Modulation of tryptophan catabolism by human leukemic cells results in the conversion of CD25- into CD25+ T regulatory cells.

Indoleamine 2,3-dioxygenase (IDO) is a novel immunosuppressive agent expressed in some subsets of normal and neoplastic cells, including acute myeloid leukemia (AML) cells. Here, we show that IDO expression correlates with increased circulating CD4+CD25+FOXP3+ T cells in patients with AML at diagnosis. In vitro, IDO+ AML cells increase the number of CD4+ CD25+ T cells expressing surface CTLA-4 and FOXP3 mRNA, and this effect is completely abrogated by the IDO inhibitor, 1-methyl tryptophan (1-MT). Purified CD4+CD25+ T cells obtained from coculture with IDO+ AML cells act as T regulatory (T(reg)) cells because they do not proliferate, do not produce interleukin (IL)-2, and inhibit naive T-cell proliferation. Coculture with IDO+AML cells results in the conversion of CD4+CD25- into CD4+CD25+ T cells, which is completely abrogated by 1-MT. Moreover, in mice, intrasplenic injection of IDO+ leukemia/ lymphoma A20 cells induces the expansion of bona fide T(reg) cells by conversion of CD4+CD25- T cells; this effect is counteracted by 1-MT treatment. These data indicate that AML cells induce T-cell tolerance by directly converting CD4+CD25- T cells into CD4+CD25+ T(reg) cells through an IDO-dependent mechanism.

Hepatocyte growth factor favors monocyte differentiation into regulatory interleukin (IL)-10++IL-12low/neg accessory cells with dendritic-cell features.

Several hematopoietic growth factors, including interleukin-10 (IL-10) and transforming growth factor-beta1 (TGF-beta1), promote the differentiation of tolerogenic dendritic cells (DCs). Hepatocyte growth factor (HGF) is a pleiotropic cytokine whose effects on human DC differentiation and function have not been investigated. Monocytes cultured with HGF (HGFMo) differentiated into accessory cells with DC-like morphology, released low amounts of IL-12p70 and up-regulated IL-10 both at the mRNA and at the protein level. Upon activation with HGFMo, allogeneic CD4+CD25- T cells expressed the T regulatory (Treg)-associated transcription factor FoxP3, proliferated poorly, and released high levels of IL-10. Interestingly, blockade of surface immunoglobulin-like transcript 3 (ILT3) on HGFMo or neutralization of secreted IL-10 translated into partial restoration of T-cell proliferation. Secondary stimulation of HGFMo-primed CD4+ T cells with immunogenic DCs differentiated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 from monocytes of the same donor resulted in measurable T-cell proliferation. HGFMo-primed CD4+ T cells significantly inhibited the proliferation of naive CD4+CD25- T cells in a cell-contact-dependent manner. Finally, DNA microarray analysis revealed a unique gene-expression profile of HGF-activated monocytes. Collectively, our findings point to a novel role for HGF in the regulation of monocyte/DC functions that might be exploited therapeutically.

Interleukin-12 production by leukemia-derived dendritic cells counteracts the inhibitory effect of leukemic microenvironment on T cells.

OBJECTIVE: Acute myeloid leukemia (AML) cells are poorly immunogenic and inhibit T-cell function. AML-derived dendritic cells (AML-DCs) have better antigen-presentation capacity than undifferentiated leukemic blasts, but may not be fully competent to stimulate T cells previously inhibited by leukemic cells. MATERIALS AND METHODS: AML-DCs were generated from AML cells and used to stimulate proliferation and cytokine production by T cells previously inhibited by AML cells. AML-DCs were also transfected with interleukin (IL)-12 gene by the nonviral method, nucleofection. RESULTS: Mature AML-DCs stimulated naive and, to a lesser extent, leukemic cell (LC)-cultured T cells more efficiently than their immature counterparts and their activity was mediated by IL-12. AML-DCs generated from CD14(-) AML samples (which represent 80% of total AML patients) were defective in IL-12 production and T-cell activation. Addition of exogenous IL-12 to LC-cultured T cells stimulated by CD14(-)-derived AML-DCs restored optimal interferon-gamma (IFN-gamma) production and Th1 skewing. IL-12 gene-nucleofected AML-DCs derived from CD14(-) cells produced significant amounts of IL-12, maintained leukemia-specific karyotype, DC-like phenotype, and function. When stimulated by IL-12-gene transduced CD14(-)-derived AML-DCs, LC-cultured T cells produced higher concentrations of IFN-gamma, thus maintaining a Th1 cytokine profile. CONCLUSION: IL-12 produced by AML-DCs plays a critical role in counteracting the inhibitory activity of LCs on T-cell function. IL-12 gene can be successfully expressed into AML-DCs defective in endogenous IL-12 production by using a novel nonviral method that does not modify their phenotypical, cytogenetic, and functional features. Genetically modified AML-DCs restore a near normal T-cell function.

Functional and kinetic characterization of granulocyte colony-stimulating factor-primed CD34- human stem cells.

We assessed the functional properties and the kinetic status in vitro, and the engraftment potential in vivo of human haematopoietic stem cells according to the expression of CD34 antigen. Lin-CD34- and Lin-CD34+ cells were isolated from granulocyte colony-stimulating factor-primed peripheral blood (PB) cells of healthy donors. The CD34- cell fraction did not contain either clonogenic cells in semisolid culture or long-term culture initiating cells (LTC-IC). However, stroma-dependent liquid cultures and cytokines induced CD34 expression on a minority of stem cells, acquisition of clonogenic capacity and generation of LTC-IC. Significantly higher percentages of quiescent G0 cells and lower percentages of cycling G1 cells were found in Lin-CD34- cells when compared with Lin-CD34+ cells. Kinetic quiescence of Lin-CD34- cells was associated with a significantly higher expression of the negative regulators of the cell cycle, p27Kip1 and p21(cip1/waf1). Cytokine-mediated induction of CD34, in vitro, resulted in cycling of stem cells and downregulation of p27. There was a higher rate of human long-term engraftment in immunocompromised non-obese diabetic (NOD)/recombination activating gene 1null and NOD/severe combined immunodeficient-beta2microglobulin(null) mice injected with CD34+ cells. Thus, our study indicated that CD34 expression on human PB stem cells was associated with haematopoietic activity, cell-cycle recruitment and downregulation of p27Kip1 in vitro and higher engraftment capacity in vivo.