Gαs-coupled receptor signaling and sleep regulate integrin activation of human antigen-specific T cells

Abstract

Efficient T cell responses require the firm adhesion of T cells to their targets, e.g., virus-infected cells, which depends on T cell receptor (TCR)-mediated activation of β₂-integrins. Gα_s-coupled receptor agonists are known to have immunosuppressive effects, but their impact on TCR-mediated integrin activation is unknown. Using multimers of peptide major histocompatibility complex molecules (pMHC) and of ICAM-1-the ligand of β₂-integrins-we show that the Gα_s-coupled receptor agonists isoproterenol, epinephrine, norepinephrine, prostaglandin (PG) E₂, PGD₂, and adenosine strongly inhibit integrin activation on human CMV- and EBV-specific CD8⁺ T cells in a dose-dependent manner. In contrast, sleep, a natural condition of low levels of Gα_s-coupled receptor agonists, up-regulates integrin activation compared with nocturnal wakefulness, a mechanism possibly underlying some of the immune-supportive effects of sleep. The findings are also relevant for several pathologies associated with increased levels of Gα_s-coupled receptor agonists (e.g., tumor growth, malaria, hypoxia, stress, and sleep disturbances).

Graphical abstract

Figure 1.

Epinephrine potently inhibits pMHC multimer-induced β₂-integrin activation on antigen-specific CD8⁺ T cells. Human whole blood cells were incubated for 5 min at 37°C in the presence of pMHC multimers (A2-NLV/PE or A2-GLC/PE) and mICAM-1. (A) Representative density plots showing the gating strategy used for defining pMHC⁺ CD8⁺ T cells. From left to right, the lymphocyte gate (on the forward scatter area [FSC-A]/side scatter area [SSC-A] plot), the FSC-A/FSC height (FSC-H) duplet exclusion, the gating of CD3⁺CD8⁺ cells, and the exclusion of CD4⁺, CD14⁺, and CD19⁺ events are shown. (B and C) CMV-specific (B) and EBV-specific (C) CD8⁺ T cells were stained with A2-NLV/PE or A2-GLC/PE multimers, respectively. Multimer binding to the TCR leads to activation of β₂-integrins (left), which was detected by staining with mICAM-1. Preincubation of the cells for 5 min with 10⁻¹⁰ M (18 pg/ml), 10⁻⁹ M (180 pg/ml), or 10⁻⁸ M (1,800 pg/ml) epinephrine partially or completely blocked the activation of the β₂-integrins. Numbers indicate the MFI of mICAM-1 binding on pMHC⁺ CD8⁺ T cells, and italic numbers in brackets indicate the frequency of mICAM-1⁺ cells among pMHC⁺ CD8⁺ T cells.

Figure 2.

Effect of Gα_s-coupled receptor agonists on pMHC-induced β₂-integrin activation on antigen-specific CD8⁺ T cells. (A–F) Whole blood cells and (for adenosine) PBMCs were preincubated in the presence or absence of the indicated Gα_s-coupled receptor agonists at the indicated concentrations for 5 min followed by staining with CMV A2-NLV/PE (A, C, E, and F) or EBV A2-GLC/PE (B and D) multimers and mICAM-1 for 5 min at 37°C. Means ± SEM of the MFI of mICAM-1 binding are shown for the different agonists as percentage of the control (i.e., the sample without Gα_s-coupled receptor agonists, which was set to 100%). The fitted standard curves were calculated by nonlinear regression; n = 5 (n = 3 for adenosine, serotonin, histamine, and dopamine).

Figure 3.

Sleep increases β₂-integrin activation on antigen-specific CD8⁺ T cells. (A–E) Means ± SEM of the MFI of mICAM-1 binding on total CMV-specific (A) and EBV-specific (B) CD8⁺ T cells, as well as on early (CD27⁺CD28⁺; C), intermediate (CD27⁺CD28⁻; D) and late (CD27⁻CD28⁻; E) differentiated CMV-specific CD8⁺ T cell subpopulations after staining with pMHC multimers and mICAM-1 are shown for a regular sleep–wake cycle (sleep, filled circles) and continuous wakefulness (wake, open circles). Gray lines illustrate adapted cosine curves for confirmed circadian rhythms in the sleep condition; the shaded area depicts bedtime. Significance is indicated for pairwise comparisons between conditions using paired t tests following significant ANOVA results. n = 10 for CMV; n = 5 for EBV; *, P < 0.05.

Figure 4.

Regulation of integrin activation by plasma collected during sleep versus wakefulness is mediated by Gα_s-coupled receptor signaling. (A and B) Means ± SEM of the MFI of mICAM-1 binding are shown on CMV-specific CD8⁺ T cells incubated with plasma collected at 2 am during sleep versus wakefulness (A) and at 6 am (during nocturnal sleep) versus 6 pm (during daytime wakefulness; B). CMV-specific T cells were incubated with plasma collected at the indicated time points and conditions (sleep vs. wake) in the presence or absence of an antagonist composite (including β₂-AR, EP₄, and DP₁ antagonists) or in the presence or absence of an agonist composite (including epinephrine, norepinephrine, PGE₂, and PGD₂). All experiments were performed in duplicates. Significance is indicated for pairwise comparisons between conditions or treatments using paired t tests. n = 6; **, P ≤ 0.01; ***, P < 0.001.