Immunomodulatory drug FTY720 induces regulatory CD4⁺CD25⁺ T cells in vitro

Abstract

As a novel immunosuppressant, FTY720 (2-amino-2-(2-[4-octylphenyl] ethyl)-1, 3-propanediol hydrochloride) has been used to prevent the allograft rejection in organ transplantation. FTY720 can prolong markedly survival of the allograft by inducing apoptosis of reactive lymphocytes and by redirecting the homing of lymphocytes. However, as the archetype of a new class of immune modulators, the potential effect of FTY720 on the immune response needs to be elucidated further. In this study, FTY720 was added into the mixed lymphocyte reaction (MLR) consisting of murine splenocytes from BALB/c and C57BL/6, to observe its direct effect on the induction of CD4⁺CD25⁺ regulatory T cells. It was demonstrated that the proportion of CD4⁺CD25⁺ and CD4⁺CD25⁺forkhead box P3 (FoxP3)⁺ T cells in MLR were increased significantly by FTY720 treatment, and the expression of FoxP3 mRNA in lymphocytes was also enhanced markedly by the drug. A synergetic effect was observed between FTY720 and co-stimulation blockades. Moreover, analysis of the function of FTY720-treated cells manifested an increased suppressive activity in an in vitro antigen-specific proliferation assay. In conclusion, FTY720 can increase the number and enhance the functional activity of CD4⁺CD25⁺ regulatory T cells in MLR, and these FTY720-treated cells possess the activity to down-regulate the alloreactivity of lymphocytes, indicating its potential use for therapeutic purposes.

Introduction

As a novel immunomodulator, FTY720 [2-amino-2-(2-[4-octylphenyl] ethyl)-1,3-propanediol hydrochloride (C₁₉H₃₃NO₂: HCl)] is a synthetic structural analogue of sphingosine related to the drug Myriocin (ISP-1), which is isolated from culture filtrates of the ascomycete Isaria sinclairii[1]. FTY720 has been used initially as an immunosuppressive agent, resulting in significant prolongation of the survival of skin, cardiac, kidney and liver allografts in animal models [2–5]. In humans, FTY720 was observed to have a stable immunosuppressive effect in renal transplant recipients, in combination with cyclosporine A or mycophenolate mofetil [6–11]. Its mechanism of action is to sequester lymphocytes to the secondary lymphoid organs, therefore preventing the migration of these cells to inflammatory sites. Despite its application as a novel immunosuppressant for allograft transplantation, many studies have been conducted to evaluate the potential of FTY720 for the treatment of other diseases, including autoimmune diseases, malignancies and ischaemia–reperfusion injury [12–18], and the preliminary results so far are encouraging.

The FTY720 impairs the circulation and the homing of effector T cells to peripheral lesions in mice, without affecting the induction and expansion of immune responses in secondary lymphoid organs [19]. Myriocin has a strong suppressive activity in mixed lymphocyte reaction (MLR), and this property is lost after its chemical modification to FTY720. FTY720 has no significant effect on T cell proliferation at doses up to 1 µM (343·9 ng/ml) in mice MLR and has no toxic effect on mononuclear cells in concentrations up to 5–10 µM [14,20]. Large-dose FTY720 exerts activity to promote splenocyte apoptosis in rat MLR [21].

Several investigators have reported that oral administration of FTY720 in animal models may be tolerogenic via the modulation of dendritic cell differentiation and migration, or based on its effect on CD4⁺CD25⁺ regulatory T cell (T_reg) [22–25]. However, despite all these results, the direct effects of FTY720 on the number and the functional activity of CD4⁺CD25⁺ T_reg were not well defined.

Unlike the commonly used immunosuppressants, FTY720 is not only an ‘immunosuppressor’ but also an ‘immunomodulator’[7,26]; therefore, the modulatory effect of FTY720 on immune response needs to be elucidated further. In this two-way MLR study, the impact of FTY720 on the induction of CD4⁺CD25⁺ T_reg was investigated, and for comparison the effect of co-stimulation blockades (Cos. blockades) was also observed.

Materials and methods

Animals

Male BALB/c (H-2^d), C57BL/6 (H-2^b) and 615 (H-2^k) mice, aged between 8 and 12 weeks, were purchased from the Experimental Animal Center of the Chinese Academy of Science (Shanghai, China) and were maintained in a specific pathogen-free mouse facility with free access to food and water. The 615 (H-2^k) mice were used as the third-party stimulator. All experiments involving animals were performed complying with the regulations set by the Institutional Animal Care and Research Advisory Committee at the Department of Animal Resources, Shanghai Jiao-Tong University School of Medicine.

Reagents

The FTY720 was purchased from Yoshitomi Pharmaceutical Corporation (Osaka, Japan). FTY720 was dissolved freshly in distilled water and diluted in RPMI-1640 medium to the final concentrations. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] dry powder was purchased from Sigma-Aldrich Corporation (St Louis, MO, USA) and was dissolved in phosphate-buffered saline (PBS) to 5 mg/ml. The anti-CD154 monoclonal antibody (mAb) (MR1, 5 mg/ml) and anti-CD80 mAb (16-10A1, 5 mg/ml) were both purchased from BD Biosciences Pharmingen (San Diego, CA, USA). All reagents were stored at 4°C.

Preparation of splenocytes

The spleen was removed aseptically from mouse and teased mechanically in 10 ml of complete RPMI-1640 medium (Gibco BRL, Carlsbad, CA, USA). Cell suspensions were filtered through 200 µM nylon mesh and then treated with red blood cell lysis buffer (Roche Applied Science, Los Angeles, CA, USA) to remove red cells. Thereafter, cells were washed and centrifuged three times at 106 g. The cells were adjusted to 1 × 10⁷ cells/ml and were cultured in complete RPMI-1640 medium containing 10% fetal bovine serum (Invitrogen, Leek, the Netherlands).

Effect of FTY720 on primary MLR

The suppressive activity of FTY720 on two-way MLR was observed. The percentage of lymphocyte apoptosis and total cell death were also measured. BALB/c splenocytes (2 × 10⁶) and C57BL/6 splenocytes (2 × 10⁶) per well were taken into 12-well flat-bottomed plates. MLRs were cultured in the presence of different concentrations of FTY720 at 5% CO₂, 95% moisture and 37°C. The FTY720 concentration was 0, 50, 100, 200, 400, 800 and 1600 ng/ml, respectively, in combination or not with Cos. blockades. The concentration of anti-CD154 mAb and anti-CD80 mAb were both 10 µg/ml. Each well contained 1 ml RPMI-1640 with 10% fetal calf serum. Five days later, cells were harvested to measure proliferation by MTT assay. Lymphocyte apoptosis was measured by detecting the annexin V phenotype of the membrane, using the annexin V-PI apoptosis detection kit (Becton Dickinson, San Diego, CA, USA) by fluorescence activated cell sorting (FACS; Coulter Epics Elite, Beckman Coulter, Miami, FL, USA). The survival rate of cultured cells was calculated with the trypan blue staining method.

The level of interleukin (IL)-2 in the supernatant of MLR was detected using the IL-2 enzyme-linked immunosorbent assay kit (eBioscience, San Diego, CA, USA).

The MTT assay

Briefly, harvested cells were washed with PBS and transferred into new 96-well flat-bottomed plates. Each sample was detected in triplicate. A total of 200 µl PBS and 20 µl MTT was added into all wells to culture for 4 h at 5% CO₂, 95% moisture in 37°. Plates were then centrifuged at 425 g for 5 min. The supernatant was discarded and 150 µl dimethylsulphoxide was added to each well to measure the optical density value at 570 nm by an enzyme immunoassay instrument (DJ-3200, Huadong Electronic Tube Company, Shanghai, China).

Percentage of T_reg

After 5 days' co-culture, all cells were harvested. For further analysis, viable cells were obtained by discontinuous density gradient centrifugation using Lympholyte-M (Cedarlane Labs, Hornby, Ontario, Canada). Cells were washed with the staining buffer, and 1 µg fluorescence antibodies were added to incubate for 30 min. Thereafter, the cells were washed twice with PBS. The percentage of CD4⁺CD25⁺ T cells and CD4⁺CD25⁺forkhead box P3 (FoxP3)⁺ T cells were measured by FACS. To determine the intracellular expression of T_reg cell-specific transcription factor FoxP3 in CD4⁺CD25⁺ cells, mix-cultured splenocytes were first surface-stained with phycoerythrin (PE)-labelled anti-mouse CD4 mAb (clone GK 1·5) and fluorescein isothiocyanate-labelled anti-mouse CD25 mAb (Clone PC61.5) as per standard practice. These cells were stained subsequently with PE-labelled anti-mouse FoxP3 mAb (clone FJK-16s, eBioscience, San Diego, CA, USA) or PE-labelled rat IgG 2a non-specific isotype control mAb.

Determination of FoxP3 mRNA expression by quantitative real-time reverse transcription–polymerase chain reaction

Total cellular RNA was extracted by treating cells in TRIzol lysis buffer (Gibco BRL). RNA concentration was measured by spectrophotometric analysis and its integrity was evaluated by assessing ultraviolet absorbance at 260 and 280 nm in 2% ethidium bromide-stained agarose gel. A total of 1 µg RNA was reverse-transcribed by Moloney murine leukaemia virus reverse transcriptase (Gibco BRL) in a final volume of 20 µl. Primers for β-actin (5′-AGAGGGAAATCGTGCGTGAC-3′; 5′-CAATAGTGATGACCTGGCCGT-3′) and FoxP3 (5′-GGAGAAAGCGGATACCAAA-3′; 5′-TCTGTGAGGACTACCGAGCC-3′) were designed and each of the primer pairs spanned at least one intron. A total of 2 µl of cDNA was used for polymerase chain reaction (PCR) containing Taq polymerase/SYBR Green PCR mastermix (Qiagen, Valencia, CA, USA). Amplification was performed and analysed using RotorGene 3000 (Corbett Research, Sydney, Australia). Real-time PCR efficiencies were detected by amplification of a dilution series of cDNA isolated from CD4⁺CD25⁺ T_reg. Standard curves for β-actin and FoxP3 were generated by serial dilution of control cDNA templates, and the amount of mRNA of β-actin and FoxP3 was calculated based on the standard curve. After data normalization, values were expressed as the ratio of FoxP3 to β-actin in each sample.

Suppressive effect and antigen-specificity of T_reg

The FTY720 and/or Cos. blockades were washed out before the cells were transferred into the secondary MLR. A total of 1 × 10⁵ viable cells/well (induced by FTY720, in combination or not with Cos. blockades) or control cells were added into the secondary MLR in 96-well flat-bottomed plates. The secondary MLR consisted of 2 × 10⁵/well fresh splenocytes from each inbred mouse, either from BALB/c + C57BL/6 or from BALB/c + 615. After 4 days' culture, cells were harvested and the lymphocytes proliferation was measured.

Statistical analysis

All experiments were repeated at least three times. Data were expressed as mean ± standard deviation. Analysis of variance or t-tests were used for the comparison when appropriate. All statistical calculations were performed using spss version 11·0 software (SPSS Inc., Chicago, IL, USA). The significant level is P = 0·05.

Results

The FTY720 suppressed MLR proliferation

In both the FTY720 group and combination group (FTY720 + Cos. blockades), the proliferation of lymphocytes was suppressed by FTY720 in a dose-dependent manner. When the FTY720 concentration was at 100 ng/ml, 200 ng/ml,400 ng/ml, 800 ng/ml and 1600 ng/ml, the proliferation of lymphocytes in the FTY720 and combination groups was 0·661 ± 0·062 versus 0·37 ± 0·029, 0·549 ± 0·056 versus 0·351 ± 0·04, 0·41 ± 0·042 versus 0·321 ± 0·036, 0·349 ± 0·036 versus 0·315 ± 0·036 and 0·321 ± 0·03 versus 0·299 ± 0·03 respectively. When the FTY720 concentration was more than 400 ng/ml in the FTY720 group and 800 ng/ml in the combination group, the suppressive effects of FTY720 on MLR were significant (P < 0·05, Fig. 1a).

Fig. 1.

FTY720 (2-amino-2-(2-[4-octylphenyl] ethyl)-1,3-propanediol hydrochloride) suppresses the proliferation of lymphocytes and decreases the level of interleukin (IL)-2 in the supernatant in mixed lymphocyte reaction (MLR). (a) The proliferation as measured by [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) on day 5 is shown on the y-axis and expressed as optical density (OD) value (mean ± standard deviation), and the data are from three experiments, each in triplicate. *Compared with control group P < 0·05; #compared with Cos. blockades group (FTY720 at 0 ng/ml) P < 0·05. (b) The secretion of IL-2 in the supernatant of MLR. The concentration of FTY720 is 1600 ng/ml. The data represent the mean levels of IL-2 from three experiments. *Compared with control group P < 0·05. Cos. B, co-stimulation blockades; F, FTY720.

When FTY720 was at a concentration of 1600 ng/ml, the IL-2 level in the FTY720, Cos. blockades and combination groups was 189·6 ± 71 pg/ml, 55·2 ± 13·3 pg/ml and 51·6 ± 15·9 pg/ml respectively. The IL-2 levels in the Cos. blockades group decreased significantly compared with those in the control group (P < 0·05), and this effect was also observed in the FTY720 and combination groups (P < 0·05). However, compared with Cos. blockades, FTY720 was less suppressive on IL-2 production (P < 0·05, Fig. 1b).

The FTY720 induced lymphocyte apoptosis

Both in the FTY720 and combination groups, lymphocyte apoptosis was enhanced. When the FTY720 concentration was at 100 ng/ml, 200 ng/ml, 400 ng/ml, 800 ng/ml and 1600 ng/ml, the lymphocyte apoptosis rate in the FTY720 and combination groups was 35·0 ± 3·18% versus 31·5 ± 3·49%, 38·9 ± 3·5% versus 36·3 ± 4·11%, 39·8 ± 3·89% versus 38·9 ± 4·2%, 45·6 ± 4·2% versus 41·6 ± 4·5% and 50·9 ± 4·37% versus 42·3 ± 5·09% respectively. When the concentration of FTY720 was greater than 200 ng/ml in the FTY720 group and 800 ng/ml in the combination group, lymphocyte apoptosis was induced significantly compared with that in the control group (P < 0·05, Fig. 2).

Fig. 2.

FTY720 (2-amino-2-(2-[4-octylphenyl] ethyl)-1,3-propanediol hydrochloride) induces apoptosis of lymphocytes in mixed lymphocyte reaction (MLR). Lymphocyte apoptosis is measured by detecting the annexin V phenotype of the membrane. *Compared with control group P < 0·05; #compared with Cos. blockades group (FTY720 at 0 ng/ml) P < 0·05.

The FTY720 induced CD4⁺CD25⁺ and CD4⁺CD25⁺FoxP3⁺ T cells in vitro

Live cells were obtained and the percentage of CD4⁺CD25⁺ T cells and CD4⁺CD25⁺FoxP3⁺ T cells was measured by FACS. The proportion of CD4⁺CD25⁺ T_reg was increased by FTY720 treatment. When the concentration of FTY720 was at 100 ng/ml, 200 ng/ml, 400 ng/ml, 800 ng/ml and 1600 ng/ml, the percentage of CD4⁺CD25⁺ T_reg in the FTY720 and combination groups was 3·2 ± 0·37% versus 4·28 ± 0·45%, 3·84 ± 0·34% versus 4·18 ± 0·45%, 4·0 ± 0·53% versus 4·6 ± 0·66%, 4·18 ± 0·38% versus 5·44 ± 0·49% and 4·82 ± 0·55% versus 6·91 ± 0·51% respectively. Compared with the control group, when FTY720 concentration was at 1600 ng/ml in the FTY720 group and 800 ng/ml in the combination group, the percentages of CD4⁺CD25⁺ T_reg were increased significantly (P < 0·05, Fig. 3). The absolute number of CD4⁺CD25⁺ T cells in MLR was calculated and found to be increased significantly from 28430 ± 2274 to 93120 ± 2924 in the FTY720 group (P < 0·05) and increased from 52904 ± 1637 to 148764 ± 5033 in the combination group (P < 0·05, Table 1).

Fig. 3.

FTY720 (2-amino-2-(2-[4-octylphenyl] ethyl)-1,3-propanediol hydrochloride) increases the proportion of CD4⁺CD25⁺ T regulatory cells (T_reg) in mixed lymphocyte reaction (MLR). Percentage of T_reg (%) = the number of CD4⁺CD25⁺ T_reg/the number of viable cultured cells harvested from the primary MLR. *Compared with control group P < 0·05; #compared with Cos. blockades group (FTY720 at 0 ng/ml) P < 0·05.

Table 1.

FTY720 (2-amino-2-(2-[4-octylphenyl] ethyl)-1,3-propanediol hydrochloride) increases the percentage and the absolute number of CD4⁺CD25⁺ T regulatory cells (T_reg) in mixed lymphocyte reaction (MLR).

FTY720 (ng/ml) Cos. blockades	0 −	0 +	200 −	200 +	800 −	800 +	1600 −	1600 +
Initial no.	4 × 106	4 × 106	4 × 106	4 × 106	4 × 106	4 × 106	4 × 106	4 × 106
Survival (%)	35·1 ± 4·11	38·91 ± 4·56	40·11 ± 4·65	46·65 ± 4·61	41·25 ± 4·33	50·21 ± 6·84	48·58 ± 7·61	53·19 ± 8·11
CD4+CD25+ Treg (%)	2·0 ± 0·31	3·47 ± 0·41	3·84 ± 0·34	4·18 ± 0·45	4·18 ± 0·38	5·44 ± 0·49	4·82 ± 0·55	6·91 ± 0·51
CD4+CD25+ Treg number	28430 ± 2274	52904 ± 1637	60800 ± 4130	76424 ± 4189	67568 ± 1833	108432 ± 2909*	93120 ± 2924*	148764 ± 5033*

^*Compared with control group P < 0·05; 2 × 10⁶ BALB/c splenocytes and 2 × 10⁶ C57BL/6 splenocytes are co-cultured in the presence of different concentration of FTY720, in combination or not with co-stimulation (Cos.) blockades. The survival rate of cultured cells is calculated with the trypan blue staining method. The data are expressed as mean ± standard deviation.

Furthermore, when FTY720 was at the concentration of 1600 ng/ml, three-colour intracellular staining flow cytometry was used to detect the expression of FoxP3 gated in CD4⁺CD25⁺ cells. The percentage of CD4⁺CD25⁺FoxP3⁺ T cells was also increased significantly from 5·98 ± 1·9% to 13·6 ± 3·1% in the FTY720 group, and from 8·9 ± 7·3% to 19·9 ± 5·9% in the combination group (P < 0·05, Fig. 4).

Fig. 4.

The expression of forkhead box P3 (FoxP3) in CD4⁺CD25⁺ T regulatory cells (T_reg) in mixed lymphocyte reaction (MLR). Three-colour, intracellular staining flow cytometry (FCM) is used to detect the expression of FoxP3 in mix-cultured splenic cells under different treatments [FTY720 (2-amino-2-(2-[4-octylphenyl] ethyl)-1,3-propanediol hydrochloride) and/or co-stimulation blockades]. (a) One representative of FCM profiles showing the FoxP3 staining gated in CD4⁺CD25⁺ cells. (b) The mean percentages of FoxP3⁺ cells gated in CD4⁺CD25⁺ cells. In each group more than three tests are performed, each one in triplicate. The concentration of FTY720 is 1600 ng/ml. The data representing the percentage of CD4⁺CD25⁺FoxP3⁺ cells in CD4⁺CD25⁺ cells (%) are expressed as the mean ± standard deviation. *P < 0·05. Cos. B, co-stimulation blockades; F, FTY720.

The FTY720 enhanced expression of FoxP3 mRNA in MLR

The mRNA expression of FoxP3 was calculated as described above. The mean ratio of FoxP3 mRNA to β-actin mRNA in the FTY720 group was 3·97 ± 1·55, and the ratio in the combination group (FTY720 + Cos. blockades) was 4·94 ± 1·22. The average level of FoxP3 mRNA expression in FTY720-treated cells or FTY720 + Cos. blockade-treated cells was enhanced significantly compared with that in control cells (P < 0·01, Table 2).

Table 2.

FTY720 (2-amino-2-(2-[4-octylphenyl] ethyl)-1,3-propanediol hydrochloride) enhances the expression of forkhead box P3 (FoxP3) mRNA in mixed lymphocyte reaction (MLR).

Group	Ratio (FoxP3/β-actin)
Control	1·00 ± 0·48
Co-stimulation blockades	1·67 ± 0·98
FTY720	3·97 ± 1·55*
FTY720 + co-stimulation blockades	4·94 ± 1·22*

^*Compared with control group P < 0·05. The expression of FoxP3 mRNA and β-actin mRNA is determined by quantitative real-time reverse transcription–polymerase chain reaction. Values are normalized to be expressed as the ratio of FoxP3 to β-actin. Each sample is detected in triplicate for each gene, and the data show the typical result from one of three comparable experiments.

Immunoregulatory cells induced by FTY720 suppressed secondary MLR

Harvested from the primary MLR, the viable cultured cells were washed and added to the secondary MLR. The 615 mouse splenocytes were used as the third-party stimulator. After a 4-day co-culture, lymphocyte proliferation in the secondary MLR (BALB/c + C57BL/6 and BALB/c + 615) was suppressed significantly by cultured cells induced by FTY720 alone or in combination with Cos. blockades (P < 0·05). When the secondary MLR consisted of BALB/c + 615 splenocytes, the suppressive effect was significantly weaker than that in BALB/c + C57BL/6 (P < 0·05, Fig. 5), to some extent indicating the antigen-specificity.

Fig. 5.

The cultured cells treated by FTY720 (2-amino-2-(2-[4-octylphenyl] ethyl)-1,3-propanediol hydrochloride) suppress the proliferation of lymphocytes in secondary mixed lymphocyte reaction (MLR). FTY720 and/or co-stimulation blockades are washed out before the cells are transferred into the secondary MLR; 1 × 10⁵ viable cultured cells/well are added into the secondary MLR. The ratio of stimulator : responder : cultured cells = 2:2:1. The optical density (OD) values (mean ± standard deviation) are from three experiments, each in triplicate. *C57BL/6 group versus 615 group, P < 0·05. Cos. B, co-stimulation blockades; F, FTY720.

Discussion

Although the pharmacological effect of FTY720 is not well defined, it has been considered that the activities of inducing apoptosis and redirecting the homing on lymphocytes were included. By the homing effect, FTY720 was used as an ‘immune modulator’ to draw lymphocytes from peripheral blood into peripheral lymphoid tissues such as peripheral lymph nodes, mesenteric lymph nodes and Peyers' patches [27,28].

In this study, it has been demonstrated that FTY720 induces lymphocyte apoptosis in a MLR setting. Induction of apoptosis by FTY720 seems not to be an effect which can be observed in allograft recipients, because this study used pharmacologically high concentrations (800–1600 ng/ml) which cannot be reached by oral administration of FTY720 in human or animal models [6,20,29]. However, such high concentrations can be achieved easily in vitro and does not result in cytotoxicity. FTY720 is less toxic to peripheral blood mononuclear cells than tumour cells [13,14]. In accordance with previous investigations, the results of this study show that at the conventional dose range in vivo it is impossible for FTY720 to induce apoptotic cell death of lymphocytes [21,27]. Although apoptosis was increased by FTY720, higher concentrations of FTY720 led to a higher percentage of cell survival in MLR. This may indicate that during the interaction between mix-cultured splenic cells in the presence of FTY720, apoptosis of some cell subsets is more likely to be induced, but in general the survival is slightly improved.

Several conventional immunosuppressive drugs have different effects on the function of T_reg, because of the differences in signalling pathways between T_reg and effector T cells. In general, it was reported that T cell-depleting antibodies, corticosteroids and rapamycin may preserve the suppressive activity of T_reg, whereas cyclosporine A and tacrolimus (FK506) inhibit FoxP3 expression significantly and possibly suppress the function of T_reg, while mycophenolic acid does not appear to have an effect on T_reg[30–33].

Several investigators have reported the effect of FTY720 on T_regin vivo. When C57BL/6 mice were treated with FTY720, an increased number of CD4⁺CD25⁺ T_reg were found in blood and spleens, and CD4⁺CD25⁺ T cells obtained from spleens of FTY720-pretreated donor mice contained T_reg capable of inhibiting an allergen-induced airway inflammation [34]. FTY720 could mediate the anti-inflammatory effect at least in part by its ability to cause the conversion of antigen-stimulated conventional T cells to become FoxP3⁺ regulators [23]. In a T helper type 1-mediated mouse colitis model, FTY720 treatment resulted in a significant increase of CD25 and FoxP3 expression in isolated CD4⁺ T cells [25]. These in vivo studies support the hypothesis that FTY720 may affect T_reg sequestration and increases T_reg functional activity. However, more direct evidence is needed to draw a causal relationship between the action of FTY720 and the induction of T_reg. It is not clear in which way FTY720 exerts its effect: to affect the recirculation of a subset of T_regin vivo, to increase the numbers of T_reg or to enhance the functional activity of this cell type. Therefore, in our study the impact of FTY720 on the induction of CD4⁺CD25⁺ T_reg was tested directly by two-way MLR consisting of splenocytes from two strains of inbred mice.

It is confirmed that FTY720 has the ability to increase the number of CD4⁺CD25⁺ T cells and CD4⁺CD25⁺FoxP3⁺ T cells in vitro. In this study, it is not likely that CD4⁺CD25⁺ cells induced in MLR could be the activated CD4⁺ T cells: IL-2 levels were decreased significantly in the supernatant of MLR, and the percentage of CD4⁺CD25⁺FoxP3⁺ T cells was also increased significantly. FoxP3, which is expressed selectively in T_reg, is regarded as the best molecular marker for T_reg and essential for their regulatory activity [35]. FoxP3 mRNA expression was enhanced markedly by FTY720, suggesting that FTY720 may have the ability to enhance the functional activity of T_reg directly. Furthermore, FTY720-treated cells have an anti-proliferative effect on secondary MLR. The anti-proliferative effect could be the result of ‘linked suppression’[36], with an extent of antigen-specificity. This suppressive effect may be attributed at least partially to the increased number and enhanced activity of CD4⁺CD25⁺ T_reg by FTY720 treatment.

The FTY720 selectively favours the induction of T_reg, which is a key element in tolerance-inducing strategies. The induction of operational tolerance depends upon the two-way interaction between donor and recipients (antigens and/or immune cells). After activation of immunoreactive cells, induction therapy may ‘educate’ these cells, resulting in a state of unresponsiveness, anergy, regulation and so on. MLR is a practical approach for the purpose of prolonging the allograft survival in vivo by adoptive transfer of these in vitro-induced cells [30,36]. It is also suggested that FTY720 may have disease-modifying potential in inflammatory disorders [34]. The use of in vitro-induced T_reg could be valuable in some clinical situations such as transplantation, autoimmune diseases and chronic infections.

Currently, the mechanism leading to the enhanced T_reg activity after FTY720 treatment is not clear. FTY720 is well known as the sphingosine 1-phosphate (S1P) receptor agonist. S1P has the ability to alter the activity of CD4⁺CD25⁺T cells and is required for the optimal activity of CD4⁺CD25⁺ T_reg[37,38]. It has been reported that CD4⁺CD25⁺ T_reg expressed lower levels of mRNA for S1P1 and S1P4 receptors and demonstrated a reduced chemotactic response to S1P [34]. The synergistic effect between FTY720 and co-stimulation blockades on T_reg is also confirmed, suggesting the potential to use these agents in combination for the treatment of diseases in a clinical setting.

It is concluded that FTY720 can increase directly the number and enhance the function of CD4⁺CD25⁺ T_regin vitro; the FTY720-treated cells possess a potent suppressive capacity with specificity to the alloantigen. The adoptive transfer of in vitro-expanded T_reg is proposed to be a therapeutic regimen for some clinical situations.