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Florins A 1 , Debacq C 1 , Gillet N 1 , Jean G 1 , Thewis A 1 , Schwartz-Cornil I 2 , Bonneau M 2 , Hay J 3 , Asquith B 4 , Burny A 1 , Kettmann R 1 and Willems L 1 1 Molecular and Cellular Biology, FUSAGx, Gembloux, Belgium; 2 INRA, Jouy-en-Josas, France; 3 Immunology, University of Toronto, Canada; 4 Dept of Immunology, Imperial College of London, United Kingdom Role of the spleen in leukemogenesis induced by bovine leukemia virus Figure 3: Percentages of CFSE positive cells in the B lymphocyte population of infected sheep (4535, 4536 and 3002) and control animals (4533, 4534, 3004). The upper (A and B) and lower (C and D) graphs correspond to percentages measured in short term (5 days) and long term (83 days) time intervals, respectively. Non splenectomized (A and C) and splenectomized (B and D) sheep were analyzed . CONCLUSION: The CFSE kinetics of the B cells is different in infected and control sheep (faster decrease in infected sheep). This difference diappears after splenectomy. Time after CFSE injection (days) Percentage of CFSE-positive cells B+/CD11b- A . D. C. B. Non splenectomized Splenectomized We characterized the role of the spleen during infection of sheep by bovine leukemia virus (BLV). Experiments based on CFSE labeling showed that B lymphocytes from infected animals disappear faster from the blood compartment compared to the controls. Mathematical modeling of these data showed that this process was associated with increased cell death rates occurring in infected sheep. This difference in dynamics seemed mainly to be due to the B-CD11b subpopulation. These cells are indeed excluded from the lymphatic compartment and migrate preferentially through the spleen. To study the involvement of the spleen in the kinetics of BLV-infected B lymphocytes, we analyzed the phenotype and proportion of different cell populations 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 cells based on the use of intravenous CFSE injection within 4 splenectomized sheep, two of them being infected with BLV. Interestingly, in the absence of spleen, the cell death rates were similar in BLV-infected and control sheep. This report thus enlightens a key role exerted by the spleen in the B cell turnover of BLV-infected animals. Non splenectomized Splenectomized 4533 4535 4534 4536 4534 3004 3002 4535 Control Infected d p p = and d ≠ Proliferation and death rates (day –1 ) A. B. Figure 6: (A) Schematic representation of a model describing CFSE labeled cell dynamics. PBMCs are assumed to proliferate at an average rate p, to disappear at an average rate d and to be replaced at an average rate L. On division, the fluorescence intensity (initially J) is assumed to halve. After five divisions, CFSE fluorescence intensity is so low that it falls below the threshold of detection and the cell is considered to be unlabeled. ( B) Graphic representations of the proliferation and death rates estimated from fitting the model to the measured B cells kinetics. CONCLUSION: The estimated death rates of the B cells are higher in infected sheep compared to the controls. This difference in death rates is abrogated by splenectomy. Figure 5: Percentages of CFSE-positive cells in the B lymphocyte population lacking (B + /CD11b - ; upper panels A and B) or expressing (B + /CD11b + ; lower panels C and D) the CD11b marker. Non splenectomized (A and C) and splenectomized (B and D) sheep were analyzed. CONCLUSION: The B+/CD11b+ and B+/CD11b- subpopulations exhibit different CFSE kinetics. This difference disappears after splenectomy . 0 0.05 0.2 0.1 0.15 0 0.05 0.2 0.1 0.15 B+/CD11b+ Non splenectomized Splenectomized 4533 4534 3004 Control 4533 4534 3004 Control 3002 4535 4536 Infected 3002 4535 4536 Infected Short term Long term Percentage of B lymphocytes labelled with CFSE Time after CFSE injection (days) Short term Long term Time after CFSE injection (days) 0 10 20 30 40 50 60 70 80 90 0 1 2 3 4 5 0 10 20 30 40 50 60 70 80 90 0 1 2 3 4 5 A. D. C. B. 0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 4533 4534 3004 Control 4533 4534 3004 Control 3002 4535 4536 Infected 3002 4535 4536 Infected Figure 2: B lymphocytes isolated from BLV-infected sheep 4535 were labelled before and after splenectomy. Analyzes were performed before and at different times after CFSE injection (15 minutes, 2 days and 3 weeks). The dot plots were obtained by flow cytometry after B cell labelling. CONCLUSION: The disappearance kinetics of CFSE-labelled B cells is different before and after splenectomy Before CFSE injection 15 minutes after CFSE injection 2 days after CFSE injection 3 weeks after CFSE injection B labelling (FL2- H) CFSE labelling (FL1-H) Before splenectomy After splenectomy <0.1% 74.3% 31.4% <0.1% 82.4% 69.4% 5.5% 22.0% 100% 25.7% 68.6% 94.5% 88.0% 17.6% 30.6% 100% 0 10 20 30 40 50 60 70 80 90 100 0 0.5 1 1.5 2 2.5 Splenic vein Jugular vein 0 10 20 30 40 50 60 70 80 90 100 0 1 2 Time after CFSE injection (hours) Percentage of B lymphocytes labelled with CFSE Splenic vein Jugular vein Figure 4: CFSE was injected into the jugular vein of a non splenectomized sheep (4544). Blood was collected at different times from the jugular and splenic veins. The percentages of CFSE-positive B cells in the total B lymphocyte population were determined by flow cytometry. CONCLUSION: The percentages of CFSE labelled cells in the splenic and jugular veins equilibrate two hours post CFSE injection Figure 1: PBMCs were isolated from the jugular vein, the splenic artery and the splenic vein of two control sheep (4533 and 4534) and two BLV-infected animals (4535 and 2672); and analyzed by flow cytometry. (A) Percentages of B lymphocytes within the PBMC population. (B) Percentages of B + /CD11b + in the B lymphocyte population. (C) Percentages of PBMCs expressing the p24 viral antigen after 16 hours of culture in complete RPMI medium supplemented with PMA/Ionomycin. (D) Average numbers of viral copies per cell. CONCLUSION: No phenotypic difference was observed in B lymphocyte populations from the splenic artery and the splenic vein. Jugular vein Splenic artery Splenic vein % of B lymphocytes in the PBMC population % of cells expressing p24 in the PBMC population 4533 4534 4535 2672 Control sheep Infected sheep 4533 4534 4535 2672 Control sheep Infected sheep C. 4535 2672 4535 2672 Average numbers of viral copies per cell Jugular vein Splenic artery Splenic vein % of B lymphocytes in the PBMC population % of B-CD11b in the B lymphocyte population % of cells expressing p24 in the PBMC population 4533 4534 4535 2672 Control sheep Infected sheep 4533 4534 4535 2672 Control sheep Infected sheep A. B. D. 4535 2672 4535 2672 Average numbers of viral copies per cell 0 10 20 30 40 50 60 70 1 2 3 4 0 10 20 30 40 50 60 70 80 90 100 1 2 3 4 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1 2 0 5 10 15 20 25 30 1 2 Control Infected p = and d =
