A stromal cell free culture system generates mouse pro‐T cells that can reconstitute T‐cell compartments in vivo

Nadine Gehre, Anja Nusser, Lilly von Muenchow, Roxane Tussiwand, Corinne Engdahl, Giuseppina Capoferri, Nabil Bosco, Rhodri Ceredig, Antonius G. Rolink

T-cell lymphopenia following bone marrow transplantation or diseases such as HIV results in immunodeficiency. Novel approaches to ameliorate this situation are urgently required. Herein, we describe a novel stromal cell-free culture system in which Lineage(-) Sca1(+) c-kit(+) bone marrow hematopoietic progenitors very efficiently differentiate into pro-T cells. This culture system consists of plate-bound Delta-like 4 Notch ligand and the cytokines SCF and IL-7. The pro-T cells developing in these cultures express CD25, CD117 and partially CD44, express cytoplasmic CD3ε and have their TCRβ locus partially D-J rearranged. They could be expanded for over 3 months and used to reconstitute the T-cell compartments of sub-lethally irradiated T-cell-deficient CD3ε(-/-) mice or lethally irradiated wild type mice. Pro-T cells generated in this system could partially correct the T-cell lymphopenia of preTα(-/-) mice. However, reconstituted CD3ε(-/-) mice suffered from a wasting disease that was prevented by co-injection of purified CD4(+) CD25(high) wild type Treg cells. In a T-cell sufficient or T-lymphopenic setting, the development of disease was not observed. Thus, this in vitro culture system represents a powerful tool to generate large numbers of pro-T cells for transplantation and possibly with clinical applications. This article is protected by copyright. All rights reserved.