Regulatory role of Epstein-Barr Virus-Infection and CD40 Ligand Stimulation on the proliferation of Mantle Cell Lymphoma Lines

Z Jin MD^a, N Teramoto MD^a, K Hayashi MD^a, T Oka PhD^a, Y-X Liu MD^a, GS Jin MD^a, K Takada MD^c, K Takahashi MD^b, T Akagi MD^a, T Yoshino MD^a

^aDepartment of Pathology, ^bDepartment of Medical Technology, Faculty of Health Sciences, Graduate School of Medicine & Dentistry; Okayama University, Okayama, Japan, ^cDepartment of Tumor Virology, Institute for Genetic Medicine, Hokka ido University, Sapporo, Japan

AIM: It has been reported that Epstein-Barr virus (EBV) resides in resting B cells in vivo. However, an ideal in vitro system for studying EBV latent infection in vivo has not yet been established. Interaction between CD40L and CD40 is important for B cell proliferation and differentiation. CD40L stimulation has been reported to induce growth arrest and/or proliferation of B cell lines according to their differentiation state. Previous reports examining the effect of stimulation via the CD40 cascade on ex vivo mantle cell lymphoma (MCL) cells have provided conflicting results. Present investigation was intended to clarify the regulatory role of EBV infection and CD40L on the proliferation of MCL cells. METHODS: A MCL cell line, SP53, was successfully infected with a recombinant EBV containing a neomycin-resistant gene. The EBV-carrying SP53 cells were obtained by selection using G418. Two MCL lines without EBV infection, SP49 and SP53, were examined for response to CD40L and/or IL-10. RESULTS: The EBV-carrying SP53 cells expressed EBER-1, EBNAs, and LMP1; this expression pattern of the EBV genes was similar to that in a lymphoblastoid cell line (LCL). However, proliferation assay showed that the EBV-carrying SP53 cells have a doubling time of 73 h, compared with 57 h of SP53 cells. Transplantation of 108 SP53 cells to nude mice formed tumors in 4 of 10 mice inoculated, but the EBV-carrying SP53 cells did not. Unexpectedly, EBV infection reduced the proliferation and tumorigenicity of SP53 cells. However, the EBV-carrying SP53 cells showed higher resistance to apoptosis induced by serum starvation than did the SP53 cells. Co-cultivation with CD40L-expressing mouse fibroblast L cells reduced the BrdU incorporation of SP49 and SP53 cells by half to one third, while BrdU incorporation of control cell lines, including Ramos, BJAB and BALL-1, was not affected or increased. Anti-CD40L antibody blocked the CD40L inhibition of SP49 cell proliferation in a dose-dependent manner in the range from 0 to 20 ng/ml. IL-10 did not affect MCL cell proliferation in the presence or absence of CD40L-expressing cells, while Ramos proliferation was promoted by CD40L and IL-10. CONCLUSIONS: Growth inhibition of MCL cell line by the EBV infection and the CD40L stimulation in vitro strongly suggest that these factors are playing an important role on the regulation of the MCL proliferation in vivo. The inhibition of proliferation and the resistance to apoptosis induced in SP53 cells by EBV infection indicate that SP53 cell line might to some extent provide a model of in vivo EBV reservoir cells.

Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in poster session 791 (Viral infections).