Tolerogenic Effect of Graft-Derived Dendritic Cells in Facial
Allograft Model
Aleksandra Klimczak, PhD, Galip Agaoglu, MD, Sakir Unal, MD, Maria
Siemionow MD,PhD
INTRODUCTION: Clinical application of composite tissue allograft
transplants opened discussion on the restoration of facial
deformities by allotransplantation. We have established an
experimental full-face and hemiface allograft transplantation model
in the rat (1-3). Hemifacial transplantation model in semi-allogeneic
and fully-allogeneic transplants served as an experimental basis to
study immunological aspects of this new composite tissue allograft.
Recently we have reported operational tolerance in fully MHC
mismatched rat hemifacial allograft model under low dose of
cyclosporine-A (CsA) monotherapy (3-5). The potential of graftderived dendritic cells (DC) on chimerism induction was tested in
hemiface allograft under CsA monotherapy across MHC barrier.
MATERIAL AND METHODS: Twenty-four hemiface transplantations were
performed in 4 groups (6 rats each). Rejection controls included
semi-allogenic LBN(RT1l+n) (Group-1) and fully-allogenic ACI(RT1a)
(Group-2) donors. Group-3 (LBN donors) and Group-4 (ACI donors)
received tapered dose of CsA monotherapy started from 16 mg/kg/day,
tapered to 2 mg/kg/day and maintained at this level thereafter.
Surgical procedure. The composite hemi-facial flaps consisted of
skin, subcutaneous fat tissue, facial muscles, parotid gland and
external ear cartilage and were elevated, based on the jugular vein
and common carotid artery. The facial skin 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.
Next, the common carotid artery and jugular vein of the allograft
were anastomosed to the common carotid artery and jugular vein of the
recipient in end-to-side fashion.
Signs of graft rejection were evaluated on daily basis. At different
time-point at days 7, 28, 63, 100 samples from lymphoid organs
(spleen, lymph nodes, thymus) and blood were harvested. Flow
cytometry monitored donor-specific chimerism for MHC class-I RT1n and
RT1a antigens. Mechanism of allograft acceptance was assessed by the
presence of donor dendritic cells (DDC) using monoclonal antibody for
DC (clone OX-62) in combination with monoclonal antibodies specific
for donor MHC class-I RT1n (clone MCA156)or RT1a (clone C3) antigens
and double immunofluorescence technique. Apoptotic cells were
detected by TUNEL technique.
RESULTS: Face transplants under CsA monotherapy from LBN and ACI
donors displayed presence of passenger leukocytes within lymphoid
organs of recipients. At day 7 post-transplant DDC and donor
leukocytes were detected within spleen and lymph nodes of recipients.
During follow-up, the number of donor-origin DC significantly
increased within spleen but only single cells were present within
lymph nodes. DDC were not detected within thymus. Donor-specific
chimerism in the peripheral blood of recipients at day 100 measured:
LBN recipients at 1.4% for CD4/RT1n, 0.5% for CD8/RT1n and 2.6% for
CD45RA/RT1n; for ACI recipients at 16.8% for CD4/RT1a, 3.7% for
CD8/RT1a and 0.2% for CD45RA/RT1a. Apoptotic cells were detected at
day 7 and during entire follow-up period (100 days) in the lymphoid
organs of recipients.
CONCLUSION: This study indicated that CsA monotherapy promoted T-cell
tolerogenicity of DDC in hemifacial allograft transplants due to
functional stabilization of DDC at the immature state. Migration and
localization of graft-derived DDC into lymphoid organs of recipient
confirmed immunomodulatory function of DDC for skin allograft
acceptance in hemifacial allograft model. Anergy of T cells,
demonstrated by the presence of apoptotic cells, contributed to longterm hemifacial skin allograft survival.
a
b
c
Figure 1. Immunofluorescene staining of the spleen of hemiface
transplant recipient for the presence of a) dendritic cells (red
staining); b) donor-origin cells RT1a positive (green staining) and c)
combination of the staining for dendritic cells and RT1a cells proved
that dendritic cells which are present within spleen of face
transplant recipients are donor origin (yellow staining, arrow).
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