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T-cell lymphopenia following BM transplantation or diseases such as AIDS result 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(+) BM 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 CD3 epsilon; and have their TCR locus partially D-J rearranged. They could be expanded for over 3 months and used to reconstitute the T-cell compartments of sublethally irradiated T-cell-deficient CD3 epsilon(-/-) mice or lethally irradiated WT mice. Pro-T cells generated in this system could partially correct the T-cell lymphopenia of pre-T-/- mice. However, reconstituted CD3 epsilon(-/-) mice suffered from a wasting disease that was prevented by co-injection of purified CD4(+) CD25(high) WT 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.

A document presenting the methods for FACS and gelatine coating. For FACS, cells isolated from fBM or cBM were resuspended in ice-cold FACS buffer and stained for 30 minutes at 4 °C in the dark with previously optimised concentrations of monoclonal antibodies listed in Additional file 1. The labelled cells were washed twice, filtered through a 40 μm filter, resuspended in FACS sorting buffer at concentrations between 10 × 106 and 20 × 106 cells/ml and sorted using a BD FACSAriaII® sorter fitted with a 70 μm nozzle for fBM and an 85 μm nozzle for cBM derived cell suspensions The BD FACSAriaII® optical configuration and corresponding fluorochomes/dyes used is defined in Additional file 4. SYTOX® blue dead cell stain (Life Technologies) was used for dead cell exclusion and added just prior to the sort (Additional file 2). Cell doublets were excluded based on FSC-A versus FSC-H and fluorescence minus one controls were used to define the position of the gates. Where appropriate, the purity of sorted cell subsets was determined with purities >95 % observed. FACS single cells were seeded on plastic or gelatine-coated 96 well plates. For gelatine coating, 96-well flat-bottom plates were coated with 0.1 % gelatine and incubated for 2 hours at room temperature. Gelatine was removed and the vessels were incubated for 1 hour at 37 °C. Gelatine-coated vessels were used immediately or stored at 4 °C in Dulbecco’s PBS for a maximum of 2 weeks. (DOCX 81 kb)

Journal article The alternative sigma factor sigma(B) of Listeria monocytogenes is responsible for regulating the transcription of many of the genes necessary for adaptation to both food-related stresses and to conditions found within the gastrointestinal tract of the host. The present study sought to investigate the influence of growth phase and temperature on the activation of sigma(B) within populations of L. monocytogenes EGD-e wild-type, Delta sigB, and Delta rsbV throughout growth at both 4 degrees C and 37 degrees C, using a reporter fusion that couples expression of EGFP to the strongly sigma(B)-dependent promoter of lmo2230. A similar sigma(B) activation pattern within the population was observed in wt-egfp at both temperatures, with the highest induction of sigma(B) occurring in the early exponential phase of growth when the fluorescent population rapidly increased, eventually reaching the maximum in early stationary phase. Interestingly, induction of sigma(B) activity was heterogeneous, with only a proportion of the cells in the wt-egfp population being fluorescent above the background autofluorescence level. Moreover, significant RsbV-independent activation of sigma(B) was observed during growth at 4 degrees C. This result suggests that an alternative route to sigma(B) activation exists in the absence of RsbV, a finding that is not explained by the current model for sigma(B) regulation. Science Foundation Ireland Research Frontiers Programme Grant no. 05/RFP/GEN0044; SFI Grant no. SRC 09/SRC/B1794; SFI Stokes Professorship