Sympathetic neural signaling via the β2-adrenergic receptor suppresses T-cell receptor-mediated human and mouse CD8(+) T-cell effector function

Abstract

Postganglionic sympathetic neurons innervate secondary lymphoid organs and secrete norepinephrine (NE) as the primary neurotransmitter. NE binds and signals through five distinct members of the adrenergic receptor family. In this study, we show elevated expression of the β2-adrenergic receptor (ADRB2) on primary human CD8(+) effector memory T cells. Treatment of both human and murine CD8(+) T cells with NE decreased IFN-γ and TNF-α secretion and suppressed their cytolytic capacity in response to T-cell receptor (TCR) activation. The effects of NE were specifically reversed by β2-specific antagonists. Adrb2(-/-) CD8(+) T cells were completely resistant to the effects of NE. Further, the ADRB2-specific pharmacological ligand, albuterol, significantly suppressed effector functions in both human and mouse CD8(+) T cells. While both TCR activation and stimulation with IL-12 + IL-18 were able to induce inflammatory cytokine secretion, NE failed to suppress IFN-γ secretion in response to IL-12 + IL18. Finally, the long-acting ADRB2-specific agonist, salmeterol, markedly reduced the cytokine secretion capacity of CD8(+) T cells in response to infection with vesicular stomatitis virus. This study reveals a novel intrinsic role for ADRB2 signaling in CD8(+) T-cell function and underscores the novel role this pathway plays in adaptive T-cell responses to infection.

Keywords: CD8+ T cells; Cytokine; Cytolysis; Norepinephrine; β2-Adrenergic receptor.

Figure 1. NE suppresses human CD8⁺ T-cell cytokine secretion and lytic activity

(A) ADRB2 mRNA expression was assessed by microarray (left, [24]) and qPCR (right) in CD8⁺CCR7^hiCXCR3^lo and CD8⁺CCR7^loCXCR3^hi cells from peripheral blood of healthy human donors. (B and C) CD8⁺ T cells were isolated and activated (B) with anti-CD3 and anti-CD28 antibodies for the indicated time points or (C) for 24 hours with increasing concentrations of NE. IFN-γ and TNF-α in the supernatants were measured by ELISA. (D) CD8⁺ T cells were isolated and incubated with ⁵¹Cr-labeled and anti-CD3-coated target cells at increasing effector to target ratios. ⁵¹Cr release was measured by scintillation counting 12 hours later. (B, D) Data are shown as mean +/− SEM of 3 replicates and are representative of 3 experiments performed with similar results. (C) The kinetic experiment was performed once. *p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001 compared to CCR7^hi or to 0 µM NE, by 2-way ANOVA with Bonferroni post-test.

Figure 2. NE suppresses human cytokine secretion in CD8⁺ T cells through the ADRB2

(A) Human CD8⁺ T cells were isolated and activated with anti-CD3 and anti-CD28 antibodies and NE for 24 hours in the presence of milliQ water (Ctrl), or a pan-α (phentolamine), or a pan-β (nadolol) adrenergic receptor antagonist. IFN-γ and TNF-α in the supernatants were measured by ELISA. (B) CD8⁺ T cells were isolated and activated as in (A), in the presence of phentolamine (10µM), or increasing concentrations of a β1-specific (atenolol, 0.1µM to 5µM), or a β2-specific (ICI-118,551, 0.1µM to 5µM) adrenergic receptor antagonist. TNF-α production was measured at 24 hours by ELISA. (C) CD8⁺ T cells were isolated and activated as in (A), in the presence of methanol (V.C.) or increasing concentrations of albuterol or NE, and IFN-γ and TNF-α production measured at 24 hours. Data shown are presented as mean +/− SEM of 3 replicates and are representative of (A and C) 2 experiments and (B) 3 experiments, all from individual donors. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to 0 µM NE or V.C, by 2-way (A and B) or 1-way (C) ANOVA with Bonferroni post-test.

Figure 3. ADRB2 signaling modulates mouse CD8⁺ T-cell function

(A) Purified CD8⁺ T cells from C57Bl/6 mice were activated with anti-CD3 and anti-CD28 antibodies and increasing concentrations of NE for 24 hours. IFN-γ and TNF-α in the supernatants were measured by ELISA. (B) CD8⁺ T cells from Cl4 mice were activated with anti-CD3 and anti-CD28 antibodies in the presence or absence of NE for 12 hours. RNA was harvested and Ifng and Tnf transcripts were quantified by qPCR. (C) Splenocytes from Cl4 mice were incubated overnight with the HA peptide IYSTVASSL, in the absence of exogenous IL-2 and in the presence or absence of NE. IL-2, IFN-γ and TNF-α were measured from the supernatants at 24 hours. (D) Splenocytes from Cl4 mice were incubated with the HA peptide IYSTVASSL in the presence or absence of albuterol or NE. IFN-γ and TNF-α in the supernatants were measured at 18 hours. (D) CD8⁺ T cells from Balb/cJ mice were activated as in (A), in the presence of albuterol or NE and in the presence of milliQ water (Ctrl), or a pan-α (phentolamine), a pan-β (nadolol), a β1-specific (atenolol), or a β2-specific (ICI-118,551) adrenergic receptor antagonist (100 nM each). IFN-γ (not shown) and TNF-α in the supernatants were measured. (E) CD8⁺ T cells from ADRB2^+/+ or ADRB2^−/− Balb/cJ mice were isolated and activated as in (A), in the presence of increasing concentrations of NE or albuterol. IFN-γ (not shown) and TNF-α in the supernatants were measured. Data are presented as mean +/− SEM of triplicate determinations and are representative of (A and D) 3 experiments, (B, E, and F) 2 experiments, and (C) 1 experiment. * p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to 0 µM NE or media, by Student’s t-test (C), or 1-way (A, B, and D) or 2-way (E and F) ANOVA with Bonferroni post-test.

Figure 4. ADRB2 signaling suppresses mouse CD8⁺ T-cell cytokine secretion independent of acetylcholine or cAMP

(A) Splenocytes from OT-I mice were isolated and incubated 23 hours with the OVA peptide SIINFEKL and NE with increasing concentrations of an α7 nicotinic receptor antagonist (methyllycaconitine) or a β2 adrenergic receptor antagonist (ICI-118,551). TNF-α in the supernatants was measured. (B) Splenocytes from Cl4 mice were incubated overnight with the HA peptide IYSTVASSL and in the presence or absence of albuterol or increasing concentrations of the cAMP stimulator forskolin. IFN-γ and TNF-α were measured. (C) Splenocytes from Cl4 mice were incubated with HA peptide in the presence or absence of NE, and with increasing concentrations of an adenylyl cyclase inhibitor (2’5’-dideoxyadenosine;) or a β2 adrenergic receptor antagonist (ICI-118,551). (D) CD8⁺ T cells from Balb/cJ mice were isolated and stimulated with anti-CD3 and anti-CD28 Abs (3 µg/mL each) for 24 hours in the presence or absence of NE and increasing concentrations of a soluble adenylyl cyclase inhibitor (KH7) or a β2 adrenergic receptor antagonist (ICI-118,551). Data are presented as mean +/− SEM of triplicate determinations and are representative of (A and D) a single experiment with pooled data from 3 mice measured separately, (B and C) 2 experiments with pooled data from 3 mice measured separately in each experiment. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 compared to 0µM NE, No Albuterol, or Ctrl, 2-way (A, C and D) or 1-way (B) ANOVA with Bonferroni post-test.

Figure 5. ADRB2 signaling does not affect CD8⁺ T-cell proliferation or programming

(A) Splenocytes from ADRB2-sufficient or -deficient Cl4 mice were isolated and incubated for 3 days in the presence or absence of the HA peptide IYSTVASSL and NE. Proliferation was measured by dilution of a proliferation dye by flow cytometry. (B) Human CD8⁺CD45RA⁺ cells were purified from peripheral blood and activated with plate-bound anti-CD3 and anti-CD28 antibodies, either neutralizing cytokines (Neut) or in the presence of rhIL-12 (IL-12) and in the presence or absence of NE for 7 days. On day 8, cells were restimulated with plate-bound anti-CD3 for 24 hours. IFN-γ and TNF-α in the supernatant were measured by ELISA. Data are presented as mean +/− SEM of triplicate determinations and are representative of (A) 3 experiments with at least 1 mouse in each experiment, and (B) 2 independent experiments from separate donors. Comparisons of NE treatments to controls were not significant as assessed by 2-way ANOVA with Bonferroni post-test.

Figure 6. ADRB2 signaling modulates cytokine secretion programming in mouse CD8⁺ T cells in vivo

(A) OT-I T cells were transferred (2000/recipient) into naïve C57Bl/6 mice followed by infection with 10⁶ PFU VSV-OVA the next day (day 0). DMSO (V.C.) or Salmeterol was given i.p. on days 0, 1 and 2. (B) Spleens were harvested on d7, and OT-I-specific cellular expansion was determined by flow cytometry. Data display percent of Kb-tet⁺ cells of the total CD8⁺ T-cell pool (left) and the total numbers of Kb-tet⁺ cells within the spleen (right). (C) IFN-γ and TNF-α secretion were measured from splenocytes by flow cytometry after 24 hours restimulation with SIINFEKL peptide. Data are presented as mean +/− SEM of triplicate determinations and are representative of 2 independent experiments with 4 mice per treatment group in each experiment.. *p<0.05 compared to V.C, 1-way ANOVA with Bonferroni post-test.