymph nodes of mice. Laloux et al. (57) have observed larger populations of NKT cells in splanchnic lymphoid tissues compared with peripheral lymph nodes. It is possible that ₄₇ expressed on NKT cells could allow a larger number of cells to enter intestinal-associated lymphoid tissues, where ₄₇/mucosal addressin cell adhesion molecule-1 interactions can mediate homing in the absence of L-selectin (58).

CXCR5 has been demonstrated on a subset of CD4⁺ T lymphocytes that can mediate B cell help in vitro and in vivo (19, 29, 30, 31). Our finding that a unique subset of NKT cells expresses CXCR5 and responds to the B cell homing chemokine BCA-1/CXCL13 suggests that NKT cell subsets could home to splenic B cell follicles and influence B cell responses. A functional role for CXCR5 in tissue localization of NKT cells is consistent with the accumulation of NKT cells in B cell zones during tolerance induction to Ags introduced via the ocular route (51), and the observation that the residual CD4⁺ T cells in MHC class II-deficient mice, presumably CD1d-restricted NKT cells, localize preferentially to the B cell zones (59). There are also several examples where NKT cells have been shown to influence B cell responses and the production of Igs. TCR transgenic mice that overexpress V14J18 have elevated numbers of NKT cells, and exhibit increases in serum IgE and IgG1 (45). Similarly, treatments with -GalCer have also been shown to induce early NKT cell-dependent B cell activation in mice, with later increases in serum IgE levels (46, 47). Furthermore, the finding that tolerance induction to Ag introduced via the eye requires both NKT cells and marginal zone B cells suggests a functional interaction between these cell types (60). We hypothesize that localizing NKT cells to B cell areas via CXCR5 is important for these interactions to take place.

Skold and Cardell (35) have previously shown that the NK markers Ly49C/I and Ly49G2 are preferentially expressed on DN rather than CD4⁺ NKT cells. We extend this observation by demonstrating that Ly49G2 is expressed predominantly by the V14i-negative NKT cell subset, while Ly49C/I is expressed on both V14i-positive and -negative NKT cells. It is clear that the mechanisms regulating the expression of Ly49 molecules differ between NK and NKT cells. For example, some Ly49 receptors are down-regulated on NKT cells in the periphery compared with the thymus (35, 61), while NK cells appear to up-regulate and express Ly49 receptors in a stable fashion (62, 63). In addition, NKT cells express members of the Ly49 receptor family that inhibit activation, but do not express activation-inducing members such as Ly49D or Ly49H (64, 65, 66). We found that Ly49G2 was expressed preferentially on V14i-negative NKT cells that responded to SLC/CCL21, while Ly49C/I was expressed on subsets of NKT cells that responded to BCA-1/CXCL13. The role for differential expression of Ly49 molecules on NKT cell subsets is unclear. Maeda et al. (64), have shown that Ly49 molecules can suppress NKT cell activation and IFN- production in response to -GalCer, but they did not distinguish between different Ly49 molecules. Ly49G2 recognizes the H-2^d haplotype of MHC class I, which is absent in C57BL/6 mice. In contrast, Ly49C/I recognizes H-2^b and has been shown to have suppressive effects on the cytotoxic activity of C57BL/6 NKT cells (65). Whether Ly49C/I regulates the activity of BCA-1/CXCL13-responsive NKT cells in the B cell zones of the spleen remains to be determined.

Interestingly, murine NKT cells exhibit significant differences in their chemotactic profile compared with human NKT cells. We have previously shown that human blood NKT cells express significant levels of CCR1, CCR2, CCR5, and CCR6, in addition to the CXCR3, CXCR4, and CXCR6 we detect on mouse NKT cells (56). Human NKT cells also exhibited significant chemotaxis to ligands for these additional receptors (except CXCR6, which was not tested) (56). In contrast to human NKT cells, mouse blood and tissue NKT cells are devoid of responses to ligands for CCR1, CCR2, CCR5, and CCR6 (see Figs. 1 and 2), and do not express CCR6 in any tissues examined (Fig. 3, and data not shown). It is possible that differences in Ag experience could influence these contrasting chemotactic patterns, with additional chemokine receptors being acquired after Ag exposure (which would be minimal in a clean animal facility). It is also possible that differences in the functional roles of human and mouse NKT cells have led to the evolution of different chemotactic targeting programs. It will be important to characterize individual subsets of human and mouse NKT cells to identify common and distinct roles in immune regulation.

In conclusion, we have characterized the chemotactic responses of murine NKT cells, and identified subsets that have the potential to home to distinct tissue sites. In addition, we revealed that chemokine responsiveness to SLC/CXCL21 is altered during maturation of V14i-positive NKT cells, providing an additional marker to identify and track these cells. In the future, it will be important to determine the functional roles of NKT cell subsets that exhibit differential migration patterns.

	Acknowledgments

 
We thank Dr. M. Lipp (Max Delbruck Center for Molecular Medicine, Berlin, Germany) for providing CXCR5-deficient mice, and Dr. D. R. Littman (New York University School of Medicine, New York, NY) for providing CXCR6-GFP knockin mice. We also thank Dr. E. Wilson for helpful comments on our manuscript.

	Footnotes

 
¹ This work was supported by grants from the National Institutes of Health and the Department of Veterans Affairs (to E.C.B.), and the FACS Core Facility of the Stanford Digestive Diseases Center. B.J. was supported by a fellowship from the Canadian Institutes of Health Research.

² Address correspondence and reprint requests to Dr. Brent Johnston, Veterans Affairs Medical Center, 3801 Miranda Avenue, MC154B, Palo Alto, CA 94304. E-mail address: wbjohnst{at}stanford.edu

³ Current address: Department of Pathobiology, Purdue University, 1243 Veterinary Pathology Building, West Lafayette, IN 47907-1243.

⁴ Abbreviations used in this paper: ₂m, ₂-microglobulin; GalCer, -galactosylceramide; SLC, secondary lymphoid-tissue chemokine; CCL, CC ligand; BCA, B cell-attracting chemokine; CXCL, CXC ligand; GFP, green fluorescent protein; eGFP, enhanced GFP; MIP, macrophage inflammatory protein; MDC, monocyte-derived chemokine; MIG, monokine induced by IFN-; IP-10, IFN--inducible protein-10; SDF, stromal cell-derived factor; TCA, T cell activation protein; TECK, thymus-expressed chemokine; CTACK, cutaneous T cell-attracting chemokine; XCL, XC ligand; DN, double-negative.

Received for publication January 29, 2003. Accepted for publication July 16, 2003.

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