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-to-end 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 T-lymphocytes 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 T-cells 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 T-lymphocytes 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.

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.

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.