Abstract
Background: Graft-versus-leukemia (GVL) effect is an essential component in the course of allogeneic stem cell transplantation (SCT). However, both prevention and treatment of established graft-versus-host disease (GVHD), including with drugs such as cyclosporine, can suppress GVL effects. Mycophenolate mofetil (MMF) is becoming a standard of care in SCT recipients for better prevention of GVHD as well as for promoting stem cell engraftment. Aims: To evaluate the effect of MMF, an immunosuppressive drug increasingly used for prevention of GVHD, on disease recurrence following SCT in a preclinical animal model. Since GVL effects may be also induced by alloreactive natural killer (NK) cells, the goal was to investigate the effects of MMF on the activity of lymphokine-activated killer (LAK) cells. Methods: MMF was administered by daily intraperitoneal injection starting at day 1 post-SCT. Cytotoxic LAK activity was measured by 5-h 35S-release assay, and GVL was tested by the appearance of BCL1 leukemia in a semi-mismatched (C57BL/6 donors to [BALB/c × C57BL/6] F1 recipients) murine model. Results: A dosage regimen of 28–200 mg/kg per day MMF had no negative effect on either cytotoxic LAK activity or GVL (as measured by finding of leukemic cells in recipient spleen by PCR or the appearance of clinical leukemia with adoptive transfer). Conclusions: These results suggest that MMF does not impair GVL effects or reduce LAK cell activity in mice.
Keywords: Graft-versus-leukemia, Lymphokine-activated killer cells, Mycophenolate mofetil
Introduction
The aim of allogeneic stem cell transplantation (SCT) induction is to combine tumor cytoreduction with chemoradiotherapy and induce host-versus-graft tolerance by engraftment of donor stem cells. This is followed by induction of graft-versus-leukemia (GVL) effects for elimination of all residual malignant cells by alloreactive immunocompetent donor cells. Graft-versus-host disease (GVHD) is the most ominous side effect of SCT. Prevention as well as treatment of established GVHD consists of various combinations of immunosuppressive and immune-modulating drugs, including steroids, cyclosporine (CSA), tacrolimus, and methotrexate. The use of these drugs can also be associated with severe immunologic failure, thus rendering the patient prone to infection [3] or secondary malignancy, and [17] reduce the efficacy of GVL, or in a broader sense, all of any anticipated graft-versus-tumor (GVT) effect [2, 5, 8, 20, 21].
Mycophenolate mofetil (MMF) (CellCept) is a prodrug of mycophenolic acid (MPA), an inhibitor of inosine monophosphate dehydrogenase (IMPDH). This is the rate-limiting enzyme in de novo synthesis of guanosine nucleotides. T and B lymphocytes are more dependent on this pathway than other cell types are. Moreover, MPA is a fivefold more potent inhibitor of the type II isoform of IMPDH, which is expressed in activated lymphocytes, than of the type I isoform of IMPDH, which is expressed in most cell types. MPA has therefore a more potent cytostatic effect on lymphocytes than on other cell types. This is the principal mechanism by which MPA exerts immunosuppressive effects. Three other mechanisms may also contribute to the efficacy of MPA in preventing allograft rejection and other applications. First, MPA can induce apoptosis of activated T lymphocytes, which may eliminate clones of cells responding to antigenic stimulation. Second, by depleting guanosine nucleotides, MPA suppresses glycosylation and the expression of some adhesion molecules, thereby decreasing the recruitment of lymphocytes and monocytes into sites of inflammation and graft rejection. Third, by depleting guanosine nucleotides, MPA also depletes tetrahydrobiopterin, a cofactor for the inducible form of nitric oxide synthase (iNOS). MPA therefore suppresses the production by iNOS of NO, and consequent tissue damage mediated by peroxynitrite [1]. MMF is generally well tolerated. Its adverse effects include back and abdominal pain, nausea, diarrhea, and hepatitis. In view of its therapeutic effects, MMF is currently being used in solid organ transplantation for rejection prevention [6]. It was also introduced into the GVHD prevention and treatment protocols, usually in combination with other drugs [9]. However, its effect on GVL was never tested. Other drugs that control GVHD, such as CSA and deoxyspergualin, had already been shown to suppress the GVL or GVT effects inducible by allogeneic donor lymphocytes.
To evaluate the influence of MMF on the GVL effect and on the in vitro antitumor alloreactivity of lymphokine-activated killer (LAK) cells, the following experiments were done.
Methods
Animals
Mice 2 to 4 months old, inbred male and female C57BL/6 (B6) and (BALB/c × C57BL/6) F1 (F1), were used as donors and recipients, respectively, purchased from Harlan Breeding Facility (Jerusalem, Israel). Mice were kept under clean (specific pathogen free [SPF]) conditions with autoclaved cages and sawdust. Food and acidified water were supplied ad libitum. All animal protocols were approved by the Institutional Committee for Animal Experimentation.
Treatment with MMF
The intravenous formula of MMF was used. After dilution in dextrose 5% (by weight), MMF was injected intraperitoneally into the animals from day +1 post-SCT in the following dosages: 28, 90, 120, and 200 mg/kg per day.
Preparation of spleen cells for SCT and for adoptive transfer
Spleens were removed aseptically from C57BL/6 mice, teased through a nylon mesh into RPMI 1640 medium (Life Technologies, Grand Island, NY, USA) to create a single cell suspension that was injected into the lateral tail vein of each recipient.
Preparation of LAK cells
Spleen lymphocytes were stimulated with rIL-2 (Chiron, Amsterdam, The Netherlands) by culturing the cells at 6,000 IU/ml at a cell concentration of 2.5–5×106/ml in 75-ml flasks (Nunc, Denmark) in RPMI 1640 medium (Biological Industries, Beit Haemek, Israel). The medium was supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate and nonessential amino acid (Biological Industries, Beit Haemek, Israel), and 10% inactivated bovine calf serum. The cells were cultured for 5 days in a humidified incubator with 5% CO2 in air at 37°C. The cells were harvested with a cell scraper and viability was determined by the trypan blue dye exclusion method.
Cytotoxicity assay
Yac cells (H-2a, NK-sensitive tumor cell line) (3–5×106) cells were incubated overnight with 5 μl 35S-methionine (cat. no. NEG-709A, 185.00 MBq [5.00 mCi] Easytag methionine, L-[35S]-, specific activity 43.48 TBq/mmol [1,175.oCi/mmol]; Perkin Elmer Life Science, Boston, MA, USA) in RPMI medium without methionine. Spleen cells from MMF-treated F1 and normal F1 mice were used for preparation of LAK cells. The cytotoxic activity of the effectors cells from different sources was measured in a 5-h 35S-release assay as previously described [12].
Total body irradiation (TBI)
Recipient mice were placed in radiation chambers on day −1 and exposed to a submyeloablative TBI dose of 700 cGy delivered by a linear accelerator (Varian Clinac 6X) at a dosage rate of 179 cGy/min, at a source-to-skin distance of 80 cm.
Stem cell transplantation
Recipient mice were conditioned with TBI on day −1, and on day 0 mice were transplanted with 20×106 splenocytes. Cells were injected into the lateral tail vein of F1 mice to a total volume of 0.25 ml.
Murine B-cell leukemia (BCL1)
BCL1 was maintained in vivo in BALB/c mice by intravenous passage of 106–107 peripheral blood lymphocytes (PBLs) obtained from tumor-bearing mice [15]. It was previously shown that even a small inoculum of BCL1 may be sufficient to cause typical leukemia in all recipients [15, 16], and all recipients of >10–100 BCL1 cells develop a typical B-cell leukemia/lymphoma characterized by marked splenomegaly, extreme peripheral blood lymphocytosis (up to 200,000/mm3), and death of all tumor-bearing mice.
Peripheral blood lymphocyte counts of all experimental animals were carried out weekly. Assay for presence of BCL1 was also analyzed by PCR at 7 days after transplantation to assess persistence of residual leukemia cells in the spleen, as previously described [13]. To determine whether or not clonogenic BCL1 were still present in the spleen of treated mice, adoptive transfer as described below was done. Onset of leukemia was defined as PBL counts exceeding 20×109/l. At peak of the disease, PBL counts usually reached more than 100×109/l.
PCR for BCL1
Genomic DNA was isolated from 300 μl of fresh whole blood with the Wizard Genomic DNA Purification Kit (Promega). DNA (0.2 μg/sample) was amplified in a 50-μl reaction buffer containing ×1 PCR buffer, 2.25 mM MgCl2, 0.1 mg/ml BSA, 0.2 mM dNTPs, 20 pmol of each primer, using primers with the following sequence: 5′ primer: 5′-ATTGCCATGGGTTGGAGCTCTA-3′; 3′ primer: 3′-AAAGTAGGTACCATAGTATCTTGCA-5′ of the murine B-cell leukemia (BCL1) rearranged Vh region [13]. Samples were vortexed, boiled 5 min, and chilled on ice before adding 1Utaq polymerase (MBI, Fermentas). Thirty-five amplification cycles were performed, each cycle consisting of a denaturation step at 94°C for 1 min, annealing step at 50°C for 1.5 min, and an extension step at 72°C for 2 min. Following these 35 cycles, an additional extension was performed at 72°C for 7 min. PCR products were separated on a 18% agarose gel and stained with ethidium bromide to assess their size.
Experimental design
In vitro studies
Normal F1 mice were treated for 8 days with high-dose i.p. MMF (200 mg/kg per day). A day later, animals were killed, LAK cells were prepared, and cytotoxic activity was tested as described above.
In vivo studies
One day after TBI, F1 animals received intravenous infusion with donor splenocytes (20×106 cells per animal) and BCL1 cells (104 cells per animal). Animals were then divided into the following groups: group a: mice treated posttransplantation with 28 mg/kg per day MMF (MMF 28) (n=16); group b: mice treated posttransplantation with 90 mg/kg per day MMF (MMF 90) (n=16); group c: mice treated posttransplantation with 120 mg/kg per day MMF (MMF 120) (n=5); group d: mice treated posttransplantation with 200 mg/kg per day MMF (MMF 200) (n=6); group e: control mice receiving no posttransplant treatment (SCT control) (n=20); and group f: mice inoculated with leukemia without allogeneic spleen cells, serving as a positive control group (leukemia control) (n=13). Animals were followed for development of leukemia by testing spleen cells for BCL1-specific PCR as detailed above and in an adoptive transfer model as judged by spleen size and PBL count on a weekly basis. Recipients were monitored for clinical signs of GVHD (ruffled fur, diarrhea), measurable weight loss, and survival (mean survival time [MST]) [7].
Adoptive transfer
Untreated BALB/c mice receiving 105 spleen cells obtained from each experimental group were used to determine by adoptive transfer whether or not clonogenic BCL1 were still present in the spleen of treated mice. Recipients were observed for the development of leukemia for at least 100 days. Splenomegaly and peripheral blood lymphocyte counts were monitored to confirm presence of leukemia and to determine if leukemia was indeed the cause of death.
Statistical analysis
Significance was determined using the Kaplan-Meier method, log rank statistic, and analysis of variance with post hoc tests using the Dunnett test. A p value of 0.05 or less was considered to indicate statistical significance.
Results
The effect of MMF on LAK cells activity
After 8 days of 200 mg/kg per day MMF treatment, the cytotoxic activity of the LAK cells generated in vitro, as measured by Y 35S release, did not significantly differ from LAK activity generated from spleen cells obtained from untreated controls (Table 1).
Table 1.
The cytotoxic activity of the effectors cells from MMF-treated mice (1–7) and controls (8–14) as measured in 5-h 35S-percentage released radioactivity assay. No difference was seen between groups with unmodified keeling in spite of MMF treatment
| Y 35S Release | |||
|---|---|---|---|
| 100:1 | 50:1 | 25.1 | |
| 1 | 19.3 | 17.2 | 22.9 |
| 2 | 14.9 | 10.5 | 17.3 |
| 3 | 25.3 | 20.6 | 29.2 |
| 4 | 19 | 17.3 | 23.7 |
| 5 | 19.3 | 13.05 | 9.5 |
| 6 | 6.2 | 10.8 | 8.4 |
| 7 | 11.8 | 7.9 | 12.3 |
| Mean ± SE | 16.5±2.5 | 13.9±2.5 | 17.6±3.2 |
| 8 | 12.3 | 12.8 | 23.4 |
| 9 | 18.7 | 15.9 | 25.4 |
| 10 | 23.2 | 8.7 | 9.9 |
| 11 | 27 | 21.6 | 10 |
| 12 | 3.81 | 10.31 | – |
| 13 | 10.07 | 1.73 | – |
| 14 | 2.84 | 9.6 | 10 |
| Mean ± SE | 14.0±3.8 | 11.5±2.5 | 15.7±3.2 |
The effect of MMF treatment on GVL effects
We have tested the effect of MMF treatment administered in different concentrations on the development of BCL1 leukemia in three separate experiments that showed identical results. None of the MMF-treated animals developed leukemia (regardless of the MMF dose used, n=43) whereas all leukemia control animals (group f, n=13) developed leukemia (p<0.0001). None of the animals in the SCT control group (group e) developed leukemia (n=20, p<0.0001).
PCR study
PCR for detection of minimal residual BCL1 in splenocytes of recipients of different experimental groups demonstrated no signs of minimal residual disease either in MMF-treated (MMF 120 and MMF 200) groups or in mice treated with spleen cells (SCT control). All leukemia control animals featured positive PCR for BCL1 (Fig. 1).
Fig. 1.
PCR of spleens from BCL1-inoculated mice harvested from 120 mg/kg per day MMF (group c, lanes 1 and 2), 200 mg/kg per day MMF (group d, lane 3), SCT control mice (group e, lane 4), leukemia control mice (group f, lanes 5 and 6), and BCL1 cells (lane 7) as a positive control. The arrow identifies the positive PCR bands for BCL1. It can be seen that the PCR is negative for leukemia in both MMF-treated and untreated groups, while positive in the controls.
Adoptive transfer
In two separate experiments, all BALB/c adoptive recipient animals receiving spleen cells from the F1 leukemia control group (group f) developed leukemia, were as only 4/32 of the BALB/c adoptive recipients receiving spleen cells from the MMF-treated F1 group developed leukemia (2/16 with MMF 120 and 2/16 with MMF 200). None of the adoptive recipient BALB/c mice receiving splenocytes from the SCT control mice developed leukemia (Fig. 2).
Fig. 2.
Appearance of BCL1 leukemia in untreated adoptive recipient BALB/c mice receiving 105 spleen cells obtained from each experimental group (i.e., leukemia control, SCT control, SCT+MMF 120 mg/kg, and SCT+MMF 200 mg/kg; n=16 for each group).
Cytokine response to MMF treatment
Mycophenolate mofetil treatment with 120 mg/kg per day (n=4) did not significantly reduce the level of interferon α in the plasma as compared to the SCT group (n=5) (1,618 pg/ml and 1,172.5 pg/ml, respectively; p=0.07). Both were significantly higher then the leukemia control (p<0.001 and p=0.003, respectively). However, treatment with 200 mg/kg per day MMF (n=2) reduced interferon-α plasma level to 225 pg/ml (Fig. 3).
Fig. 3.
IFN-α (mean ± SD) in plasma of F1 mice transplanted with C57BL/6 spleen cells and treated daily with MMF (either 120 mg/kg per day [n=4] or 200 mg/kg per day [n=2]) for 7 days. SCT control (n=5) and leukemia control (n=5) are F1 mice transplanted with C57BL/6 spleen cells plus BCL1 cells or BCL1 cells alone, respectively, and which received no further treatment. MMF120 and SCT group were significantly higher than the leukemia control (p<0.001 and p=0.003, respectively).
The effect of MMF treatment on GVHD
Mycophenolate mofetil had no beneficial therapeutic effect on GVHD. Mean survival time (MST) of mice with GVHD treated with MMF was significantly shorter than in the SCT control (group e) (Fig. 4). There was no significant difference in weight loss between mice treated with MMF (regardless of dosage) and SCT controls (Fig. 5), since MST was shorter in the MMF-treated group.
Fig. 4.
Survival in percentage following induction of GVHD in animals with or without treatment with mycophenolate mofetil (MMF). Death was a result of GVHD. MST of mice with GVHD treated with MMF was significantly shorter than in SCT control. GVHD control control mice receiving no posttransplant treatment (n=20); MMF 28 treated with 28 mg/kg per day MMF (n=16); MMF 90 treated with 90 mg/kg per day MMF (n=16); High dose MMF treated with a combination of 120 and 200 mg/kg per day MMF (n=11).
Fig. 5.
Mean body weight following induction of GVHD in animals with or without treatment with mycophenolate mofetil (MMF). Weight loss is similar for all groups. SCT control control mice receiving no posttransplant treatment (n=8); MMF 28 treated with 28 mg/kg per day MMF (n=8); MMF 90 treated with 90 mg/kg per day MMF (n=8); HD MMF high-dose MMF representing treated with combination of 120 and 200 mg/kg per day MMF (n=11).
Discussion
The main goal of SCT is the establishment of GVL effects for the eradication of minimal residual disease. The use of posttransplant immunosuppressive agents for GVHD prophylaxis and treatment is limited in part by the impairment of the GVL effect as shown in mice [20] and in clinical practice [2, 8]. Thus, it is important to assess whether any new anti-GVHD agent suppresses the GVL effect. Mycophenolate mofetil (CellCept) is a prodrug of mycophenolic acid (MPA), an inhibitor of inosine monophosphate dehydrogenase (IMPDH). In the past, after being tested in solid organ transplantation for prevention of graft rejection, it was introduced to allogeneic stem cell transplantation as a GVHD prophylaxis and treatment agent [10, 19] since GVHD significantly contributes to morbidity and mortality after transplantation. It is used as a third immunosuppressive agent by adding MMF to standard GVHD prophylaxis with methotrexate and CSA [10] or as a methotrexate and methylprednisolone-sparing regimen in a tacrolimus-based protocol [11]. Yet, its effect on GVL was never tested, especially while shown to have no negative effect on tolerance development [4].
In this study we have evaluated the effect of MMF on the in vitro activity of LAK cells produced from spleens of animals treated with a high dose of MMF for 8 days, as well as in vivo evaluation of the GVL effect in a murine model of SCT and leukemia. The model used in this study is of a murine spontaneous, transplantable BALB/c B-cell leukemia. Extreme leukemia and splenomegaly develop in H-2d–compatible recipients of tumor cells. Tumor cells are medium to large lymphocytes. Karyotypic analysis of transformed tumor cells revealed 36 chromosomes with several monosomies and seven marker chromosomes [16]. This spontaneous murine leukemia provides a useful model for the study of various aspects of human bone marrow–derived malignant disorders including the response to immunotherapy. The MMF in this study was administered intraperitoneally, an easier, yet proven method of MMF administration with documented effective systemic immunomodulatory effects [14]. Induction of GVHD/GVL was done after submyeloablative dose of TBI (700 cGy) followed by SCT with C57BL/6 splenocytes. We have found that despite treatment with MMF, the activity of LAK cells was not interfered with (Table 1). It was also found that in spite of the very high dose of MMF used (up to seven times more than the therapeutic dose used in humans), the GVL was not suppressed in all doses tried. Unfortunately, MMF as a single agent, did not suppress GVHD as previously reported in a murine model of GVHD [14]. Therefore, it is not anticipated that the final goal—induction of GVL with no GVHD—could be induced with MMF alone, although it appears that adding MMF for prevention or treatment of GVHD, unlike cyclosporine, is relatively safe, at least from maintaining the GVL effects induced by the allograft’s aspect.
We conclude that our results suggest that MMF does not reduce either GVL effects, which are mediated primarily by T lymphocytes, or the activity of LAK cells. Therefore, the use of MMF for GVHD prevention and treatment may be relatively safe in patients at high risk of relapse. Human clinical correlation of these results is warranted.
Acknowledgments
This work was done at the Danny Cunniff Leukemia Research Laboratory. We wish to thank the Gabrielle Rich Leukemia Research Foundation; the Cancer Treatment Research Foundation; the Novotny Trust; the Szydlowsky Foundation; The Fig Tree Foundation; and the Ronne & Donald Hess and the Silverstein families for their continuous support of our basic and clinical research.
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