Diapositive 1

1
Prevention of murine sclerodermatous chronic
graft-versus-host disease by Rapamycin Belle L,
Binsfeld M, Caers J, Dubois S, Briquet A, Hannon
M, Beguin Y, Humblet S and Baron F Hematolgy
Research Unit, GIGA-I3, University of Liege,
Liege, Belgium
Background
A
B
C
Classical cGvHD
Chronic GvHD occurs up to 50 in long-term
survivors and is the main cause of late mortality
and morbidity after allo-HSCT. Despite its high
frequency, current treatment of sclerodermatous
cGvHD remains unsatisfactory and based on a long
treatment with relatively high-dose
corticosteroids. The most widely used mice model
of chronic graft-versus-host disease (cGvHD) is
an MHC-matched bone marrow transplantation model
of sclerodermatous cGvHD. However, the mortality
is low, making challenging to study the impact of
potentially therapeutic compounds. The aim was to
develop a more severe model of cGVHD and to
assess the impact of Rapamycin administration.
B
A
Severe cGvHD
Methods
Figure 1. Mortality and cGvHD clinical score were
higher and occurred earlier in the severe model.
(A). Survival curves., all mice with a score of x
8 were sacrificied. (B). Clinical scores were
calcultated by evaluating 5 criteria Weight
loss, Posture, Skin Lesions,, Hair loss and
activity (0-1-2 points for each criteria). (C)
Representative pictures
The well-described MHC-matched bone marrow
transplantation model of sclerodermatous cGvHD
use Balb/C and B10.D2 mice. Lethally irradiated
Balb/C mice were injected with 10x106 bone marrow
cells and 70x106 splenocytes from B10.D2 donor
mice. Twenty-one days later, all mice developed
cGvHD. For the severe model, donor B10.D2 mice
were injected with 0.5x106 splenocytes from
Balb/C twenty-one days before transplantation.
Sensitized B10.D2 donor mice were then
sacrificied. B10.D2 splenocytes and bone marrow
cells were then injected into Balb/C recipient
mice. For the experiments where preventive
effects of rapamycin were investigated,
allo-transplanted recipient Balb/C were treated
from D7 post-transplantation with rapamycin 1
mg/Kg/Day ip.  
A
D
C
B
Figure 2. Impact of severe cGvHD on absolute
numbers per microliter of Regulatory T Cells
(FoxP3 CD4 T cells) and CD4 T and CD8 T cell
subpopulation at day21 after allo-HSCT. (A).
Focus on the CD4 T cells (B). Comparison of CD8
T cell numbers in each group (C). Absolute number
of Regulatory T cells (D). Tregs/CD4 of Central
Memory T cells. Median.. Mann Whitney T test..
Results
Figure 3. Rapamycin prevented deaths due to
cGvHD. Seven days after receiving allo-HSCT,
Balb/cJ mice were divided in two groups. One
group receives daily injection of Rapamycin 1
mg/kg/day after day 7, one group with daily
injection of PBS (placebo). (A) Survival for the
severe model. (B) Survival for the classical
model. (C) Representative pictures.
A
C
B
PBS
All mice from the severe model died a median of
32 days while 3 of 11 mice in the classical model
survived beyond day 52. Mean survival was
decreased in the severe model compared to the
classical model (Fig. 1A). Recipient mice in the
severe group experienced higher weight loss, hair
loss and skin fibrosis (Fig. 1B-C). Numbers of
CD8 T lymphocytes and CD4 T cells per
microliter of blood at day 21 were lower in the
severe group than in the classical model.
Moreover, number and frequency of regulatory T
cells (Tregs) was decreased in the severe model
(Fig. 2). We then investigated whether rapamycin
administration could prevent GVHD in the severe
model. All mice treated with PBS died a median of
32 days after transplantation, while 7 of 13 mice
given 1mg/kg/day i.p. rapamycin survived beyond
day 52 (Fig. 3). Number of Tregs/µl was higher at
day 21 in rapamycin-treated mice than in mice
given PBS (Fig. 4A). Moreover, number of naïve
CD4T, effector memory CD4 T cells (EMT) and
central memory CD4 T cells (CMT) were higher in
rapamycin mice. Finally, proliferation of EMT
(assessed by flow cytometry using Ki-67) and CMT
was higher in PBS than in rapamycin mice (Fig.
4B).  
A
Rapamycin
A
B
Figure 4. CD4 T cell subpopulations and Tregs in
blood. Blood was collected from cGvHD mice and a
flow cytometry staining for CD4 T cells was
performed. Cells were labelled with antibodies
conjugated with fluorochromes. Data acquisition
was realized on a FACSCantoII. (A). Focus on the
Tregs cells (B). Central memory CD4 T cells in
each group.
Conclusion
We have developed a mice model of severe cGVHD.
Interestingly, rapamycin prevented death from
cGVHD in that model, perhaps through in vivo
expansion of Treg.