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Many human myeloid leukemia-derived cell lines possess the ability to acquire a dendritic cell (DC) phenotype. However, cytokine responsiveness is generally poor, requiring direct manipulation of intracellular signaling mechanisms for differentiation. In contrast, the CD34 ؉ human acute myeloid leukemia cell line MUTZ-3 responds to granulocyte macrophagecolony-stimulating factor (GM-CSF), interleukin 4 (IL-4), and tumor necrosis factor alpha (TNF␣), cytokines known to be pivotal both in vivo and in vitro for DC generation from monocytes and CD34 ؉ stem cells. In all respects, MUTZ-3 cells behave as the immortalized equivalent of CD34 ؉ DC precursors. Upon stimulation with specific cytokine cocktails, they acquire a phenotype consistent with either interstitial-or Langerhans-like DCs and upon maturation (mDC), express CD83. MUTZ-3 DC display the full range of functional antigen processing and presentation pathways. These findings demonstrate the unique suitability of MUTZ-3 cells as an unlimited source of CD34 ؉ DC progenitors for the study of cytokineinduced DC differentiation. 3,4 However, the currently defined culture protocols require long expansion periods, given the relative scarcity of blood DC precursors, and involve the use of extensive cytokine cocktails. [5][6][7][8] Therefore, a human cell line exhibiting the characteristics of CD34 ϩ -derived DC precursors would allow for the detailed study of DC differentiation without the associated problems of donor variability and DC precursor cell availability. It has been observed that cell lines derived from tumors of lymphoid or myeloid lineage may also share a potential for differentiation to DC-like APCs, thus providing a ready supply of DC precursors from which DCs can be easily and routinely generated. However, many leukemia cell lines are often refractory to cytokine treatment, 9,10 requiring pharmacologic agents to induce a DC-like phenotype in myeloid cells, bypassing important checkpoints in the differentiation of DCs. 9,10 In contrast, it has been reported that the human cytokine-dependent myeloid cell line MUTZ-3 downregulates CD14 in response to interleukin 4 (IL-4) and low-level granulocyte macrophage-colony-stimulating factor (GM-CSF). 11,12 Here we demonstrate that this cell line is unique in its capacity to acquire a cytokine-induced interstitial and LC iDC phenotype, thus providing a rapid, logistically reproducible model for studies of the immunomodulatory capacity of DCs and such DC-related processes as antigen processing and presentation. Study design Generation of iDC-and mDC-like cells from leukemia cell linesThe cytokine-dependent human cell line MUTZ-3 (Deutsche Sammlung von Mikroorganismen und Zellkulturen [DSMZ], Braunschweig, Germany), and the cytokine-independent human cell lines, HL-60, KG-1, THP-1 U937, and K562 (American Type Culture Collection [ATCC], Manassas, VA) were cultured at 1 ϫ 10 5 /mL (total volume of 2.5 mL) in 12-well tissue culture plates (Costar, Cambridge, MA) in the presence of GM-CSF (100 ng/mL; Novartis/Scheri...

The repertoire of human cytotoxic T-lymphocytes (CTL) in response to influenza A viruses has been shown to be directed towards multiple epitopes, with a dominant response to the HLA-A2-restricted M158–66 epitope. These studies, however, were performed with peripheral blood mononuclear cells (PBMC) of individuals selected randomly with respect to HLA phenotype or selected for the expression of one HLA allele without considering an influence of other HLA molecules. In addition, little information is available on the influence of HLA makeup on the overall CTL response against influenza viruses. Here, the influenza A virus-specific CTL response was investigated in groups of HLA-A and -B identical individuals. Between groups the individuals shared two or three of the four HLA-A and -B alleles. After in vitro stimulation of PBMC with influenza virus, the highest CTL activity was found in HLA-A2+ donors. A similar pattern was observed for the precursor frequency of virus-specific CTL (CTLp) ex vivo, with a higher CTLp frequency in HLA-A2-positive donors than in HLA-A2-negative donors, which were unable to recognize the immunodominant M158–66 epitope. In addition, CTL activity and frequency of CTLp for the individual influenza virus epitopes were determined. The frequency of CTLp specific for the HLA-B8-restricted epitope NP380–388 was threefold lower in HLA-B27-positive donors than in HLA-B27-negative donors. In addition, the frequency of CTLp specific for the HLA-A1-restricted epitope NP44–52 was threefold higher in HLA-A1-, -A2-, -B8-, and -B35-positive donors than in other donors, which was confirmed by measuring the CTL activity in vitro. These findings indicate that the epitope specificity of the CTL response is related to the phenotype of the other HLA molecules. Furthermore, the magnitude of the influenza virus-specific CTL response seems dependent on the HLA-A and -B phenotypes

Here, we describe a new HLA-B*3501-restricted cytotoxic T lymphocyte (CTL) epitope in the influenza A virus (H3N2) nucleoprotein, which was found to exhibit a high degree of variation at nonanchor residues. The influenza virus variants emerged in chronological order, and CTLs directed against old variants failed to recognize more recent strains of influenza A virus, indicating an escape from CTL immunity.

Apigenin and its structural analogues chrysin and luteolin were used to evaluate their capacity to inhibit the production of pro-inflammatory cytokines by lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMC). Furthermore, flowcytometric analysis was performed to compare the effects of apigenin, chrysin, luteolin, quercetin and naringenin on the different cell types present in PBMC. LPS-stimulated PBMC were cultured in the presence of the flavonoids and TNFalpha, IL-1beta and IL-6 were measured in the supernatants. In parallel, metabolic activity of the PBMC was determined by measuring succinate dehydrogenase activity. Apigenin, chrysin and luteolin dose-dependently inhibited both pro-inflammatory cytokine production and metabolic activity of LPS-stimulated PBMC. With increasing concentration of apigenin, chrysin or luteolin the monocytes/macrophages disappeared as measured by flowcytometry. This also appeared to occur in the non-LPS-stimulated PBMC. At the same time there was an increase in dead cells. T- and B-lymphocytes were not affected. Quercetin and naringenin had virtually no effects on cytokines, metabolic activity or on the number of cells in the studied cell populations. In conclusion, monocytes were specifically eliminated in PBMC by apigenin, chrysin or luteolin treatment in vitro at low concentrations (around 8 microM), in which apigenin appeared to be the most potent.