Poster Board III-462
Recipients of allogeneic hematopoietic transplantation experience a slow reconstitution of donor-derived B and T cell number and function. This post-transplant period of immunodeficiency is associated with an increased risk of infection and malignant relapse. The developement of these complications notably correlates with the recovery of CD4+T cell subset. We proposed a strategy to enhances in vivo reconstitution by promoting donor-derived T cell development in the recipient's thymus. Recently Notch1-based ex-vivo system have been established to mature cord blood- or bone marrow-derived human HSCs into committed T-cell precursors. We used this system for the generation of T-cell precursors starting from G-CSF mobilized human HSCs. We cultured mobilized human CD34+ hematopoietic stem cells (HSCs) (2.5 × 105) in vitro on OP9 mouse stromal cells expressing the Notch 1 ligand Delta-like-1 (OP9-DL1) in the presence of rhFLT3-ligand (5ng/ml) and rhIL7 (5 ng/ml). After 6 weeks of co-culture we obtained a 3 log increase of human T-linage precursors of CD45RA+CD7high phenotype. Further co-colture (7-9 weeks) leed to the generation of CD4+ and CD8+ double-positive (DP) T cells and even mature CD4+ and CD8+ single positive (SP) ab-TCR lymphocytes.
Experiments were designed in order to evaluate whether human CD45RA+CD7high T cell precursors could 1) engraft into NOD-SCID IL2 rg-/− mice 2) leed to in vivo expansion and maturation along T cell developmental pathway.
Control mice were irradiated and transplanted with G-CSF-mobilized human CD34+ (dose 5×106 i.v.). 4 weeks after transplant more than 20% human CD45 positive cells engrafted in the bone marrow. Thymic engraftment occured at 8 weeks after transplant, with 80% human CD45 positive cells (thymic cellularity: 2.7×105 cells), mostly with T cell-immature phenotype of CD3-CD4-CD8 triple negative (95%) (TN) and CD4+CD8+double positive (5%) (DP).
Co-transplant of CD45RA+CD7high T cell precursors (106 cells i.v.) along with CD34+HSC leed to an accelerated thymic engraftment (95% human CD45 positive cells; thymic cellularity 2.5 × 106 cells) already at 6 wks after transplant. Thymocytes were CD3-CD4-CD8 triple negative (51%) (TN) and CD4+CD8+double positive (DP) (42%) cells and at 8 weeks after transplant matured into CD3+CD4+ and CD3+CD8+ single positive (SP) T cells. Spectratyping analyses revealed a broad diversity of the T-cell receptor (TCR) repertoire. This occured in the complete absence of Graft versus Host Disease (GvHD) suggesting that adoptively transferred ex vivo-generated T-cell precursors developed into host-tolerant mature T cells. Ongonig experiment are needed to clarify the beneficial effect of adoptive immunotherapy with human T cell precursors on peripheral T cell reconstitution and control of infection in the humanized mouse system.
We conclude that ex-vivo generation of human T-linage precursors is feasible from the G-CSF-mobilized HSCs and that can be succesfully tranfered in-vivo as a new strategie to enhance T-cell reconstitution after allogeneic HSCT with no risk of GvHD.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.