Summary. Blood monocytes and CD341 haemopoietic progenitor cells, as well as certain leukaemic cell lines, acquire characteristics of mature dendritic cells (DC) after stimulation with calcium ionophore (CI). We studied whether the in vitro treatment of primary human acute myelogenous leukaemia (AML) cells with CI leads to differentiation towards DC. Blast cells derived from nine AML patients were cultured in the presence of either CI or an established differentiation cocktail consisting of granulocyte-macrophage colony-stimulating factor plus interleukin 4 and tumour necrosis factor-a for 5±7 d. Microscopic examination revealed that under both conditions, AML cells were shifted along the DC pathway. In seven out of nine cases, CI-cultivation led to a higher proportion of cells with dendritic morphology. The percentage of CD40 and CD86 expressing cells was significantly increased upon CI treatment compared with cytokine-cultured cells. DC molecules as CD80 and CD83 were up-regulated upon calcium mobilization of AML cells in four out of nine samples. In four cases, CI-treated stimulator cells induced an enhanced proliferative allogeneic T-cell response compared with cytokine-treated stimulator cells. In conclusion, these data demonstrate that CI treatment is an alternative in vitro strategy to differentiate human AML cells into DC.
A substantial fraction of the genome is transcribed in a cell-type-specific manner, producing long non-coding RNAs (lncRNAs), rather than protein-coding transcripts. Here, we systematically characterize transcriptional dynamics during hematopoiesis and in hematological malignancies. Our analysis of annotated and de novo assembled lncRNAs showed many are regulated during differentiation and mis-regulated in disease. We assessed lncRNA function via an in vivo RNAi screen in a model of acute myeloid leukemia. This identified several lncRNAs essential for leukemia maintenance, and found that a number act by promoting leukemia stem cell signatures. Leukemia blasts show a myeloid differentiation phenotype when these lncRNAs were depleted, and our data indicates that this effect is mediated via effects on the MYC oncogene. Bone marrow reconstitutions showed that a lncRNA expressed across all progenitors was required for the myeloid lineage, whereas the other leukemia-induced lncRNAs were dispensable in the normal setting.
Staphylococcal superantigens (sAgs), such as toxic shock syndrome toxin 1 (TSST-1), induce massive cytokine production, which may result in toxic shock syndrome (TSS) and sepsis. Recently, we reported that in vitro studies in human peripheral blood mononuclear cells (PBMC) do not reflect the immunological situation of the host, because after exposure to superantigens (sAgs) in vivo, mononuclear cells (MNC) leave the circulation and migrate to organs, e.g., the spleen, liver and lung. Our experimental model of choice is the rabbit because it is comparable to humans in its sensitivity to sAg. T cell activation has been assessed by lymphocyte proliferation and IL-2 gene expression after in vivo challenge with TSST-1 and the mutant antigens; expression of the genes of proinflammatory cytokines were taken as indicators for the inflammatory reaction after the combined treatment with TSST-1 and LPS. The question as to whether the biological activities of TSST-1, e.g., lymphocyte extravasation, toxicity and increased sensitivity to LPS, are mediated by T cell activation or activation by MHC II-only, are unresolved and results are contradictory. We have addressed this question by studying these reactions in vivo, with two TSST-1 mutants: one mutated at the MHC binding site (G31R) with reduced MHC binding with residual activity still present, and the other at the T cell binding site (H135A) with no residual function detectable. Here, we report that the mutant G31R induced all the biological effects of the wild type sAg, while the mutant with non-functional TCR binding did not retain any of the toxic effects, proving the pivotal role of T cells in this system.
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