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Comparative Study
. 2005 Feb;114(2):204-12.
doi: 10.1111/j.1365-2567.2004.02076.x.

Human monocyte isolation methods influence cytokine production from in vitro generated dendritic cells

Eyad Elkord  1 , Paul E Williams, Howard Kynaston, Anthony W Rowbottom
Affiliations
Comparative Study

Human monocyte isolation methods influence cytokine production from in vitro generated dendritic cells

Eyad Elkord et al. Immunology. 2005 Feb.

Abstract

There is growing interest in the in vitro generation of dendritic cells (DC) from peripheral blood monocytes, but the effect of the method chosen to isolate CD14+ monocytes for subsequent DC generation is poorly documented. The method used to isolate monocytes may have an impact on the subsequent function of DC by affecting their ability to express costimulatory molecules (CD80/86), maturation marker (CD83) and/or to produce important immunomodulatory cytokines. In this study, we show that the positive selection of monocytes by anti-CD14-coated microbeads inhibits the lipopolysaccharide (LPS)-induced production of interleukin (IL)-12, IL-10 and tumour necrosis factor-alpha (TNF-alpha) from human DC. However, when DC were grown from monocytes isolated by plastic adherence, LPS induced the production of much higher levels of these cytokines. DC derived from adherence-isolated monocytes induced the development of potent cytotoxic T lymphocytes of the Tc1 subset specific for influenza matrix protein, as confirmed by interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot-forming cell assay (ELISPOT), cytotoxicity assay, major histocompatibility complex (MHC)-peptide tetrameric complexes and T helper 1/T helper 2 (Th1/Th2) cytokine production assays.

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Figures

Figure 1
Figure 1
Immunophenotype profile of monocytes, immature dendritic cells (DC) and mature DC. Monocytes were isolated by adherence, and generated immature DC were matured by incubation with 0·5 µg/ml of lipopolysaccharide (LPS), 1 µg/ml of CD40L or 20 ng/ml of tumour necrosis factor-α (TNF-α) for the last 3 days of culture. All cells were stained with fluorochrome-conjugated monoclonal antibodies and analysed by flow cytometry. Data shown are representative of four separate experiments. Black lines represent cells stained with monoclonal antibodies and red lines represent cells stained with isotype-matched control antibodies.
Figure 2
Figure 2
Effect of the monocyte isolation method [adherence or magnetic activated cell sorting (MACS)] on the cell-surface expression of CD80, CD86 and CD83. The expression of these markers was measured, by using flow cytometry, on the surface of immature and mature adherence or MACS/monocyte-derived dendritic cells (MoDC). Data are shown as the percentage of cells that express any of these markers. Data represent the mean values ± standard deviation (SD) of three separate experiments. LPS, lipopolysaccharide; TNF-α, tumour necrosis factor-α.
Figure 3
Figure 3
Effect of the monocyte isolation method [adherence or magnetic antibody cell sorting (MACS)] on cytokine secretion. Monocytes were isolated by either adherence or MACS, dendritic cell (DC)-generated and matured by lipopolysaccharide (LPS), tumour necrosis factor-α (TNF-α) or CD40L. Then, interleukin (IL)-12p70, IL-10, TNF-α, IL-2 and interferon-γ (IFN-γ), released from unstimulated or stimulated adherence or MACS/monocyte-derived dendritic cells (MoDC), were measured in culture supernatants by using the Bio-Plex protein array system. Cytokines released from adherence/MoDC are shown as black columns, while cytokines from MACS/MoDC are shown as white columns. The graphs shown are representative of experiments performed in quadruplicate from four separate donors.
Figure 4
Figure 4
Interferon-γ (IFN-γ) enzyme-linked immunosorbent spot-forming cell assay (ELISPOT), performed with the IMP-CD8+ clone as an effector and allogeneic dendritic cells (DC) derived from adherence-isolated monocytes as stimulators. The table shows the description for each column. The negative control was obtained by incubating effectors alone and the positive control by incubating effectors overnight with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) and 500 ng/ml ionomycin. For column (c), clone cells were incubated with peptide in the absence of DC. Results are expressed as the number of spot-forming cells (SFC) per 1 × 104 cells. Data represent the mean value ± standard deviation (SD) of tests repeated three times.
Figure 5
Figure 5
Interferon-γ (IFN-γ) enzyme-linked immunosorbent spot-forming cell assay (ELISPOT) for IMP58–66-specific CD8+ cells expanded for one round of in vitro stimulation (IVS) by using IMP58–66-pulsed autologous mature dendritic cells (DC) derived from adherence-isolated monocytes and activated by lipopolysaccharide (LPS). The actual number of peptide-specific CD8+ cells can be obtained by subtracting the number of spot-forming cells (SFC) when unpulsed DC are used as stimulators from the number of SFC when pulsed DC are used as stimulators. Data represent the mean value ± standard deviation (SD) of tests repeated three times. One representative experiment of five, performed from five different donors, is shown. IL-2, interleukin-2; NC, negative control; PC, positive control.
Figure 6
Figure 6
Percentage specific lysis of T2A2 cells by IMP58–66-expanded peripheral blood lymphocyte (PBL) effectors in a 4-hr PKH26-labelling flow cytometric assay. Effectors were expanded for 11 days by using IMP58–66-pulsed autologous dendritic cells (DC) derived from adherence-isolated monocytes and matured by lipopolysaccharide (LPS). In this experiment, interleukin-2 (IL-2) was added on day 4. The expanded effectors were then incubated for 4 hr with unpulsed or peptide-pulsed T2A2 targets, and the percentage of target killing was determined flow cytometrically. The graph shown is representative of experiments performed in triplicate from three separate donors.
Figure 7
Figure 7
Tetramer staining of unexpanded (a) and expanded (b) CD8+ T lymphocytes. Peripheral blood lymphocytes from HLA-A2-positive normal donors were expanded by using mature adherence/monocyte-derived dendritic cells (MoDC) pulsed with IMP58–66 peptide. Lymphocytes were gated by using their forward- and side-scatter properties, and CD8+ T cells were gated if positive for anti-CD8 monoclonal antibody (mAb). Only 0·25% of CD8+ T cells stained positive for the tetramer before stimulation (a), while 77·8% of these cells became tetramer positive after one round of in vitro stimulation (IVS) (b). PE, phycoerythrin.
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