Adenosine and cAMP signalling skew human dendritic cell differentiation towards a tolerogenic phenotype with defective CD8(+) T-cell priming capacity

Abstract

Multiple endogenous mechanisms that regulate immune and inflammatory processes contribute to the maintenance of peripheral tolerance and prevent chronic inflammation in mammals. Yet pathogens and tumours are able to exploit these homeostatic pathways to foster immunosuppressive microenvironments and evade immune surveillance. The release of adenosine in the extracellular space contributes to these phenomena by exerting a broad range of immunomodulatory effects. Here we document the influence of adenosine receptor triggering on human dendritic cell differentiation and functions. We show that the expression of several immunomodulatory proteins and myeloid/monocytic lineage markers was affected by adenosine receptors and the cAMP pathway. These changes were reminiscent of the phenotype associated with tolerogenic dendritic cells and, functionally, translated into a defective capacity to prime CD8(+) T-cells with a common tumour antigen in vitro. These results establish a novel mechanism by which adenosine hampers CD8(+) T-cell immunity via dendritic cells that may contribute to peripheral tolerance as well as to the establishment of immunosuppressive microenvironments relevant to tumour biology.

Figure 1

Differential expression of lineage and differentiation markers on monocyte-derived dendritic cells (moDCs) differentiated in the presence of vehicle, N-ethylcarboxamidoadenosine (NECA) or forskolin/1-methyl-3-isobutylxanthine (FSK/IBMX). Typical differentiation status determined by monitoring CD14 and CD1a expression on moDCs generated with granulocyte–macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) in the presence of vehicle control (a), NECA (b) or FSK and IBMX (c). Cell numbers in each quadrant are shown as insets in each dot plot. The total numbers of acquired events were 10 080 (a), 12 090 (b) and 13 425 (c). The expression levels of three different myeloid and monocytic markers (d–f), as well as CD95/Fas (g), were differentially regulated by differentiation in presence of NECA or FSK and IBMX. The moDCs were stained with the indicated antibodies after 7 days of differentiation. Data are representative of at least five experiments for each staining.

Figure 2

Up-regulation of proteins involved in immune regulatory mechanisms on the surface of monocyte-derived dendritic cells (moDCs) differentiated in the presence of N-ethylcarboxamidoadenosine (NECA) or forskolin/1-methyl-3-isobutylxanthine (FSK/IBMX). The moDCs were stained with the indicated antibodies after 7 days of differentiation in the presence of granulocyte–macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) with vehicle control, NECA or FSK/IBMX, as indicated in the key (a–c). Data are representative of at least six experiments for each staining.

Figure 3

Comparison of the cytokine profiles of monocyte-derived dendritic cells (mo-DCs) differentiated in the presence of N-ethylcarboxamidoadenosine (NECA) or vehicle control upon lipopolysaccharide (LPS) maturation. The moDCs were differentiated with granulocyte–macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 6–8 days with vehicle control, in the presence of NECA or forskolin/1-methyl-3-isobutylxanthine (FSK/IBMX). Cells were then treated with LPS for 24 hr in the absence of any other compound. (a) Cytokine concentrations in supernatants were determined by cytometric bead arrays and ELISA for the IL-12 cytokine family or (b) and (c) by using the 27-plex Luminex bead combination. Assays were performed in triplicate for six donors in the case of IL-12, IL-23 and IL-27 ELISAs and for three donors in the case of the 27-plex Luminex bead arrays. Representative data are shown. P values were calculated using the two-tailed paired student t test (*P < 0·05; **P < 0·01; ***P < 0·001; ns: non-significant).

Figure 4

Assessment of the stimulatory and CD8⁺ T-cell priming capacities of mature monocyte-derived dendritic cells (moDCs) differentiated in the presence of N-ethylcarboxamidoadenosine (NECA) or forskolin/1-methyl-3-isobutylxanthine (FSK/IBMX) compared with bona fide moDCs. (a) The CD8⁺ T-cell clone Mel5 was stimulated in intra-cellular cytokine staining assays, as described in the Materials and methods section, using mature moDCs without peptide or moDCs, moDC NECA and moDC FSK/IBMX pulsed with agonist peptide as indicated. Staining is representative of seven different experiments. (b) Proportions of antigen-specific CD8⁺ T-cells following an 8-day priming experiment using peptide-pulsed and lipopolysaccharide (LPS) -matured moDCs, moDC NECA or moDC FSK/IBMX as antigen-presenting cells. The percentage of tetramer-positive cells is indicated in each plot for cells gated as follows: Forward-scatter (FSC-H) and side-scatter (SSC-H) lymphocyte gate/Singlet determined by FCS-A/FSC-H analysis/Live CD8⁺ cells negative for the Aqua viability dye. The baseline frequency of tetramer-positive CD8⁺ cells, assessed in a staining performed before priming, was below 0·1% (data not shown). Absolute cell counts were as follows: DCs 0 (85 726 gated events and 8144 tetramer-positive cells); NECA DCs (81 731 gated events and 1365 tetramer-positive cells); and F/I DCs (71 826 acquired events and 232 tetramer-positive cells). (c) Comparison of the priming capacities of mature, peptide-pulsed moDCs, moDC NECA or moDC FSK/IBMX (as indicated) for seven different donors. Bars represent mean values (11·23% for DCs 0, 2·21% for NECA DCs and 0·75% for F/I DCs). The ranges of tetramer-positive frequency values were: 3·3–29·6% for moDCs, 0·7–4·35% for moDC NECA and 0·32–1·1% for moDC FSK/IBMX. P-values were calculated using the two-tailed unpaired Student's t-test.