Hypoxia induces T-cell apoptosis by inhibiting chemokine C receptor 7 expression: the role of adenosine receptor A(2)

Abstract

Hypoxia is a major characteristic of the tumor microenvironment, and its effects on immune cells are proposed to be important factors for the process of tumor immune escape. It has been reported that hypoxia affects the function of dendritic cells and the antitumor function of T cells. Here we discuss the effects of hypoxia on T-cell survival. Our results showed that hypoxia induced apoptosis of T cells. Adenosine and adenosine receptors (AR) are important to the hypoxia-related signaling pathway. Using AR agonists and antagonists, we demonstrated that hypoxia-induced apoptosis of T cells was mediated by A(2a )and A(2b) receptors. Furthermore, we are the first, to our knowledge, to report that hypoxia significantly inhibited the expression of chemokine C receptor 7 (CCR7) of T cells via the A(2)R signal pathway, perhaps representing a mechanism of hypoxia-induced apoptosis of T cells. Collectively, our research demonstrated that hypoxia induces T-cell apoptosis by the A(2)R signaling pathway partly by suppressing CCR7. Blocking the A(2)R signaling pathway and/or activation of CCR7 can increase the anti-apoptosis function of T cells and may become a new strategy to improve antitumor potential.

Figure 1

Hypoxia induces apoptosis of peripheral T cells. T cells were freshly isolated from heparinized blood of healthy donors as described in the section on ‘Materials and Methods'. Next, the isolated T cells were cultured with RPMI 1640 medium (supplemented with 10% fetal bovine serum and 5 µg/ml phytohemagglutinin) under normoxic (N) or hypoxic (H) conditions with or without LPS for 24 h. The percentage of apoptosis was assayed by flow cytometry and one representative result is shown (a). (b) Statistical analysis of apoptosis percentage (n=14). Data were the mean±SD of 14 independent experiments. *P<0.05, compared to the normoxic condition. LPS, lipopolysaccharide; PI, propidium iodide.

Figure 2

Effects of hypoxia on the expression of AR subtypes in T cells. Total RNA was isolated from human peripheral blood T cells cultured under normoxic (N) or hypoxic (H) conditions. RNA samples isolated from four different donor-derived T cells were reverse transcribed and tested for adenosine receptors A₁, A_2a, A_2b, A₃ and β-actin mRNA expression by RT-PCR analysis. Relative levels of adenosine receptors mRNA transcripts were calculated in relation to the values obtained in parallel for the reference gene (β-actin). Data represent mean±SD of four independent experiments. *P<0.05, compared to the normoxic condition. AR, adenosine receptor; RT-PCR, reverse transcription polymerase chain reaction.

Figure 3

Hypoxia induces apoptosis of T cells through adenosine receptors A_2a and A_2b. The freshly isolated T cells were cultured under normoxic conditions with adenosine receptor agonists and hypoxic conditions with A₂R antagonists for 24 h and the apoptosis was determined by flow cytometry. (a) T cells were treated with adenosine receptor agonists under normoxia. Agonists used were specific for A₁ receptor (CPA), A_2a receptor (CGS21680), A₃ receptor (IB-MECA), or nonspecific for A_2b receptor (NECA). (b) T cells were treated with adenosine receptor antagonists under hypoxia. The antagonists used were specific for A_2a receptor (SCH58261) or A_2b receptor (MRS1706). CGS21680, 2-[p-2-(carboxy-ethyl)-phenylethylamino]-5′-N-ethylcarboxamidoadenosine; CPA, N⁶-cyclopentyladenosine; IB-MECA, N⁶-(3-iodobenzyl)adenosine-5'-N-methylluronamide; MRS1706, N-(4-acetylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl) phenoxy] acetamide; NECA, 5′-N-ethyl-carboxamidoadenosine; SCH58261, 5-amino-2-(2-furyl)-7-phenylethyl-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c] pyrimidine.

Figure 4

Hypoxia inhibits the expression of CCR7, and CCR7 mediates the apoptosis of T cells. T cells (n=14) were cultured under normoxic and hypoxic conditions for 24 h and the CCR7 expressions were determined by flow cytometry. (a) The thin line represents staining with a control isotype-matched antibody. The bold line and grey solid histograms represent normoxia and hypoxia, respectively. (b) T cells were cultured under normoxic condition with anti-CCR7 antibody or isotype control for 24 h and the apoptosis of T cells was assayed by flow cytometry. CCR7, chemokine C receptor 7; H, hypoxia; N, normoxia.

Figure 5

Hypoxia downregulates the expression of CCR7 through the adenosine receptor A₂. The freshly isolated T cells (n=14) were cultured under normoxic conditions with adenosine receptor agonists and under hypoxic conditions with adenosine receptor antagonists for 24 h. The expression of CCR7 was assayed by flow cytometry. (a) T cells were treated with adenosine receptor agonists under normoxia. Agonists used were specific for the A₁ receptor (CPA), the A_2a receptor (CGS21680), the A₃ receptor (IB-MECA), and nonspecific for A_2b receptor (NECA). (b) Statistical analysis of effects of adenosine receptor agonists on the expression of CCR7 under normoxia. (c) T cells were treated with A_2aR (SCH58261) and A_2bR (MRS1706) antagonists under hypoxia. (d) Statistical analysis of effects of A₂ agonists on the expression of CCR7 under hypoxia. CCR7, chemokine C receptor 7; CGS21680, 2-[p-2-(carboxy-ethyl)-phenylethylamino]-5′-N-ethylcarboxamidoadenosine; CPA, N⁶-cyclopentyladenosine; IB-MECA, N⁶-(3-iodobenzyl)adenosine-5'-N-methylluronamide; MRS1706, N-(4-acetylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl) phenoxy] acetamide; NECA, 5′-N-ethyl-carboxamidoadenosine; SCH58261, 5-amino-2-(2-furyl)-7-phenylethyl-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c] pyrimidine.