Azusa Kaneko

A method to identify cDNAs based on localization of green fluorescent protein fusion products

We previously established a high-efficiency, retrovirus-mediated expression cloning method. Using this system, we now have developed an expression cloning method (FL-REX; fluorescence localization-based retrovirus-mediated expression cloning) in which cDNAs can be isolated based on the subcellular localization of their protein products. Complementary DNAs generated from mRNA using random hexamers were fused to the cDNA of green fluorescent protein (GFP) in the pMX retrovirus vector. The resulting cDNA-GFP fusion library was transfected into retrovirus-packaging cells, and the derived retroviruses were used to infect NIH 3T3 cells. Infected cells then were screened to identify cDNAs of interest through the subcellular localization of the GFP-fusion products. Using FL-REX, we have identified 25 cDNAs, most of which showed reasonable subcellular localization as GFP-fusion proteins, indicating that FL-REX is useful for identification of proteins that show specific intracellular localization.

Interleukin-7 inhibits pre-T-cell differentiation induced by the pre-T-cell receptor signal and the effect is mimicked by hGM-CSF in hGM-CSF receptor transgenic mice.

We have previously reported that human granulocyte–macrophage colony‐stimulating factor (hGM‐CSF) causes a stage‐specific inhibition of T‐cell receptor (TCR) αβ cell development in the thymus of transgenic mice constitutively expressing the hGM‐CSF receptor. Since it has been reported that the addition of interleukin‐7 (IL‐7) to fetal thymic organ culture (FTOC) has similar effects, we compared the effects of IL‐7 and hGM‐CSF on TCRαβ cell development in hGM‐CSF receptor transgenic mice. We reconstituted fetal lobes with sorted pre‐T, or post pre‐T CD4−CD8− precursor cells. The addition of either IL‐7 or hGM‐CSF to these cultures suppressed further differentiation of pre‐T cells but not post pre‐T cells. At the same time, the cell number was increased, suggesting that pre‐T‐cell proliferation is stimulated by these cytokines. Furthermore, the differentiation of recombination‐activating gene‐1 (RAG‐1)‐deficient pre‐T cells in response to anti‐CD3 antibody stimulation was suppressed by either IL‐7 or hGM‐CSF, suggesting that these cytokines inhibit the pre‐T‐cell receptor (pre‐TCR) signal. This inhibition is unexpected because the pre‐TCR signal and the IL‐7 signal have previously been considered to be co‐operative. Recent analysis of the downstream events of IL‐7 receptor and GM‐CSF receptor revealed that they share common signal transduction molecules. Our results show that IL‐7 is able to promote pre‐T cell proliferation and to suppress differentiation induced by the pre‐TCR signal. GM‐CSF can mimic these biological activities of IL‐7 when the pre‐T cells express GM‐CSF receptors. Our data suggest that both timing and level of activation of the IL‐7 signalling pathway must be precisely regulated to facilitate the differentiation of thymocytes.

Cytokine requirement for the development of T-lymphoid lineage potential in clonal lymphohaematopoietic progenitors in vitro: Development of T Cells from Lymphohaematopoietic Progenitors

The early process of T‐cell development prior to thymic colonization has been poorly investigated because of the lack of a sensitive assay. We have developed a two‐step in vitro culture system by combining a clonal culture with a fetal thymus organ culture (FTOC) and analysed the early development of T cells from lymphohaematopoietic progenitors. Cells of immature colonies derived from bone marrow cells of 5‐fluorouracil (5FU)‐treated mice using various combinations of early acting cytokines were transferred into a FTOC. All the combinations of stem cell factor (SCF), interleukin (IL)‐3 and IL‐6 capable of inducing colony formation supported T‐cell generation. IL‐11 and the Flt3 ligand possessed T‐lineage promotional effects similar to IL‐6 and SCF respectively. However, there were some quantitative differences in the final T‐cell yield among cytokine combinations. Thus, the commitment towards T lineage in lymphohaematopoietic progenitors may be an event determined intrinsically rather than induced by specific stimuli, but there may be a hierarchy between the activity of cytokines in further development. Furthermore, we examined the T‐lineage potential of individual colonies derived from Lin−c‐Kit+Sca‐1+ cells clone‐sorted from post‐5FU marrow cells. No colonies that contained only myelocytic progenitors showed T‐lineage potential, but 23·3% of colonies with a haematopoietic multipotentiality did. Therefore, the divergence of the T lineage from other lineages such as myeloid potential may occur at an early stage of the hierarchy of haematopoiesis. The proposed method should prove valuable for exploring the molecular and cellular changes that occur during early T‐cell development before thymic colonization.