Use of IL3 and chromatin-modifying reagents valproic acid and 5-aza-2'-deoxycytidine to affect mobilized peripheral blood CD34+ cell fate decisions.

BACKGROUND AND OBJECTIVES: Culture of blood CD34(+) cells with chromatin-modifying agents can lead to an increase in marrow repopulating cells and in the case of valproic acid increased erythroid cell colony formation. We undertook research to help understand what effects these reagents have on mobilized peripheral blood (MPB) CD34(+) cells. MATERIALS AND METHODS: Mobilized peripheral blood was obtained under informed consent and ethics committee approval from nine patients and allograft donors. Epigenetic modifiers valproic acid and 5-aza-2'-deoxycytidine were used singly or in combination with each other and with IL3 when culturing mobilized peripheral blood CD34(+) cells. Cultured cells were subsequently used in flow cytometry and colony-forming unit assay experiments. RESULTS: Addition of IL3 to the in vitro cell growth medium improved the expansion and maintained the functionality of CD34(+) cells. Valproic acid and IL3 also work synergistically to increase the numbers of CD34(+) /CD36(+) double-positive cells. We found that an apparent increase in red cell colony formation was a result of a decrease in white cell colonies, with no overall increase in red cell colonies when equivalent numbers of CD34(+) cells are plated. CONCLUSIONS: Mobilized peripheral blood CD34(+) stem and progenitor cells are affected by chromatin-modifying agents and IL3 giving higher numbers of CD34(+) /CD36(+) double-positive erythroid progenitors.

Analysis of Wnt pathway genes during ex vivo expansion and neutrophil differentiation of umbilical-cord-blood-derived CD34 cells.

Previous work has shown that optimal ex vivo expansion and differentiation of CD34(+) progenitor cells into neutrophils is by addition of Flt3-L, SCF and G-CSF. Here we report that a variety of genes involved in the WNT pathway are transcriptionally active in both undifferentiated and differentiated umbilical cord blood CD34(+) cells, however statistically significant changes in gene expression are not always consistent across UCB samples.

Blood donor derived dendritic cells and cytotoxic T cells for specific fusion-gene adoptive immunotherapy.

BACKGROUND AND OBJECTIVES: Therapeutic immunological reagents tailored to individual patients have been shown to be a viable treatment strategy for some forms of leukaemia. This work investigates the possibility of using blood donations as a source of leukaemia-specific immune therapeutics. MATERIALS AND METHODS: The acute promyelocytic cell line NB4 carrying the PML-RAR alpha fusion was used as a target for cytotoxic T lymphocytes (CTL) stimulated to recognize the fusion. Stimulation of CTL was by production of dendritic cells pulsed with plasmid vectors containing polymerase chain reaction (PCR)-generated sequences of PML-RAR alpha derived from NB4 cells. PCR primer design included a Kozak consensus sequence to allow correct translation of the nucleic acid into protein. Identification of specific cytotoxicity was by both Granzyme B ELISPOT and by (51)Cr-release assays. RESULTS: Specific CTL activity targeting NB4 cells can be generated from donor-derived peripheral blood mononuclear cells. However, individual donors appear to respond differently to the length of stimulatory sequence encoded in the vector. Use of an internal methionine in the PML gene, which also satisfies the Kozak rules, allows translation in vitro and, thus, might provide a suitable start site for stimulation using acute promyelocytic leukaemia-specific sequence. CONCLUSION: The work presented here suggests that blood donor derived dendritic cells can be used to stimulate leukaemia-specific CTL from the same donation ex vivo. This would enable the generation of patient-specific therapeutics from major histocompatibility (MHC)-matched allogeneic donors. However, different MHC-matched donors might vary in their response depending on the length of the antigenic sequence.