Abstract

DO.14.07

Effect of low dose Rapamycin and/or Cylosporin A on regulatory T cells in experimental keratoplasty 

Uwe Pleyer1, Svetlana Stanojlovic1, Stephan Schlickeiser2, Christine Appelt2, Thomas Ritter3, Birgit Sawitzki2
1Charité - Universitätsmedizin Berlin, Augenklinik, 2Institute of Medical Immunology, Charité University Medicine, Berlin, Germany, 3Regenerative Medicine Institute (REMEDI), Galway, Ireland

Objective
The involvement of CD4(+)CD25(+) regulatory T cells (Treg) in immune homeostasis and protection from immune mediated disorders is now well established. Recent experimental studies indicate that commonly used immunosuppressive drugs have detrimental effects on the induction and function of Tregs. Therefore we analyzed the efficiency of systemic treatment with rapamycin (Rapa) alone or in combination with cyclosporin A (CsA) to prevent corneal allograft rejection and investigated their impact on Tregs.
Methods
A total of 70 corneal transplantations were performed using C57BL/6 mice as recipients of BALB/c allografts. Recipients were treated with CsA 3mg/kg/day or Rapa 0.5mg/kg/day monotherapy or with a combination of both Rapa 0.5mg/kg/day plus CsA 3mg/kg/day. Immunomodulatory treatment as intraperitoneal injections were started on the day of corneal transplantation and continued for 14 days. In addition, corneal samples were subjected to real time RT-PCR analysis for cytokine transcription. The frequency of CD4+CD25+Foxp3+ T regulatory cells (Treg) in secondary lymphoid organs were measured by flow cytometry. Memory T cells were estimated by Elispot.
Results
Monotherapy with Rapa moderately delayed allograft rejection (p=0.03).  However, a combination of low dose Rapa and CsA significantly prolonged corneal allografts survival (MST=17.1 ± 1.37 days, p=0.000). Addition of CsA to Rapa resulted in down-regulation of intragraft CD3, IL-2, IFN-γ and IL-10 transcription (p=0.028, p= 0.027, p=0.028 and p= 0.027 respectively). Rapa alone increased the frequency of CD4+CD25+Foxp3+Treg in draining lymph nodes (drLN), whereas addition of CsA reduced the Treg frequency. Monotherapy with Rapa as well as combined treatment prevented development of alloantigen specific IFN-γ producing memory T cells in spleen.
Conclusions
Combined treatment with low dose CsA and Rapa effectively modulates mRNA expression of inflammation and infiltration markers which resulted in significant prolongation of corneal allograft survival. Rapa, but not CsA had an impact on Tregs.
Supported in part by DFG (Pl 150/14-2) and SFB 650 TP14.

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