The pyrimidin analogue cyclopentenyl cytosine induces alloantigen-specific non-responsiveness of human T lymphocytes

Abstract

Cyclopentenyl cytosine (CPEC) has been shown to induce apoptosis in human T lymphoblastic cell lines and T cells from leukaemia patients. In this study we have addressed the question of whether CPEC is able to decrease proliferation and effector functions of human alloresponsive T lymphocytes and induce T cell anergy. The proliferative capacity of human peripheral blood mononuclear cells in response to allogeneic stimulation was measured by 5,6-carboxy-succinimidyl-diacetate-fluorescein-ester staining. Flow cytometric analysis was performed using surface CD4, CD8, CD25, CD103 and intracellular perforin, granzyme A, granzyme B, caspase-3 and forkhead box P3 (FoxP3) markers. The in vivo immunosuppressive capacity was tested in a murine skin graft model. Addition of CPEC at a concentration of 20 nM strongly decreased the expansion and cytotoxicity of alloreactive T cells. Specific restimulation in the absence of CPEC showed that the cells became anergic. The drug induced caspase-dependent apoptosis of alloreactive T lymphocytes. Finally, CPEC increased the percentage of CD25^high FoxP3⁺ CD4⁺ and CD103⁺ CD8⁺ T cells, and potentiated the effect of rapamycin in increasing the numbers of alloreactive regulatory T cells. Treatment with CPEC of CBA/CA mice transplanted with B10/Br skin grafts significantly prolonged graft survival. We conclude that CPEC inhibits proliferation and cytotoxicity of human alloreactive T cells and induces alloantigen non-responsiveness in vitro.

Introduction

Cyclopentenyl cytosine (CPEC) is a carbocyclic analogue of cytidine which inhibits cytidine triphosphate (CTP) synthetase, an enzyme that catalyses the conversion of uridine triphosphate (UTP) into CTP [1]. In addition, it serves as an alternative substrate of uridine/cytidine kinase, the first enzyme of the pyrimidine salvage pathway, and acts as a competitive inhibitor for uridine and cytidine phosphorylation [1,2]. The drug was shown to have anti-tumour [3–7] and anti-viral [8–10] activity both in vitro and in vivo.

Treatment of mice suffering from lymphocytic leukaemia with CPEC led to an increased life span of 58–129%, depending on the treatment duration [11]. In athymic mice, inoculated with human colon carcinoma cells, treatment with CPEC induced a reduction of tumour size [12]. In a phase I clinical trial in human adults with solid tumours, administration of low doses of CPEC was quite well tolerated, but was complicated by cardiovascular and moderate haematological toxicity at the higher dosages (plasma levels of CPEC 3·1 mM) [13].

Studies into the mechanism of action of the anti-tumour effect of CPEC have been performed in myeloid cell lines and leukaemic cells obtained from patients [6,14] and in some solid tumour cells [3,15,16]. In malignant cells, CTP synthetase activity was shown to be up-regulated [6,17,18]. By inhibiting CTP synthetase activity and thereby decreasing the CTP pool, CPEC reduced the ability of tumour cells to proliferate. Increased CTP synthetase activity in malignant T lymphocytes appeared to be a cell cycle-independent feature, whereas the increase in activity of CTP synthetase in normal human T lymphocytes following a growth stimulus appeared to be most prominent in the S-phase of the cell cycle [19]. In the human T lymphoblastic MOLT-3 cell line, CPEC was already shown to inhibit proliferation at a concentration of 25 nM and to induce apoptosis and secondary necrosis of T lymphoblasts [3,5]. Until now, the effect of CPEC on proliferation and differentiation of normal human T lymphocytes has not been studied.

In the present study, we evaluated the effects of CPEC on activation, proliferation and expression of cytotoxic effector molecules of alloantigen-stimulated human T cells in vitro. In particular, we studied the potential of CPEC to induce apoptosis and anergy of alloreactive T lymphocytes as well as its influence on their cytotoxic capacity. In addition, the interference of CPEC with the formation of regulatory CD25^bright forkhead box P3 (FoxP3)⁺ CD4⁺ T cells and CD103⁺ CD8⁺ T cells during the allogeneic response was examined. Finally, we studied its immunosuppressive effect in vivo, using a murine model of skin transplantation.

Materials and methods

Cells

Peripheral blood mononuclear cells (PBMC) were isolated from heparinized whole blood obtained from healthy donors by Ficoll-Paque density centrifugation (Pharmacia Biotech AB, Uppsala, Sweden).

Monoclonal antibodies

Anti-CD4-peridinin chlorophyll (PerCP) (dilution used: 1/20), CD8-PerCP (1/20), CD25-PE (1/20), active caspase-3-phycoerythrin (PE) (1/50) and mIgG1-PE (1/33) were all purchased from BD Immunocytometry systems BD PharMingen (San Jose, CA, USA). Anti-FoxP3-antigen-presenting cells (APC) (1/5) were purchased from eBioscience (San Diego, CA, USA) and anti-CD103-PE (1/50) from Caltag (Burlingame, CA, USA). Anti-perforin-PE (1/100) was obtained from Hölzel Diagnostika (Köln, Germany) and anti-granzyme B-PE (1/133) from Sanquin (Amsterdam, the Netherlands).

Cell culture

All culture experiments were performed in Iscove's modified Dulbecco's medium (IMDM) (Life Technologies, Gaithersburg, MD, USA) containing 10% heat-inactivated autologous human serum, antibiotics (100 U/ml sodium penicillin G (Brocades Pharma BV, Leiderdorp, the Netherlands) and 100 μg/ml streptomycin sulphate (Gibco brl, Paisley, Scotland, UK) and 2-mercapto-ethanol (2-ME) (0·0035%; Merck) (culture medium). Mixed lymphocyte cultures (MLCs) were performed with responder–stimulator combinations that gave high proliferative responses in classical MLCs, as detected by [³H]-thymidine incorporation. Responder PBMC were 5,6-carboxy-succinimidyl-diacetate-fluorescein-ester (CFSE)-labelled and cultured with irradiated allogeneic or autologous stimulator PBMCs in a 1 : 1 ratio for 6 days. For secondary MLCs, cells were washed and restimulated specifically for 5 days with the same stimulator cells as used previously in primary MLC. The precursor frequency was calculated as follows: [Σ_{n ≥ 1}(P_n/2ⁿ)]/[Σ_{n ≥ 0}(P_n/2ⁿ)], where n is the division number that cells have gone through and P_n is the number of the cells in division n[20].

CFSE labelling

Cells were washed once with phosphate-buffered saline (PBS)-penicillin/streptomycin at room temperature prior to staining; 1 μl of CFSE (5 μM stock concentration; Molecular Probes Europe BV, Leiden, the Netherlands) was added in 1 ml PBS-penicillin/streptomycin per 10⁷ cells. Cells were incubated for 13 min at 37°C, washed three times in PBS/penicillin/streptomycin at 4°C and resuspended in 1 ml culture medium.

Immunofluorescent staining and flow cytometry

For surface staining, 3 × 10⁵ PBMC were incubated with fluorescent-labelled monoclonal antibodies for 30 min at 4°C protected from light. Intracellular staining of FoxP3 was performed using the FoxP3 staining set (eBioscience). For detection of intracellular perforin, granzyme B and caspase-3, cells were first labelled with surface antibodies and then fixed in 1% (w/v) paraformaldehyde (PFA; Merck) in PBS for 5 min at 4°C, protected from light. Cells were permeabilized in PBS–bovine serum albumin (BSA)–NaN3 supplemented with 0·1% (w/v) saponin (Sigma Chemicals, St Louis, MO, USA) and 50 mmol D-glucose. Cells were washed in saponin buffer and incubated with anti-perforin, anti-granzyme B or anti-caspase-3 monoclonal antibody or appropriate isotype control for 30 min at 4°C protected from light. Next, cells were washed in saponin buffer and analysed directly using a fluorescence activated cell sorter (FACS)Calibur flow cytometer and CellQuestPro software (BD PharMingen).

Effect of addition of CPEC to culture

CPEC was provided by the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment (National Cancer Institute, Bethesda, MD, USA).

To assess the effect of CPEC on T lymphoblasts, PBMC of healthy individuals were incubated in the presence of 1 μg/ml CD3 monoclonal antibodies (mAb) and 2·5 μg/ml CD28 mAb for 72 h. After 3 days, different concentrations of CPEC were added to the lymphoblasts. To detect if apoptosis, caused by addition of CPEC, was caspase-dependent, control samples were incubated with 50 μM of the pan-caspase inhibitor N-carbobenzoxy-Val-Ala-Asp fluoromethyl ketone (zVAD-fmk) (Calbiochem, San Diego, CA, USA).

FACS cytotoxicity assay

MLCs consisting of responder PBMC and irradiated stimulator cells were used to generate effector cells after 6 days. The FACS cytotoxicity assay was employed to assess apoptosis of allogeneic or autologous target cells at varying effector : target (E : T) ratios. After 6 days of culture, the target lymphocyte cells were washed and labelled with DDAO-SE dye for 5 min at 37°C. Subsequently, target cells were washed and incubated for 6 h together with effector cells at varying E : T ratios at 37°C. Mitochondrial transmembrane potential (ΔΨ_m) was assessed using DiOC₆ (40 nM in PBS) (Molecular Probes, Leiden, the Netherlands). The DioC6 dye was added to the cells for the last 15 min of incubation and cells were analysed on FACS without prior washing [21]. Analysis was conducted within DDAO-SE-positive (target) cells; cytotoxicity was determined as percentage of apoptotic target cells. Cells which had undergone apoptosis during incubation with effector cells and lost their membrane potential became DioC6 dull or negative, live cells remained DioC6 bright. As a control, target cells were also labelled with DioC6 and measured by FACS before incubation with effector cells. To assess antigen non-specific cytotoxicity, cytotoxic T lymphocyte (CTL) activity was determined in a CD3 mAb (mouse IgG1)-mediated cytotoxicity assay. In brief, FcR-bearing P815 target cells were labelled with DDAO-SE dye for 5 min at 37°C. After that, target cells were washed and incubated for 6 h together with effector cells at varying E : T ratios at 37°C. At the last 15 min of incubation the DioC6 dye was added to the cells and cells were analysed on FACS without prior washing. Cytotoxicity was determined as described above.

Mice and surgical procedure

Eight-week-old CBA/Ca (H-2K^k), B10/Br (H-2K^k) and BL6 (H-2K^b) mice were purchased from Harlan Olac (Bicester, UK) and maintained at the animal facility of the Academic Medical Centre, Amsterdam, the Netherlands. All animals were treated in accordance to the rules of the local animal ethical committee and the principles of laboratory animal care. The research protocol was approved by the local animal ethical committee. Skin grafting was performed according to a modified procedure by Medawar [22]. Full-thickness donor tail skin, approximately 0·7 × 1·0 cm, was transplanted onto the lateral thoracic wall of the recipients. Skin grafts were monitored al least three times a week after removal of the bandages at day 8 after transplantation. Rejection was complete when no viable graft tissue was seen on the thoracic wall.

Experimental model

CBA/Ca recipient mice were grafted with either B10/Br (multiple minor differences) or BL6 skin (multiple minors and H2 differences). CPEC was administered intraperitoneally (i.p.) in a dose of 0·15 mg/kg, 0·5 mg/kg and 1·5 mg/kg five times a week for 3 weeks. Control mice received saline i.p. Each experimental group contained eight mice.

Statistical analysis

The Mann–Whitney U-test was used for comparison of two independent groups of observations. Kruskal–Wallis and one-way analysis of variance (anova) tests were used for comparisons of more than two means. P-values below 0·05 were considered statistically significant.

Results

CPEC inhibits proliferative capacity of alloreactive T cells

To study whether CPEC affects the proliferative capacity of alloreactive T cells, we labelled responder cells with CFSE and performed MLCs in the presence or absence of CPEC in culture. T cell proliferation was already decreased significantly at a concentration of 20 nM (Fig. 1a).

Fig. 1.

Cyclopentenyl cytosine (CPEC) inhibits proliferation of alloreactive CD4⁺ and CD8⁺ T cells. (a) CPEC inhibits proliferation of alloreactive CD4⁺ and CD8⁺ T cells in a dose-dependent manner. The effect of addition of different concentrations of CPEC, ranging from 0 nM to 1 μM, on proliferation of CD4⁺ (black bars) and CD8⁺ (white bars) alloantigen-stimulated T cells was studied. The mean ± standard deviation of seven experiments is shown. (b) CPEC also inhibits the proliferation of both CD4⁺ and CD8⁺ alloreactive T cells when added at 24 or 48 h after initiation of mixed lymphocyte culture. The effect of delayed addition of 20 nM CPEC on the proliferation of alloantigen-stimulated CD4⁺ and CD8⁺ T cells was studied. On the x-axis the mean fluorescence intensity of CFSE staining is shown; on the y-axis cell counts are given. Proliferation of CD4⁺ T cells is depicted. One of three representative experiments is shown.

At this concentration precursor frequencies of alloreactive CD4⁺ T cells decreased from 7·5 ± 2·1% in the control cultures to 1·7 ± 0·53% in the cultures where CPEC was present (P = 0·002). In CD8⁺ T cells, addition of CPEC decreased the precursor frequencies from 6·34 ± 1·1% to 1·5 ± 0·38% (P = 0·001). Hardly any proliferation was observed when CPEC was used at a concentration of 30 nM and higher.

When CPEC was added 24 or 48 h after initiation of mixed lymphocyte culture, the proliferation of both CD4 and CD8 positive T cells was still inhibited as much as in the cultures where the drug was added immediately (Fig. 1b, shown on CD4⁺ T cells).

This is in line with previous data, showing that CPEC has an inhibiting effect especially on the cells in the late G1-S phase of the cell cycle [19].

The proliferative capacity of alloreactive T cells is not restored once CPEC is eliminated from the alloreactive culture

To test whether the inhibition of proliferative capacity of alloreactive T cells by CPEC was reversible, cells from primary MLCs cultured in the presence of CPEC were washed after 6 days of culture and restimulated specifically in the absence of the drug. We observed that CPEC-treated cells proliferated poorly in the secondary MLC (Fig. 2, upper panel; shown on CD4⁺ T cells). Proliferation of cells against a third-party donor was also diminished, although to a much lesser extent (data not shown). On the contrary, cells from primary MLC cultured in the absence of CPEC showed an increased proliferative response after specific restimulation (Fig. 2, lower panel).

Fig. 2.

Cyclopentenyl cytosine (CPEC)-treated cells do not respond to restimulation with alloantigen after the drug has been washed out of culture. Cells from primary mixed lymphocyte cultures (MLCs) were cultured in the presence of CPEC in a final concentration of 20 nM (upper panel, left). After 6 days of MLC, cells were washed and restimulated specifically for 5 days in the absence of the drug. CPEC-treated cells showed low proliferation in the secondary MLCs (upper panel, right). As a control, cells from primary MLC cultured in the absence of CPEC (lower panel, left) were also washed after 6 days of culture and restimulated specifically for 5 days (lower panel, right). Proliferation of CD4⁺ T cells is shown. The x-axis shows the mean fluorescence intensity of 5,6-carboxy-succinimidyl-diacetate-fluorescein-ester dye dilution. On the y-axis cell counts are given. One of three representative experiments is shown.

CPEC induces apoptosis in divided CD4⁺ and CD8⁺ alloreactive T cells

To investigate whether CPEC could also limit T cell expansion by inducing apoptosis, we measured the expression of active caspase-3 on T cells after 6 days of MLC. In the undivided CFSE^high population, the percentage of apoptotic (caspase-3-positive) CD4⁺ and CD8⁺ T cells did not change once CPEC was added to the culture (data not shown). On the contrary, in divided CD4⁺ T cells the number of active caspase-3-positive cells increased from 6·03 ± 4·21% in untreated MLC to 25·65 ± 15·26% in CPEC-treated cultures (P = 0·037). The same picture was observed within the divided CD8 population, where the percentage of caspase-3-positive cells increased from 6·6 ± 5·08 to 26·25 ± 12·53% after addition of CPEC (P = 0·02). An example of active caspase-3 staining within divided CFSE^low CD4⁺ and CD8⁺ T cells in the presence or absence of CPEC in MLC is shown in Fig. 3a.

Fig. 3.

Cyclopentenyl cytosine (CPEC) induces caspase-dependent apoptosis. (a) CPEC induces apoptosis in divided alloreactive CD4⁺ and CD8⁺ T cells. Intracellular expression of active caspase-3 in CD4⁺ T cells (left panel) and CD8⁺ T cells (right panel) in the absence (filled histogram) or presence (grey line) of 20 nM CPEC in mixed lymphocyte culture was measured on day 6. One representative experiment of five is shown. (b) CPEC-induced apoptosis of T cell blasts is caspase-dependent. Human peripheral blood mononuclear cells were stimulated in the presence of anti-CD3 and anti-CD28 monoclonal antibodies. After 3 days of culture, cells were labelled with 5,6-carboxy-succinimidyl-diacetate-fluorescein-ester (CFSE), and CPEC in a concentration of 100 nM was added alone or in the presence of N-carbobenzoxy-Val-Ala-Asp fluoromethyl ketone (zVAD-fmk) for 24 h. Intracellular expression of active caspase-3 in CD4⁺ T cells (left panel) and CD8⁺ T cells (right panel) was measured on day 4 of culture. Filled histogram shows caspase-3 staining in the absence of CPEC; the grey line shows caspase-3 staining after addition of CPEC; the black line shows caspase-3 staining after addition of zVAD-fmk simultaneously with CPEC. The histogram plots are gated on divided CFSE^low T cells. One representative experiment of three is shown.

CPEC decreases proliferation and induces caspase-dependent apoptosis of T cell blasts

To check whether CPEC can also limit proliferation and induce apoptosis of lymphoblastic cells, we stimulated PBMC with a combination of CD3 and CD28 mAb. After 3 days of stimulation cells were labelled with CFSE and CPEC was added to the culture at different concentrations, ranging from 20 nM to 1 mM. We did not observe significant changes in proliferation and apoptosis in concentrations up to 50 nM, whereas in alloantigen-stimulated cultures a significant decrease in proliferation had already appeared at a CPEC concentration of 20 nM. However, at concentrations of 100 nM and higher, CPEC induced almost complete inhibition of lymphoblast proliferation (data not shown). There was also a significant increase in numbers of apoptotic CD4⁺ and CD8⁺ T cells. The maximum percentage of caspase-3-positive cells was observed after treatment of cells with 100 nM CPEC, when 25·42 ± 3·45% of CD4⁺ T cells became apoptotic versus 6·29 ± 1·12% in untreated cultures (P = 0·015). This was also true for the CD8 population, where 51·98 ± 5·6% of cells became apoptotic in the presence of CPEC versus 15·82 ± 3·17% in untreated cultures (P = 0·02). We did not observe a further increase in the amount of caspase-3-positive T cells when higher CPEC concentrations were used.

To obtain more insight into apoptosis induced by CPEC, we examined the effect of simultaneous addition of the pan-caspase inhibitor zVAD-fmk and CPEC to the CD3/CD28-stimulated cultures. If zVAD-fmk was present in CPEC-treated cultures, the percentage of caspase 3-positive CD4⁺ T cells and CD8⁺ T cells was 10·31 ± 2·35 and 21·3 ± 4·1%, respectively, which is comparable to untreated cultures (Fig. 3b).

CPEC decreases the cytotoxic potential of alloreactive T cells

The presence of 20 nM CPEC in 6 days' mixed lymphocyte culture did not decrease the number of perforin and granzyme B-producing alloreactive CD4⁺ and CD8⁺ T cells (Fig. 4a). To examine whether these cells also retained their cytotoxic capacity, we tested their ability to decrease the membrane potential and cause apoptosis in the allogeneic target cells.

Fig. 4.

Cyclopentenyl cytosine (CPEC) does not change the expression of effector molecules but inhibits cytotoxic capacity of alloreactive CD4⁺ and CD8⁺ T cells. (a) Intracellular expression of granzyme B and perforin in CD4⁺ T cells and CD8⁺ T cells in the absence (upper row) or presence (lower row) of CPEC in a concentration of 20 nM in mixed lymphocyte culture was measured on day 6. Dot plots are gated on divided 5,6-carboxy-succinimidyl-diacetate-fluorescein-ester (CFSE)^low cells. In the upper right corner of the dot plots percentage of granzyme B or perforin positive cell is shown. One of five experiments is shown. (b, c) CPEC inhibits cytotoxic capacity of alloreactive T cells. Alloantigen-dependent (b) and -independent (c) cytotoxicity was measured by fluorescence activated cell sorter cytotoxicity assay. Different numbers of alloreactive effector cells are depicted on the x-axis. The number of DiOC6 negative (apoptotic) target cells is shown on the y-axis. Alloreactive cytotoxic T lymphocytes were generated in mixed lymphocyte cultures after 5 days of allogeneic stimulation in the presence (grey line) or absence (black line) of 20 nM CPEC. The punctured line in (c) depicts the amount of apoptotic P815 cell line target cells after anti-CD3 stimulation without addition of effector cells. One representative experiment of three is shown.

As shown in Fig. 4b, CPEC strongly inhibited the alloreactive cytotoxic capacity. The antigen-independent cytotoxicity against P815 cell line was also diminished (Fig. 4c), indicating a general defect in cytotoxic effector function of the T cells.

The effect of CPEC on regulatory T cells formation

To investigate if the addition of CPEC affects regulatory T cell formation during the allogeneic response, we looked at the presence of different regulatory subsets after 6 days of MLC performed in the presence or absence of CPEC.

Although the total number of CD25⁺CD4⁺ T cells within the divided CFSE^low cell population did not change in the presence of CPEC, the mean fluorescence intensity (MFI) of CD25 staining was significantly higher in CD4⁺ T cells from CPEC-exposed cultures, 1315·4 ± 293·53 in CPEC-treated compared to 779·5 ± 280·99 in CPEC-untreated cultures (Fig. 5a, P = 0·013). Interestingly, when cells were treated with the combination of 20 nM CPEC and 10 ng/ml ofrapamycin, which has been shown to favour the formation of regulatory CD4⁺ T cells [23], the MFI of CD25 staining was even higher, 3446·67 ± 1217·00 compared to 2109·75 ± 611·39, when rapamycin alone was used (P = 0·023).

Fig. 5.

Cyclopentenyl cytosine (CPEC) does not prevent the formation of regulatory CD4⁺ T cells during allogeneic stimulation. (a) The mean fluorescence intensity (MFI) of CD25 in CD25^bright CD4⁺ T cells within divided CD4⁺ T cells after 6 days of mixed lymphocyte culture (MLC) in the presence of CPEC, rapamycin or the combination of CPEC and rapamycin is significantly higher than in the absence of these immunosuppressive drugs. Mean ± standard deviation of five experiments is shown. (b) Forkhead box P3 (FoxP3) expression within divided 5,6-carboxy-succinimidyl-diacetate-fluorescein-ester (CFSE)^low CD4⁺ T cells after 6 days of MLC in the absence or presence of CPEC and rapamycin, a combination of CPEC and rapamycin. CPEC was used at the concentration of 20 nM, rapamycin at a concentration of 10 ng/ml. In the upper right corner of the dot plots the percentage of FoxP3⁺ cells and the MFI of FoxPp3 staining within the divided CFSE^low CD4⁺ T cells are given. One representative experiment of five is shown.

We also looked at FoxP3 expression on alloreactive CD4⁺ T cells. When cells were incubated with CPEC, an increased percentage of CD25^bright FoxP3⁺ cells (15·19 ± 4·65% in CPEC- treated versus 9·84 ± 5·05 in untreated cultures; P = 0·014) was observed. Adding CPEC into the combination with rapamycin further increased the percentage of FoxP3⁺ CD4⁺ T cells up to 27·18 ± 4·5%. An example of FoxP3 staining on alloreactive CD4⁺ T cells is shown in Fig. 5b.

Recently, CD103 was shown to be a marker for alloreactive regulatory CD8⁺ T cells [24]. We observed that CPEC increased the amount of CD103⁺ CD8⁺ alloreactive T cells slightly but consistently from 33·66 ± 10·5% in control cultures to 41·2 ± 10·25% in MLCs in which CPEC was present (P = 0·043). We were also interested in the combined effect of CPEC and rapamycin, because the latter has been shown recently to induce CD103 expression on alloreactive CD8⁺ T cells [25]. Indeed, when used in combination with CPEC, rapamycin increased significantly both the percentage of CD103⁺ CD8⁺ T cells and the MFI of CD103 expression compared to cultures to which rapamycin alone was added. Interestingly, when used in combination with 100 ng/ml prednisolone, the drug which has been shown to exert a negative effect on the development of alloreactive CD103⁺ CD8⁺ T cells during the alloresponse [25], CPEC could not overcome this inhibiting effect and did not influence the amount of CD103⁺ CD8⁺ T cells (Fig. 6).

Fig. 6.

Cyclopentenyl cytosine (CPEC) does not prevent formation of alloreactive regulatory CD103⁺ CD8⁺ T cells during allogeneic stimulation. CD103 expression within CD8⁺ T cells after 6 days of mixed lymphocyte culture in the absence or presence of CPEC, rapamycin and prednisolone and combinations of these drugs. CPEC was used in a concentration of 20 nM; rapamycin was used in a concentration of 10 ng/ml; prednisolone was used in a concentration of 100 ng/ml. Dot plots are gated on divided 5,6-carboxy-succinimidyl-diacetate-fluorescein-ester (CFSE)^low CD8⁺ T cells. In the upper right corner of the dot plots the percentage of CD103⁺ cells is given. One representative experiment of five is shown.

Significant immunosuppressive effect of CPEC in a mouse skin transplantation model

To examine the in vivo efficacy of CPEC, we tested it in CBA/CA mice transplanted with B10/Br or BL6 skin graft. B10/Br graft survival was slightly longer in the CBA/CA recipients treated with CPEC compared to the saline-treated group: the median graft survival was 16, 16 and 19 days in the 0·15, 0·5 and 1·5 mg/kg CPEC group, respectively, versus median graft survival of 13 days in the saline-treated group (P< 0·05, Fig. 7a). BL6 skin graft survival in all three CPEC groups was comparable to the control group (Fig. 7b). Drug toxicity in the highest-dose group was cumulative and considerable. In all three groups treated with CPEC mice showed general malaise, weight loss, anaemia and leucopenia. These effects were dose-dependent and most prominent in the group treated with 1·5 kg/mg CPEC. In the group with the highest dose of CPEC, the mice were also found to be suffering from peritonitis and only 50% of the recipients were alive after 3 weeks.

Fig. 7.

Cyclopentenyl cytosine (CPEC) has a mild immunosuppressive effect in CBA/CA mice. (a) Prolonged graft survival in CBA/CA mice grafted with B10/Br donor skin. (b) Graft survival in CBA/CA mice grafted with BL6 donor skin and treated with CPEC is comparable to the control mice. Mice were treated intraperitoneally with either saline (▪), 0·003 mg CPEC (▾), 0·01 mg CPEC (•) or 0·03 mg CPEC (▾) 5 days a week for 3 weeks. Cumulative graft survival curves of eight mice per group are shown.

Discussion

In the present study we show that CPEC in low concentrations strongly inhibited the alloantigen-induced proliferative capacity of human CD4⁺ and CD8⁺ T lymphocytes in vitro. This effect was mediated by a decrease in precursor frequency of alloreactive T cells and also by diminished cell expansion. CPEC also exerted its effect when it was added up to 48 h after initiation of the cultures. This suggests its efficacy in conditions where antigen stimulation has already taken place, as occurs in most clinical situations. Remarkably, the inhibitory effect on alloantigen-induced proliferation was not reversible after the drug has been washed out of the cell cultures. Subsequent activation of responder cells by the original stimulator cells induced hardly any proliferation compared to that in control cultures. This implies that CPEC is able to induce anergy, which is uncommon for a proliferation inhibitor, and has not been recognized for azathioprine or mycophenolate.

CPEC also induced apoptosis of alloreactive T cells, which appeared to be caspase-dependent, as the addition of the pan-caspase inhibitor zVAD-fmk completely reversed the effect. The latter is in agreement with previous data showing that CPEC in a concentration of 100 nM induced caspase-dependent apoptosis of the T lymphoblastic MOLT-3 cell line [7].

Because approximately 25% of both CD4- and CD8-positive alloreactive T cells expressed active caspase-3 in the presence of CPEC during primary MLC, we cannot exclude the possibility that the diminished restoration of proliferative capacity of alloreactive T cells is not only the result of anergy but also the result of apoptosis. Apoptosis becomes especially prominent with higher doses of CPEC. Ford et al. [26] have shown, in an example of MOLT-4 lymphoblast T cell line, that approximately 50% of lymphoblasts became apoptotic when 150 nM CPEC had been present in culture for just 2 h. The same authors showed that after 24 h of incubation with 200 nM CPEC, lymphocyte proliferation was diminished by 80%, and 24 h after washing the drug out of culture none of the cells restored their proliferation.

Although the content of effector molecules perforin and granzyme B by alloreactive T cells was not affected by the presence of CPEC in culture, the effector function of cytotoxic T lymphocytes (CTL) was impaired. The latter was demonstrated not only for allospecific, but also for non-antigen-specific cytotoxic capacity, implicating impairment in functional maturation of cytotoxic effector cells.

The decrease in both proliferative and cytotoxic capacity of alloreactive T lymphocytes by CPEC may be explained not only by its ability to induce apoptosis, but also by its effect on regulatory T cell formation. We found that CPEC did not interfere with and even promoted the induction of regulatory CD4⁺ T cells after allogeneic stimulation. The drug appeared to increase the numbers of CD25^bright FoxP3⁺ CD4⁺ T cells as well as the MFI of CD25 and FoxP3 expression. In contrast, this effect was not observed when cells were stimulated by alloantigen in the presence of other nucleotide synthesis inhibitors, such as 6-mercaptopurine and mycophenolate mofetil (MMF) (data not shown).

The numbers of alloreactive regulatory CD3⁺ CD108⁺ T cells were increased slightly but consistently after the addition of CPEC. The fact that CPEC increased CD103 expression on CD8⁺ divided T cells might also explain partially the decreased T cell cytotoxicity. Previously, we showed that CFSE^low CD8⁺ CD103⁺ T cells, induced during allogeneic stimulation, although positive for granzyme B, are not cytotoxic and, moreover, possess a regulatory suppressive function [24].

As the mTOR (mammalian target of rapamycin)-inhibitor rapamycin has also been shown to increase the numbers of regulatory CD25^bright FoxP3⁺CD4⁺ T cells [23] and CD103⁺ CD8⁺ T cells after allogeneic stimulation [25], we questioned whether the combination of CPEC and rapamycin will have a synergistic effect. Indeed, CPEC seemed to potentiate the action of rapamycin in increasing the formation of both types of regulatory T cells in vitro.

The target of rapamycin is an evolutionary conserved protein. The Saccharomyces cerevisiae yeast TOR1 and TOR2 genes were identified originally as the targets of rapamycin [27]. Subsequently, the structurally and functionally conserved mammalian counterpart of yeast TOR, mTOR, was discovered based on its ability to bind to the FK506 binding protein (FKBP)–rapamycin complex [28,29]. After formation of the rapamycin–FKBP complex, rapamycin acts by inhibition of mTOR activity. One of the lesser-known components of the mTOR signalling network is URA7, or CTP synthetase, the same enzyme whose activity is inhibited by CPEC. Indeed, rapamycin was shown to induce a decrease in transcription of CTP synthetase [30], which may explain partly the synergistic effect of the combination of rapamycin and CPEC on regulatory T cell formation during alloresponse observed in this study.

mTOR is known to induce phosphorylation of two protein kinase C (PKC) isotypes with their subsequent activation. In turn, rapamycin, by blocking mTOR, was found to interfere with phosphorylation of PKC [31]. In vitro studies in S. cerevisiae, where PKC is required for the cell cycle and plays a role in maintaining cell wall integrity, showed that CTP synthethase is also phosphorylated and stimulated by PKC [32].

Based on these literature data we can therefore propose a model of synergistic action of CPEC and rapamycin in anergy induction.

That this pyrimidine–analogue unexpectedly induces anergy and increases the number of regulatory T cells may thus be explained by its role in the mTOR signalling complex.

The mild immunosuppressive effect exerted by CPEC as observed in vivo is comparable to that found for other inhibitors of purine/pyrimidine synthesis, such as azathioprine. Used alone, azathioprine only weakly prolongs graft survival, whereas in combination with other immunosuppressive drugs such as prednisolone or FK506, it exerts a strong effect [33,34]. The ability of CPEC to potentiate the effect of other immunosuppressive drugs in vivo, in particular that of rapamycin, remains to be tested.

In conclusion, our data show that CPEC in low concentrations is a potent inhibitor of both allogeneic-induced expansion and alloreactive cytotoxic capacity of human T cells in vitro. Next to this, it induces low-responsiveness of alloantigen-stimulated T cells and favours the formation of alloantigen-induced regulatory T cells. Regarding the latter, a synergistic effect with the drug rapamycin was shown. In vivo, CPEC induces prolongation of skin graft survival in mouse.