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. 2009 Sep;128(1 Suppl):e728-37.
doi: 10.1111/j.1365-2567.2009.03075.x. Epub 2009 Feb 9.

Adenosine regulates CD8 T-cell priming by inhibition of membrane-proximal T-cell receptor signalling

Carsten Linnemann  1 , Frank A Schildberg, Anna Schurich, Linda Diehl, Silke I Hegenbarth, Elmar Endl, Svenja Lacher, Christa E Müller, Jürgen Frey, Luca Simeoni, Burkhart Schraven, Dirk Stabenow, Percy A Knolle
Affiliations

Adenosine regulates CD8 T-cell priming by inhibition of membrane-proximal T-cell receptor signalling

Carsten Linnemann et al. Immunology. 2009 Sep.

Abstract

Adenosine is a well-described anti-inflammatory modulator of immune responses within peripheral tissues. Extracellular adenosine accumulates in inflamed and damaged tissues and inhibits the effector functions of various immune cell populations, including CD8 T cells. However, it remains unclear whether extracellular adenosine also regulates the initial activation of naïve CD8 T cells by professional and semi-professional antigen-presenting cells, which determines their differentiation into effector or tolerant CD8 T cells, respectively. We show that adenosine inhibited the initial activation of murine naïve CD8 T cells after alphaCD3/CD28-mediated stimulation. Adenosine caused inhibition of activation, cytokine production, metabolic activity, proliferation and ultimately effector differentiation of naïve CD8 T cells. Remarkably, adenosine interfered efficiently with CD8 T-cell priming by professional antigen-presenting cells (dendritic cells) and semi-professional antigen-presenting cells (liver sinusoidal endothelial cells). Further analysis of the underlying mechanisms demonstrated that adenosine prevented rapid tyrosine phosphorylation of the key kinase ZAP-70 as well as Akt and ERK1/2 in naïve alphaCD3/CD28-stimulated CD8 cells. Consequently, alphaCD3/CD28-induced calcium-influx into CD8 cells was reduced by exposure to adenosine. Our results support the notion that extracellular adenosine controls membrane-proximal T-cell receptor signalling and thereby also differentiation of naïve CD8 T cells. These data raise the possibility that extracellular adenosine has a physiological role in the regulation of CD8 T-cell priming and differentiation in peripheral organs.

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Figures

Figure 1
Figure 1
Adenosine inhibits early activation processes in naïve CD8 T cells after αCD3/CD28 activation. Naïve CD8 T cells were activated in the absence or presence of adenosine with αCD3/CD28 beads for 24 hr. (a) Phenotypic analysis of activation markers on CD8 T cells by flow cytometry (shaded: no adenosine; thick black line: 1 mm adenosine; black line: 0·1 mm adenosine; dashed: unstimulated). (b) Interleukin-2 (IL-2) concentration was determined in cell culture supernatants after 24 hr by enzyme-linked immunosorbent assay and calculated to the numbers of viable T cells at the end of the experiment. (c) Intracellular staining for interferon-γ (IFN-γ) in CD8 T cells after 24 hr of stimulation. (d) Flow cytometric analysis of CD8 T-cell size at 24 hr post-activation. (e) Uptake of the fluorescently labelled glucose analogue 6-NBDG (30 μm) was determined by flow cytometry in living CD8 T cells. (f) Lactate release into the cell culture supernatant over a period of 16 hr was determined from 105 CD8 T cells isolated at 24 hr post-activation with αCD3/CD28 beads in the absence or presence of adenosine. *P < 0·05, ***P < 0·001.
Figure 2
Figure 2
Adenosine inhibits expansion and effector differentiation of naïve CD8 T cells after αCD3/CD28 activation. Naïve carboxyfluorescein succinimidyl ester (CFSE)-loaded CD8 T cells were activated with αCD3/CD28 beads in the absence or presence of adenosine. (a) After 48 hr proliferation was analysed by flow cytometry. (b) Percentage of divided cells, division index and total number of living CD8 cells. (c) Intracellular staining of CD8 T cells for Ki-67 protein after stimulation with αCD3/CD28 beads in the absence or presence of adenosine for 48 hr (shaded: no adenosine; black line: 1 mm adenosine). (d) Restimulation of living CD8 T cells 72 hr post-activation with αCD3 in adenosine-free culture media. After 16 hr the cell culture supernatant was assessed for interferon-γ (IFN-γ) and interleukin-2 (IL-2) concentrations. *P < 0·05, **P < 0·01, ***P < 0·001.
Figure 3
Figure 3
Adenosine blocks tolerogenic and immunogenic priming of naïve CD8 T cells. Antigen-presenting liver sinusoidal endothelial cells (LSEC; 106) or splenic dendritic cells (DC; 5 × 105) were cultured with 106 CD8 DesTCR transgenic T cells in the absence or presence of adenosine. (a) Flow cytometric analysis of activation markers after 24 hr (shaded: no adenosine; black line: 1 mm adenosine; dashed line: 0·1 mm adenosine) and (b) proliferation of DesTCR T cells after 48 hr of stimulation. (c) Percentage of divided cells, division index and total number of living DesTCR cells after 48 hr. (d) Ovalbumin (OVA) -loaded splenic DCs (5 × 105) were cultured with 106 CD8 OT-I T-cell receptor transgenic T cells in the absence or presence of adenosine for 48 hr. After 24 hr adenosine in indicated concentrations was added again. After 48 hr living CD8 T cells were restimulated with αCD3 in adenosine-free culture media for 4 hr and stained intracellularly for interferon-γ (IFN-γ). **P < 0·01, ***P < 0·001.
Figure 4
Figure 4
Increase in intracellular cAMP levels interferes with T-cell activation. (a) 4 × 106 naïve CD8 T cells were treated with adenosine (1 mm) for 5 min and intracellular cAMP levels were analysed. (b) T cells were activated with αCD3/CD28 beads in the absence or presence of adenosine or db-cAMP. After 24 hr, expression levels of CD25 and CD44 on living CD8 T cells were analysed by flow cytometry (shaded: no adenosine; black line: 1 mm adenosine; dashed line: 50 μm db-cAMP) (c) T cells from (b) were stained intracellularly for interferon-γ (IFN-γ). (d) Naïve carboxyfluorescein succinimidyl ester (CFSE) -loaded 106 CD8 T cells were activated with αCD3/CD28 beads in the presence of db-cAMP (50 μm). After 48 hr CFSE profiles of living CD8 cells were analysed by flow cytometry. (e) Naïve CD8 T cells were treated with adenosine (1 mm) or CGS21680 (100 μm) and activated with αCD3/CD28 beads for 24 hr. Activation markers (CD25, CD44 and PD1) on CD8 T cells were analysed by flow cytometry and are shown as relative expression levels compared with untreated cells. *P < 0·05.
Figure 5
Figure 5
Adenosine-induced inhibition of membrane-proximal signalling events in T-cell receptor (TCR)/CD28-induced naïve CD8 T-cell activation. (a) Naïve OT-I/RAG−/− CD8 T cells were stimulated with cross-linked αCD3/CD28 antibodies for the times indicated immediately or after 5 min pre-incubation with 1 mm adenosine. Afterwards, T cells were lysed and specific protein-phosphorylation was determined. (b) Flow cytometric analysis of calcium influx in fluo-4-loaded naïve CD8 T cells after stimulation with streptavidin cross-linked αCD3/CD28 antibodies (red line: αCD3/CD28 / no adenosine; blue line: αCD3/CD28/adenosine (1 mm); black line: isotype control). Equal loading with Fluo-4 was controlled by control stimulation with 1·5 μm ionomycin.
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