Supportive Therapy with Donor Bone Marrow Cells and Presence of Regulatory
CD4+/CD25+ T-Cell Promotes Face Transplant Survival
Aleksandra Klimczak,PhD, Mehmet Unal,MD, Yavuz Demir,MD, Maria Siemionow, MD, PhD
INTRODUCTION: We have formerly achieved functional tolerance in MHC mismatched rat
facial allotransplantation model under cyclosporine-A (CsA) monotherapy (1). In
this study we tested the effect of supportive therapy with donor bone marrow
transplantation (DBMT) under short-term alpha-betaTCRmAb and CsA 7-day protocol on
development of chimerism, allograft survival, and presence of regulatory T-cells in
face allograft model across MHC barrier.
METHODS: Thirty six hemiface allotransplantation were performed between LBN(RT1l+n)
donors and LEW(RT1l) recipients in 6 groups (6 rats each) using our standard
surgical procedure (2,3). Controls: Group 1 isograft and Group 2 allograft without
treatment, Group 3 received intraosseous DBMT only. Groups 4, 5 and 6 received
alpha-betaTCR/CsA 7-day therapy. Additionally Groups 5 and 6 were augmented with
intraosseous DBMT of 35x106, and 100x106 of bone marrow cells (BMC) respectively.
Before transplantation BMC were stained with PKH dye to evaluate engraftment and
migratory process of donor BMC.
Flow cytometry assessed immunodepletion of T–lymphocytes, donor-specific chimerism
for MHC class I (RT1n) antigens and presence of regulatory T-cells CD4+/CD25+.
Grading of graft rejection was assessed by H+E staining.
RESULTS: Isograft controls survived indefinitely, whereas controls without
immunosuppression rejected transplanted allograft within 5 to 8 days. Group 3
treated with DBMT only accepted transplant up to 13 days. Median survival time
(MST) of facial allografts(Group 4) under alpha-betaTCR/CsA therapy was 35 days. In
both Groups 5 and 6 augmented with donor BMC MST was 48 days. However, the longest
survival time in Groups receiving 35x106 or 100x106 BMC was 465 and 498 days
respectively. In long-term survaivals split tolerance was found as presented by
acceptance of skin and rejection of hair components.
PKH-positive cells of donor origin were present within lymphoid organs and skin of
recipients. In rejected allografts T-cell chimerism declined <1%, and B-cell
chimerism was at 4.5% of CD45RA/RT1n. In contrast, in long-term survival animals Tcell chimerism was 2%-4% (RT1n) during follow-up period and B-cell chimerism reached
12.4% CD45RA/RT1n at day 465 post-transplant. At day 7 post-transplant, regulatory
CD4+/CD25+ T-cells initially were detected at 1.8%, however in long-term survivals
reached 4.0%, whereas rejected allografts reveled 2.2% of CD4+/CD25+ T-cells at the
time of euthanasia.
CONCLUSION: Long-term face allograft survival was achieved under 7-day alpha/betaTCRmAb/CsA protocol augmented with DBMT, without chronic immunosuppression, and was
associated with maintenance of donor T-cell and B-cell chimerism. Increased level
of regulatory T-cells in the peripheral blood of face allograft recipients was
associated with long-term allograft survival (4).
REFERENCES
1. Siemionow M, Demir Y, Mukherjee AL, Klimczak A. Development and maintenance of
donor-specific chimerism in semi-allogenic and fully major histocompatibility
complex mismatched facial allograft transplants. Transplantation 79:558-567, 2005.
2.Gozel-Ulusal B, Ulusal AE, Ozmen S, Zins JE, Siemionow M. A new composite facial
and scalp transplantation model in the rat. Plast Reconstr Surg 112:1302-1311,
2003.
3. Demir Y, Ozmen S, Klimczak A, Mukherjee AL, Siemionow M. Tolerance Induction in
Composite Facial Allograft Transplantation in the Rat Model, Plast Reconst Surg
114: 1790-1801, 2004.
4. Kingsley CI, Karim M, Bushell AR, Wood KJ. CD25+CD4+ regulatory T cells prevent
graft rejection: CTLA-4- and IL-10-dependent immunoregulation of alloresponses. J
Immunol 168:1080-1086, 2002