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
Results
Conclusion
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.
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 CD4+T,
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).
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.
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.
Methods
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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.
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 D+7 post-transplantation with rapamycin 1 mg/Kg/Day ip.
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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
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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 day+21 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.. PBS
Rapamycin
