T-Regulatory Cells Population and Tolerogenic Factors Gene Expression Patterns in Face Allograft Model Augmented with Donor-Recipient Chimeric Cells Therapy
Joanna Cwykiel, MSc, Fatih Zor, MD, Aleksandra Klimczak, PhD, Maria Siemionow, MD PhD DSc.
Cleveland Clinic, Cleveland, OH, USA.
PURPOSE: Skin is the major unit of most composite face allograft models and is considered as the most antigenic tissue. Face allograft transplantation model supported with donor-recipient chimeric cells therapy, under immunosuppressive protocol for 7 days only was used to determine gene expression of factors (Il-10, Tgfβ1, FoxP3) which play a role in allograft survival. Expression of these factors was correlated with changes in T regulatory cell population.
METHODS: Nineteen hemiface transplantations were carried out in 5 groups. Group 1, 2- isotransplants were performed between Lewis rats (RT1l) with or without immunosuppressive treatment. Hemiface allotransplantations were performed between ACI (RT1a) donors and Lewis (RT1l) recipients. Recipients were treated either with continuous CsA protocol- group 3 or anti- αβTCR/CsA protocol for 7 days only- group 4. Short-term immunosuppressive protocol was augmented with 4x106 in vivo created donor-recipient chimeric cells (DRCC) - group 5. Blood samples and skin biopsies from donor face allograft and adjacent recipient skin were harvested at various post-transplant time points. Gene expression of tolerogenic cytokines (Il-10, Tgfβ1) and factor FoxP3 was evaluated in skin biopsies using Taqman® real-time PCR. Flow cytometry analysis was used to determine changes in T regulatory cell population (CD4/25).
Increased Il-10 gene expression was observed up to 35 days in allograft group under short-term immunosuppressive protocol supported with DRCC therapy- group 5 and in group 3 under continuous CsA protocol. The levels of Tgfβ1 were comparable in all assessed allograft groups.
Continuous up-regulation of FoxP3 gene expression was observed in groups under continuous CsA protocol and under short-term immunosuppressive protocol augmented with DRCC therapy up to 35 days post-transplant.The number of T regulatory cells in blood was found to be significantly increased (22,84%) at early stage post-transplantation only in group 5 supported with DRCC therapy under 7 days immunosupressive protocol of anti- αβTCR/CsA.
CONCLUSION: In skin biopsies, in group supported with DRCC therapy, up-regulated gene expression of Il-10 was correlated with gene expression of FoxP3 and increased number of T regulatory cells in the peripheral blood at day 7 post-transplantation. The longest allograft survival without continuous immunosuppression was observed in group supported by DRCC. These results might indicate protective effect at early stage post-transplantation of DRCC therapy under 7day immunosuppressive protocol.