Differential Development Of Donor Bone Marrow-Derived Thymocytes After Allogeneic In Utero Hematopoietic Cell Transplantation In The Murine Model

In Utero Hematopoietic Cell Transplantation (IUHCT) is a promising therapeutic strategy for congenital hematopoietic disorders. While mixed allogeneic hematopoietic chimerism with associated donor specific tolerance is routinely achieved by a predominant mechanism of central deletion, the critical events of donor and host thymocyte development have not been determined. In this study, we utilized the murine model of allogeneic IUHCT to analyze donor and host thymocyte development during postnatal ontogeny.

Bone marrow (BM) cells (10x10⁶) from Foxp3GFP C57/BL6 (B6, H2k^b) mice were injected intravenously into Foxp3GFP Balb/c (H2k^d) fetuses at embryonic day 14 (E14). At indicated postnatal ages the thymocytes were delineated by multi-color flow cytometry. Cell apoptosis and proliferation were determined by Annexin V staining and in vivo BrdU incorporation, respectively.

Our findings demonstrate that the thymic processing of donor BM-derived thymocytes differs significantly from host thymocyte processing and from thymocyte development in normal control mice. While the phenotypic development of host thymocytes remained comparable with that of normal control Balb/c mice, the four major subsets of donor thymocytes showed altered distribution, with significantly higher proportions of immature TCRβ-CD4+CD8- and TCRβ-CD4-CD8+ single positive (SP) cells, a slightly higher proportion of CD4-CD8- (DN) cells, and a dramatically lower proportion of CD4+CD8+ double positive (DP) cells, compared to their host-derived counterparts and B6 controls. The extent of the alteration is directly related to both BM chimerism levels and age. Higher levels of chimerism and/or older age are associated with more profound alterations in donor thymocyte distribution. Moreover, DP cells of donor origin show higher apoptosis and lower proliferation than those of the host. Donor DN cells and TCR gamma/delta cells which do not require positive selection based on MHC recognition are increased compared to the host. Donor T-regulatory cells were present at a higher frequency in the thymus and secondary lymphoid organs of chimeric animals compared to T-regulatory cells in host and normal strain controls.

Our data suggests that in an allogeneic IUHCT system the immune reconstitution of the donor bone marrow-derived thymocytes differs from that of the host cells and that of normal mice. The data supports a mechanism of impaired MHC based positive selection of donor cells by the predominantly host thymic stroma resulting in lack of progression of a higher proportion of donor cells from the DN to DP stage of thymocyte development. In contrast, generation of donor T-regulatory cells was not impaired and appeared to be increased in chimeric animals. The alloreactivity, regulatory suppression, and MHC restriction of the donor derived T cells, and the potential importance of these observations for clinical IUHCT remain to be determined.

No relevant conflicts of interest to declare.