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Departments of
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Pharmacology and
Immunology and Rheumatology, Merck Research Laboratories, Rahway NJ 07065
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell-mediated immune responses include initial T cell activation by APCs in the lymph nodes and subsequent trafficking of activated T cells to sites of inflammation. Numerous studies have demonstrated that FTY720 is efficacious in prolonging allograft rejection and preventing development of autoimmune diseases (14). However, few studies have been focused on dissecting the mechanisms for FTY720-mediated immunosuppression. It is known that for immune surveillance, naive T cells circulate from lymph node to lymph node until they encounter their Ag in the specialized lymph node microenvironment. With FTY720 treatment, T cell recirculation is inhibited to a large extent as T cells are sequestered in individual lymph nodes and Peyer’s patches. Whether defective T cell circulation can affect T cell responses in the lymph nodes remains unclear. Pinschewer et al. (15) have reported that FTY720 has no measurable effect on the induction or expansion of cytotoxic cells in a systemic virus infection model. We reason that T cell circulation is more critical in a localized immune response because it ensures that those T cells that carry the right TCR have the chance to encounter their Ag in a particular region of the body. Therefore, the effect of FTY720 on T cell responses in the draining lymph node (DLN) needs to be further examined in a local Ag-challenged model.
Differential migratory properties have been directly demonstrated for naive, effector, and memory T cells (16, 17). Naive T cells are thought to be incapable of homing to inflammatory tissues due to the absence of appropriate adhesion molecules that are essential for transendothelial migration at peripheral sites. On the contrary, activated T cells express adhesion molecules, including functional selectin ligand and chemokine receptors as a result of Ag and cytokine stimulation and therefore, are capable of migrating to peripheral sites of Ag (16) (data not shown). Infiltrated lymphocytes at peripheral sites further proliferate and produce cytokines in response to specific Ag to amplify local inflammatory responses. Thus, preventing activated T cells from homing to the peripheral site of Ag can lead to immunosuppression in diseases that are T cell mediated and directed against peripherally located tissues, such as allotransplantation and organ-specific autoimmune diseases. To date, few studies have focused on the roles of FTY720 in altering the trafficking of T effector/memory populations. Pinschewer and colleagues (15) have shown that the number of adoptively transferred Ag-specific V
2+V
8+ cells after 8-day viral Ag stimulation is greatly reduced with FTY720. However, V2+V8+ cells are a mixed population of naive and effector T cells in peripheral blood. Furthermore, the dose requirement for reducing the number of effector vs naive T cells in peripheral blood is not known.
In the studies presented here we have examined the effect of FTY720 on T cell-mediated responses, including T cell expansion in the DLN and subsequent release of activated T cells to the peripheral blood compartment. By adoptively transferring naive DO11.10 TCR transgenic CD4+ cells to syngeneic recipients followed by footpad injection of Ag and adjuvant, we showed that FTY720 treatment led to reduced expansion of Ag-specific T cells in the DLN. We also demonstrated that FTY720 caused a significant reduction in the number of activated T cells in peripheral blood when T cell priming occurs under FTY720 treatment.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 OVA323–339 in the context of the MHC class II molecule I-Ad. 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 / TCR (V11, V3), specific for pigeon cytochrome c88–104 (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 x 106/ml) from C57BL/6 mice were cultured with BALB/c splenic cells (100 µl at 5 x 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 x 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 x 107/mouse). In some cases, naive splenic cells from AND TCR mice were adoptively transferred to irradiated B10.BR mice (5 x 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 x 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 x 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 x 107 BALB/c splenocytes. The draining popliteal nodes were harvested 3 days after alloantigen challenge, and DLN cells were counted with a hemocytometer.
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Results |
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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. 1A) 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. 1A).
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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 x 107 BALB/c splenocytes. Three days after alloantigen stimulation the number of DLN (draining popliteal node) cells was increased to 6.76 ± 0.68 x 106 (Fig. 2). However, with FTY720 treatment the number of DLN cells was only increased to 1.77 ± 0.17 x 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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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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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. 1A. 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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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. 5A). 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. 5A). Correspondingly, there were fewer CD4+KJ126+ cells in the DLN of mice treated with rapamycin (Fig. 1A).
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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. 6A). 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. 6A).
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1 cell division) T cell populations (Fig. 6B). 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. 6B). 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. 6C). 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. 6B 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. 6B). 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. 6B 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. 6B). 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-
, 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. 7A). In contrast, the percentage of GT+CD4+ Th1 cells in peripheral blood remained largely unchanged following FTY720 treatment (Fig. 7B). In the same mice the percentage of endogenous naive GT-CD4+ T cells was dramatically decreased by FTY720 treatment (Fig. 7B, 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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Discussion |
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Apoptosis has been proposed to be the possible mechanism of action for FTY720 (5, 20, 21). However, this is mainly based on in vitro studies in which the concentrations of FTY720 needed for causing apoptosis are several orders of magnitude greater than the blood concentrations in mice given FTY720 at a therapeutic dose of 0.5 mg/kg (21, 22). We, as well as others (13, 15), have shown that FTY720-induced lymphopenia is reversible after cessation of the compound, suggesting that FTY720 probably causes sequestration of lymphocytes rather than significantly affecting their viability. Whether the degree of lymphocyte apoptosis can be altered by FTY720 in Ag-challenged mice needs to be further evaluated.
We think that altered trafficking of naive T cells by FTY720 is the likely explanation for the reduced number of Ag-specific T cells in the DLN compared with the vehicle control. T cell expansion is caused by the proliferation of naive Ag-specific T cells that have migrated to the DLN. Normally, there is a constant recirculation of naive T cells, including naive CD4+KJ126+ cells, from lymph node to lymph node, but only the CD4+KJ126+ cells that traffic to the DLN would be retained and proliferate. With FTY720 treatment, the circulation of naive T cells is greatly inhibited, as the egress of T cells out of lymph nodes is blocked. As a result, naive CD4+KJ126+ cells are permanently distributed to individual lymph nodes by FTY720, and therefore, the total number of naive CD4+KJ126+cells migrated to the regional DLN is expected to be reduced. Lack of continuous entrance of naive CD4+KJ126+ cells to the DLN caused by FTY720 treatment is indicated by the absence of CD4+KJ126+ cells with no or a few divisions after 3 days of Ag stimulation (Fig. 5A). Our results suggest that the defective recirculation of naive T cells impairs the efficiency of T cell responses in the DLN, highlighting the importance of T cell circulation as a way of immunosurveillance.
Although the number of Ag-activated T cells in the DLN is dramatically reduced, our preliminary data indicate that the CD4+ T cells primed under FTY720 are functional, as they produce comparable amount of IFN- upon Ag restimulation in vitro when the same number of DLN cells is plated (data not shown). Furthermore, DLN T cells from FTY720-treated mice, when adoptively transferred to syngeneic recipients, are capable of homing to an inflammatory tissue in a CFA-induced peritonitis model (data not shown). However, with FTY720 we show these Ag-activated T cells are not released to the blood compartment. Thus, homing to inflammatory tissues is prevented. It should be noted that FTY720 reduces the number of blood T effector cells only when T cell priming occurs in the presence of FTY720. FTY720 is not effective in inducing lymphopenia of pre-existing T effector cells in peripheral blood, e.g., adoptively transferred Th1 cells. A previous report from von Andrian’s group (17) demonstrates that T effector cells are not efficient in homing to lymph nodes compared with naive T cells, probably due to the low expression of CD62L and CCR7. Therefore, the effectiveness of FTY720 in keeping T cell subsets in the lymph nodes, away from sites of inflammation, relies on the ability of T cells to home to lymph nodes. It would be interesting to determine whether FTY720 can alter trafficking of in vitro generated central memory-like T cells with either IL-15 for CD8+ or under nonpolarized conditions for CD4+ T cells, as these cells are more efficient in homing to the lymph nodes compared with T effector cells (17, 23).
Taken together, S1P receptor agonism by FTY720 induces immunosuppression through inhibition of recirculation of naive T cell and release of Ag-activated T cells from DLN to lymph and the blood compartment. It should be noted that in the present study Ag was administered after a few hours of FTY720 treatment. Aside from altering the trafficking of T cells in the periphery, FTY720 has been reported to inhibit emigration of mature thymocytes from the thymus to the periphery (evident after 1 wk of treatment) (24). How chronic pretreatment of FTY720 affects T cell-mediated immune responses needs to be addressed in the future.
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Acknowledgments |
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Footnotes |
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2 Current address: The Scripps Research Institute, ICND-118, La Jolla, CA 92037.
3 Abbreviations used in this paper: S1P, sphingosine-1-phosphate; BrdU, 5-bromodeoxyuridine; CMFDA, 5-chloromethylfluorescein diacetate; DLN, draining lymph node; GT, Green Tracker; PCC, pigeon cytochrome c88–104.
Received for publication October 7, 2002. Accepted for publication January 21, 2003.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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S. Han, X. Zhang, G. Wang, H. Guan, G. Garcia, P. Li, L. Feng, and B. Zheng FTY720 suppresses humoral immunity by inhibiting germinal center reaction Blood, December 15, 2004; 104(13): 4129 - 4133. [Abstract] [Full Text] [PDF]
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 U. Kunzendorf, E. Ziegler, and D. Kabelitz FTY720--the first compound of a new promising class of immunosuppressive drugs Nephrol. Dial. Transplant., July 1, 2004; 19(7): 1677 - 1681. [Full Text] [PDF]
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 M. Forrest, S.-Y. Sun, R. Hajdu, J. Bergstrom, D. Card, G. Doherty, J. Hale, C. Keohane, C. Meyers, J. Milligan, et al. Immune Cell Regulation and Cardiovascular Effects of Sphingosine 1-Phosphate Receptor Agonists in Rodents Are Mediated via Distinct Receptor Subtypes J. Pharmacol. Exp. Ther., May 1, 2004; 309(2): 758 - 768. [Abstract] [Full Text] [PDF]
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M. G. Sanna, J. Liao, E. Jo, C. Alfonso, M.-Y. Ahn, M. S. Peterson, B. Webb, S. Lefebvre, J. Chun, N. Gray, et al. Sphingosine 1-Phosphate (S1P) Receptor Subtypes S1P1 and S1P3, Respectively, Regulate Lymphocyte Recirculation and Heart Rate J. Biol. Chem., April 2, 2004; 279(14): 13839 - 13848. [Abstract] [Full Text] [PDF]
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E. Sawicka, C. Zuany-Amorim, C. Manlius, A. Trifilieff, V. Brinkmann, D. M. Kemeny, and C. Walker Inhibition of Th1- and Th2-Mediated Airway Inflammation by the Sphingosine 1-Phosphate Receptor Agonist FTY720 J. Immunol., December 1, 2003; 171(11): 6206 - 6214. [Abstract] [Full Text] [PDF]
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 H. Rosen, C. Alfonso, C. D. Surh, and M. G. McHeyzer-Williams Rapid induction of medullary thymocyte phenotypic maturation and egress inhibition by nanomolar sphingosine 1-phosphate receptor agonist PNAS, September 16, 2003; 100(19): 10907 - 10912. [Abstract] [Full Text] [PDF]
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