Abstract
In vitro generation of mature T cells from human hematopoietic stem and progenitor cells (HSPC) could fulfill two existing needs. First, it could enhance and quicken T cell immune reconstitution after stem cell transplantation, which is very slow and generates a skewed TCR repertoire. Second, by generation of tumour antigen specific T cells it could provide an efficient therapy for numerous malignancies and could enhance GVT effect in the context of allogeneic SCT, without aggravating GVHD. T cells can be generated from human HSPC by culturing them on the murine stromal cell line OP9-transduced with the Notch ligand Delta-like-1 (OP9-DL1). Notch receptor activation is essential for T cell development. However, it is unclear whether Notch activation is sufficient for end maturation into functionally and phenotypically mature TCR positive cells. It was shown that human CD34+ cells cultured on OP9-DL1 differentiate to T cells which can proliferate and produce interferon-g upon polyclonal stimulation. The nature of the mature cells generated in these cultures, however, has not been well studied. CD34+ HSPC from postnatal thymus (PNT) or cord blood were cocultured with OP9-DL1, in the presence of the cytokines Flt-3L (5 ng/ml), SCF (2.5 ng/ml) and IL-7 (5 ng/ml). Every 3–5 days cells were harvested and transferred to fresh OP9-DL1 cells. At repetitive timepoints, an aliquot of the cells was analysed phenotypically. In some experiments, IL-15 was added to the culture. For some experiments, cells harvested from OP9-DL1 at the timepoint mature T cells were observed (usually about d 40 of culture), were transferred to feeder cells, consisting of JY cell line (5.104 cells/ml irradiated with 50 Gy and PBMC (5.105/ml irradiated with 40 Gy), in the presence of PHA (1 mg/ml). After 7 days, IL-2 (50 IU/ml) was added to the culture. Every 14 days, cells were restimulated with new feeders (irradiated JY and PBMC) and new addition of PHA. After 3 weeks of stimulation cells were stimulated overnight with 15 ng/ml PMA and 1500 ng/ml ionomycin, and 18 hours later cells were checked for intracellular presence of cytokines. We investigated whether the T cell population generated in these cultures contains mature cells with the characteristics of TCRγδ cells and of positively selected CD8 or CD4 single positive (SP) TCRαβ cells as observed in the human thymus. We found that under the described conditions, HSPC mature into CD1-CD27+ phenotypically mature T cells, with the TCRγδ fraction maturing faster and more efficiently compared to the TCRαβ fraction. Consistent with a mature phenotype, TCRγδ cells were mostly CD8αα or double negative (DN). No mature CD4 SP TCRαβ cells were observed and the mature CD8 SP cells co-expressed variable ratios of CD8αβ and CD8αα dimers, suggesting that these cells are not conventional positively selected TCRαβ cells. In support of this hypothesis, both mature CD1- TCRαβ and TCRγδ cells expressed the IL2Rβ receptor consitutively and both populations proliferated on IL-15 without prior antigen stimulation, CD8αα (TCRαβ and TCRγδ) cells being the most IL-15 responsive. Mature activated T cells secreted IFN-γ and TNFα, little or no IL-2 and IL-4, with no difference observed between TCRαβ and TCRγδ cells. These data suggest that CD8 TCRαβ cells generated in these cultures are unconventional CD8 cells possibly maturated through agonist selection. However, when cells harvested after 40 days of culture on OP9-DL1 were stimulated with PHA and IL-2 for 3 weeks, conventional appearing CD8αβ cells emerged, with a cytokine production profile similar to that of thymic CD8αβ TCRαβ T cells, with the majority of cells secreting IFN-γ and IL-2. We can conclude from these data that OP9-DL1 supports the development of both unconventional and conventional CD8+ TCRαβ cells, of which the generation and selection process are currently being investigated. Also the in vitro anti-tumor capacities of both populations need to be addressed.
Notch ligand activity is modulated by glycosphingolipid membrane composition in Drosophila melanogaster