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T Regulatory Recruitment To Vascularized Composite Allograft Tissue Using Ccl22 Chemokine: Development Of A Model
Ariel C. Johnson, BS, Bing Li, MD, Charles Owens, BS, Yong Wang, MD, Christene A. Huang, PhD, Nalu Navarro-Alvarez, PhD, An-Jey Su, PhD, Kia Washington, MD, C. Bruce Verchere, PhD, David W. Mathes, MD.
University of Colorado, Aurora, CO, USA.

PURPOSE: Patients who receive vascularized composite allografts (VCA) are required to take a multitude of immunosuppressive drugs to prevent rejection. This is due to the high immunogenicity of the components included in a VCA, particularly skin. We are working to develop a model using the chemokine CCL22 to recruit T regulatory cells to the transplanted tissue. Chemokines are responsible for cell signaling and the homing of cells in the body. Once bound to their respective receptors, they signal cell homing and migration. CCL22 specifically binds to the CCR4 receptor on T-regulatory cells and homes these cells to designated areas to decrease T-effector cell activity. This process has been used in transplant immunology to induce immune tolerance in transplanted tissue using cell vectors expressing CCL22. The known immunomodulatory potential of mesenchymal stem cells (MSCs) within bone marrow makes them an attractive cell vector in vascularized composite allografts. We aim to assess the fate of the locally injected MSCs expressing CCL22, and their ability to recruit T-regs.
METHODS: Bone marrow cells (BMCs) from rat hind limbs were isolated and cultured in a standard fashion. The percentage of MSCs in culture after passage three was evaluated by flow cytometry, based on CD45-, CD54+, and CD90+ surface markers. Cultured cells were then transduced with ds-adenovirus encoding CCL22 and green fluorescent protein (GFP). Once transfection was confirmed by visualization of GFP, cells were injected into non-surgical skin and the wound bed receiving a syngeneic skin graft (n=5). Tissue samples were collected from the skin graft, non-surgical skin with cell injection, and naive skin with no treatment on postoperative days 1 and 5. GFP expression was assessed by paraffin sections and examined under a fluorescent microscope. T-reg cell recruitment was evaluated by immunohistochemistry of internal marker FoxP3.
RESULTS: A total of 73.8% of our cultured BMCs had the characteristic markers of MSCs. Cultured cells were transduced ex vivo with an efficiency of 99%. Unstained paraffin sections of tissue at postoperative days 1 and 5 showed no difference in fluorescence when compared to untreated controls. FoxP3 immunohistochemistry was negative for T-regs in the skin graft tissue and non-surgical tissue with injected cells at postoperative days (PODs) 1 and 5.
CONCLUSION: We were able to transduce bone marrow cells with adenovirus carrying CCl22 and GFP gene sequence as GFP was visible under fluorescent microscope Transfected cells were successfully injected into the rat skin and wound bed of a skin graft. However, no T-regs detected in our tissue samples at PODs 1 and 5. We are unable to state the fate of the MSCs at this time. As such, anti-GFP staining for the detection of the MSCs is currently being optimized. We are also unable to determine if the cells transduced secreted CCL22, as our ELISA results were inconclusive and require further optimization. Thus far, these results have given no conclusive answer to the experimental question. We believe it is essential to determine which step of the model is unsuccessful to tailor the model and make necessary changes.


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