Combination Of Regulatory T Cells and Rapamycin As Treatment For Experimental Chronic Graft-Versus-Host Disease

Chronic graft-versus-host disease (cGvHD) is one of the main complications of allogeneic hematopoietic cell transplantation (allo-HCT). Donor CD4⁺ conventional T cells (T_conv) as well as regulatory T cells (T_reg) are the key-players in its pathogenesis. Rapamycin (sirolimus, rapa) is a mTor inhibitor that can suppress activation and proliferation of T_conv without inhibiting T_reg.

To assess the impact of a combined treatment with T_regand rapa on experimental cGvHD.

Lethally irradiated BALB/c mice were injected with 10x10⁶ bone marrow cells and 70x10⁶ splenocytes from B10.D2 donor mice. Mice were divided in four groups on day -1 and treatments were started on day 20 with either PBS, rapa 1 mg/kg/Day, T_reg 1.10⁶ cells (D+20), or rapa 1 mg/kg/Day + T_reg 1.10⁶ cells (D+20). T_reg (CD4⁺ CD25⁺ cells) were purified from spleen cells from donor B10.D2 mice using the CD4⁺ CD25⁺ regulatory T cell isolation kit (Miltenyi Biotec, GmbH, Germany). T_reg purity (defined as CD4⁺ FoxP3⁺ cells) was ≥ 70% of total cells and > 92% of CD4⁺T cells.

The severity of sclerodermatous cGvHD was assessed with the following clinical scoring system. Briefly, animals were individually scored every 3 days for five parameters: weight loss (1, 10-20%; 2, > 20%), posture (1, kyphosis only at rest; 2, severe kyphosis when the animal moved), activity (1, moderate activity impairment; 2, no move unless stimulated), skin (1, erythema or scaling tail; 2, open lesion on the body surface) and hair loss (1, > 1 cm²; 2, > 2 cm²). Mice which reached a score of 8 were estimated to have terminal GvHD and were sacrificed. Terminal GvHD-free survivals between the 4 groups were compared using the Log-rank test.

The effects of the treatments were evaluated 7 days after starting the treatments (D+27 post-transplantation). Numbers of total and central memory and effector memory CD4⁺ T cells/µL were significantly (p<0.05) decreased in rapa-, T_reg-, and T_reg + rapa- treated mice compared to PBS mice, while numbers of naïve CD4⁺ T cells/µL were significantly (p<0.05) decreased in T_reg-, and T_reg + rapa- treated mice. Further, proliferation of CD4⁺ T cells (assessed by Ki67 expression) was significantly decreased in rapa- (p<0.05) and T_reg + rapa- treated mice (p<0.05). In addition, counts and proliferation of CD8⁺ T cells/µL were significantly (p<0.05) lower in rapa- and T_reg+ rapa- treated mice compared to PBS mice.

Importantly, terminal-GvHD-free survival was significantly shorter in PBS mice than in T_reg (P=0.03), rapa (P=0.04), and T_reg+ rapa mice (P=0.02).

Our results showed that T_reg and rapa administration improved cGvHD in this model of cGvHD.

No relevant conflicts of interest to declare.