Prevention of Murine Sclerodermatous Chronic Graft-Versus-Host Disease by Rapamycin

Background. The most widely used mice model of chronic graft-versus-host disease (cGvHD) is an MHC-matched bone marrow transplantation model of sclerodermatous cGvHD. A limitation of that model is that mortality is relatively low, making difficult to study the impact of potentially therapeutic compounds.

Aims. To develop a more severe model of cGVHD and to assess the impact of Rapamycin administration in that model.

Results. 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. All mice from the severe model (n=8) died a median of 32 days while 3 of 7 mice in the classical model survived beyond day 52. Mean survival was decreased in the severe model compared to the classical model (32 days versus 37 days; p=0.0185). Recipient mice in the severe group experienced higher weight loss, hair loss and skin fibrosis. Numbers of T lymphocytes (251.9 ± 151.4 versus 626.3 ± 532.8; p=0.0004) and CD4⁺ T cells (61.57 ± 41.93 versus 125.0 ± 14.39; p=0.0008) per microliter of blood at day 21 were lower in the severe group than in the classical model. Moreover, number of regulatory T cells (Tregs) was decreased in the severe model (0.9870 ± 0.8864 versus 7.979 ± 6.753; p=0.0062).

We then investigated whether rapamycin administration could prevent GVHD in the severe model. All (n=8) mice treated with PBS (placebo) died a median of 32 days after transplantation, while 6 of 8 mice given 1mg/kg/day i.p. rapamycin survived beyond day 52 (p=0.0012). Number of Tregs/µl was higher at day 21 in rapamycin-treated mice than in mice given PBS (3.532 ± 1.195 versus 1.958 ± 0.8864; p=0.0796). Moreover, number of naïve CD4⁺T (7.798 ± 4.192 versus 25.71 ± 5.185; p= 0.0276), effector memory CD4⁺ T cells (EMT) (40.50 ± 3.180 versus 25.17 ± 7.881; p= 0.0392) and central memory CD4⁺ T cells (CMT) (53.58 ± 3.180 versus 26.29 ± 7.881; p= 0.0060) were higher in rapamycin mice. Finally, proliferation of EMT (assessed by flow cytometry using Ki-67) (34.70% ± 4.084 versus 30.80% ± 2.003; p=0.0221) and CMT (33.25% ± 4.084 versus 27.50% ± 2.003; p=0.0183) was higher in PBS than in rapamycin mice.

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.