Development and Maintenance of Donor-Specific Chimerism in SemiAllogenic 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 hemifacial 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 Tlymphocyte 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
Group 1 (n=6)
Group 2 (n=6)
Group 3 (n=6)
Group 4 (n=6)
Surgical procedure
Isotransplantation
Semi-allogenic
transplantation
from LBN donor
Fully-allogenic
transplantation
from ACI donor
Semi-allogenic
Treatment
No
No
Survival (days)
Indefinite
5,6,7,6,7,7,
No
6,8,7,5,6,7
CsA (Tapered
>400†, >373*,>345*,
Group 5 (n=6)
transplantation
from LBN donor
Fully-allogenic
transplantation
from ACI donor
dose)
>331*,>206*,>206*
CsA (Tapered
dose)
>330*,>310*,>306*,
>200†,>198*,196†,
* Face transplants are still alive and under observation
†
20
20
18
18
16
16
14
12
RT1n/CD4
10
RT1n/CD8
8
RT1n/CD45RA
6
Chimerism Level [%]
Chimerism Level [%]
Face transplants showing mild signs of rejection reversed by CsA dosage adjustment
and still alive
14
12
RT1a/CD4
10
RT1a/CD8
8
4
4
2
2
0
RT1a/CD45RA
6
0
21
63
Time (Days)
160
21
63
160
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 posttransplantation.
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.