Re-expression of CD14 in Response to a Combined IL-10/TLR Stimulus Defines Monocyte-Derived Cells With an Immunoregulatory Phenotype

Abstract

Interleukin 10 is a central regulator of the antigen-presenting function of myeloid cells. It exerts immunomodulatory effects in vivo and induces a regulatory phenotype in monocyte-derived cells in vitro. We analyzed phenotype and function of monocytic cells in vitro in relation to the cytokine milieu and the timing of TLR-based activation. In GM-CSF/IL-4 cultured human monocytic cells, we identified two, mutually exclusive cell populations arising from undifferentiated cells: CD83⁺ fully activated dendritic cells and CD14⁺ macrophage like cells. Re-expression of CD14 occurs primarily after a sequential trigger with a TLR signal following IL-10 preincubation. This cell population with re-expressed CD14 greatly differs in phenotype and function from the CD83⁺ cells. Detailed analysis of individual subpopulations reveals that exogenous IL-10 is critical for inducing the shift toward the CD14+ population, but does not affect individual changes in marker expression or cell function in most cases. Thus, plasticity of CD14 expression, defining a subset of immunoregulatory cells, is highly relevant for the composition of cellular products (such as DC vaccines) as it affects the function of the total product.

Keywords: IL-10; MDSC; macrophages; monocyte-derived DC; regulatory dendritic cells.

Figure 1

IL-10 in combination with R848 induces re-expression of CD14 in GM-CSF/IL4-cultured monocytic cells (A). Individual plots of cells on d5 of culture after 24 h-incubation R848 (2 μg/ml) without and with IL-10 (40 ng/ml) pre-incubation (1 h), or the combination (B). Summary of 19 different experiments from different healthy donors. (Two-way ANOVA for multiple comparisons; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001) (C). IL-10 dose dependent increase of the percentage of CD14⁺ cells in combination with a fixed dose of R848 (2 μg/ml) (D). Left: Downregulation of CD14 on monocytes during culture in GM-CSF/IL-4 (before experimental treatment): %CD14⁺: black solid: d1 (94%); dotted: d2 (71%); dashed: d3 (12%); thin solid, tinted: d5 (without activation) (8.6%) (one of 3 experiments). Right: Upregulation of CD14 on day 5 of culture in cells, after treatment on day 4: dotted: IL-10/R848 (33%); solid blue: IL-10/R848 treated, after CD14 depletion on d4 (27%); dashed: R848 only (15%), light blue,tinted: R848(only) after CD14-depletion on d4 (10%) (E). Comparison of %CD14⁺ cells (left) and %CD83⁺ cells (right) after the respective treatment following a 4 day (black) culture or a 7 day (gray) culture in GM/IL-4 (n = 3) (F). Effect of IL-10 blockade on CD14 re-expression. Functional grade anti-IL10-antibody and anti-IL10R-antibody were added prior to preincubation with IL-10 or prior to R848 addition. CD14 and CD83 expression were measured 16 h later. Examplary plots and a summary from 7 different donors are shown.

Figure 2

CD14 upregulation depends on IL-10 and the maturation stimulus (A). Monocytes, cultured in GM-CSF/IL-4, were either preincubated with IL-10 or not, and subsequently stimulated for 16–18 h with the indicated substances. Cells were evaluated for CD14 and CD83 expression the following day (B). Summary from n = 3 experiments (C). TNFα-preincubation for 24 h prior to adding IL10 hinders CD14 upregulation. Upper row: no IL10 addition, lower row with IL-10 (D). Summary of 5 independent experiments, showing the absolute % of CD14 depending on TNFα-preincubation without or with exogenous IL-10 (left, Two-way-ANOVA for multiple comparisons, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). The right panel shows the relative inhibition of CD14 expression by TNFα, taking the %CD14 without TNFα of each individual experiment as the reference point (100%). % Inhibition is calculated as: [1-(%CD14(sample)/%CD14(ref.point)] × 100. As these are calculated values from the original data shown in the left panel, no statistical test is shown in this panel.

Figure 3

Phenotypic changes of moC depending on IL-10 pre-treatment. moC were pre-treated with IL-10 (40 ng/ml) or not. Cells were then activated using R848 and stained 16 h later. (A) Representative histograms for individual markers. Solid lines represent cell populations from the control group without IL-10. Dotted line/tinted filling represent cell populations from the IL-10 treated group. The first panel represents analysis of total cells according to the live scatter gate. The middle panel shows histograms from cells within the CD14⁺ gate. The right panels show histograms from the CD83⁺ population. Numbers in each plot indicate the Median fluorescence intensity; —indicates the control group without IL-10, + indicates the IL-10 group. (B) Median fluorescence of individual markers (indicated on the left of the figure), in relation to the IL-10 concentration. Black circles indicate analysis of total cells, red triangles indicate CD83⁺ cells and blue squares indicate CD14⁺ cells. The left panels show mean absolute values from 3 independent experiments. The right panels show the change from the respective baseline (0 ng/ml) in percent. (C) Mean with SD from 5 independent experiments using 40 ng/ml IL-10. Black columns (left) represent total cells, blue columns (middle) represent the CD14⁺ population, red columns (right) represent CD83+ cells. (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Figure 4

Phenotypic changes in response to other protocols used for generating regulatory DCs. moC were incubated either with IL10 (40 ng/ml), rapamycin (100 ng/ml) or dexamethasone (100 nM) (for 16 h) or left alone. All groups were then activated with R848 (and a second addition of the modulating substance) and stained 24 h later. The light pink population represents CD83⁺ cells, the dark blue population represents CD14⁺ cells. CD14⁻CD83⁻ non-committed cells were excluded in this analysis. Examplary plots of three experiments are shown.

Figure 5

IL-10-mediated effects on the function of moC. (A) IL-12 production is reduced. moC were preincubated with IL-10 (or not) and activated either with R848 or a cytokine cocktail. 1 h later, brefeldin A was added for 4 h and cells subsequently stained for intracellular IL-12 and IL-8. Distinction between CD14 and CD83 in the same samples is not possible, as CD14 upregulation is hindered by brefeldin A. CD14(y-axis)- and CD83(x-axis)-staining of a corresponding parallel sample (without brefeldin A) is shown as an inserted dot plot. (n = 3) (B) IL-6 concentration in the supernatant of differentially treated and activated moC, —pooled data from 5 experiments (*p < 0.05) (C). APC-dextran uptake over time in different cell populations. The left panel shows the MFI for Dextran after 1 h of _GM/IL4moC with or without IL-10 (7 experiments). The right panel shows the analysis of activated cells with or without IL-10. Analysis was either done on total cells, or gated on CD14⁺ or CD83⁺ cells, respectively. 1–5 indicates the duration of dextran incubation (1 = 0 min, 2 = 20 min, 3 = 40 min, 4 = 60 min, 5 = 90 min). Each value is normalized to the MFI of _GM/IL4moC at 1 h within the individual experiment (7 experiments). (D) Transmission of the CD14⁺ phenotype onto non-committed bystander moC. Two ‘effector' populations were generated either by using LPS/IFNγ as a full type 1- stimulus or IL-10 followed by R848 to induce a CD14⁺ population. After 16 h they were stained with cell tracker dye and mixed at a 1:1 ratio with non-committed, autologous _GM/IL4moC. Twenty four hours later R848 was added to this co-culture. Cells were then analyzed the next day and separated on the basis of the membrane dye. Examplary plot of 1 out of 3 experiments.

Figure 6

Effects of IL-10-treated moC on antigen-specific priming of naive T-cells. (A) moC were differentially treated (left panel) and pulsed with Melan-A peptide. They were then used to prime naive CD8⁺ T-cells. After 10 days of culture using IL-21, IL-7, and IL-15, MHC-multimer⁺ cells were determined and phenotypically characterized. T-cells were then restimulated with peptide-pulsed moC and MHC-multimer⁺ cells were re-evaluated 7 days later. Right panels depict the cell expansion in absolute numbers. (B) Summary of three different experiments from different donors. Depending on the cell numbers available (number of APC and number of naïve T-cells), experiments were set up in 1–4 parallel wells. The left panel shows the absolute numbers of antigen-specific (MHC-multimer+) cells per well after 10 days of expansion (based on the precursor frequency the starting cell number in each well varies between 20 and 200 cells). The middle panel summarizes MFI-values for CD62L of the resulting MHC-multimer⁺ cells. The right panel shows the ratio of the CD62L MFI of specific vs. non-specific CD8+ cells within the same sample.