In Vitro Generation of Human T-Cell Precursors From Bone Marrow CD34+ Cells by Short Exposure to Immobilized Notch-Ligand Delta-Like 4.

Abstract 3532

Poster Board III-469

Prolonged posttransplant immune deficiency is a major complication following hematopoietic stem cell transplantation, particularly in the T-lymphoid compartment. Accelerating T-cell development by injecting donor derived T-cell precursors has been proposed as a novel strategy to shorten the immune deficient phase. Several research groups have successfully generated T-cell precursors from murine and human HSC in vitro by transitory exposure to the Notch-ligand presenting murine OP9DL1-cell line. Transfer of the in vitro generated murine T-cell precursors into irradiated NOD/SCID/γcnull-mice accelerated T-cellular reconstitution. However, the clinical application of the OP9DL1-system is limited. Recent studies have demonstrated that short exposure of cord blood CD34+ cells to Notch-ligand Delta-like 4 is sufficient to promote human T-cell differentiation in vitro. Here, we modified this technique to better characterize and ameliorate T-cell development in vitro, with the objective of eventually transferring this method to a clinical phase.

Towards this aim, we exposed human CD34+ HSC derived from any available source to immobilized Notch-ligand Delta-like 4 in the presence of different cytokine combinations implicated in human haematopoiesis (IL-7, SCF, Flt3-ligand and TPO). Within 7 days a population of CD34+CD7+ and CD34-CD7++ T-cell precursors emerged in the presence of Delta-like 4, but not under control conditions. After 7 days the CD34+CD7+ population subsequently declined while further amplification of the CD34-CD7++ population was observed. Two distinct progenitor subsets emerged within the CD34-CD7++ population, namely CD34-CD7++CD5+ and CD34-CD7++CD5-. The CD34-CD7++CD5+ subset further acquired CD1a and, thus, adopted a pre-T-cell phenotype. Between days 7 and 14 the CD34-CD7++CD5- acquired a NK-cell phenotype, as indicated by CD16 and CD56 expression. Beyond 14 days no further expansion of the pre-T-cell fraction was observed, while the NK-cell fraction continued proliferating. More advanced stages T-cell development, such as immature single positive CD4+ cells as observable in OP9DL1 co-cultures, did not arise after exposing cells only to immobilized Delta-like4. Intermittent emergence of a CD13+CD14+CD7- myeloid population was observed within the first 14 days of culture on Delta-like 4; however, this population disappeared spontaneously and did not preserve its common myeloid progenitor.

Selecting a more immature CD34+CD38- population resulted in a two-fold increase of the frequency of CD34+CD7+ and CD34-CD7++ cells as compared to the whole CD34+ population, while myeloid differentiation was inhibited. A further increase was obtained by replanting cultured cells to freshly coated plates with Delta-like 4 every 3 days of culture. T-cell precursors cells derived after 7 days of culture were injected into NOD/SCID/γcnull mice. The in vivo-experiments are ongoing and results are pending.

Our results provide further evidence that human T-cell precursors can be generated in vitro, not only in co-culture with murine OP9DL1-cells but also by short exposure to immobilized Notch-ligand Delta-like 4. These ongoing experiments are an important prerequisite for the potential clinical application of this method.