Azacytidine prevents experimental sclerodermic chronic graft-versus-host disease

Introduction: Graft-versus-host disease (GVHD) remains one major complication of allogeneic hematopoietic stem cell transplantation (HSCT). Following unmanipulated peripheral-blood stem cell transplantation, 60% of the patients experience chronic GVHD while approximately 15% of them develop a sclerodermic form of chronic GVHD characterized by multiple organ fibrosis and loss of skin elasticity. Regulatory T cells (Tregs) play a pivotal role in the pathology of chronic GVHD by inhibiting alloreactive conventional T cells. Several studies have shown the hypomethylating agent Azacytidine (Aza) can demethylate the master transcription factor of Treg (Forkhead box protein 3 factor, FoxP3), thus promoting Treg differentiation of conventional T cells. This work investigates the impact of Aza in a classical murine model of sclerodermic chronic GVHD (B10.D2  BALB/cJ).

Methods: Lethally irradiated BALB/cJ recipient mice were injected with 107 bone marrow cells + 7.107splenocytes from B10.D2 donor mice. Recipients were treated with subcutaneous injections of Aza at the dose of 0,5 or 2 mg/kg every two days from day 10 to 30 following transplantation. Mice GVHD was evaluated with five criteria (weight loss, activity, fibrosis, hair loss and mice posture; 0-1-2 points/criteria). Mice were sacrificed at a score of 8/10 (or > 20% weight loss).

Results: Mice treated with Aza 0.5 mg/kg (n = 14) or 2 mg/kg (n = 17) had significant lower clinical scores compared to control ones (n = 15) after treatment. FACS analysis showed a higher proportion of Treg among CD4+ T cells in the blood of Aza 2 mg/kg mice than in control mice (P = 0.047), as well as a higher percentage of Tregs expressing the KI67 proliferative marker on the same day (P = 0.0005). Finally, analyses of the cellular blood components with Cell-dyn demonstrated that Aza 2 mg/kg treated mice were significantly lymphopenic as compared to control mice (P = 0.05).

Conclusion : Aza prevented sclerodermic GVHD in this classical murine model of chronic GVHD.