Adoptive transfer of in vitro generated T cell precursors improves T cell reconstitution and mediates graft-versus-tumor activity without graft-versus-host disease in allogeneic hematopoietic stem cell transplantation recipients

Abstract

Immunoincompetence after allogeneic hematopoietic stem cell transplantation (HSCT) particularly affects the T cell lineage resulting in significant morbidity and mortality from opportunistic infections. Recent studies have shown that murine T cells and their precursors can be generated from hematopoietic stem cells (HSC) in vitro using OP9 bone marrow stromal cells (H2Kk/H2Kb) expressing the Notch 1 ligand Delta-like 1 and growth factors. In this study we determined the effects of adoptively transferred in vitro generated T cell precursors on T cell reconstitution after allogeneic HSCT. We selected HSC (Lin-Sca-1hi c-kit+) from mouse bone marrow (BM) of various strains and cultured these cells on a monolayer of OP9-DL1 cells in the presence of growth factors. T lineage cell development proved to be strain independent and is therefore not MHC restricted in this culture system. C57BL/6 dervied HSC expanded 2000–5000-fold within 3–4 weeks and consisted of 90–95% CD4-CD8-double negative (DN) T cell precursors after 16–28 days of culture. We infused these cells (8 ×106) with T cell depleted (TCD) BM (5 × 106) or purified HSC into allogeneic recipients using minor antigen mismatched and MHC class I/II mismatched transplant models. Progeny of OP9-DL1 derived T cell precursors were found in the thymus and the periphery significantly improving thymic cellularity, and thymic and splenic donor T cell chimerism. Combination of T cell precursor administration and pre-conditioning with keratinocyte growth factor (KGF) further improved thymic engraftment of OP9-DL1 derived T cell precursors. T cell receptor repertoire and proliferative response to foreign antigen of OP9-DL1 derived mature T cells were intact. Moreover, Th1-type cytokine secretion of OP9-DL1 derived splenic T cells after stimulation with PMA/ionomycin was better than that of BM or HSC derived donor T cells. Progeny of OP9-DL1-derived T cell precursors remained detectable for at least 60 days after transplant with intact cytokine production. Administration of in vitro generated T cell precursors did not induce GVHD but mediated significant graft-versus-tumor (GVT) activity (determined by in vivo bioluminescence imaging) resulting in a subsequent significant survival benefit. We conclude that the adoptive transfer of OP9-DL1 derived T cell precursors significantly enhances donor T cell reconstitution in allogeneic HSCT recipients and results in GVT activity in the absence of GVHD.