Purpose: Induction of chimerism and phenotype of bone marrow (BM) cells were assessed following vascularized bone marrow transplantation (VBMT) across MHC barriers under combined ab T-cell receptor monoclonal antibody and cyclosporine A (αβ-TCR mAb/CsA) protocol. Method: Eighteen VBM transplants were performed between BN(RT1ⁿ) donors and Lewis(RTl^l) recipients in three groups, of six animals each. Group I-isograft controls between Lewis rats, group II-allografts rejection controls without treatment and Group III-allografts under 7 day of αβ-TCR/CsA protocol. 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 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). FC analysis revealed similar level of stem 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% 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). The level of CD90+ cells among all nucleated BM cells in the recipient bone was 38.5% but in donor femur grafts was lower and revealed 14.3%. In αβ-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, 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 were evaluated at 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 compartment. Recipient specific cells CD90/RT1^l colonized donor bone and peak level was observed at day 21 (16.2 – 25.7%). At day 7, in allograft rejection group chimerism level in peripheral blood was below 1%. In VBM transplants, under αβ-TCR/CsA, T- cell depletion was significant 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 follow-up. In contrast, in αβ-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 recipient femoral bone. Donor specific cells CD90+/RT1ⁿ were 1.2-1.5% in recipient BM compartment. In peripheral blood the chimerism declined to 1% for both T-cell populations but was stable for B-lymphocytes. H+E staining revealed normal architecture of grafted bone in isograft during follow-up and up to 21 days in treatment group. At day 63 in treatment group fibrotic changes was seen. Grade-III rejection was assessed in non-treated transplants. Conclusion: Vascularized bone marrow allograft transplantation resulted in chimerism induction under αβ-TCR mAb/CsA protocol. Interestingly, VBMT was characterized by over 50 % higher engraftment of donor specific B-cell lineage. 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.
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