Abstract
BackgroundThe role of IL-7 and pre-TCR signaling during T cell development has been well characterized in murine but not in human system. We and others have reported that human BM hematopoietic progenitor cells (HPCs) display poor proliferation, inefficient double negative (DN) to double positive (DP) transition and no functional maturation in the in vitro OP9-Delta-like 1 (DL1) culture system.ResultsIn this study, we investigated the importance of optimal IL-7 and pre-TCR signaling during adult human T cell development. Using a modified OP9-DL1 culture ectopically expressing IL-7 and Fms-like tyrosine kinase 3 ligand (Flt3L), we demonstrated enhanced T cell precursor expansion. IL-7 removal at various time points during T cell development promoted a slight increase of DP cells; however, these cells did not differentiate further and underwent cell death. As pre-TCR signaling rescues DN cells from programmed cell death, we treated the culture with anti-CD3 antibody. Upon pre-TCR stimulation, the IL-7 deprived T precursors differentiated into CD3+TCRαβ+DP cells and further matured into functional CD4 T cells, albeit displayed a skewed TCR Vβ repertoire.ConclusionsOur study establishes for the first time a critical control for differentiation and maturation of adult human T cells from HPCs by concomitant regulation of IL-7 and pre-TCR signaling.
Highlights
The role of IL-7 and pre-TCR signaling during T cell development has been well characterized in murine but not in human system
We found that LmDL1-FL7 provided a proliferative advantage to adult bone marrow (BM) CD34+hematopoietic progenitor cells (HPCs) over LmDL1 cell line supplemented with soluble recombinant hIL-7 and hFlt3L
To examine the differentiation and expansion potential of adult human BM CD34+ HPCs co-cultured with LmDL1 exogenously supplemented with recombinant human Fms-like tyrosine kinase 3 ligand (Flt3L) (5 ng/mL) and IL-7 (5 ng/mL), or co-cultured with LmDL1-FL7, we determined the proliferation rate of the incubated cells by counting total number of suspension cells at various time points in three independent experiments
Summary
The role of IL-7 and pre-TCR signaling during T cell development has been well characterized in murine but not in human system. We and others have reported that human BM hematopoietic progenitor cells (HPCs) display poor proliferation, inefficient double negative (DN) to double positive (DP) transition and no functional maturation in the in vitro OP9-Delta-like 1 (DL1) culture system. Generation of mature human T cells from adult bone marrow (BM) CD34+HPCs in vitro may overcome two major limitations in T cell therapy, namely HLA disparity and immune tolerance. Hematopoietic stem cell transplant (HSCT) has been used to reconstitute the immune system in such patients [2]. T cells take the longest time to recover after HSCT [2]. Ex vivo differentiation of T cells using an in vitro OP9 stromal cell line expressing Notch ligand, Delta like-1 (DL1), has been of tremendous interest [3,4,5].
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