The immunosuppressive macrolide RAD inhibits growth of human Epstein-Barr virus-transformed B lymphocytes in vitro and in vivo: A potential approach to prevention and treatment of posttransplant lymphoproliferative disorders

Abstract

Whereas the standard immunosuppressive agents foster development of posttransplant lymphoproliferative disorders (PTLDs), the impact of RAD, a macrolide with potent immunosuppressive properties, and other immunosuppressive macrolides on these disorders remains undetermined. We found that RAD had a profound inhibitory effect on in vitro growth of six different PTLD-like Epstein-Barr virus+ lymphoblastoid B cell lines. Similar to normal T cells, RAD blocked cell-cycle progression in PTLD-like B cells in the early (G(0)/G(1)) phase. Furthermore, RAD increased the apoptotic rate in such cells. The drug also had a profound inhibitory effect on the growth of PTLD-like Epstein-Barr virus+ B cells xenotransplanted s.c. into SCID mice. The degree of the RAD effect varied among the three B cell lines tested and was proportional to its effects on the cell lines in vitro. In this in vivo xenotransplant model, RAD markedly delayed growth or induced regression of the established tumors. In one line, it was able to eradicate the tumor in four of eight mice. When RAD treatment was initiated before tumor cell injection, a marked inhibition of tumor growth was seen in all three lines. In two of them, the drug prevented tumor establishment in approximately 50% of mice (5/11 and 5/8). In summary, RAD is a potent inhibitor of PTLD-like cells in vitro and in vivo. These findings indicate that, in contrast to the standard immunosuppressive agents, macrolides such as RAD may be effective in prevention and treatment of PTLDs.

Figures

Figure 1

RAD-mediated inhibition of in vitro proliferation of PTLD-like EBV+ B cells. BC-1 is an EBV+/HSV8+ B cell line derived from primary effusion lymphoma. The other cell lines are in vitro EBV-transformed B cell lines derived from patients with low-grade B cell lymphoma (15A and 20A), a patient with T cell lymphoma (LCL), or healthy individuals (A1 and A2D6). HTLV-I+ malignant T cell lines HUT-102, C10MJ, and ATL-2 served as controls. The cell lines were pulsed for 18 h with tritiated thymidine (TdR) after 32 h culture with 0–10 nM of RAD.

Figure 2

RAD-mediated inhibition of cell-cycle progression in PTLD-like B cells. Four EBV+ B cell lines were cultured for 48 h with 0–10 nM of RAD, labeled with propidium iodine, and analyzed by flow cytometry.

Figure 3

RAD-mediated increase in apoptotic rate of PTLD-like B cells. Three EBV+ B cell lines were cultured for 24 h with 0–10 nM of RAD, labeled with propidium iodine and anti-annexin V antibody, and analyzed by flow cytometry.

Figure 4

Morphology and phenotype of the 20A cell line, xenotransplanted into a SCID mouse. (A) Hematoxylin-eosin stain showing large cell lymphoma with high mitotic rate. (B) Immunoperoxidase stain with antibody against human CD20 (B cell antigen) showing cell-membrane staining in all lymphoma cells. (C) Immunoperoxidase stain with an antibody against Ki-67 (cell proliferation-related antigen) showing nuclear staining in 50–80%. (D) In situ hybridization for EBV-encoded RNA1 (EBER1) showing nuclear positivity in all lymphoma cells.

Figure 5

RAD-mediated inhibition of in vivo growth of PTLD-like B cells; treatment of established tumors. Fragments of tumors derived from EBV+ B cell lines 15A, 20A, and A2D6 were implanted into recipient SCID mice. Treatment with 5 mg/kg/day of the drug was started when the tumors reached 5 mm in diameter (50 mm3 in volume). The numbers of mice per group are shown in parentheses; lines depicting results from some of the mice are superimposed, particularly in the RAD-treated mice.

Figure 6

RAD-mediated inhibition of in vivo growth of a malignant PTLD-like B cells; prevention of tumor growth. Fragments of tumors derived from EBV+ B cell lines 15A, 20A, and A2D6 were implanted into recipient SCID mice. The treatment with 5 mg/kg/day of the drug was started 3 days before the tumor implantation. The numbers of mice per group are shown in parentheses; lines depicting results from some of the mice are superimposed.