FTY720 exerts differential effects on CD4+ and CD8+ T-lymphocyte subpopulations expressing chemokine and adhesion receptors
Torsten Böhler,
Johannes Waiser,
Manuela Schuetz,
Hans H. Neumayer and
Klemens Budde
Department of Nephrology, Charité, Campus Mitte, Humboldt-University, Berlin, Germany
Correspondence and offprint requests to: Dr Torsten Böhler, Department of Nephrology, Charité, Humboldt-University Berlin, Schumannstrasse 20/21, 10117 Berlin, Germany. Email: torsten.boehler{at}charite.de
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Abstract
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Background. FTY720 (FTY), a novel immunomodulator with the potential to improve immunosuppressive therapy after organ transplantation, is currently under clinical investigation. Previous experimental animal studies have shown that FTY has a unique mechanism of action associated with altered lymphocyte recirculation.
Methods. Participating in a phase I clinical trial, we studied the pharmacodynamic effects of FTY in stable renal allograft recipients. We analysed the effect of FTY on surface marker expression on T-cell subpopulations by flow cytometry.
Results. A single oral dose of FTY (0.253.5 mg) significantly reduced peripheral lymphocyte counts by 3070%. FTY reduced all T-lymphocyte subsets, CD4+ cells more than CD8+ cells. However, we observed that lower doses of FTY (0.252 mg, n = 11) did not affect peripheral CD4+CCR5+ T-lymphocyte counts, while the highest FTY dose of 3.5 mg (n = 2) exerted a rapid reduction of CD4+CCR5+ cells. Peripheral CD8+CCR5+ T-lymphocyte counts were reduced by either low (0.252 mg) or high (3.5 mg) doses of FTY. In contrast to CCR5+ cells, cells expressing CD62L were preferentially reduced after administration of FTY. In particular, CD4+CD62L+ T cells declined after treatment. CD4+ and CD8+ T-lymphocyte subpopulations expressing the other chemokine and adhesion receptors (CXCR4, CD11a and CD49d) were reduced to a similar extent as compared with overall CD4+ or CD8+ T-lymphocyte counts.
Conclusions. Despite the limited number of patients, especially in the placebo (n = 3) and the high-dose groups (n = 2), our observations suggest that FTY exerts differential effects on T-cell subpopulations. FTY predominantly reduces CD4+CD62L+ cells in the peripheral blood suggesting increased migration into lymph nodes. It seems that only FTY doses above 2 mg are able to reduce peripheral CD4+CCR5+ T lymphocytes, which are potentially capable of infiltrating into the allograft during rejection.
Keywords: CCR5; CD62 ligand; FTY; human; lymphocyte subpopulation; renal transplantation
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Introduction
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FTY720 (FTY) is a derivative of the sphingolipid myriocin, produced by the fungus Isaria sinclairii [1]. In several animal models, FTY prevents allograft rejection with high efficacy [2]. Administration of FTY together with subtherapeutic concentrations of cyclosporine A (CyA) exerts synergism in allograft survival without increased toxicity. In contrast to classical immunosuppressive drugs, FTY does not inhibit lymphocyte proliferation or IL-2 synthesis [3,4]. The predominant feature of FTY is a reversible peripheral lymphopenia caused by an altered lymphocyte trafficking [2,5]. A recent report shows that the phosphorylated form of FTY binds to G-protein coupled receptors of the endothelial-differentiation gene family, which are expressed on lymphocytes [6]. However, the exact mechanism of action is still under investigation.
Currently, FTY is tested in humans for its use in renal transplantation. In the first human trial we investigated pharmacokinetics and pharmacodynamics of FTY in stable renal allograft recipients [7]. Confirming preclinical data, we observed that FTY induced a transient lymphopaenia by an apoptosis-independent mechanism [8]. All lymphocyte subsets were reduced, T cells more than B cells and CD4+ T cells to a higher extent than CD8+ T cells [9]. Interestingly, we observed that FTY had only minor effects on T cells expressing CCR5, while lymphocytes expressing CD62L were reduced to a much greater extent [10]. It is believed that FTY prevents transplant rejection through the inhibition of lymphocyte-infiltration into the allograft by increased lymphocyte homing [11]. The fact that CCR5+ T cells, which are capable of infiltrating into the graft [12], remain in the peripheral blood after FTY treatment, stimulated us to reinvestigate this phenomenon in more detail. In the present study, we investigated whether FTY exerts differential effects on the subpopulations of CD4+ and CD8+ T cells expressing specific chemokine and adhesion receptors.
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Subjects and methods
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Patient population and clinical study design
Our centre participated in a randomized, double-blind, placebo-controlled, time-lagged, ascending single-oral-dose phase 1 study of FTY. The patient characteristics have been previously described in detail [7]. In brief, all patients were at least 12 months post-transplantation and had therapeutic levels of CyA with a maximum serum creatinine level of 3.0 mg/dl. Except one, all patients received methylprednisolone (4 mg/day). The study was approved by the local Ethics Committee, and in concordance with the guidelines of the Declaration of Helsinki. A total of 10 subjects were enrolled, with six re-enrolling in later dose groups, for a total of 16 pharmacodynamic profiles (placebo, n = 3; 0.25 mg FTY, n = 3; 0.5 mg FTY, n = 4; 0.75 mg FTY, n = 1; 1.0 mg FTY, n = 2; 2.0 mg FTY, n = 1; 3.5 mg FTY, n = 2). Renal transplantation had been performed 8.4±3.9 years (range 3.614.4) before. Concomitant therapy remained unchanged throughout the dosing interval. Except for clinically insignificant abnormalities, the safety laboratory data were unremarkable in pretreatment evaluations with normal lymphocyte counts (2.02±0.38 x 109/l).
Subjects were domiciled from 24 h before dosing with FTY until 96 h following dosing. Subjects fasted overnight prior to oral administration of study medication and consumed an average of 120 ml of water per hour prior to dosing and 24 h after dosing. Unless performing a study assessment, subjects had to rest quietly in an upright position for 4 h after administration of the drug. Strenuous physical exercise was prohibited for 7 days before dosing until study completion.
Determination of peripheral lymphocyte counts
Whole blood was drawn by venipuncture from a peripheral forearm vein or indwelling venous cannula immediately pre-dose, and at 2, 6, 12, 24, 72 and 96 h post-dosing into EDTA-containing tubes for assessment of differential blood counts. Absolute leucocyte counts were analysed with a Micro-Diff-II cell counter (Coulter-Immunotech Diagnostics, Hamburg, Germany). Additionally, 20 ml of citrate anti-coagulated blood was drawn immediately pre-dose and at 4, 8, 12, 24 and 96 h after FTY administration. The absolute lymphocyte counts 4 and 8 h post-dosing were calculated as the individual mean value of the lymphocyte count after 2 and 6 h, or after 6 and 12 h, respectively. Absolute lymphocyte subpopulation counts were calculated by the formula: absolute subpopulation counts = (% lymphocyte subset gate x lymphocyte counts)/100%.
Isolation of peripheral blood mononuclear cells (PBMCs)
PBMCs were isolated by Ficoll density centrifugation as previously described [13]. Briefly, 20 ml of citrate anti-coagulated blood was diluted with 20 ml of PBS (Dulbecco's phosphate-buffered saline; PAA Laboratories, Linz, Austria). Then, 4 ml of Ficoll (Ficoll-PaqueTM Plus; Amersham Pharmacia Biotech, Uppsala, Sweden) was overlaid with 10 ml of blood/PBS. After centrifugation (Megafuge-2.0R; Haraeus Instruments GmbH, Berlin, Germany) for 20 min at 1650 g, PBMCs were collected from the interface between the plasma and the Ficoll layer, and washed with PBS. PBMCs were resolved in RPMI medium (Biochrom, Berlin, Germany) for cell culture or in FACS-Flow (Becton-Dickinson, Heidelberg, Germany) for immediate flow cytometric analysis.
Flow cytometry for human cell surface markers
Aliquots of 100 µl of PBMCs (106/ml) were analysed by flow cytometry after staining with monoclonal antibodies (5 µl) against lymphocyte subset markers (CD3/CD4, CD3/CD8) in combination with 5 µl of monoclonal antibodies against CCR5, CXCR4, CD11a, CD49d and CD62-L according to the manufacturer's instructions. All monoclonal antibodies and the corresponding isotype controls were purchased from Becton-Dickinson. Antibodies were conjugates with peridinin chlorophyll protein (PerCP), fluorescin isothiocyanate (FITC) or phycoerythrin (PE). After 45 min incubation at 4°C, PBMCs were centrifuged and cell pellets were diluted in Cytofix/Cytoperm (Becton-Dickinson) solution for 20 min at 4°C. After washing with 400 µl of FACS-Flow (Becton-Dickinson), PBMCs were resolved in 100 µl of FACS-Flow (Becton-Dickinson) and analysed with a flow cytometer equipped with an argon laser emitting light at 488 nm for three-colour analysis (Becton-Dickinson Facs-Calibur).
In vitro experiments
The effect of different concentrations of FTY (1 nM to 1 µM) on the expression of T-cell surface markers was investigated in vitro. FTY (Novartis AG, Basel, Switzerland) was freshly dissolved in distilled water and diluted in RPMI (Biochrom) to the final concentration. PBMCs from healthy volunteers (n = 4) were isolated by Ficoll density centrifugation and cultured under standard conditions (37°C, 5% CO2, humidified air) in complete RPMI medium (Biochrom) as described above. After 12 h, cultured PBMCs were harvested and reconstituted in FACS solution. PBMCs (106/100 µl) were stained with 5 µl of mAbs against CD3 (CD3-PerCP) and CD4 (CD4-PE) or CD8 (CD8-PE) in combination with 5 µl of FITC-labled mAbs against CCR5 (or CXCR4 or CD11a or CD49d or CD62-L) as described above. After washing with CellWASH (Becton-Dickinson) and fixation with CellFix (1x) (Becton-Dickinson), PBMCs were analysed by flow cytometry as described above. The in vitro data are expressed as the mean of four independently performed experiments.
Statistical analysis
Analysis of all FACS data was performed with CellQuest (Becton-Dickinson) and SPSS software. Because we did not observe a clear doseresponse relationship of FTY-induced lymphopaenia [7,9], mean values and standard deviation were calculated for all patients (n = 13) treated with FTY.
Statistically significant differences between pre-dose and post-dose values were assessed by Student's paired t-test following testing for normal distribution of the data according to KolmogorovSmirnov. A P-value of <0.05 was considered to be statistically significant. Due to the small number of patients, no statistics were performed for the placebo group (n = 3) and the 3.5 mg dose group (n = 2). Note that standard deviations of the placebo group are low.
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Results
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Effect on CD4+ and CD8+ T cells
As described in our initial report [9], all lymphocyte subsets, except NK cells, declined after a single oral dose of FTY. In our patient cohort, we observed a reversible reduction of lymphocyte counts reaching a nadir at 8 h after dosing. Lymphocyte numbers returned to baseline after 96 h. T cells decreased by 45% (from 1600±340 to 890±440/µl; P<0.001), and the reduction of CD4+ cells (by 64%, from 830±310 to 300±240/µl; P<0.001) was more pronounced compared with the decline of CD8+ cells (by 42%, from 430±210 to 250±140 µl; P<0.001). As a consequence, the percentage of CD4+ cells in the peripheral blood compartment decreased, while the percentage of CD8 cells remained stable (Table 1). In the placebo group, neither the absolute number nor the percentage of CD4+ cells changed during the observation period. However, the number of peripheral CD8+ cells (absolute and relative number) showed a diurnal increase after 8 h.
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Table 1. Effect of FTY on the percentage of peripheral blood lymphocyte subsets in human renal allograft recipients
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Effect on CD4+CCR5+ and CD8+CCR5+ T cells
Because the chemokine receptor CCR5 is involved in chemotaxis, migration and infiltration of lymphocytes into the allograft during transplant rejection [12], we investigated CCR5+ lymphocyte subpopulations in the peripheral blood after single oral doses of FTY. Following administration of FTY, the absolute number of CD4+CCR5+ T lymphocytes did not change during the entire monitoring period of 96 h (Figure 1A). In contrast to CD4+CCR5+ T cells, absolute CD8+CCR5+ T lymphocyte counts decreased by 27% from 145±100 to 85±70/µl (P<0.05) 8 h after administration of FTY (Figure 1B). The number of CCR5+ cells returned slowly back to baseline after 96 h. While CD4+CCR5+ cell numbers remained stable in the placebo group, the CD8+CCR5+ T-cell numbers exhibited some variability, with higher values after 8 and 96 h (Figure 1).

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Fig. 1. The effect of FTY on (A) absolute peripheral (squares) CD4+CCR5+ and on (B) absolute peripheral (triangles) CD8+CCR5+ T-lymphocyte subpopulation counts after administration of FTY or placebo. PBMCs were analysed by flow cytometry at the indicated time points. Data represent the absolute counts of 0.253.5 mg FTY (n = 13) and placebo (n = 3) treated renal transplant patients. Statistics were only performed for the 0.253.5 mg FTY (n = 13) treatment group: *P<0.05, **P<0.01, #P<0.001.
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Because CD4+CCR5+ cell counts remained stable after FTY administration, whereas other CD4+ T-cell populations decreased, the percentage of peripheral CD4+CCR5+ cells increased (Table 1).
More detailed data analysis revealed that in patients (n = 2) receiving the highest dose FTY (3.5 mg), CD4+CCR5+ cells declined by 72.6% (from 230 to 60 cells/µl) (Figure 2A). In these patients the number of CD4+CCR5+ cells did not return to baseline over the entire 96 h observation period. In the lower dose group (0.252.0 mg) the absolute number of CD4+CCR5+ T lymphocytes did not change over the entire monitoring period (Figure 2A). In low-dose FTY-treated patients the relative number of CD4+CCR5+ cells normalized after 2496 h, while the percentage of CD4+CCR5+ cells remained elevated in the highest dose group (Table 1).

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Fig. 2. The dose-dependent effect of FTY on absolute peripheral CCR5+ T-lymphocyte subpopulation counts. (A) CD4+CCR5+ T-lymphocyte counts were compared with (B) CD8+CCR5+ T-lymphocyte counts in patients receiving 0.252 mg FTY (n = 11) (circles), 3.5 mg FTY (n = 2) (triangles) or placebo (n = 3) (squares) after administration of FTY or placebo. PBMCs were analysed by flow cytometry at the indicated time points. Data represent the absolute counts of 0.252 mg FTY (n = 11) and placebo (n = 3) treated renal transplant patients. Statistics were only performed for the 0.252 mg FTY (n = 11) treatment group: *P<0.05, **P<0.01, #P<0.001.
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In contrast to CD4+CCR5+ T cells, absolute CD8+CCR5+ T-lymphocyte counts decreased in all dosing cohorts. We observed a reduction by 38.3% (from 150±110 to 90±70/µl; P<0.05) in the 0.252.0 mg FTY group after 8 h (Figure 2B). In the highest dosing group, the decline was more pronounced (by 67.9%, from 100 to 30/µl after 8 h) and more sustained, as CD8+CCR5+ cells did not return to baseline after 96 h (Figure 2B). The percentage of peripheral CD8+CCR5+ increased slightly in the low-dose FTY treatment group, and remained stable in the highest FTY group (Table 1). Placebo-treated patients exhibited no change in the relative amount of CD4+CCR5+ or CD8+CCR5+ lymphocytes.
Effect on CD4+CXCR4+ and CD8+CXCR4+ T cells
During transplant rejection, infiltrating lymphocytes have been shown to express CXCR4, another chemokine receptor involved in the chemotactic response of lymphocytes [12]. Therefore, we investigated absolute and relative numbers of CD4+ and CD8+ cells expressing CXCR4. In FTY-treated renal transplant patients, CD4+CXCR4+ T-lymphocyte counts decreased (P<0.01) by 61% (from 650±350 to 260±220/µl) 8 h after intake. Cell numbers returned back to baseline after 96 h (Figure 3A). Absolute CD8+CXCR4+ T-lymphocyte counts also decreased 8 h after administration of FTY, but to a lesser extent (P<0.01), by 36% (from 300±150 to 210±110/µl), reaching baseline already after 24 h (Figure 3B). Again, 8 h after administration of placebo we observed an increase in peripheral CD8+CXCR4+ T lymphocytes, while CD4+CXCR4+ cell numbers were stable. The percentage of both CD4+CXCR4+ and CD8+CXCR4+ cell numbers remained stable in the placebo-treated patients, while the percentage of CXCR4+ lymphocytes after FTY treatment increased slightly (not significant) after 4 h. No differences were found between low- and high-dose FTY cohorts.

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Fig. 3. The effect of FTY on (A) absolute peripheral (squares) CD4+CXCR4+ and on (B) absolute peripheral (triangles) CD8+CXCR4+ T-lymphocyte subpopulation counts after administration of FTY or placebo. PBMCs were analysed by flow cytometry at the indicated time points. Data represent the absolute counts of 0.253.5 mg FTY (n = 13) and placebo (n = 3) treated renal transplant patients. Statistics were only performed for the 0.253.5 mg FTY (n = 13) treatment group: *P<0.05, **P<0.01, #P<0.001.
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Effect on CD4+CD62L+ and CD8+CD62L+ T cells
CD62L is involved in lymphocyte homing by facilitating the migration of lymphocytes through the endothelial barrier of high endothelial venules (HEV) into lymph nodes. As FTY may promote the homing of lymphocytes, we were interested in lymphocyte populations expressing CD62L. Absolute CD4+CD62L+ T lymphocytes sharply declined by 90%, from 570±260 to 60±160/µl (P<0.001) after 8 h in FTY-treated cohorts, and did not return to baseline within 96 h (Figure 4A). Absolute CD8+CD62L+ T lymphocytes decreased by 60%, from 160±70 to 70±60/µl after 8 h (P<0.05), returning back to baseline after 24 h (Figure 4B).

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Fig. 4. The effect of FTY on (A) absolute peripheral (squares) CD4+CD62L+ and on (B) absolute peripheral (triangles) CD8+CD62L+ T-lymphocyte subpopulation counts after administration of FTY or placebo. PBMCs were analysed by flow cytometry at the indicated time points. Data represent the absolute counts of 0.253.5 mg FTY (n = 13) and placebo (n = 3) treated renal transplant patients. Statistics were only performed for the 0.253.5 mg FTY (n = 13) treatment group: *P<0.05, **P<0.01, #P<0.001.
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The percentage of peripheral CD4+CD62L+ counts decreased significantly between 4 and 8 h after intake of FTY, returning back to baseline after 24 h (Table 1). Again, both patients receiving the highest FTY dose exhibited a stronger and more sustained decline in the percentage of CD4+CD62L+ cells. The percentage of CD8+CD62L+ T lymphocytes was reduced after 8 h, which was more pronounced in the highest dosing cohort. Except for the diurnal increase after 8 h, the absolute number as well as the percentage of cells expressing CD62L+ was rather stable in placebo-treated subjects (Figure 4 and Table 1).
Effect on CD4+CD11a+ and CD8+CD11a+ T cells
CD11a is an adhesion receptor on lymphocytes, which might be relevant for FTY-induced lymphocyte migration. In the FTY group, CD4+CD11a+ T-lymphocyte counts decreased by 44%, from 160±70/µl pre-dose to 90±60/µl after 8 h (P<0.05). CD4+CD11a+ T-lymphocyte counts did not return to baseline within 96 h (P<0.05) (Figure 5A). Throughout the observation period, the percentage of CD4+CD11a+ T cells remained stable between 35 and 45%, with exceptionally low pre-dose values in the 3.5 mg dosing group (Table 1). CD8+CD11a+ T-lymphocyte counts decreased by 21%, from 280±160/µl pre-dose to 210±120/µl after 8 h (P<0.05) and returned to baseline within 24 h (Figure 5B). The percentage of CD8+CD11a+ cells did not change significantly, and higher doses of FTY exhibited similar kinetics compared with lower doses. Absolute and relative numbers of peripheral CD11+ T lymphocytes were stable in the placebo group (Table 1).

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Fig. 5. The effect of FTY on (A) absolute peripheral (squares) CD4+CD11a+ and on (B) absolute peripheral (triangles) CD8+CD11a+ T-lymphocyte subpopulation counts after administration of FTY or placebo. PBMCs were analysed by flow cytometry at the indicated time points. Data represent the absolute counts of 0.253.5 mg FTY (n = 13) and placebo (n = 3) treated renal transplant patients. Statistics were only performed for the 0.253.5 mg FTY (n = 13) treatment group: *P<0.05, **P<0.01, #P<0.001.
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Effect on CD4+CD49d + and CD8 +CD49d + T cells
Next, we investigated CD49d, a cell surface marker known to facilitate migration of lymphocytes through the endothelial barrier of vessels. The numbers of CD4+CD49d+ T lymphocytes decreased by 60%, from 460±240 to 190±150/µl after 8 h (P<0.05) and did not return to baseline within 96 h (P<0.05) (Figure 6). Because CD4+CD49d+ T lymphocytes declined to a similar extent as the overall CD4 count, the relative amount of CD4+CD49+ cells in the peripheral blood was not affected (Table 1). Again, we could not detect relevant differences between the different dosing cohorts. Similar to the other markers, the absolute and relative number of peripheral CD4+CD49d+ T lymphocytes did not change in the placebo group (Figure 6A).

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Fig. 6. The effect of FTY on (A) absolute peripheral (squares) CD4+CD49d+ and on (B) absolute peripheral (triangles) CD8+CD49d+ T-lymphocyte subpopulation counts after administration of FTY or placebo. PBMCs were analysed by flow cytometry at the indicated time points. Data represent the absolute counts of 0.253.5 mg FTY (n = 13) and placebo (n = 3) treated renal transplant patients. Statistics were only performed for the 0.253.5 mg FTY (n = 13) treatment group: *P<0.05, **P<0.01, #P<0.001.
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The absolute CD8+CD49d+ T-lymphocyte counts (P<0.05) decreased by 38%, from 270±150 to 190±120/µl after 8 h and returned to baseline after 24 h (Figure 6B). The percentage of CD8+CD49d+ cells showed a high inter-individual variability, especially in the lower dosing cohorts, so we could not detect any significant changes after FTY dosing. Similarly, we observed some variability of CD8+CD49+ T cells in the placebo group without significant changes in the relative composition (Figure 6 and Table 1).
Effect on surface marker expression of human lymphocytes in vitro
Parallel to the clinical study, we investigated the effect of FTY on lymphocyte chemokine receptors and adhesion molecules in vitro. After incubation of PBMCs for 12 h with different concentrations of FTY (0.1 nM to 1 µM), we determined the percentage and the mean expression level of these surface markers. In general, we did not detect any direct effect of FTY on the percentage and mean expression level of these chemokine receptors and adhesion molecules on CD4+ and CD8+ T cells (Tables 2 and 3). Only CD11 showed an slightly increased percentage (1 nM FTY), which was not dose dependent (Table 2). CD62L+ cells exerted a decreased mean expression level with high inter-individual variability after treatment with FTY in a range which cannot explain the strong decrease of these cell counts observed in vivo.
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Table 3. Mean expression level of different surface markers on CD4+ and CD8+ T lymphocytes after incubation with FTY in vitro
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Discussion
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In the present study, we investigated the effect of the novel immunomodulator FTY on different lymphocyte subpopulations. Previous studies in animals [14] as well as our own data [9] in humans demonstrate that FTY affects predominantly CD4+ cells whereas CD8+ cells are reduced to a lesser extent. In order to characterize this effect in more detail, we analysed CD4+ and CD8+ T-cell populations with respect to their expression of chemokine receptors and adhesion molecules. Interestingly, T lymphocytes expressing CCR5 chemokine receptors decreased only marginally, while the decline of CD62L+ T cells was far more pronounced. Thus, CD4+CD62L+ T cells appear to be the main target of FTY, because up to 90% of this cell type disappeared from the peripheral blood compartment after single oral doses of FTY. In contrast, CD4+CCR5+ T cells were only influenced by high (>2.0 mg) doses of FTY. CD8+CCR5+ T cells were reduced upon administration of high and low doses of FTY. T cells expressing CXCR4, CD11a or CD49d seemed not to be specifically regulated by FTY, and FTY had no direct effect on the expression on cell surface adhesion receptors and chemokine receptors on CD4+ or CD8+ T cells.
This study extends the existing knowledge on this interesting new immunosuppressive compound, as this is the first study to investigate the effect of FTY on the expression of receptors for inflammatory chemokines, CCR5 and CXCR4. It has been proposed that FTY modulates lymphocyte migratory responses to secondary lymphoid-tissue chemokine (SLC) and other chemokines [15]. Mice deficient for the CC chemokine receptor CCR7, the receptor for SLC, exerted only a delayed lymphopaenia after treatment with FTY [12]. In plt/plt mutant mice, which are deficient for SLC, FTY-induced lymphopaenia was less pronounced [16], providing evidence for the involvement of chemokine receptors in the mechanism of action of FTY. Here, we observed that low-dose FTY treatment had no effect on CD4+CCR5+ T cells in the peripheral blood. Because other subpopulations (e.g. CD62L+ T cells) are reduced to a much greater extent, the net effect was an increased percentage of CCR5+ peripheral T cells, especially a doubling of CD4+CCR5+ T cells. CCR5 is expressed by inflammatory cells infiltrating the allograft and may mediate chemotaxis of T cells at sites of rejection [12,17]. Individuals homozygous for the delta32 mutation resulting in an impaired expression of CCR5 show a lower rate of acute rejections and prolonged graft survival [18]. In this context, it is surprising that this cell type is modulated only to a minor extent by FTY, as FTY has potent anti-rejection properties in all animal species tested [2] as well as in humans [19]. From our data it is tempting to speculate that cells expressing CCR5 are the predominant cell type in rejecting grafts under FTY treatment. Thus, blockade of CCR5-infiltrating cells by specific antibodies may be highly synergistic in combination with FTY.
The observation that only the highest dose of FTY effectively reduced the number of CD4+CCR5+ T-cell counts suggests that higher FTY doses might exert qualitatively different effects on the composition of lymphocyte subpopulations in the peripheral blood. In general, the pharmacodynamic effect of FTY on lymphocyte counts has no clear dose relationship, at least for doses between 0.25 and 2 mg [7,14]. However, at the highest dose of FTY (3.5 mg) there appeared to be a more pronounced and more sustained response for most cellular markers investigated, but the small number of patients in this group makes it difficult to draw any clear conclusion. Our observations may favour the use of FTY doses higher than 2 mg for effective rejection prophylaxis. This hypothesis is supported by the results of a recent clinical trial [19]. Consequently, ongoing phase III trials are using doses of 2.5 and 5 mg in combination with cyclosporine and steroids. Certainly, more studies in larger populations under FTY maintenance therapy are needed to address this interesting observation in more detail.
In contrast to T cells expressing CCR5, T cells expressing CXCR4 decreased to a similar extent as the total T-cell population. This observation clearly documents that FTY does not exert its effects uniformly on all T-cell subpopulations, but has specific effects on certain subpopulations. Our in vitro data demonstrate that FTY has no direct effect on the expression of chemokine receptors and adhesion molecules. We conclude that the observed changes in the composition of these surface markers in vivo may be caused by an altered lymphocyte homing affecting specific subpopulations more than others.
Our current understanding of the immune-modulating effects of FTY leading to allograft survival is still incomplete. It is believed that FTY prevents allograft rejection by inhibition of lymphocyte infiltrates in the transplant via altered lymphocyte trafficking. Chiba et al. [5] observed in a skin allograft model that peripheral lymphopaenia was associated with an increased number of lymphocytes in secondary peripheral lymphoid tissues. Co-treatment with monoclonal antibodies against CD62L, CD49d and CD11a blocked FTY-induced lymphopaenia [7]. Interestingly, FTY led to an increased frequency of CD62L+ T cells in Peyer's patches, suggesting increased lymphocyte homing [20]. The critical role for CD62L in lymphocyte homing has been well established [21]. Extending the animal data we observed a dramatic reduction of CD62L+ T cells in the periphery, CD4+CD62L+ decreased by 90% and CD8+CD62L+ by 60%. Thus, our finding supports the hypothesis that CD62L+ T cells are the main target of FTY. Because the direct effect of FTY on the expression of this surface marker in vitro is rather low, the FTY-mediated depletion of CD62L+ T cells from the peripheral blood in transplant patients probably was due to enhanced homing to the secondary lymphoid organs, consistent with previous experimental data [8]. Cells bearing CD62L on their surface may easily respond to FTY because CD62L facilitates the migration of CD4+ and CD8+ lymphocytes through the endothelial barrier of HEV into lymph nodes.
The dramatic decrease in CD4+CD62L+ counts support the hypothesis that FTY increases the migration of naïve T cells into secondary lymphoid organs. This suggestion is supported by our earlier report demonstrating an increased reduction of CD45RA+ cells while CD45RO+ memory cells were less effected [9]. Further studies investigating other markers of naïve T cells, like CCR7, are needed to further substantiate this hypothesis. Recently, Mandala and colleagues have shown that FTY favours the sequestration of lymphocytes into lymph nodes and inhibits the lymphocyte egress into lymphatic sinus [22]. In the view of this report, our clinical observation might be alternatively explained by FTY-mediated preferential trapping of CD62L+ T cells in the lymph nodes. On the contrary, CCR5+ T cells may be less retained in lymph nodes after administration of FTY. Studies with other markers, like CXCR3+, which is expressed on activated TH1 cells, may help to further clarify whether mature or activated lymphocytes are generally less affected by FTY.
In the experimental setting, the number of T cells expressing CD49d and CD11a did not change in lymph nodes, indicating a differential regulation of lymphocyte subpopulations by FTY [20]. Extending the experimental data, we observed in human renal allograft recipients that T cells expressing the adhesion molecules CD11a+ and CD49d+ declined to a similar extent as total CD4+ and CD8+ lymphocytes, suggesting that CD11a and CD49d are not specifically involved in the response to FTY. However, it is important to point out that repeated administration of FTY may have different effects compared with those observed in the present single-dose study.
In summary, the present study in humans adds some important new information to our understanding of the new immunomodulator FTY. The results of the present descriptive study suggest that CD4+CD62L+ T cells are a predominant target of FTY, while T cells bearing CCR5 are less affected. Further studies are needed to elucidate the exact mechanism by which FTY modulates lymphocyte trafficking. The investigation of these events may additionally shed some light on the interaction between lymphocyte recirculation and immune response.
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Acknowledgments
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Torsten Böhler is a recipient of a grant from the Novartis Stiftung für therapeutische Forschung.
Conflict of interest statement. The authors have declared no conflict of interest.
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References
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Received for publication: 6. 8.03
Accepted in revised form: 10.10.03