Influence of combined treatment of low dose rapamycin and cyclosporin A on corneal allograft survival.

Objective: To analyze the immune modulatory effect of low-dose systemic treatment with rapamycin (Rapa) alone or in combination with cyclosporin A (CsA) in a high-responder corneal allograft model.

Methods: A total of 80 C57BL/6 mice received corneal grafts from BALB/c donors. Recipients were treated with either CsA 3 mg/kg/day or Rapa 0.5 mg/kg/day monotherapy or received combined treatment. Immunomodulatory treatment was started on the day of surgery, and continued for 14 days. The frequency of CD4(+)CD25(+)Foxp3(+) T regulatory cells (Treg) in secondary lymphoid organs was measured by flow cytometry. Development of IFN-gamma producing alloreactive T cells was estimated by Elispot. In addition, corneal samples were subjected to real-time RT-PCR analysis for cytokine transcription.

Results: Monotherapy with Rapa significantly delayed allograft rejection (13.4 +/- 1.34 days, p = 0.03). However, the combination of both, low-dose Rapa and CsA prolonged corneal allograft survival at a significantly higher level (MST = 17.1 +/- 1.37 days, p = 0.0001) than in the control group (MST = 11.2 +/- 1.91 days). Rapa monotherapy increased the frequency of CD4(+)CD25(+)Foxp3(+)Treg in draining lymph nodes, whereas addition of CsA reduced Tregs. Monotherapy with Rapa as well as combined treatment prevented development of IFN-gamma producing alloreactive T cells in spleen. Combined treatment resulted in down-regulation of intragraft CD3, IL-2, IFN-gamma and IL-10 transcription (p = 0.028, p = 0.027, p = 0.028 and p = 0.027 respectively).

Conclusions: Combined treatment with low-dose CsA and Rapa resulted in superior graft survival, and effectively modulated mRNA expression of inflammation and infiltration markers.