Title: Experimental Model of Vascularized Bone Marrow Transplantation
for Chimerism Induction
Authors: Aleksandra Klimczak, PhD, Galip Agaoglu, MD, Sakir Unal, MD,
Alper Sari, MD, Maria Siemionow, MD,PhD
Purpose: We have achieved a remarkably stable chimerism in the
peripheral blood of limb allograft transplant recipients suggesting
that the presence of certain tissue components (vascularized bone
marrow is an integral part of limb allograft) may facilitate
efficient engraftment of the donor specific cells.1
This study was designed to evaluate induction of chimerism following
vascularized bone marrow transplantation (VBMT) across MHC barriers
under combined  T-cell receptor monoclonal antibody and cyclosporine
A (-TCR/CsA) protocol. The phenotype of bone marrow (BM) cells was
also studied
Method: Eighteen VBM transplants were performed between BN(RT1n)
donors and Lewis (RT1l) recipients in three groups, of six animals
each. Isograft controls - Group I were performed between isogenic
Lewis rats. Rejection controls - Group II without treatment, were
transplanted between BN and LEW rats. Group III allograft recipients
were subjected to 7 day protocol of TCR/CsA therapy.
Transplantation procedure. The vascularized femoral bone was
harvested based on the external iliac vessels. In the recipient the
anastomoses were performed to the femoral artery and vein in end-toend fashion. The inset of the vascularized bone in the inguinal
region of the recipient completed the procedure.
Flow cytometry (FC) was used for evaluation of immunomodulation of Tlymphocytes and donor-specific chimerism for MHC class-I-RT1n antigens
up to day 100. Phenotype of BM cells in grafted and host bones was
assessed. H+E staining assessed bone for changes in architecture and
signs of rejection.
Results: At day 7, isograft controls showed similar number of BM
cells in grafted and host bones (47.5x106 and 43.75x106 cells
respectively) (Fig.1). FC analysis revealed similar level of immature
bone marrow cells (CD90+) in both grafted and recipient bones (38.4%
vs 48.7% respectively). Double staining showed 1.1% of CD90/CD4 Tcells and 11.0% of CD90/CD45RA B-cells in grafted and host bones.
In no treatment Group II after VBMT, 55x106 viable BM cells were
present in the recipient femoral bone, but the number significantly
decreased in vascularized allotransplanted bone and revealed 7x106 of
viable cells (p=0.049)(Fig. 1). The level of CD90+ cells among all
nucleated BM cells in the recipient bone was 38.5% but in donor
femoral grafts was lower and revealed 14.3%.
In -TCR/CsA treatment Group III, total number of viable cells in
grafted and recipient bone was equal and revealed 33.9x106 and
31.6x106 BM cells (Fig. 1), and was significantly higher compared to
grafted bone in no treatment Group II (p= 0.025). The number of CD90+
cells was higher in grafted bone (64.4%) compared to the CD90+ level
in host bone (28.0%). Analysis of BM cell phenotype revealed 1.0% of
CD90/CD4 cells in grafted and host bone. Bone marrow cells with B-
cell phenotype CD90/CD45RA revealed 4.2% in the recipient bone and
10.0% in grafted bone.
The percentage of donor-specific CD90/RT1n cells in host bone was
3.5%-4.0%, indicating migration of donor cells to the recipient bone
marrow compartment. Recipient specific cells CD90/RT1l colonized donor
bone and peak level was observed at day 21 (16.2 - 25.7%) (Fig. 2).
At day 7, in allograft rejection group chimerism level in peripheral
blood was below 1%.
In VBM transplants, under TCR/CsA, significant T- cell depletion was
seen at day 7 (>95%). Multilineage donor-specific chimerism, in
peripheral blood, revealed 37.0% CD4/RT1n and 25.7% CD8/RT1n of Tlymphocytes and 1.6% CD45RA/RT1n of B-lymphocytes.
In isograft control, viable bone marrow cells were seen in grafted
femoral bone during entire follow-up. In contrast, in -TCR/CsA
treatment Group III, viability of bone marrow cells in transplanted
bone declined to 1.7 x 106 cells at day 63 without any changes in the
recipient’s femoral bone. 1.2-1.5% of donor specific cells CD90+/RT1n
were observed in the recipient BM compartment. In peripheral blood
chimerism declined to 1% for both T-cell populations but was stable
for the B-lymphocytes.
H+E staining revealed normal architecture of grafted bone in the
isografts during entire follow-up and up to 21 days in the treatment
group. At day 63 in treatment group fibrotic changes were seen.
Grade-III rejection was assessed in non-treated transplants.
Conclusion: Transplantation of the vascularized bone under 7 day
protocol of immunosuppression provides a substantial source of bone
marrow derived hematopoietic cells within its natural
microenvironment, leading to the development of donor specific
chimerism. Migratory potential of BM cells was confirmed by the
presence of donor specific cells in the recipient bone and recipient
cells in the grafted bone. Interestingly, VBMT was characterized by
over 50 % higher engraftment of the donor specific B-cell lineage.
Total number of bone marrow
cells (x106)
50
45
40
35
30
25
Host bone
20
Graft bone
15
10
5
0
Isograft control Allograft VBMT Allograft VBMT
no treatment
TCR/CsA
Fig 1. Total number of bone marrow cells in grafted and host bone at
day 7 after vascularized bone transplantation revealed high number of
viable cells in grafted bone in transplants under TCR/CsA protocol
and low number of bone marrow cells in allograft transplants without
treatment.
Migratory Potential Between Donor And Recipient Bone Marrow
Compartment
25
20
15
[%]
Host bone RT1n TCR/CsA
10
Graft bone RT1l TCR/CsA
5
0
7
21
63
100
Days After Transplantation
Fig 2. Migratory potential between donor and recipient bone marrow
compartment. Donor cells (RT1n) were present at the same level in the
recipient bone during entire follow-up, whereas presence of host bone
marrow cells in grafted bone was transient.
References:
1. Siemionow, M.Z.,Izycki, D.M., and Zielinski, M. Donor-specific
tolerance in fully major histocompatibility complex-mismatched limb
allograft transplants under an anti- T-cell receptor monoclonal
antibody and Cyclosporine A protocol. Transplantation, 76:1662-1668,
2003.