Ganciclovir Inhibits Lymphocyte Proliferation by Impairing DNA Synthesis

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1 Biology of Blood and Marrow Transplantation 13: (2007) 2007 American Society for Blood and Marrow Transplantation /07/ $32.00/0 doi: /j.bbmt Ganciclovir Inhibits Lymphocyte Proliferation by Impairing DNA Synthesis Minoo Battiwalla, 1 Yiyuan Wu, 1 Rajinder P.S. Bajwa, 1 Marija Radovic, 1 Nikolaos G. Almyroudis, 1 Brahm H. Segal, 1 Paul K. Wallace, 2 Ryotaro Nakamura, 3 Swaminathan Padmanabhan, 1 Theresa Hahn, 1 Philip L. McCarthy, Jr. 1 1 Department of Medicine and the 2 Department of Pathology and Laboratory Medicine, Roswell Park Cancer Institute, Buffalo, New York; and 3 Division of Hematology/Hematopoietic Cell Transplantation, City of Hope, Duarte, California Correspondence and reprint requests: Minoo Battiwalla, MD, Roswell Park Cancer Institute, Buffalo, NY ( minoo.battiwalla@roswellpark.org). Received December 14, 2006; accepted March 20, 2007 ABSTRACT Cytomegalovirus (CMV) disease-related mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients has dramatically declined because of ganciclovir prophylaxis and preemptive therapeutic strategies. However, ganciclovir has not improved overall survival in randomized studies despite effectively preventing overt CMV disease. Moreover, recurrent posttransplant CMV antigenemia, associated with prolonged ganciclovir exposure, is a predictor of increased relapse of malignancy. We examined the hypothesis that ganciclovir itself may have a negative impact on immune reconstitution by testing the effect of ganciclovir on normal human lymphocytes in vitro. T-lymphocyte activation and proliferation, as measured by PHA-induced 3 H- thymidine uptake, was greatly reduced at therapeutic concentrations of ganciclovir (10 g/ml) but not for foscarnet (300 M/L). Moreover, ganciclovir impaired bromodeoxyuridine incorporation in proliferating lymphocytes, but did not impair lymphocyte survival or induce lymphocyte apoptosis. Collectively, these results show that ganciclovir suppresses T-lymphocyte proliferation in vitro by inhibiting DNA synthesis; with implications for T-lymphocyte function following allogeneic BMT American Society for Blood and Marrow Transplantation KEY WORDS CMV Ganciclovir DNA polymerase INTRODUCTION Cytomegalovirus (CMV) disease has historically been a major contributor to mortality after allogeneic hematopoietic stem cell transplantation (HSCT) [1]. With the introduction of ganciclovir, prophylactic, and preemptive antiviral strategies have greatly reduced the incidence of breakthrough CMV organ disease and mortality to about 5% [2-4]. Despite impressive reduction in CMV-related mortality, several studies have shown that CMV seropositive patients continue to have higher mortality than patient-donor pairs who are both CMV seronegative [5-9]. Reasons cited for this observation include: (1) predisposing graft-versus-host disease (GVHD); (2) antiviral toxicities (myelosuppression with ganciclovir; renal dysfunction with foscarnet and cidofovir); (3) infections related to ganciclovir-induced neutropenia; and (4) CMV itself disabling immune reconstitution. Ganciclovir is a nucleoside analog of guanosine that competitively inhibits the incorporation of dgtp by viral DNA polymerase [10]. However, this effect is only partially specific for the viral polymerase because ganciclovir also inhibits DNA synthesis in hematopoietic progenitors [11]. Although cytopenias are a wellrecognized complication of ganciclovir therapy, the possibility of direct lymphocyte inhibition has escaped attention, perhaps because ganciclovir is usually employed in subjects with a preexisting quantitative or qualitative impairment of cellular immunity. Ganciclovir has been previously shown to inhibit in vitro proliferative responses to cytomegalovirus antigen and to phytohemagglutinin by greater than 50% [5]. The purpose of our study is to define the mecha- 765

2 766 M. Battiwalla et al. nism by which ganciclovir inhibits T cell proliferation. MATERIAL AND METHODS Peripheral Blood Mononuclear Cells (PBMCs) Blood samples were obtained from consenting normal adult volunteers and PBMCs extracted by Ficoll-Paque centrifugation. This study was approved by the Roswell Park Cancer Institute Review Board. Lymphocyte Proliferation Assays Peripheral blood mononuclear cells were suspended in RPMI-CM supplemented with heat-inactivated 10% AB-serum at a concentration of cells/ml. Lymphocytes were stimulated with 10% PHA (T-Stim, BD Biosciences, Bedford, MA), or with 5 L/mL cytomegalovirus lysate (ABI, Columbia, MD), and incubated for 5 days at 37 C with 5% CO 2. Ganciclovir (Roche, Nutley, NJ), foscarnet (Abbot Labs, Chicago, IL) or tacrolimus (Astellas, Tokyo, Japan) were added to achieve final concentrations equal to their peak therapeutic concentrations achieved in vivo (10 g/ml, 300 M/L, and 10 ng/ml, respectively), and concentrations 5-fold lower or 5-fold higher. One hundred microliters of cellular suspension were placed in quadruplicate in wells of a 96-well plate. Lymphocyte proliferation in response to these stimulators was assessed by a standard [ 3 H] Thymidine uptake assay. Experiments were performed on PBMCs collected from 3 different healthy voluntary donors on 2 separate occasions. Survival and Apoptosis The survival of unstimulated PBMCs in the presence or absence of ganciclovir was assessed using an automated cell counter (Hematology System ADVIA120, Bayer). Fresh PBMCs were incubated in RPMI-CM supplemented with 10% AB-serum at 37 C with 5% CO 2 for 4 days. Cells were counted for total and % lymphocytes on days 0, 1, 3, and 4. Apoptosis was assessed using the Raji Lymphoblastic Leukemia cell line and with normal human PBMCs. Raji cells (unstimulated) or PBMCs induced to proliferate with 10% PHA (T-Stim, BD Biosciences), or with 5 L/mL Epstein Barr Virus lysate (ABI), were treated with varying concentrations (25 or 125 g/ml) and durations (12, 24, and 48 hours) of ganciclovir. HD10 antibody (10 mg/ml for 2 hours) was used with the Raji cells as a positive control. Cells were stained with Annexin V-FITC and Propidium Iodide (PI) and analyzed by flow cytometry. (BD Biosciences apoptosis kit). Cell Cycle Analysis Resting or PHA-stimulated PBMCs, were incubated for 5 days. Ganciclovir ( g/ml) was added after days 1, 3, or 4. At the end of a 5-day incubation, cells were pulsed with 20 M of bromodeoxyuridine (BrdU) for 30 minutes. Cell cycle analysis was performed using the BrdU-Flow Kit using manufacturer s instructions (BD-Pharmingen, San Diego, CA). The experiment was repeated using cells from 3 different individuals on separate occasions for confirmation. RESULTS Lymphocyte Proliferation Lymphocyte proliferation experiments demonstrated dose-dependent inhibition of thymidine uptake by ganciclovir in all individuals (Figure 1). However, foscarnet did not inhibit lymphocyte proliferation even at 1500 M/L, 5-fold the therapeutic concentration. Tacrolimus uniformly suppressed proliferative responses at all concentrations (2-50 ng/ ml). The extent of inhibition by GCV when lymphocytes were stimulated with CMV antigen was 59.2% (95% confidence interval [CI], %) (P.001 by the paired t-test) compared with PHA-stimulation 37% (95% CI, %P (P.001 by the paired t-test). The difference between the inhibition of proliferation using CMV antigen and PHA as stimuli was statistically significant. Survival and Apoptosis Assays Ganciclovir had no effect on the survival of unstimulated PBMCs, either total or % lymphocytes at any time point compared to controls. Ganciclovir did not induce apoptosis of Raji cells or of stimulated normal peripheral blood lymphocytes even at 5-fold the therapeutic concentration (Figure 2). Cell Cycle Analysis Bromodeoxyuridine incorporation into PHA-stimulated lymphocytes was greatly reduced by ganciclovir. Reduction of S-phase BrdU incorporation ranged between 56%-64%. The effect was not seen for periods of incubation with ganciclovir for 24 hours or less and was pronounced for ganciclovir exposures greater than 72 hours (Figure 3). DISCUSSION We found that ganciclovir directly inhibited T cell proliferation induced by various stimuli at therapeutic concentrations in a dose dependent fashion as previously reported. We observed that CMV-antigen driven lymphocyte proliferation was inhibited to a greater extent than PHA-induced proliferation. This is probably related to the relative strength of the stimuli; powerful stimuli like PHA can overwhelm the

3 Ganciclovir Inhibits Lymphocyte DNA Synthesis 767 Figure 1. Effect of ganciclovir, foscarnet, and tacrolimus on 3H incorporation in normal human PBMCs stimulated with PHA (A) or CMV antigen (B). Values are medians; error bars represent SEM. The stimulation index (SI) was determined by dividing the average of the experimental results of each quadruplicate by the average of the unstimulated cells on the same plate. moderate immunosuppressive property of ganciclovir. Ganciclovir did not reduce survival of unstimulated PBMCs, nor did it induce apoptosis. We demonstrate that the mechanism by which ganciclovir inhibits T cell proliferation is by inhibition of DNA synthesis. Ganciclovir triphosphate competitively inhibits the incorporation of deoxyguanosine-triphosphate (dgtp) into viral DNA, resulting in the intranuclear accumulation of short DNA fragments [12,13]. Selectivity for viral DNA polymerase relates to the 10- fold higher concentration achieved in CMV-infected cells [14]. The inhibitory effect of ganciclovir on BrdU incorporation demonstrated in the present study can be interpreted in light of the above mechanism as evidence that ganciclovir affects lymphocyte function by impairing DNA synthesis at the therapeutic concentrations achieved in vivo. Ganciclovir is the traditional first-line agent for CMV reactivation, partly because of its familiar and relatively favorable safety profile. However, in a large randomized multicenter trial of 203 patients comparing foscarnet with ganciclovir as preemptive therapy for CMV-reactivation, foscarnet was shown to be equally effective with no greater toxicity [15]. Longterm survival data were not presented in that study. In contrast to ganciclovir, foscarnet directly inhibits DNA polymerase in a noncompetitive manner; selectivity for viral DNA polymerase results from its 100- fold greater inhibitory effect on viral rather than cellular DNA polymerase [16,17]. This finding may explain the neutral effect of foscarnet in the proliferation assay in the present study. The consequences of the immunomodulatory effect of ganciclovir are potentially serious in the HSCT setting. Ultimately, suppression of CMV reactivation requires the expansion of donor-derived CMVspecific T cells from the stem cell graft [18-20]. Prolonged antiviral therapy is known to impair the acquisition of full CMV immunity by suppressing antigen production resulting in late CMV reactivation [21]. Although this effect is not confined to ganciclovir, the inhibition of lymphocyte proliferative responses by ganciclovir could contribute to the late CMV reactivations. Of greater concern is the potential impairment of the graft-versus-leukemia response accounting for the higher incidence of relapse seen in patients with multiple reactivations of CMV [22]. The impact of the immunosuppressive effect of GCV relative to traditional risk factors for disease relapse such as the remission status prior to transplant, extent of immune

4 768 M. Battiwalla et al. Figure 2. Effect of ganciclovir on apoptosis. Raji cell line with media alone, ganciclovir, or HD10 (A,B). Normal human PBMCs either resting or stimulated by PHA or EBV-antigen with varying concentrations of ganciclovir (C). Values are medians; error bars represent SEM. suppression, and the presence of GVHD remains to be conclusively determined. The previously unsuspected impairment of immune reconstitution by ganciclovir may explain another inconsistency in the HSCT literature. Ganciclovir is several-fold more active than acyclovir in vitro and in vivo against CMV. Prophylactic and preemptive ganciclovir have resulted in reduced CMVinfection related mortality, but no benefit on overall survival (OS). However, high-dose acyclovir prophylaxis has led to improved OS, although it is less effective than ganciclovir in suppressing CMV reactivation [23,24]. Although this is an intriguing observation, high-dose acyclovir has not been compared in a randomized fashion to ganciclovir. In conclusion we show that ganciclovir exerts significant inhibitory effects on lymphocyte proliferation by inhibiting DNA synthesis. Further studies are required to determine whether these in vitro effects translate into impaired immune reconstitution in patients. Medications should be carefully evaluated for immunological effects in the clinical practice of HSCT. ACKNOWLEDGMENTS This work was independent of commercial support. Flow cytometry was performed at Roswell Park Cancer Institute s Flow Cytometry Laboratory, which was established in part by equipment grants from the NIH Shared Instrument Program, and receives support from the Core Grant (5 P30 CA ) from the National Cancer Institute to the Roswell Park Cancer Institute. We thank Francisco Hernandez,

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