Sphingosine-1-Phosphate Receptor Agonism Impairs the Efficiency of the Local Immune Response by Altering Trafficking of Naive and Antigen-Activated CD4⁺ T Cells

Mice

BALB/c mice were purchased from Taconic Farms (Germantown, NY). DO11.10 TCR transgenic mice (18) were purchased from The Jackson Laboratory (Bar Harbor, ME). DO11.10 mice express the DO.11.10 TCR, specific for the chicken OVA peptide OVA_323–339 in the context of the MHC class II molecule I-A^d. They have been backcrossed 15 generations onto the BALB/c background and are histocompatible with normal BALB/c mice. The AND TCR transgenic mice expressing an α/β TCR (Vα11, Vβ3), specific for pigeon cytochrome c_88–104 (PCC) (19), were purchased from The Jackson Laboratory, where they have been backcrossed to the B10.BR strain for five generations. Mice were further backcrossed to the B10.BR mice for seven generations at Merck Research Laboratories (Rahway, NJ).

In vitro MLR

Irradiated splenic cells (800 rad; 100 μl at 5 × 10⁶/ml) from C57BL/6 mice were cultured with BALB/c splenic cells (100 μl at 5 × 10⁶/ml) in RPMI 1640 medium (Cellgro; Mediatec, Washington, D.C.) supplemented with 2 mM l-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 50 μg/ml gentamicin, and 10% FCS. Cells were treated with various doses of FTY720 or FTY720-phosphate. Three days later, cells were pulsed with [³H]thymidine (2 μCi/well), and the next day incorporation of [³H]thymidine was measured with an LKB 1205 Betaplate liquid scintillation counter (Wallac, Gaithersburg, MD).

Generation of in vitro polarized Th1 cells

T cell differentiation was induced by culturing splenic cells from AND TCR mice (at 2 × 10⁶/ml) with1 μM PCC peptide plus 2 ng/ml IL-2 and 10 ng/ml IL-12. Cultures were fed with fresh medium (supplemented with peptide and appropriate cytokines) on days 2 and 4. Cells were harvested on day 6 and labeled with 1 μM Green Tracker (GT; 5-chloromethylfluorescein diacetate (CMFDA); Molecular Probes, Eugene, OR) before adoptive transfer to irradiated (750 rad) B10.BR mice through tail vein injection (1.5 × 10⁷/mouse). In some cases, naive splenic cells from AND TCR mice were adoptively transferred to irradiated B10.BR mice (5 × 10⁷/mouse). After 24 h mice were treated with vehicle or FTY720 (1 mg/kg orally). Peripheral blood was collected 18 h later, and the percentage of CMFDA-positive CD4⁺ T cells was determined by flow cytometry.

Adoptive transfer and Ag challenge

Splenocytes from DO11.10 mice were adoptively transferred into nonirradiated BALB/c mice by i.v. injection (3 × 10⁷/mouse). On the following day the BALB/c recipients were treated with various doses of FTY720 orally or with 1 mg/kg rapamycin (Wyeth Laboratories, Philadelphia, PA) i.p. After 4 h mice were given footpad injections of 100 μg of OVA (Sigma-Aldrich, St. Louis, MO) or PBS emulsified in the same volume of CFA (Sigma-Aldrich) in a total volume of 50 μl. FTY720 (orally) and rapamycin (i.p.) were administered daily until 1 day before harvest. Draining popliteal nodes (Ag-challenged side), nondraining popliteal nodes (contralateral side), and peripheral blood were collected at various times after Ag challenge. Cell suspensions were prepared, counted with a hemocytometer, stained with Abs, and then analyzed by flow cytometry.

Flow cytometric analysis

Popliteal node cells were stained with allophycocyanin-labeled anti-CD4 mAb (BD PharMingen, San Diego, CA) and PE-labeled KJ126 mAb (Caltag, Burlingame, CA), which binds exclusively to the DO11.10 TCR. Twenty thousand events were collected for each sample on a FACSCalibur flow cytometer (BD Bioscience, Mountain View, CA) and analyzed using CellQuest (BD Bioscience). The number of DO11.10 T cells that were identified as CD4⁺KJ126⁺ cells was calculated by multiplying the total lymph node cells counted with a hemocytometer by the percentage of CD4⁺KJ126⁺ cells determined by flow cytometry.

Peripheral blood samples were diluted 1/5 with PBS, layered on the same volume of Lymphocyte Separation Medium (ICN Biomedical, Aurora, OH), and centrifuged at 400 × g for 30 min. PBMC were resuspended in PBS, counted with a hemocytometer, and stained with allophycocyanin-labeled anti-CD4 mAb.

Measurement of cell cycle progression by CFSE in vivo

In some experiments DO11.10 splenocytes were labeled with 5 μM CFSE (Molecular Probes) before adoptive transfer to monitor cell cycle progression. FL-1 fluorescence was measured in CD4⁺KJ126⁺ T cells at various times after stimulation, and cells demonstrated a 2-fold decrease in FL-1 fluorescence with each successive round of cell division. Two hundred thousand events were collected for lymph node and PBMC samples using a FACSCalibur flow cytometer.

Measurement of in vivo T cell proliferation by 5-bromodeoxyuridine (BrdU) incorporation

BrdU (1 mg) was injected i.p. on day 2 after OVA CFA injection in the DO11.10 adoptive transfer model described above. The draining popliteal node was collected the next day and stained with anti-CD4-allophycocyanin, KJ126-PE, and anti-BrdU-FITC according to the manufacturer’s instructions (BD PharMingen).

In vivo MLR

FTY720 (0.5 mg/kg/day) was administered orally to C57BL/6 mice. After 4 h mice were given footpad injections of 1.5 × 10⁷ BALB/c splenocytes. The draining popliteal nodes were harvested 3 days after alloantigen challenge, and DLN cells were counted with a hemocytometer.

Reduced expansion in number of Ag-specific T cells by FTY720 in DLN of Ag-challenged mice

In this study Ag-mediated T cell responses in the DLN were examined by adoptive transfer of DO11.10 splenocytes to syngeneic BALB/c mice, followed by footpad injection with OVA Ag emulsified in CFA. The donor DO11.10 T cells were detected by flow cytometry using the anticlonotypic mAb, KJ126. Clonal expansion of CD4⁺KJ126⁺ cells occurred after Ag stimulation. The percentage of CD4⁺KJ126⁺ cells in the DLN (popliteal node from the Ag-injected side) was typically between 8–10% by day 3 after Ag challenge compared with 0.5% from nontreated mice (data not shown). The absolute number of CD4⁺KJ126⁺ cells, calculated by the total lymph node cell count multiplied by the percentage of CD4⁺KJ126⁺ cells, was typically increased by 50- to 80-fold (66-fold shown in Fig. 1⇓A) compared with nontreated mice. This T cell response required Ag, as adjuvant alone increased the number of CD4⁺KJ126⁺ T cells by only 2-fold (Fig. 1⇓A).

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FIGURE 1.

Reduced expansion of the number of Ag-specific T cells by FTY720 in DLN of Ag-challenged mice. Splenocytes from DO11.10 mice were adoptively transferred into unirradiated BALB/c mice by i.v. injection (3 × 10⁷/mouse). On the following day the BALB/c recipients were treated with rapamycin (1 mg/kg i.p.; A) or FTY720 (0.5 mg/kg orally in A and B, and various doses in C). After 4 h, mice were given footpad injections of 100 μg of OVA protein or PBS emulsified in same volume of CFA. FTY720 (FTY) and rapamycin (Rap) were given daily until 1 day before harvest. Draining popliteal nodes were harvested after 3 days of Ag challenge (A and C) or after 1, 2, 3, or 4 days of Ag challenge (B) and then stained with CD4-allophycocyanin and KJ126-PE. The percentage of CD4⁺KJ126⁺ cells in the DLN was determined by flow cytometry and multiplied by the total number of DLN cells to obtain the absolute number of CD4⁺KJ126⁺ cells. A, The number of CD4⁺KJ126⁺ T cells was reduced by 74% with FTY720 and by 77% with rapamycin treatment compared with the vehicle control after 3 days of Ag challenge. The expansion of CD4⁺KJ126⁺ cells was 74% less (∗∗, p < 0.005; n = 4) with FTY720 and 77% less with rapamycin treatment on day 3 after Ag challenge. The numbers of CD4⁺KJ126⁺ cells from mice with no injection or with PBS-CFA are also shown. One of eight comparable experiments is shown. B, The number of CD4⁺KJ126⁺ cells in FTY720-treated mice was reduced by 64% on day 1, 78% on day 2, 74% on day 3, and 68% on day 4 after Ag challenge compared with the vehicle controls (∗, p < 0.05 for days 2, 3 and 4; n = 4). One of two comparable experiments is shown. C, The expansion of CD4⁺KJ126⁺ cells was inhibited by 17, 37, 78, or 87% with 0.01, 0.03, 0.1, or 0.3 mg/kg FTY720, respectively. (∗, p < 0.05; ∗∗, p < 0.005; n = 5). One of two comparable experiments is shown. Values indicate the mean ± SEM.

To investigate the effect of FTY720 on T cell expansion in this Ag-induced model, mice were treated orally with 0.5 mg/kg FTY720 4 h before footpad injection of OVA CFA and then daily with the same dose until 1 day before harvest. We first examined the number of Ag-specific T cells 4 h post-FTY720 treatment without Ag challenge. As expected, they were depleted from the peripheral blood compartment but present in all secondary lymphoid organs, including the popliteal lymph node, which is the regional node for an Ag delivered through the footpad (data not shown). After 3 days of Ag stimulation, the total number of DLN cells in FTY720-treated mice was significantly less than that in vehicle-treated mice (data not shown). Although the percentages of CD4⁺KJ126⁺ cells in vehicle- and FTY720-treated animals were similar (data not shown), the absolute number of CD4⁺KJ126⁺ cells in FTY720-treated mice was 74% less compared with that in vehicle-treated groups (Fig. 1⇑A). Treatment with rapamycin, a cell cycle blocker, also reduced CD4⁺KJ126⁺ cells by 77% in the DLN (Fig. 1⇑A). To investigate the possibility that FTY720 simply alters the kinetics of the immune response in the DLN, we conducted a time-course study to examine T cell expansion in the DLN after 1, 2, 3, or 4 days of Ag stimulation. As shown in Fig. 1⇑B, for each day examined, the number of CD4⁺KJ126⁺ cells in the DLN of FTY720-treated mice was less (ranging from 64–78%) compared with that in the vehicle controls. A similar reduction was observed with FTY720 treatment on day 7 after Ag challenge in a separate experiment (data not shown). Furthermore, the number of Ag-specific T cells was reduced by FTY720 in a dose-dependent fashion, with a 50% inhibitory dose between 0.03 and 0.1 mg/kg (Fig. 1⇑C).

We also locally introduced another type of Ag, alloantigen and examined the T cell response in DLN after FTY720 treatment. C57BL/6 mice were treated daily with vehicle or FTY720 (0.5 mg/kg orally), with the first dose given 4 h before footpad injection of 1.5 × 10⁷ BALB/c splenocytes. Three days after alloantigen stimulation the number of DLN (draining popliteal node) cells was increased to 6.76 ± 0.68 × 10⁶ (Fig. 2⇓). However, with FTY720 treatment the number of DLN cells was only increased to 1.77 ± 0.17 × 10⁶, which was 74% less compared with the vehicle control value. Without alloantigen stimulation, 3 days of FTY720 treatment did not significantly change the number of popliteal node cells (Fig. 2⇓). In conclusion, we found that T cells respond to Ag or alloantigen stimulation in DLN of FTY720-treated mice, but the final number of responding T cells in DLN is significantly less after FTY720 treatment.

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FIGURE 2.

Reduced expansion in the number of DLN cells by FTY720 in alloantigen-challenged mice. FTY720 (0.5 mg/kg/day) was administered orally to C57BL/6 mice. Four hours later mice were given footpad injections of 1.5 × 10⁷ BALB/c splenocytes. The draining popliteal nodes were harvested 3 days after alloantigen challenge and counted by hemocytometer. The expansion of LN cells was inhibited by 74% (∗∗, p < 0.005; n = 4). No alloantigen injection control was included. One of three comparable experiments is shown.

T cell proliferation in a MLR in vitro

To investigate whether T cell proliferation was inhibited by FTY720, we examined in vitro T cell responses in a MLR. Upon stimulation with the alloantigen provided by irradiated C57BL/6 splenic cells, BALB/c T cells were activated and proliferated, as judged by thymidine incorporation (Fig. 3⇓). FTY720, at doses up to 1 μM, had no effect on thymidine incorporation of BALB/c T cells. At higher doses (>1 μM), FTY720 caused a dramatic decrease in thymidine incorporation, but we also observed a high percentage of TUNEL-positive apoptotic T cells (data not shown), suggesting that the decrease in thymidine incorporation was due to the cytotoxicity of FTY720 (6). We further evaluated FTY720-phosphate, the active FTY720 metabolite that acts as a full agonist for the S1P receptors (13), in this proliferation assay. We found that FTY720-phosphate had no significant effect on T proliferation at doses up to 1 μM (Fig. 3⇓). The blood concentration of FTY720-phosphate required to cause lymphopenia in vivo is several orders of magnitude less than 1 μM. Therefore, we conclude neither FTY720 nor its active metabolite interferes with T cell proliferation in vitro.

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FIGURE 3.

In vitro T cell proliferation. Irradiated splenic cells from C57BL/6 mice (100 μl at 5 × 10⁶/ml) were cultured with BALB/c splenic cells (100 μl at 5 × 10⁶/ml) in RPMI 1640 medium. Cells were treated with various doses of FTY720 or FTY720-phosphate. Three days later cells were pulsed with [³H]thymidine, and the incorporation of [³H]thymidine was determined after 16–18 h of pulsing. One of two comparable experiments is shown.

In vivo T cell proliferation analyzed by BrdU incorporation

In vivo T cell proliferation requires interaction of T cells and APC in the lymph node microenvironment. We examined in vivo T cell proliferation by injecting BrdU i.p. 2 days after OVA CFA injection in the DO11.10 adoptive transfer model described in Fig. 1⇑. On the following day DLN were collected and stained with CD4 and KJ126 Abs for tracking Ag-specific T cells. In addition, cells that incorporated BrdU in vivo were detected by anti-BrdU Ab. Again, we found that the number of CD4⁺KJ126⁺ T cells was significantly less with FTY720 treatment, as shown in Fig. 1⇑A. BrdU-incorporated cells were analyzed on the gated CD4⁺KJ126⁺ population. As shown in Fig. 4⇓, 54.6 ± 1.2% of CD4⁺KJ126⁺ cells incorporated BrdU in the vehicle-treated group. FTY720 treatment (0.5 mg/kg/day) resulted in a modest increase in the percentage of CD4⁺KJ126⁺ cells that were BrdU positive (p < 0.005). However, since the number of CD4⁺KJ126⁺ cells in DLN was dramatically less after FTY720 treatment (Fig. 1⇑), the absolute number of CD4⁺KJ126⁺ cells that incorporated BrdU was decreased by FTY720 treatment (data not shown).

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FIGURE 4.

In vivo T cell proliferation analyzed by BrdU incorporation. DO11.10 splenocytes were adoptively transferred to BALB/c mice. The recipients were administered vehicle (veh) or FTY720 (0.5 mg/kg/day) and then challenged with OVA CFA in the footpad as described in Fig. 1⇑. BrdU (1 mg) was injected to mice after 2 days of Ag challenge. On the following day the DLN or the contralateral popliteal node (Non-DLN) was harvested and stained with CD4-allophycocyanin, KJ126-PE, and anti-BrdU-FITC. BrdU-incorporated cells were analyzed after gating on CD4⁺KJ126⁺ cells. The upper right quadrant represents the percentage of CD4⁺KJ126⁺ cells that incorporated BrdU (A), which was then quantified as shown in B. One of two comparable experiments is shown.

In vivo T cell proliferation analyzed by CFSE fluorescence intensity

We next used CFSE to obtain information on the number of cell divisions in vivo. DO11.10 splenocytes were labeled with CFSE before adoptive transfer to BALB/c mice. FTY720 (0.5 mg/kg orally) and rapamycin (1 mg/kg i.p.) were given 4 h before Ag challenge and then daily until 1 day before harvest. DLN and non-DLN (contralateral popliteal node) were collected after 1, 2, or 3 days of OVA CFA challenge, and their cell cycle status was evaluated by green fluorescence intensity on a flow cytometer after gating on the CD4⁺KJ126⁺ population. In DLN, CD4⁺KJ126⁺ T cells proliferated in response to Ag stimulation, as evidenced by the loss of CFSE tracker dye over time. Separate peaks, representing different numbers of cell divisions, were observed (Fig. 5⇓A). In vehicle-treated mice, some CD4⁺KJ126⁺ cells completed approximately one cell division on day 1, three divisions on day 2, and six or seven divisions on day 3. Ag-induced cell cycling was inhibited in mice that received rapamycin, as indicated by less cell division and higher mean fluorescence intensity (Fig. 5⇓A). Correspondingly, there were fewer CD4⁺KJ126⁺ cells in the DLN of mice treated with rapamycin (Fig. 1⇑A).

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FIGURE 5.

Kinetic analysis of T cell division of Ag-specific T cells in the lymph nodes. DO11.10 splenocytes were labeled with CFSE and transferred into syngeneic BALB/c recipients. FTY720 and rapamycin were administered at doses of 0.5 mg/kg/day (orally) and 1 mg/kg/day (i.p.), respectively, with the first dose given 4 h before Ag challenge. After 1, 2, or 3 days of Ag stimulation, DLN (A) and non-DLN (B; contralateral popliteal node) were collected, and CFSE fluorescence intensity was detected at the FL-1 channel after gating on CD4⁺KJ126⁺ cells using a flow cytometer. The numbers appearing on each histogram (0–7) denote each division population, with the undivided T cells residing in the rightmost peak, and the T cells that have divided seven times residing in the leftmost peak. The mean fluorescence intensity (MFI) is also indicated. One of three comparable experiments is shown.

With FTY720 treatment, the maximal number of cell divisions that CD4⁺KJ126⁺ cells completed was similar to that in the vehicle controls (Fig. 5⇑A). Furthermore, the T cell response seemed to be more synchronized in FTY720-treated groups, and this was particularly evident on day 3 (Fig. 5⇑A). Almost all CD4⁺KJ126⁺ cells completed five to seven cell divisions by day 3 in FTY720-treated groups, whereas a relatively heterogeneous pattern was seen in vehicle-treated groups with cells undivided or divided fewer than five cycles. The more synchronized T cell cycling status correlates well with FTY720 function, which is to sequester T cells in individual LNs and to limit continuous trafficking of T cells, including Ag-responding T cells, to the DLNs. The cell cycling status was also evaluated for CD4⁺KJ126⁺ cells from the non-DLN as a control (Fig. 5⇑B). After 2 days of Ag stimulation, CD4⁺KJ126⁺ T cells in the non-DLN showed no evidence of cell division. Limited cell division was observed on day 3 as the immune response to Ag began to be evident systemically. In conclusion, our results show that T cell proliferation per se is not hampered by FTY720 in vivo.

Reduction in the number of Ag-activated T cells by FTY720 in peripheral blood

We next examined whether FTY720 prevents the release of Ag-activated CD4⁺ T cells from DLN to the peripheral blood compartment by analyzing peripheral blood from OVA CFA-challenged BALB/c mice, adoptively transferred with D011.10 T cells. We found that the number of total PBMC was reduced by 80% with FTY720 treatment (data not shown). The depletion was more pronounced in T cells as the percentage of total CD4⁺ cells was decreased from 47 to 10% by FTY720 (Fig. 6⇓A). The absolute number of CD4⁺ cells, therefore, was decreased by 96% with FTY720 treatment (data not shown). Rapamycin (1 mg/kg), at a dose that inhibited cell cycling, reduced the number of CD4⁺KJ126⁺ cells in the DLN, but did not alter the total PBMC count or the percentage of CD4⁺ cells in peripheral blood (Fig. 6⇓A).

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FIGURE 6.

Reduction in the number of naive and activated CD4⁺ cells in peripheral blood of Ag-challenged mice. DO11.10 splenocytes were labeled with CFSE before adoptive transfer to BALB/c mice, followed by injection of OVA CFA in the footpad. Mice were treated daily with vehicle (veh), FTY720 (orally; 0.5 mg/kg for A and B, and various doses for C) or rapamycin (rap; 1 mg/kg i.p.), with the first dose started 4 h before Ag challenge. After 3 days of Ag stimulation, peripheral blood was collected and pooled (n = 5). PBMCs were prepared from peripheral blood, counted using a hemocytometer, and stained with allophycocyanin-labeled anti-CD4 mAb. Shown in A is the percentage of PBMCs that were CD4⁺. In B, the R3 gate represents naive CD4⁺KJ126⁺ cells; the R2 gate represents activated CD4⁺KJ126⁺ cells that have divided at least once. C, Mice were treated with 0.01, 0.03, 0.1, or 0.3 mg/kg FTY720. The R2 and R3 gated populations represent activated and naive CD4⁺KJ126⁺ cells, respectively. D, The numbers of total CD4⁺, naive or activated CD4⁺KJ126⁺ cells, were determined by the percentage of total CD4⁺ cells, naive or activated CD4⁺KJ126⁺ cells, obtained from flow cytometry, multiplied by the total number of PBMC counted with a hemocytometer. Shown here is the percent reduction for total CD4⁺ cells, naive and activated CD4⁺KJ126⁺ cells, over the baseline value for vehicle-treated mice. One of three comparable experiments is shown.

To study the effect of FTY720 on the trafficking of Ag-activated T cells, we divided CD4⁺KJ126⁺ cells in peripheral blood into naive (R3 gate, with 0 cell division) and activated (R2 gate, with ≥1 cell division) T cell populations (Fig. 6⇑B). We found that the number of naive CD4⁺KJ126⁺ cells in the R3 gate was reduced by 96% with FTY720 treatment, similar to the aforementioned reduction in total CD4⁺ cells. Again, rapamycin did not reduce the number of naive CD4⁺KJ126⁺ cells in peripheral blood (Fig. 6⇑B). Without Ag stimulation, the activated CD4⁺KJ126⁺ cells in the R2 gate were not detectable in peripheral blood, as they were simply not generated in the lymph nodes (Fig. 6⇑C). Our kinetics study showed that it took 3 days of Ag stimulation before activated Ag-specific T cells were detected in peripheral blood of vehicle-treated mice (Fig. 6⇑B and data not shown). FTY720 (0.5 mg/kg/day) reduced the number of activated CD4⁺KJ126⁺ cells in peripheral blood of 3-day Ag-challenged mice by 99% (Fig. 6⇑B). Furthermore, the absence of activated CD4⁺KJ126⁺ T cells in peripheral blood was observed after 1, 2, 3, 4, or 7 days of Ag stimulation (Fig. 6⇑B and data not shown). Activated Ag-specific T cells generated in the DLNs are first released to lymphatic vessels, from which they reach the blood vessels, and then they home to peripheral tissues. Our results from the kinetics study suggest that the reduction in the number of activated T cells in peripheral blood caused by FTY720 is due to T cell sequestration in the DLN rather than homing to the tissues. The number of activated CD4⁺KJ126⁺ T cells in peripheral blood was also reduced in rapamycin-treated mice, probably due to impaired T cell activation in the DLN (Fig. 6⇑B). Depletion of total CD4⁺, naive and activated CD4⁺KJ126⁺ cells was further evaluated with different doses of FTY720. As shown in Fig. 6⇑, C and D, the maximal degree of inhibition was achieved for both naive and activated T cells with FTY720 at doses as low as 0.1 mg/kg/day. These results demonstrate that FTY720 treatment leads to similar reductions in both naive and Ag-activated T cells in peripheral blood, suggesting that the compound blocks the egress of both naive and activated T cells from the DLN to lymph.

FTY720 on preactivated T effector cells in peripheral blood

We also examined whether FTY720 has any effect on effector T cells that already exist in peripheral blood. AND mice, another class II-restricted and PCC-specific TCR transgenic mouse strain, were used to generate Th1 cells in vitro. After treatment with IL-12 and the PCC peptide, splenic cells from AND TCR mice were differentiated into polarized Th1 cells that secrete IFN-γ, but not IL-4 (data not shown). Naive splenic or Th1 cells were labeled with GT CMFDA before adoptive transfer to irradiated AND TCR mice. The percentage of GT⁺CD4⁺ donor cells in peripheral blood was evaluated after overnight treatment with FTY720 (1 mg/kg). As expected, the percentage of naive GT⁺CD4⁺ T cells was dramatically decreased by FTY720, from 6.2 ± 1.8 to 0.2 ± 0.08% % (p < 0.005; Fig. 7⇓A). In contrast, the percentage of GT⁺CD4⁺ Th1 cells in peripheral blood remained largely unchanged following FTY720 treatment (Fig. 7⇓B). In the same mice the percentage of endogenous naive GT⁻CD4⁺ T cells was dramatically decreased by FTY720 treatment (Fig. 7⇓B, see arrows), indicating that the drug coverage was adequate. FTY720 also showed minimal effect on the distribution of adoptively transferred Th2 cells (data not shown). Our results indicate that FTY720 has no significant role in redistributing preactivated T cells in peripheral blood.

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FIGURE 7.

Effects of FTY720 on adoptively transferred naive (A) or Th1 (B) cells. Naive splenic cells (5 × 10⁷) from AND TCR mice or Th1 cells (1.5 × 10⁷) were labeled with GT CMFDA and transferred i.v. into irradiated AND TCR mice. On the following day recipient mice were treated with FTY720 (1 mg/kg orally). After 24 h peripheral blood was collected, stained with CD4-PE (A) or CD4-allophycocyanin (B), and analyzed by flow cytometry. A dot plot example of CD4 vs GT is shown for naive (A) and Th1 (B) cell-transferred mice. GT⁺CD4⁺ cells represent donor T cells and GT⁻CD4⁺ cells represent endogenous CD4⁺ cells. Shown adjacent are the quantified results of the percentages of GT⁺CD4⁺ naive (A) and Th1 (B) T cells in peripheral blood. One of two comparable experiments is shown.