Maria Z. Siemionow, MD, Phd, Dsci; Yavuz Demir,MD; Abir Mukherjee,MD; Aleksandra Klimczak, Phd

Development and Maintenance of Donor-Specific Chimerism in Semi- Allogenic and Fully MHC Mismatched Facial Allograft Transplants

Maria Z. Siemionow, MD, PhD, Dsci; Yavuz Demir,MD; Abir Mukherjee,MD; Aleksandra Klimczak, PhD

Clinical application of composite tissue allograft transplants opened discussion on the restoration of facial deformities by allotransplantation. Reconstruction of the face, which is the most important aesthetic unit of our body, both the functional and aesthetic outcome is essential. When reconstructing facial deformities secondary to severe burn or trauma injuries, it is possible to obtain optimal coverage but aesthetic outcome is usually unsatisfactory. Therefore, face reconstruction with a facial allograft from the human donor could provide optimal aesthetic and functional restoration. This approach, however is challenging, since skin, which is the major component of the facial flap may generate high immunological response compared to solid organs and other CTA transplants. Recently we have established full-face allograft transplantation model in the rat (1, 2). This model is however, technically demanding and challenging in small animals. Therefore as an experimental basis to study immunological aspects of this new composite tissue allograft, we have developed a hemi-facial transplantation model in semi-allogeneic and fully-allogeneic transplants. Here we introduce hemi-facial allograft transplant model to investigate rationale for development of chimerism across MHC barrier.

Material and Methods: Thirty rats were studied in five groups of six animals each (Table 1). Composite hemi-facial isograft transplantations were performed in group 1. Allograft rejection controls included semi-allogenic transplantations from LBN (RT1l+n) donors to LEW (RT1l) recipients in group 2 and fully-allogenic transplantations from ACI (RT1a) donors in group 3. In allograft treatment groups, recipients of LBN (RT1l+n) donors in group 4 and of ACI (RT1a) donors in group 5 were treated with standard immunosuppressive protocol of CsA monotherapy at dose of 16 mg/kg/day, tapered to 2 mg/kg/day and maintained at this level thereafter. Surgical procedure. Preparation of hemi-facial flap: composite hemi- facial flaps including the neck, face and scalp on the left sides of the donors were elevated. The facial flap consisted of skin, subcutaneous fat tissue, facial muscles, parotid gland and external ear cartilage was elevated, based on the jugular vein and common carotid artery. Preparation of the recipient: The facial skin on left side of the recipient was removed as a full-thickness skin graft of the same dimensions including external ear but sparing the periorbital and perioral skin. The jugular vein and common carotid artery was isolated in the neck for the recipient vessels. Venous anastomosis was performed using standard end-to-side technique between the jugular veins of the donor and recipient. The common carotid artery of the facial allograft was anastomosed to the common carotid artery of the recipient in end-to-side fashion. Signs of graft rejecting were checked on daily basis. Flow cytometry was used to evaluate donor specific chimerism for MHC class I - RT1n and RT1a specific antigens. The effect of immunosuppression of CD4+ and CD8+ T-cell repertoire behavior was evaluated at days 21, 63 and 160. Mixed lymphocyte reaction (MLR) for donor specific tolerance in vitro was tested at day 160 post-transplant. Following H+E staining histological grading of graft rejection was evaluated.

Results: Isograft controls survived indefinitely. All non-treated allografts rejected within 5 to 8 days post-transplant. Long-term survival was achieved in 100% of LBN recipients and in 67% of ACI recipients. Five out of six face transplants (83%) from LBN donors and 2 out of 6 flaps (67%) from ACI donors did not show any signs of rejection at long-term follow-up 400 days and 330 days respectively. The rejection signs were reversed by CsA dose adjustments in remaining allografts. At day 160 donor specific chimerism was achieved in both LBN recipients (10.14% CD4/RT1n, 6.38% CD8/RT1n T- lymphocyte subpopulation and 10.02% CD45RA/RT1n B-lymphocyte, and in ACI recipients (17.54% CD4/RT1a and 9.28% CD8/RT1a) (Fig.1). MLR assay at day 160 post-transplant revealed suppressed response against donor (LBN) antigens in semi-allogenic transplant recipients (group 4) but moderate reactivity to donor (ACI) antigens in fully mismatched transplant recipients (group 5). Skin biopsies taken from animals showing sign of rejection demonstrated Grade II histopathological lesions in LBN and ACI recipients at days 140 and 180 post-transplant.

Conclusion: This study indicated that functional tolerance and better outcome of graft survival can be achieved in semi-allogenic transplants, whereas strong genetic barrier between the donor (ACI) and recipients (Lewis) resulted in moderate responsiveness to the host antigen, however, functional tolerance was still maintained under low dose of CsA monotherapy. Tolerance was associated with presence and maintenance of donor specific multilinege chimerism. Encouraging results achieved in our new hemi-face transplantation model, may serve as a basis for continuation of experimental studies on tolerance induction with varying protocols of immunosuppression, before clinical application of this challenging procedure will be justified.

Table 1. Study design and survival of the hemi-facial flaps

Groups Surgical procedure Treatment Survival (days) Group 1 (n=6) Isotransplantation No Indefinite Group 2 (n=6) Semi-allogenic No 5,6,7,6,7,7, transplantation from LBN donor Group 3 (n=6) Fully-allogenic No 6,8,7,5,6,7 transplantation from ACI donor Group 4 (n=6) Semi-allogenic CsA (Tapered >400†, >373*,>345*, transplantation dose) >331*,>206*,>206* from LBN donor Group 5 (n=6) Fully-allogenic CsA (Tapered >330*,>310*,>306*, transplantation dose) >200†,>198*,196†, from ACI donor

* Face transplants are still alive and under observation † Face transplants showing mild signs of rejection reversed by CsA dosage adjustment and still alive

20 20 18 18 16 16 14 14

12 RT1n/ CD4 12 RT1a/ CD4 10 RT1n/ CD8 10 RT1a/ CD8 8 RT1n/ CD45RA 8 RT1a/ CD45RA 6 6

4 4 2 2 0 0 21 63 160 21 63 160

Time (Days) Time (Days)

Figure 1. The kinetics of donor-specific chimerism in peripheral blood for the presence of MHC class I antigens of the donor RT1n antigens in semi-allogenic (LBN) (left panel) and RT1a antigens in fully allogenic (ACI)(right panel hemi-face allograft transplant recipients, under CsA monotherapy at 21, 63 and 160 days post- transplantation.

References

1.Siemionow, M., Gozel-Ulusal, B., Ulusal, A., Ozmen, S., Izycki, D., Zins, J.E. Functional tolerance following face transplantation in the rat. Transplantation, 75:1607-1609, 2003.

2.Gozel-Ulusal. B., Ulusal, A.E., Ozmen, S., Zins, J.E., Siemionow, M. A new composite facial and scalp transplantation model in the rat. Plast Reconstr Surg, 112: 1302-1311, 2003.