Signaling by sphingosine 1-phosphate (S1P) through its receptor S1P1 has recently been shown to promote thymocyte egress. In the periphery, S1P1 is expressed on naive T cells but lost upon T cell activation. To determine the significance of S1P1 down-regulation and function of S1P1 in peripheral T cells, we developed transgenic mice that constitutively express S1P1 in T cells. Mature T cells from these mice exhibited enhanced chemotactic response toward S1P, and preferentially distributed to the blood rather than secondary lymphoid organs. S1P1-transgenic mice showed significant delay in the onset of experimental autoimmune encephalomyelitis, and had defective contact hypersensitivity reaction and local Ag-induced responses. These impairments were associated with reduced numbers of Ag-activated T cells in the draining lymph nodes. Our studies demonstrate that S1P1 signaling affects systemic trafficking of peripheral T cells and immune responses and highlight that levels of S1P1 expression represent an important mechanism of immune regulation.

Sphingosine 1-phosphate (S1P),2 a bioactive lipid mediator found in high levels in blood (0.1–1 μM), signals through G protein-coupled receptors (1). Among the five receptors for S1P, S1P1 and S1P4 are highly expressed in T cells and S1P1 is involved in regulating chemotactic responses of T cells in vitro (2, 3). Recent studies have revealed a critical function of S1P1 in promoting thymocyte egress in vivo. In mice whose T cell lineage lacks S1P1, there are no T cells in the periphery because mature T cells are unable to exit the thymus (4, 5). Adoptively transferred S1P1-deficient thymocytes readily accumulate in secondary lymphoid organs but fail to egress into blood (4). Thus, signaling by S1P1 is required for emigration of developing T cells into blood. FTY720, a novel immunosuppressive drug for transplantation and autoimmunity, is able to induce aberrant internalization and functional inactivation of S1P1. This renders the cells unresponsive to S1P, depriving them of an obligatory signal to egress from lymphoid organs (6, 7, 8).

Despite these recent developments, the role of S1P1 in regulating mature T cell function has not been addressed, because mice deficient in S1P1 lack T cells in the periphery (4). The results obtained from the FTY720 studies are informative but not conclusive: for example, in addition to causing S1P1 inactivation, phosphorylated FTY720 is a potent agonist for multiple S1P receptors (6, 7). FTY720 also targets other cell types including vascular endothelial cells, which may contribute to its immunoregulatory function (9). Furthermore, although T cell activation is associated with dynamic changes in S1P1 expression (2, 4), whether S1P1 modulates immune responses is not known. From functional genomics studies, we found that S1P1 levels are drastically reduced after peripheral T cell activation; we were therefore interested in determining the biological relevance of such down-regulation. To this end, we developed transgenic mice with constitutive expression of S1P1 in T cells. We found that levels of S1P1 expression influence trafficking of peripheral T cells, with a direct effect on the magnitude of T cell-mediated immune response and disease.

The mouse S1P1 cDNA was amplified by RT-PCR from T cell RNA and subcloned into the human CD2 promoter construct. To generate transgenic mice, the expression cassette was excised and injected into fertilized eggs of C57BL/6 (B6) mice. Founder mice were identified by PCR and maintained in B6 background.

CD4 T cells were isolated from peripheral lymph nodes (PLN) using CD4-coupled beads (Miltenyi Biotec). The response of T cells toward S1P was examined using 6.5-mm Transwell inserts with a 5-μm pore size (Corning), as described previously (5). T cell suspension (100 μl at 107/ml) in RPMI 1640 medium plus 0.4 mg/ml fatty acid-free BSA (Sigma-Aldrich) was added to each insert in a well containing 600 μl of medium alone (control), or 10 nM S1P (Sigma-Aldrich) in the same medium. In some experiments, a reverse gradient of S1P was used in that T cell suspension in medium containing S1P was added to an insert in a well with medium alone. After 3 h at 37°C, cells migrated to the lower chamber were harvested and counted using a hemocytometer.

S1P1-transgenic (Tg) mice were crossed with CD45.1+ mice for one generation. CD45.1+ S1P1-Tg or wild-type (WT) CD4 T cells were mixed with approximately same number of CFSE-labeled B6 (CD45.2+) T cells, and injected i.v. into B6 recipients. Five hours later, lymphocytes were prepared from blood, spleen, PLN (including inguinal, axillary, and brachial lymph nodes (LN)), and mesenteric lymph nodes (MLN) of recipient mice, stained with CD45.1 and CD4 Abs, and analyzed by flow cytometry. The results were expressed as a ratio between CD45.1+ T cells and the cotransferred CFSE+ internal control cells. An aliquot of the input population was analyzed by flow cytometry to correct for variability in the relative frequency of the cotransferred cells.

Mice were sensitized on day 0 by epicutaneous application to shaved abdominal skin of 25 μl of 0.5% 2,4-dinitrofluorobenzene (DNFB) in a mixture of acetone:olive oil (4:1). On day 5, after measuring baseline ear thickness with an engineer’s micrometer, mice were challenged by applying 10 μl of 0.2% DNFB to each side of each ear. The ear thickness was measured daily over the next 3 days. Results were expressed as the ear swelling response above baseline ± SD. For adoptive transfer of CHS, 5 days after sensitization, single-cell suspensions were prepared from draining lymph nodes (DLN), and 25 × 106 DLN cells were injected i.v. into naive B6 mice. Two hours later, recipient mice were challenged with 0.2% DNFB, and ear thickness was measured as above.

OT-II CD4 T cells purified from PLN of S1P1-Tg or WT mice were injected i.v. into CD45.1+ recipient mice (1.5 × 106 cells/mouse). After 24 h, they were injected s.c. with 1 mg of OVA (Sigma-Aldrich) in CFA in two flanks. Mice were sacrificed on various days after immunization, and the draining inguinal LN were isolated and stained with Abs: donor-derived Ag-specific cells were identified as CD4+, TCR Vα2+, and CD45.2+, and normalized as percentages of the total CD4 T cells in the DLN of the recipient mice. In some experiments, donor cells were labeled with 5 μM CFSE before adoptive transfer.

EAE was induced by s.c. flank injections of 50 μg of myelin oligodendrocyte glycoprotein (MOG)35–55 peptide (synthesized in the Keck Facility at Yale University) in CFA (Difco) with 500 μg of Mycobacterium tuberculosis on day 0, supplemented by i.p. injections of 200 ng of pertussis toxin on day 0 and day 2. The mice were observed daily for clinical signs and scored on a scale of 0–5: 0, no clinical signs; 1, flaccid tail; 2, wobbly gait; 3, partial hindlimb paralysis; 4, complete hindlimb paralysis; 5, complete hindlimb paralysis and forelimb weakness or paralysis.

All of the results were expressed as means ± SD with at least three mice per group. Values of p were determined using Student’s t test. A value of p < 0.05 was regarded as statistically significant (indicated by the asterisk in the graphs).

In the analyses of gene expression profiles of T cell differentiation (10), we identified that S1P1 mRNA was highly expressed in naive T cells but lost upon T cell activation. We confirmed the gene array results with a Northern blot analysis: 24 h after activation with anti-CD3 and anti-CD28, S1P1 mRNA was essentially undetectable (Fig. 1,A). This finding was consistent with recent studies that showed activation-induced reduction of S1P1 expression (2, 4). To determine the significance of S1P1 down-regulation and function of S1P1 in peripheral T cells, we developed transgenic mice that express S1P1 in the T cell compartment (S1P1-Tg) under the control of the human CD2 promoter. Under these conditions, we anticipated that activation-induced down-regulation of S1P1 would be prevented. Two founder mice were obtained in which expression levels of the S1P1 transgene were similar to those of the endogenous gene. More importantly, after TCR stimulation, although the endogenous S1P1 expression was minimal, the transgene expression was maintained at similar levels as in unstimulated cells (Fig. 1 A).

FIGURE 1.

Generation and analysis of S1P1-Tg mice. A, Northern blot analysis of S1P1 mRNA in resting (rest) and 24 h-activated (act) WT and S1P1-Tg CD4 T cells. Endogenous and transgenic S1P1 mRNAs were indicated by the upper and lower arrows, respectively. Ethidium bromide (EtBr)-stained 18s and 28s RNA were used as loading controls. B, Altered cell numbers in S1P1-Tg PLN, spleen, and blood (expressed as cell number per milliliter of blood). Total lymphocytes in these tissues were enumerated and the numbers of CD4 and CD8 were calculated based on the total lymphocyte number multiplied by the percentage of each subset.

FIGURE 1.

Generation and analysis of S1P1-Tg mice. A, Northern blot analysis of S1P1 mRNA in resting (rest) and 24 h-activated (act) WT and S1P1-Tg CD4 T cells. Endogenous and transgenic S1P1 mRNAs were indicated by the upper and lower arrows, respectively. Ethidium bromide (EtBr)-stained 18s and 28s RNA were used as loading controls. B, Altered cell numbers in S1P1-Tg PLN, spleen, and blood (expressed as cell number per milliliter of blood). Total lymphocytes in these tissues were enumerated and the numbers of CD4 and CD8 were calculated based on the total lymphocyte number multiplied by the percentage of each subset.

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We analyzed the lymphoid system of S1P1-Tg mice. Thymocytes of S1P1-Tg and WT mice had similar distributions of various markers, including CD4, CD8, CD3, CD62L, and CD24 (data not shown). Compared with WT littermates, PLN in S1P1-Tg mice appeared slightly smaller, and the total number of lymphocytes was reduced by ∼30%. In contrast, the S1P1-Tg spleens were moderately enlarged, albeit with a normal architecture (data not shown). The total cell number of splenocytes was increased by 70% (Fig. 1,B). Flow cytometry analysis showed that the numbers of CD4 and CD8 T cells were reduced in the PLN but increased in the spleen and peripheral blood (Fig. 1 B). To determine effects of S1P1 expression on homeostatic status of T cells, we analyzed expression of surface markers CD62L and CD44. S1P1-Tg spleens had more CD62LlowCD44high memory-like T cells (data not shown), suggesting that these cells may have undergone spontaneous activation in vivo. Given the complex mechanisms that could be involved in such a process, in our following analyses, we used CD4 T cells isolated from PLN of WT and S1P1-Tg mice, which had comparable ratios of memory vs naive T cells (data not shown). We reasoned that this would allow us to evaluate the intrinsic differences between these two groups of cells.

S1P is known to induce a chemotactic response of peripheral T cells in vitro (2). To examine effects of increased S1P1 expression on the S1P responsiveness, we performed Transwell migration experiments (Fig. 2 A). WT CD4 T cells exhibited a modest chemotactic response toward S1P. In contrast, S1P1-Tg cells showed a significantly greater migratory response. When the gradient of S1P was reversed by placing S1P together with T cells in the upper chamber, both WT and S1P1-Tg cells had a similar low degree of migration. Thus, increased expression of S1P1 rendered T cells more responsive to S1P-induced chemotaxis, indicative of enhanced S1P-mediated signaling in these cells. These results also validated the use of the transgenic mice as a “gain-of-function” system to study the role of S1P1.

FIGURE 2.

Altered migration of S1P1-Tg CD4 T cells. A, CD4 T cells from WT or S1P1-Tg PLN were added to the Transwell inserts (with or without S1P) and allowed to respond to medium alone, or 10 nM S1P in the lower chamber of the Transwell. Results are shown as the percentage of input cells that migrated after 3 h of incubation. ∗, p < 0.05 when compared with WT response to medium alone; #, p < 0.05 when compared with S1P1-Tg response to medium alone, and compared with WT response to S1P. B, CD45.1+ CD4 T cells from WT or S1P1-Tg mice were cotransferred with equal numbers of CFSE-labeled T cells into B6 mice. Five hours later, PLN, MLN, spleen, and blood were examined for the presence of donor cells by flow cytometry. The results are expressed as normalized ratios between WT or S1P1-Tg cells (CD45.1+) and the cotransferred control cells (CFSE+).

FIGURE 2.

Altered migration of S1P1-Tg CD4 T cells. A, CD4 T cells from WT or S1P1-Tg PLN were added to the Transwell inserts (with or without S1P) and allowed to respond to medium alone, or 10 nM S1P in the lower chamber of the Transwell. Results are shown as the percentage of input cells that migrated after 3 h of incubation. ∗, p < 0.05 when compared with WT response to medium alone; #, p < 0.05 when compared with S1P1-Tg response to medium alone, and compared with WT response to S1P. B, CD45.1+ CD4 T cells from WT or S1P1-Tg mice were cotransferred with equal numbers of CFSE-labeled T cells into B6 mice. Five hours later, PLN, MLN, spleen, and blood were examined for the presence of donor cells by flow cytometry. The results are expressed as normalized ratios between WT or S1P1-Tg cells (CD45.1+) and the cotransferred control cells (CFSE+).

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Whether S1P1 regulates mature T cell migration in vivo has not been shown. We used an adoptive transfer approach to evaluate involvement of S1P1 in this process. Donor CD4 T cells from PLN of CD45.1+ S1P1-Tg or WT mice were transferred i.v. into WT recipients. An equal number of CFSE-labeled CD45.2+ cells were cotransferred as an internal standard. Five hours later, the frequency of donor T cells populating the blood and secondary lymphoid organs was determined by flow cytometry. As shown in Fig. 2 B, transferred S1P1-Tg CD4 cells showed a reduced distribution to the PLN, and also had a moderate reduction in populating the MLN and spleen. In contrast, compared with WT cells, there was a 60% increase in the number of transferred S1P1-Tg cells in the blood of recipient mice. Therefore, enforced expression of S1P1 favors distribution of peripheral T cells to the blood rather than secondary lymphoid organs, suggesting that S1P1 expression is sufficient to affect systemic trafficking of mature T cells.

Having established that S1P1-Tg T cells had altered migration in vivo, we next determined the consequences of such a change in a model of T cell-mediated immune response. CHS is a T cell-mediated cutaneous immune/inflammatory reaction to haptens. Defective T cell migration is known to cause impaired CHS responses (11, 12). Mice were sensitized with DNFB, and ear swelling was assessed upon rechallenge with the same allergen 5 days later. The ear swelling was significantly lower in S1P1-Tg mice than WT mice over a period of 3 days (Fig. 3,A). CHS involves two distinct phases: during initiation or sensitization, naive T cells in the DLN undergo clonal expansion and differentiate into effector cells; during elicitation, effector T cells accumulate at sites of allergen reapplication where their activation initiates a proinflammatory cascade. To distinguish whether S1P1 regulates CHS initiation and/or elicitation, we isolated DLN cells from DNFB-immunized S1P1-Tg and WT mice. Cell yields from S1P1-Tg mice were only 40% of WT mice, suggesting that S1P1-Tg mice had reduced responses during the initiation phase (Fig. 3,B). However, the functionality of these cells appeared to be normal, because when the same numbers of DLN cells were transferred into nonsensitized recipients, both WT and S1P1-Tg cells induced comparable levels of responses upon Ag exposure (Fig. 3 C).

FIGURE 3.

Impaired CHS response in S1P1-Tg mice. A, DNFB-sensitized mice were rechallenged, and ear thickness was measured. B, Five days after sensitization, DLN were isolated, and total lymphocyte numbers were counted. C, Adoptive transfer of DLN cells from DNFB-sensitized WT and S1P1-Tg mice to naive recipients resulted in equivalent ear-swelling responses after DNFB challenge.

FIGURE 3.

Impaired CHS response in S1P1-Tg mice. A, DNFB-sensitized mice were rechallenged, and ear thickness was measured. B, Five days after sensitization, DLN were isolated, and total lymphocyte numbers were counted. C, Adoptive transfer of DLN cells from DNFB-sensitized WT and S1P1-Tg mice to naive recipients resulted in equivalent ear-swelling responses after DNFB challenge.

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To further address the effects of S1P1 on primary responses of T cells, we used a TCR-transgenic mouse model, which allowed us to visualize activation of Ag-specific T cells directly. The S1P1 transgene was bred onto OT-II TCR transgenic mice, in which CD4 T cells express a TCR specific for OVA323–339. S1P1-Tg or WT OT-II T cells were purified from PLN, labeled with CFSE, and separately transferred into congenic CD45.1+ mice. At 24 h after T cell transfer, mice were immunized s.c. with OVA in CFA. The percentages of donor-derived CD4 T cells (CD45.2+Vα2+) in the DLN of the recipient mice were determined before and after immunization. Before immunization, donor-derived WT and S1P1-Tg OT-II cells constituted 0.58 and 0.37% of the total CD4 T cells of the recipient mice, respectively. Both groups of cells expanded in response to immunization, but the numbers of Ag-activated WT cells were significantly higher than those of S1P1-Tg cells at all times examined (Fig. 4,A). These results demonstrated that constitutive expression of S1P1 in T cells leads to reduction in the number of Ag-activated cells and the efficiency of T cell responses in the DLN. However, the activated cells in each group went through the same number of cell divisions in the CFSE labeling analysis (Fig. 4 B). Also, cell death was comparable between WT and S1P1-Tg T cells (data not shown). Thus, there were no intrinsic defects in cell proliferation or death of S1P1-Tg T cells.

FIGURE 4.

Reduced Ag-induced immune responses and EAE disease in S1P1-Tg mice. A, WT or S1P1-Tg OT-II CD4 T cells were transferred into naive CD45.1+ recipients. At 24 h (day 0), the recipient mice were immunized with OVA. The number of Ag-specific donor T cells (Vα2+CD45.2+ CD4 T cells) in the DLN was determined by flow cytometry before and after immunization. B, WT or S1P1-Tg OT-II cells were labeled with CFSE before transfer, and 3 days after immunization, CFSE fluorescence was analyzed on the donor cells in the DLN. C, WT and S1P1-Tg were immunized with MOG/CFA, and development of EAE disease was scored daily.

FIGURE 4.

Reduced Ag-induced immune responses and EAE disease in S1P1-Tg mice. A, WT or S1P1-Tg OT-II CD4 T cells were transferred into naive CD45.1+ recipients. At 24 h (day 0), the recipient mice were immunized with OVA. The number of Ag-specific donor T cells (Vα2+CD45.2+ CD4 T cells) in the DLN was determined by flow cytometry before and after immunization. B, WT or S1P1-Tg OT-II cells were labeled with CFSE before transfer, and 3 days after immunization, CFSE fluorescence was analyzed on the donor cells in the DLN. C, WT and S1P1-Tg were immunized with MOG/CFA, and development of EAE disease was scored daily.

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We then examined the importance of S1P1 in an organ-specific autoimmune disease. EAE is a mouse model for multiple sclerosis, which depends upon activation of CD4 T cells specific for one of the proteins in myelin. We tested whether the impaired immune response in S1P1-Tg mice could affect disease progression of EAE. Following immunization with MOG peptide, WT mice rapidly developed EAE, whereas S1P1-Tg mice showed substantially slower kinetics in disease development (Fig. 4 C). At later time points, S1P1-Tg mice developed similar clinical scores as WT, probably due to the accumulation of self-reactive cells in the CNS.

In this report, we have established that S1P1 signaling plays an important role in modulating systemic trafficking of peripheral T cells in vivo. T cells with increased expression of S1P1 and concurrent enhanced S1P-induced migratory response were preferentially distributed to the blood rather than secondary lymphoid organs. Accordingly, S1P1-Tg mice had reduced T cell numbers in PLN and a corresponding increase in the blood. Notably, these findings are consistent with either reduced entry of S1P1-Tg T cells into secondary lymphoid organs or accelerated exit into the blood. However, S1P1-Tg T cells exhibited enhanced chemotactic response toward S1P in vitro, whereas reversing this S1P gradient by placing T cells in a “high” concentration of S1P had no effects on the rate of migration (Fig. 2 A). Considering the higher concentration of S1P in the blood relative to various tissues, it is likely that, in response to such an S1P gradient, S1P1-Tg T cells have enhanced egress into the blood rather than reduced entry into lymphoid organs. This interpretation is in agreement with the loss-of-function studies using S1P1−/− thymocytes, which are able to enter lymphoid organs but unable to egress into blood (4, 5).

We have further demonstrated that enforced expression of S1P1 on T cells results in an impaired immune response. This conclusion is based on the results from three different models of T cell-mediated immunity: CHS, local Ag-induced activation, and an autoimmune model. In these assays, activated T cells were substantially reduced but not completely eliminated in the S1P1-Tg mice, consistent with the “gain-of-function” approach in which the effects of increased gene expression are likely to be quantitative relative to WT mice. Importantly, the S1P1-Tg T cells that were activated appeared to be functional: they underwent normal cell division and death, and upon transfer to naive mice, were able to confer Ag reactivity. Thus, in contrast to the in vitro observation that the S1P1 pathway may transduce negative signals for T cell proliferation (13), there were no intrinsic proliferative defects in S1P1-Tg T cells in vivo. Most likely, the impaired trafficking and insufficient retention of S1P1-Tg T cells in the LN is responsible for the defective immune responses. Similar deficiencies in T cell-mediated immune responses have been observed in mice with reduced LN homing, for example, in CD62L−/− mice (11, 12).

Following T cell activation, expression of S1P1 is significantly reduced (2, 4). Our data have provided direct evidence for the significance of S1P1 down-regulation, because constitutive expression of S1P1 results in compromised immune reaction. Thus, levels of S1P1 expression represent an important mechanism of T cell regulation to ensure that a productive immune response could occur. It will be interesting to identify factors that regulate differential S1P1 expression in naive and activated cells.

The authors have no financial conflict of interest.

We thank S. V. Kim, B. Lu, R. E. Tigelaar, E. H. Tran, and Y. Y. Wan for valuable advice and discussions.

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.

2

Abbreviations used in this paper: S1P, sphingosine 1-phosphate; S1P1, S1P receptor 1; LN, lymph node; PLN, peripheral lymph node; MLN, mesenteric lymph node; DLN, draining lymph node; Tg, transgenic; WT, wild type; CHS, contact hypersensitivity; DNFB, 2,4-dinitrofluorobenzene; EAE, experimental autoimmune encephalomyelitis; MOG, myelin oligodendrocyte glycoprotein.

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