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Published as doi: 10.1096/fj.07-9087com.
 (The FASEB Journal. 2008;22:307-315.)
© 2008 FASEB
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(The FASEB Journal. 2008;22:307-315.)
© 2008 FASEB

S1P1 receptor expression regulates emergence of NKT cells in peripheral tissues

Maria L. Allende*,1, Dapeng Zhou{dagger}, Danielle N. Kalkofen*, Sonia Benhamed*, Galina Tuymetova*, Christine Borowski{ddagger}, Albert Bendelac{ddagger} and Richard L. Proia*,1

* Genetics of Development and Disease Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA;

{dagger} Department of Melanoma Medical Oncology, MD Anderson Cancer Center, University of Texas, Houston, Texas, USA; and

{ddagger} Committee on Immunology, University of Chicago and Howard Hughes Medical Institute, Chicago, Illinois, USA

1Correspondence: R.L.P. and M.L.A., National Institute of Diabetes and Digestive and Kidney Diseases, Bldg. 10, Room 9D-06, 10 Center DR MSC 1821, Bethesda, MD 20892-1821, USA. E-mail: R.L.P., proia{at}nih.gov; M.L.A., mariaa{at}intra.niddk.nih.gov


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The S1P1 receptor, on the surface of lymphocytes and endothelial cells, regulates the unique trafficking behavior of certain lymphocyte populations. We have examined whether the S1P1 receptor also dictates the distinctive tissue distribution of V{alpha}14-J{alpha}18 natural killer T (NKT) cells, whose trafficking pattern is not well understood. Mice (TCS1P1KO) were established with a conditional deletion of the S1P1 receptor in thymocytes that included precursors of NKT cells. Within the thymus, NKT cells were found at normal or increased levels, indicating that S1P1 receptor expression was dispensable for NKT cell development. However, substantially reduced numbers of NKT cells were detected in the peripheral tissues of the TCS1P1KO mice. Short-term S1P1 deletion after NKT cells had established residence in the periphery did not substantially alter their distribution in tissues, except for a partial decrease in the spleen. FTY720, a S1P1 receptor ligand that has potent effects on the trafficking of conventional T cells, did not alter the preexisting distribution of NKT cells within peripheral tissues of wild-type mice. Our results indicate that the S1P1 receptor expression on NKT cells is dispensable for development within thymus but is essential for the establishment of their tissue residency in the periphery.—Allende, M. L., Zhou, D., Kalkofen, D. N., Benhamed, S., Tuymetova, G., Borowski, C., Bendelac, A., Proia, R. L. S1P1 receptor expression regulates emergence of NKT cells in peripheral tissues.


Key Words: lipid signaling • G-protein coupled receptors • lymphocyte trafficking • conditional knockout mice


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE S1P1 receptor, also known as Edg1, is pivotal for regulating the trafficking patterns of lymphocyte populations (1 2 3 4 5) . Deletion of the lymphocyte S1P1 gene has demonstrated that S1P1 receptor expression is required for controlling egress of B and T cells from secondary lymphoid organs (6 , 7) , localization of B cells to the marginal zone in the spleen (8) , plasma cell homing (9) , and exit of maturing CD4 and CD8 single-positive lymphocytes from the thymus (6 , 10) . Deletion of the S1P1 receptor within the endothelium has revealed its important role in regulating the endothelial barrier function (11 , 12) , which is an additional point of control for lymphocyte trafficking (4) .

The natural ligand for the S1P1 receptor is sphingosine-1-phosphate, a sphingolipid metabolite produced by the action of sphingosine kinase 1 and 2 (13 , 14) . Sphingosine-1-phosphate is secreted by hematopoietic and other cells (15 16 17 18) and is found in relatively high concentrations within blood and lymph, where it provides a signal for lymphocyte egress from lymphoid organs (19 20 21) . S1P1 receptor agonists are potent immunosuppressants. They produce lymphopenia through sequestration of the majority of T and B cells within lymph nodes and Peyer’s patches and by blocking T cell egress from the thymus (22 23 24 25 26 27 28 29) . As a result, lymphocytes cannot circulate to peripheral sites of inflammation. The most well-studied and clinically important of these compounds is FTY720, which is phosphorylated primarily by sphingosine kinase 2 in vivo to produce the multi-S1P receptor-active metabolite, FTY720-P (30 31 32 33) . Interestingly, FTY720 shows differential activity on some T cell subsets (34 , 35) ; notably, CD4+/CD25+ T-regulatory cells are relatively spared from lymph node sequestration by FTY720 (34 , 35) .

Natural killer T (NKT) cells are a sublineage of T cells with a T cell receptor (TCR) that in mice is predominantly a product of a single variant TCR {alpha} chain (the variable region V{alpha}14 joined to J{alpha}18) and a variable β chain (36 , 37) . These V{alpha}14-J{alpha}18 NKT cells are autoreactive for the MHC class I-like CD1d protein and respond to glycolipids produced both endogenously, such as the lysosomally generated glycosphingolipid, isoglobotrihexosylceramide (38) , and from exogenous sources, including marine sponge-derived {alpha}-galactosylceramide ({alpha}GalCer) (39 , 40) and microbial cell-wall glycosylceramides (41 , 42) . After CD1d-mediated positive selection within the thymus, precursors for V{alpha}14-J{alpha}18 NKT cells expand prior to NK1.1 expression (43) . Maturation of NKT cells is coincident with the expression of NK1.1 and the determination of their long-residency either in the thymus or in the periphery, where NKT cells are exported as NK1.1- and later become NK1.1+ (43 , 44) . Outside the thymus, NKT cells are found in liver, spleen, blood, and bone marrow, but they are scarce in peripheral lymph nodes (36 , 37 , 45 , 46) . Through recognition of glycolipids, NKT cells function in tumor responses and control of bacterial and viral infections, as well as in suppression of autoimmune diseases. After TCR ligation, NKT cells secrete large amounts of Th1- and Th2-type cytokines, leading to the rapid activation of many cell types of the innate and adaptive immune system (reviewed in (36 , 37 , 47) .

Because of the fundamental importance of S1P1 receptor signaling in several lymphocyte trafficking patterns, we have studied whether the S1P1 receptor plays a role in the establishment NKT cells in their major tissues of residence. Our results demonstrate that S1P1 receptor expression is not required for NKT cell development in the thymus but is necessary to attain their normal tissue distribution.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of the S1P1loxP/loxP Lck-Cre knockout mice
We previously generated the S1P1loxP/loxP mice (10 , 11) , which carry the S1P1 gene with loxP sequences flanking exon 2. To specifically delete the S1P1 receptor from T cells, S1P1loxP/loxP mice were bred with a Lck-driven Cre transgenic mouse strain, as described in ref. 10 . S1P1loxP/loxP mice carrying the Lck-Cre gene (thymocyte-specific S1P1KO or TCS1P1KO) and littermate S1P1loxP/loxP mice (without the Lck-cre transgene, used as controls) were on a C57Bl/6–129Sv mixed background. These mice were genotyped by polymerase chain reaction (PCR) using Lck-Cre specific primers: lck1 on the lck-proximal promoter: 5'-CCTCCTGTGAACTTGGTGCTTGAG-3'; lck2 on the Cre gene coding region: 5'-TGCATCGACCGGTAATGCAG-3'. Inducible S1P1 KO mice on a C57Bl/6 background (backcrossed 10 times) were generated by crossing the S1P1loxP/loxP mice to a strain carrying the Cre recombinase gene under the inducible promoter of the mouse Mx1 gene (48) . S1P1loxP/loxP carrying Mx-cre transgene and control S1P1loxP/loxP mice (without the Mx-cre transgene) were injected intraperitoneally with polyinosinic-polycytidylic acid (pIpC; 250 µg per injection) three times at 2-day intervals. Animals were analyzed either at 2 wk, 10 wk, or 6–7 months after the first pIpC injection. All experiments were conducted in accordance with the guidelines of animal user and care committee of National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health.

Lymphocyte preparation
Lymphocytes from thymus, spleen, and peripheral lymph nodes (lingual, axillary, branchial, and inguinal) from 8- to 12-wk-old mice were dissected and mechanically disaggregated. Where noted, 1-year-old mice were used. Single-cell suspensions were obtained using a 40 µm cell strainer. Red blood cells were removed by NH4Cl lysis. Liver tissue was cut and pressed through a 100 µm-pore mesh. Lymphocytes were separated on a Ficoll gradient performed at room temperature. Peripheral blood mononuclear cells were obtained after red cell lysis of ~400 µl of total blood. NKT cells were sorted from thymus and spleen for RNA analysis. Briefly, the NKT cells were stained by CD1d/{alpha}GalCer tetramers as described (38) , in combination with antibodies against NK1.1 and TCRβ (BD Bioscience, San Jose, CA, USA). CD1d/{alpha}GalCer+TCRβ+ NKT cells were sorted for further analysis from control and TCS1P1KO thymi (n=4). The CD1d/{alpha}GalCer+TCRβ+NK1.1+ and CD1d/{alpha}GalCer+TCRβ+ NK1.1- populations were sorted separately from pooled C57BL/6 thymi (n=3–5). Alternatively, CD1d/{alpha}GalCer+ NKT cells were bound to anti-PE microbeads and separated using autoMACS columms (Miltenyi Biotec, Auburn, CA, USA).

Real-time PCR
Total RNA was extracted with a RNAqueous-Micro kit (Ambion, Austin, TX, USA) or Trizol (Invitrogen, Carlsbad, CA, USA). mRNA expression levels of the mouse Edg1 (S1P1), Edg5 (S1P2), Edg3 (S1P3), and Edg6 (S1P4) genes were quantified by real-time PCR using Assay-on-Demand probe and primers (Applied Biosystems, Foster City, CA, USA) on a ABI Prism 7700 Sequence Detection System (Applied Biosystems). The relative level of glyceraldehyde 3-phosphate dehydrogenase (Gadph) mRNA, which was used as an internal control, in each sample was set to 1.

Flow cytometry
Cells were diluted in 1% bovine serum albumin–phosphate buffered saline (PBS) and incubated with anti-Fc{gamma}R antibody (BD Bioscience) to block binding of conjugated antibodies to Fc{gamma}R. Anti-mouse CD44, NK1.1, B220, CD62L, and TCRβ antibodies were purchased from BD Bioscience. Anti-mouse CCR7 antibody was purchased from eBioscience (San Diego, CA, USA) and from BioLegend (San Diego, CA, USA). NKT cells were stained with mouse CD1d:mouse Ig fusion protein (BD Bioscience), loaded with {alpha}GalCer (kindly provided by Dr. Masaru Taniguchi), and labeled according to the manufacturer’s protocol. In some experiments cells were labeled with CD1d/{alpha}GalCer tetramers (39) loaded with PBS57, an analog of {alpha}GalCer (49) . After cells were labeled with the conjugated antibodies, they were washed in PBS, fixed in 1% paraformaldehide in PBS, and then analyzed by flow cytometry using a FACScalibur flow cytometer (BD Bioscience). Dead cells were excluded based on forward and side scatter. Data analysis was performed using the FlowJo software (Tree Star, Ashland, OR, USA). NKT cells were identified as CD1d/{alpha}GalCer+ TCRβ+ after comparing to cells stained with unloaded CD1d:mouse Ig fusion protein and anti-βTCR. Conventional T cells were identified as CD1d/{alpha}GalCer- TCRβ+. The appropriate isotype control antibodies were used for each staining to determine background. Absolute lymphocyte numbers for thymus, spleen and liver were obtained using a Z1 Coulter Particle Counter (Beckman Coulter, Fullerton, CA, USA). Absolute blood cell counts were obtained using Caltag counting beads (Invitrogen). Expression of S1P1 was determined by flow cytometry using a rabbit polyclonal antibody against the first 49 amino acids of the mouse S1P1 (generously provided by Dr. Jason Cyster) followed by anti-rabbit IgG biotin (BD Biosciences) and streptavidin-APC (Molecular Probes, Eugene, OR, USA) (7) .

Annexin V labeling of apoptotic cells
Lymphocytes were stained with mouse CD1d:mouse Ig fusion protein loaded with {alpha}GalCer and anti-TCRβ as described above. Apoptotic cells were detected using the Annexin V apoptosis detection kit from BD Biosciences Briefly, cells were washed and incubated with 2 µl of 7-amino-actinomycin D and 5 µl of Annexin V-FITC as recommended by the manufacturer for 15 min at room temperature. Cells were analyzed by flow cytometry as described above. Cells that stained positive for Annexin V and negative for 7-amino-actinomycin D were considered for the quantification.

FTY720 treatment
FTY720 (Cayman, Ann Arbor, MI, USA) was dissolved in ethanol:PBS (1:2). C57Bl/6 male mice were injected intraperitoneally with 1 mg/kg and euthanized after 12 h. Lymphocytes were isolated from thymus, spleen, liver, and blood, and the numbers of NKT cells and conventional T cells were determined by flow cytometry as described above.

Statistical analysis
Statistical significance was determined using Student’s t test for two samples assuming unequal variance that was corroborated by the single-factor ANOVA test or the Mann-Whitney test. In all cases, values of P < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of the S1P receptors by NKT cells
To investigate the role of the S1P1 receptor signaling in NKT cell development in the thymus, we first determined the expression profile of the S1P receptors expressed in the immune system (1) within normal thymic NKT cells by real-time PCR (Fig. 1 A). Thymic NKT cells expressed S1P1, S1P2, and S1P4, whereas S1P3 was expressed at very low levels. We then examined the expression of the S1P1 receptor mRNA in developing NKT cells from normal thymus by real-time PCR (Fig. 1B ). Thymic NKT cells were sorted by their NK1.1 expression into NK1.1– cells (immature plus emigrating NKT cells) and NK1.1+ cells (more mature, thymic-resident NKT cells) (43 , 44 , 50) . Within the thymic NKT population, S1P1 receptor mRNA was more highly expressed in NK1.1- NKT cells compared to resident NK1.1+ NKT cells. These results indicate that S1P1 receptor expression is developmentally regulated on NKT cells and suggest that lower S1P1 receptor expression levels might be associated with NKT cell residence within the thymus.


Figure 1
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  		Figure 1. Expression of S1P receptors in NKT cells. A) NKT cells from the thymi of control (white bars) and TCS1P1KO mice (red bars) were isolated, and the expression of S1P1 (Edg1), S1P2 (Edg5), S1P3 (Edg3), and S1P4 (Edg6) mRNA was determined by real-time PCR. The relative level of GAPDH mRNA expression in each sample was set to 1. The results were expressed as mean value ± SD. (Student’s t test, *P=0.021, n=4). B) S1P1 (Edg1) receptor mRNA expression in NKT populations from normal mice was determined by real-time PCR. NKT cells from 3–5 pooled C57BL/6 thymi and spleens were sorted, and RNA was purified from the pooled samples (mean±SD of 2 real-time PCR determinations). C) Flow cytometry analysis of S1P1 receptor expression on NK1.1– and NK1.1+ NKT cells from thymus, spleen, liver, and blood from control mice. The profile in red indicates background staining using a control antibody (anti-GST), and the profile in blue indicates the staining with the anti-S1P1 receptor antibody.

We studied the expression of S1P1 on NKT cells by flow cytometry using a rabbit polyclonal antibody against the extracellular, N-terminal 49 amino acids of mouse S1P1 (7) (Fig. 1C ). Within thymus, a higher percentage of NK1.1– cells expressed S1P1 compared with NK1.1+ cells in basic agreement with the real-time PCR results. Within the spleen and liver a substantial fraction of NKT cells were S1P1 positive, regardless of their NK1.1 expression level. However, a relatively small fraction of blood NK1.1+ and NK1.1– NKT cells expressed S1P1. In blood, concentrations of S1P ligand are high, which has been shown to down-modulate S1P1 receptor on T cells (7) .

NKT cells are present within thymus but are reduced in the periphery in thymocyte-specific S1P1 receptor knockout mice
The global deletion of the S1P1 receptor results in embryonic lethality (12) . To delete the S1P1 receptor in thymocytes of adult mice, we generated thymocyte-specific knockout mice (TCS1P1KO) by crossing S1P1loxP/loxP mice with Lck-Cre transgenic mice (10) . Lck-Cre transgenic mice express the Cre recombinase under the control of the Lck proximal promoter, which drives expression specifically in immature thymocytes (51) . Both conventional T and NKT cells differentiate in the thymus from CD4+ CD8+ double-positive precursors (52 53 54) . Thus, NKT precursors should express the Cre recombinase in the TCS1P1KO mice, which would lead to the deletion of the S1P1 receptor expression (10) . Indeed, S1P1 receptor mRNA expression level in NKT cells isolated from the thymi of TCS1P1KO mice was reduced by greater than 97% compared to cells from littermate controls (Fig. 1A ). The expression levels of S1P2, S1P3, and S1P4 were not affected in the S1P1-deficient cells (Fig. 1A ).

We next determined the abundance of NKT cells in tissues from the TCS1P1KO mice. Within the TCS1P1KO mice, we found a substantial reduction of NKT cells that bound CD1d/{alpha}GalCer complexes in spleen, liver, and peripheral blood compared to littermate controls (Fig. 2 A, B). In contrast to the large reduction of NKT cells within the peripheral tissues of the TCS1P1KO mice, the proportion and number of NKT cells within the thymi of 8- to 12-wk-old TCS1P1KO mice compared to littermate controls were not reduced (Fig. 2A, B ). In 12-month-old TCS1P1KO mice, however, we detected a significant increase in thymic NKT cell numbers (determined as CD1d/{alpha}GalCer+ NKT cells and NK1.1+ NKT cells) compared with age-matched controls, (Fig. 2C ). As with younger animals, older TCS1P1KO mice showed a significant decrease in the number of NKT cells in spleen, liver, and peripheral blood compared to controls (data not shown).


Figure 2
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  		Figure 2. Distribution of NKT cells in TCS1P1KO mice. A, B) NTK cell proportions and numbers were reduced in peripheral tissues from TCS1P1KO mice. Lymphocytes from thymus, spleen, liver, and blood from 8- to 12-wk-old control and TCS1P1KO mice were analyzed by flow cytometry using PE-labeled CD1d/{alpha}GalCer complexes and FITC-conjugated anti-TCRβ antibody to identify NKT cells (CD1d/{alpha}GalCer+ TCR+). A) Plots show percentages of NKT cells (red gates) in each organ from control and TC1P1KO mice (mean±SD; *P=0.020, **P=0.048, *** =0.001, t test; n=6 to 9 mice of each genotype). B) NKT cell absolute cell numbers were calculated in the different organs from control (white bars) and TC1P1KO mice (red bars) (mean±SD; *P=0.011, **P=0.005, t test; n=6 to 9 mice of each genotype). C) NTK cell numbers were increased in thymus from 12-month-old TCS1P1KO mice. Lymphocytes from the thymi of control and TCS1P1KO mice were isolated and analyzed by flow cytometry using PE-labeled CD1d/{alpha}GalCer complexes, and FITC-conjugated anti-TCRβ antibodies were used to identify NKT cells. Results are expressed as percentage of the control sample (mean±SD; *P=0.05; n=4). D) CD44highNK1.1+ NKT cells are not reduced in TC1P1KO thymus. Left: NKT cells from control and TC1P1KO thymus (boxed) were identified by staining with APC-conjugated CD1d/{alpha}GalCer tetramers, in combination with CyChrome-conjugated anti-B220 and CyChrome-conjugated unloaded CD1d tetramer (to increase specificity of CD1d/{alpha}GalCer staining). Right: CD1d/{alpha}GalCer+ NKT cells were analyzed for expression of FITC-conjugated CD44 and PE-conjugated NK1.1. Percentages of CD44highNK1.1+ NKT cells are indicated. Results shown are the representative staining of n = 3 of each genotype. E, F) Spleen and liver NKT cells from control (white bars) and TC1P1KO (red bars) mice were identified by staining with PE-labeled CD1d/{alpha}GalCer complexes and FITC-conjugated anti-TCRβ antibody (CD1d/{alpha}GalCer+ TCR+) and were analyzed for the expression of NK1.1 by flow cytometry. Results are expressed as absolute number of NKT cells in each organ (mean±SD; *P=0.035, **P<0.008, t test; n=3 mice of each genotype).

In the thymus, early precursor CD44lowNK1.1- NKT cells give rise to CD44highNK1.1– and, subsequently, to CD44highNK1.1+ NKT cells (43 , 50) . The results in Fig. 2D show that TCS1P1KO thymus contained normal levels of CD44highNK1.1– and CD44highNK1.1+ NKT cells, indicating the NK1.1+ maturation of NKT cells in the thymus occurred normally even with a peripheral deficiency of NKT cells.

These results show that, in the absence of S1P1 receptor expression, NKT cells develop and are present in normal or, in the case of older animals, increased numbers within the thymus but are substantially decreased in amounts within peripheral tissues. If NKT cells were blocked in their exit from the thymus due to the absence of S1P1 receptor expression, as suggested by their distribution in the TCS1P1KO mice, an expectation would be that NKT cells that have recently emigrated from the thymus (NK1.1–) would also be decreased in the periphery. We, therefore, quantified the peripheral NK1.1– NKT cell population and found that, within the spleen and liver, NK1.1– NKT cells were substantially decreased in TCS1P1KO mice (Fig. 2E, F ). As would be expected, the peripheral NK1.1+ NKT cells, which are derived from the recently emigrated NK1.1– population (43) , were also largely depleted (Fig. 2E, F ).

We next determined whether the S1P1 receptor is involved in the maintenance of NKT cell tissue distribution after their establishment in the periphery. Adult inducible S1P1 KO mice (S1P1loxP/loxP carrying the Mx-Cre transgene) were treated with pIpC and NKT cells were examined 2 wk and 6–7 months after Cre induction. S1P1 receptor expression was significantly decreased in NKT cells after Cre induction (Fig. 3 A). At 2 wk after Cre induction, the proportions and numbers of NKT cells in blood and liver of the adult inducible S1P1 KO mice were not significantly different from controls, suggesting that NKT survival and residency in these organs were not affected by the S1P1 receptor deletion (Fig. 3B, C ). We did, however, detect a decrease in NKT cell proportion and number within the spleen of adult inducible S1P1 KO mice (Fig. 3B, C ). In mice in which Cre had been induced 6–7 months prior to analysis, both NKT (Fig. 3D, E ) and conventional T cell (not shown) proportions and numbers were decreased significantly in the peripheral tissues, consistent with a long-term block in thymic emigration.


Figure 3
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  		Figure 3. Effect of S1P1 deletion in adult inducible S1P1 KO mice on NKT cells. A–E) Distribution of NKT cells in adult inducible S1P1 KO mice. Adult controls (S1P1loxP/loxP) (white bars) and inducible S1P1 KO mice (S1P1loxP/loxP carrying Mx-cre) (red bars) were analyzed 2 wk (A–C) or 6–7 months (D, E) after pIpC treatment. A) S1P1 receptor mRNA expression in liver NKT cells is shown 2 wk after pIpC treatment. The proportions (B, D) and absolute numbers (C, E) of NKT cells in thymus, spleen, liver, and blood were determined as described in Fig. 2 . Data are expressed and analyzed as in Fig. 2 . A) n = 4 mice of each genotype; **P = 0.03 (t test). B, C) n = 7 mice of each genotype; **P = 0.03***; P < 0.002 (Mann-Whitney test). D, E) n = 5 of each genotype; *P = 0.05; **P = 0.03***; P < 0.002 (Mann-Whitney test). F) Expression of Annexin V was determined on NKT cells from thymus, spleen, liver, and blood of control (white bars) and inducible S1P1 KO mice (red bars) 10 wk after pIpC injection. Results are shown as percentage of Annexin V+ NKT cells (mean ± SD; n=3).

It was also possible that NKT cells were more susceptible to apoptotic death in peripheral organs in the S1P1-deficient mice, which explains their deficiency. Thus, we determined the apoptosis levels by Annexin V staining on NKT cells from adult inducible S1P1 KO mice 10 wk after Cre induction. Percentages of Annexin V+ NKT cells in thymus, spleen, blood, or liver of adult inducible S1P1 KO mice were not significantly different when compared with control littermates (Fig. 3F ).

Because surface CD69 expression is up-regulated in T and B lymphocytes lacking the S1P1 receptor (6 , 7) , we determined whether deletion of the S1P1 receptor also resulted in altered levels of surface CD69 on NKT cells. Unlike the TCR high-expressing thymocytes from TCS1P1KO mice, which showed higher levels of CD69 expression compared to controls, the CD69 expression on thymic NKT cells from the same TCS1P1KO mice did not change (data not shown).

FTY720 does not alter peripheral distribution of NKT cells
The S1P1 receptor ligand FTY720, after a 12 h treatment, provokes a substantial deficiency of T cell levels in blood by blocking the emigration of conventional T cells from lymph nodes (22 , 24 , 30 31 32) . We investigated whether FTY720 could also affect NKT cell peripheral distribution by treating wild-type mice with FTY720 for 12 h. A relatively short exposure with FTY720 was utilized because longer-term treatments have reportedly produced significant T cell apoptosis (55 56 57) . Within the thymi of FTY720-treated mice, NKT cells showed a very slight but statistically significant increase compared to vehicle-treated mice consistent with a block in egress (Fig. 4 A, B), while the percentage of conventional T cells did not vary significantly after FTY720 treatment (Fig. 4A, C ). In the periphery, FTY720 did not significantly change NKT cell numbers in blood, spleen, and liver from treated mice when compared to vehicle-treated animals (Fig. 4A, B ). By contrast, FTY720 treatment produced a substantial reduction of conventional T cell proportion and numbers in blood and liver (Fig. 4A, C ). The splenic T cell numbers were not significantly altered by FTY720 treatment (Fig. 4A, C ). In the peripheral lymph nodes, the total cell number increased by ~2-fold after FTY720 treatment. However, NKT cells remained within the background range after treatment (data not shown).


Figure 4
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  		Figure 4. FTY720 treatment does not alter peripheral distribution of NKT cells. C57Bl/6 mice were injected with 1 mg/kg FTY720, and tissues were removed after 12 h. Lymphocytes were stained with PE-labeled CD1d/{alpha}-GalCer complexes and FITC-conjugated anti-TCRβ antibody to identify NKT cells (CD1d/{alpha}GalCer+ TCR+) and conventional T cells (CD1d/{alpha}GalCer– TCR+) by flow cytometry. Values are presented as percentage of the total lymphocyte population (A), and absolute number of cells of NKT (B) and conventional T cells (C) in thymus, spleen, liver, and blood from FTY720-treated mice (red bars) compared with those from vehicle-treated mice (white bars). Data are expressed as mean ± SD from 9–11 mice for each treatment; A) * P = 0.003, ** P = 3 x 10–5, *** P = 1 x 10–9; B, C) * P = 0.04, ** P = 1 x 10–4, ** P = 1 x 10–7 (Mann-Whitney test). NKT: red gates, T cells: black gates.

The inability of FTY720 to alter the peripheral distribution of NKT cells may be due to a relatively low rate of entry of NKT cells into peripheral lymph nodes and, thus, a lack of FTY720-induced sequestration. Indeed, NKT cells are rare in peripheral lymph nodes (36 , 37 , 45 , 46 , 58 59 60 61) . Lymphocyte entry from blood into lymph nodes requires the expression of CD62L (or L-selectin) on the lymphocyte, which mediates tethering and rolling over high endothelial venules (62 63 64) . We found that blood NKT cells expressed levels of CD62L that were barely above background (Fig. 5 A, B). Since the chemokine receptor CCR7 also plays an important role in the migration of lymphocytes through high endothelial venules into lymph nodes (65) , we analyzed the expression of CCR7 on blood NKT cells. In this case, only a small fraction of NKT cells expressed CCR7 (Fig. 5C ). It has been shown that CCR7 expression is also generally low on human NKT cells in blood (61 , 66 , 67) . After 12 h treatment with FTY720, the profile of CCR7 and CD62L expression on blood NKT cells did not significantly change (not shown).


Figure 5
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  		Figure 5. CD62L and CCR7 expression on NKT cells. Blood NKT cells from control mice were analyzed by flow cytometry. NKT cells were gated using PE-labeled CD1d/{alpha}GalCer complexes and FITC-conjugated anti-TCRβ antibody staining (CD1d/{alpha}GalCer+ TCR+, red gate) (A) and further analyzed using either APC-conjugated CD62L (B) or APC-conjugated CCR7 antibodies (C). Data shown are representative of 9 mice for CD62L staining and 6 mice for CCR7 staining.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
NKT cells display a distinctive tissue distribution pattern. Within the thymus, separate populations of NKT cells exist, including one that will emigrate and another that will become resident. In the periphery, NKT cells populate the blood, bone marrow, liver, and spleen but are rare in peripheral lymph nodes (36 , 37 , 45 , 46) . The basis for NKT cell trafficking and homing is not well understood, however, the "activated" phenotype displayed by NKT cells might exclude them from peripheral lymph nodes and instead guide them into peripheral tissues (61) . For the most part, NKT cells express a profile that is similar to Th1 inflammatory homing cells (high levels of the chemokine receptors CCR5 and CXCR3 and lower levels of CXCR5 and CCR7, together with high expression of the activation marker CD69 and glycosaminoglycan hyaluronan receptor CD44), suggesting that these cells perform their function in peripheral tissues rather than in secondary lymphoid organs (58 59 60 61) . Moreover, NKT cells that are found in splanchnic lymph nodes are in a less activated state than those in spleen (59) .

Our results with S1P1 receptor knockout mice indicate that the expression of the S1P1 receptor on thymic NKT cells is essential for their normal tissue distribution pattern, with normal or, as the mice age, increased numbers within the thymus and substantially reduced levels in the periphery when S1P1 receptor expression is absent. Within the periphery, the NK1.1- NKT cells, comprising recent emigrants, were reduced under these same experimental conditions. Such a pattern is consistent with a block in NKT cell emigration from thymus as has previously been observed for conventional T cells in the absence of S1P1 expression (6 , 10) . Our finding that the NKT cell population representing long-term residents within the thymus expressed lower S1P1 receptor levels than the emigrating NKT population supports a role for S1P1 receptor expression in the decision of NKT cells to leave the thymus. An alternative possibility is that S1P1 receptor deficient NKT cells exit the thymus but are defective in their homing to tissues, which explains their absence in the periphery. In this case, one might expect an increase in apoptotic NKT cells in the S1P1 knockout mice, which was not detected. We did, however, find an eventual accumulation of thymic NKT cells in S1P1 knockout mice as they aged consistent with a block in egress.

It has been unclear whether the CD44highNK1.1+ subset in the thymus and in the periphery were independent products of the CD44highNK1.1– NKT cells or, alternatively, whether some CD44highNK1.1– NKT cells that mature in periphery and acquire NK1.1 re-circulate back to the thymus. A block in thymic egress in the S1P1 receptor knockout mice would support the conclusion that maturation to NK1.1 NKT cells can occur within the thymic compartment without first entry into the periphery.

NKT cells and conventional T cells share the requirement for S1P1 expression while maturing within the thymus in order to emerge into the periphery. In contrast, the signaling of lymphotoxin {alpha}β through the LTβ receptor was shown to be indispensable for NKT cells, but not for conventional T cells, to emigrate from the thymus (68) . The critical LTβ receptor signaling event that regulates NKT cell exit occurs in thymic stromal cells, a mechanism distinct form the intrinsic lymphocyte requirement demonstrated for S1P1 receptor function.

Once established in peripheral tissues, short-term deletion of S1P1 receptor expression did not alter tissue distribution of NKT cells except for a partial decrease in numbers within the spleen, suggesting that the S1P1 receptor may function in homing or retention of splenic NKT cells. A role for the S1P1 receptor in the retention of B cells to the splenic marginal zone has also been described (8) . After long-term deletion of the S1P1 receptor, a more generalized NKT cell deficiency was apparent consistent with a block in the exit of cells from thymus to the periphery.

FTY720 blocks the egress of conventional T cells from thymus and lymph nodes through an interaction with the S1P1 receptor, although the precise mechanism is not entirely clear. Suggestions have included i) the down-regulation of the lymphocyte S1P1 receptor (69 70 71) , which chemotactically directs lymphocytes into blood or lymph; and ii) the closing of a stromal (endothelial cell) gate (4 , 72) . Unlike the potent effect of FTY720 on the peripheral distribution of conventional T cells due to sequestration into lymph nodes (22 23 24 25 26 27 28 29) , the peripheral NKT cell distribution was not altered by exposure to FTY720. These results, together with experiments using the S1P1 adult conditional knockout mice, indicate that NKT and conventional T cells share a requirement for S1P1 receptor for peripheral emergence. Once in the periphery, however, NKT and convention T cell distributions are distinctly affected by the S1P1 receptor signal. Possibly, the "activated" phenotype displayed by NKT cells, which includes low CD62L and CCR7 expression, might exclude NKT cells from peripheral lymph nodes (58 59 60 61) , making them less susceptible to the effects of S1P receptor ligand, whether its site of action is on the lymphocyte or endothelial S1P1 receptor. Similarly, a subpopulation of CD4+/CD25+ T-regulatory cells expressing low levels of CD62L was more resistant than conventional T cells to sequestration from blood into lymph nodes by FTY720 (34 , 35) .

Our results indicate that, like conventional T cells, S1P1 receptor expression on NKT cells is necessary for emergence from thymus and appearance in peripheral tissues. However, once within the periphery, the two cell populations diverge in their reliance on the S1P1 receptor for regulation of trafficking. The possibility that S1P signaling may control aspects of peripheral NKT cell function other than trafficking is open.


   ACKNOWLEDGMENTS
 
We thank Dr. Jason Cyster for providing the S1P1 antibody and Dr. Masaru Taniguchi for the {alpha}GalCer. This research was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, and by National Institutes of Heath grant AI038338 to A.B. A.B. is a Howard Hughes Medical Institute Investigator. D.Z. was supported by the M.D. Anderson Cancer Center

Received for publication May 22, 2007. Accepted for publication August 2, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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