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Role of the spleen in leukemogenesis induced by bovine leukemia virus. 3 weeks after CFSE injection. Before CFSE injection. 15 minutes after CFSE injection. 2 days after CFSE injection. 68.6%. 94.5%. 5.5%. 31.4%. < 0 . 1 %. 25.7%. 7 4. 3 %. 100%. Before splenectomy. - PowerPoint PPT Presentation
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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
1Molecular and Cellular Biology, FUSAGx, Gembloux, Belgium; 2INRA, Jouy-en-Josas, France; 3Immunology, 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)
Perc
enta
ge o
f C
FSE
-pos
itive
cel
ls B+
/CD
11b-
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 ≠
Prol
ifer
atio
n an
d de
ath
rate
s (d
ay –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+
/CD
11b+
Non splenectomized Splenectomized
4533 4534 3004
Control
4533 4534 3004
Control
30024535 4536
Infected
30024535 4536
Infected
Shor
t ter
mL
ong
term
Perc
enta
ge o
f B
lym
phoc
ytes
labe
lled
with
CFS
E
Time after CFSE injection (days)
Shor
t ter
mL
ong
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
30024535 4536
Infected
30024535 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 la
belli
ng (
FL2-
H)
CFSE labelling (FL1-H)
Bef
ore
sple
nect
omy
Aft
er s
plen
ecto
my
<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)
Perc
enta
ge o
f B
lym
phoc
ytes
labe
lled
with
CFS
E
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
% o
f B
lym
phoc
ytes
in
the
PBM
C p
opul
atio
n%
of
cells
exp
ress
ing
p24
in th
e PB
MC
pop
ulat
ion
4533 4534 4535 2672
Control sheep Infected sheep
4533 4534 4535 2672
Control sheep Infected sheep
C.
4535 2672 4535 2672
Ave
rage
num
bers
of
vira
l co
pies
per
cel
l
Jugular vein Splenic artery Splenic vein
% o
f B
lym
phoc
ytes
in
the
PBM
C p
opul
atio
n
% o
f B
-CD
11b
in th
e B
ly
mph
ocyt
e po
pula
tion
% o
f ce
lls e
xpre
ssin
g p2
4 in
the
PBM
C p
opul
atio
n
4533 4534 4535 2672
Control sheep Infected sheep
4533 4534 4535 2672
Control sheep Infected sheep
A. B.
D.
4535 2672 4535 2672
Ave
rage
num
bers
of
vira
l co
pies
per
cel
l
0
10
20
30
40
50
60
70
1 2 3 40
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 20
5
10
15
20
25
30
1 2
Control Infected
p = and d =