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Florins A. *1, Gillet N. 1, Jean G. 1, Thewis A. 1, Schwartz-Cornil I. 2, Bonneau M. 2, Hay J.B. 3, Kettmann R. *1, Willems L *1. 1 Molecular and Cellular Biology, FUSAGx, Gembloux, Belgium; 2 INRA, Jouy-en-Josas, France; 3 University of Toronto, Toronto, Canada; * are members of the FNRS. Role of the spleen in leukemogenesis induced by bovine leukemia virus In this work, we aimed at characterizing the role of the spleen during infection of sheep by bovine leukemia virus (BLV). Experiments based on CFSE labelling showed that B lymphocytes from infected animals disappear faster from the blood compared to the controls. This difference in dynamics seemed to be mainly due to the B-CD11b subpopulation. These cells are excluded from the lymphatic compartment and migrate preferentially towards the spleen. We thus hypothesized that the spleen plays a major role in the kinetics of B lymphocytes within BLV-infected sheep. In order to study the involvement of the spleen, we analyzed the percentages of various cellular populations (B, B-CD11b, and cells expressing the p24 viral antigen after brief culture) as well as the proviral loads in the splenic artery and vein of 2 infected sheep. No difference was observed amongst all measured parameters. We next performed a kinetic analysis of the B cell population based on the use of intravenous CFSE injection within 4 splenectomized sheep. We observed that the difference in kinetics between infected animals and the controls disappeared after splenectomy. This report thus enlighten a key role exerted by the spleen in the B cell turnover of BLV-infected animals. Figure 1: Study of the PBMCs from jugular vein, splenic artery and splenic vein of two control sheep (4533 and 4534) and two infected animals (4535 and 2672). (A) Percentages of B lymphocytes within the PBMCs population. (B) Percentages of B + /CD11b + in the B lymphocyte population. (C) Percentages of PBMCs expressing p24 viral antigen after 16 hours of culture in complete RPMI medium supplemented with PMA/Ionomycin. (D) Average numbers of viral copies per cell. Figure 2: Dot plots obtained by flow cytometry after PBMC labelling, 5 days after CFSE injection (sheep 3002, infected by BLV). Labelling of the p24 viral protein was performed after 16 hours of culture in complete RPMI medium supplemented with PMA/Ionomycin Figure 3: Percentages of CFSE-positive cells in the T lymphocyte population of four splenectomized sheep (two infected and two controls). (A) T + /CD4 + lymphocytes. (B) T + /CD8 + lymphocytes. (C) T + /γδ + lymphocytes. Figure 4: Percentages of CFSE positive cells in the B lymphocyte population of infected sheep (4535, 4536 and 3002) and controls (4533, 4534, 3004). (A) Short term, non splenectomized. (B) Short term, splenectomized. (C) Long term, non splenectomized. (D) Long term, splenectomized. Figure 5: Percentages of CFSE-positive cells in the B lymphocyte population within the jugular and splenic veins (sheep 4544). Figure 6: Percentages of CFSE-positive cells in the B + /CD11b - lymphocyte population for (A) non splenectomized and (B) splenectomized sheep and among B + /CD11b + lymphocytes for (C) non splenectomized and (D) splenectomized sheep. Non splenectomizedSplenectomized