Hypoxia-induced and A2A adenosine receptor-independent T-cell suppression is short lived and easily reversible

Abstract

Tissue hypoxia plays a key role in establishing an immunosuppressive environment in vivo by, among other effects, increasing the level of extracellular adenosine, which then signals through A2A adenosine receptor (A2AR) to elicit its immunosuppressive effect. Although the important role of the adenosine--A2AR interaction in limiting inflammation has been established, the current study revisited this issue by asking whether hypoxia can also exert its T-cell inhibitory effects even without A2AR. A similar degree of hypoxia-triggered inhibition was observed in wild-type and A2AR-deficient T cells both in vitro and, after exposure of mice to a hypoxic atmosphere, in vivo. This A2AR-independent hypoxic T-cell suppression was qualitatively and mechanistically different from immunosuppression by A2AR stimulation. The A2AR-independent hypoxic immunosuppression strongly reduced T-cell proliferation, while IFN-γ-producing activity was more susceptible to the A2AR-dependent inhibition. In contrast to the sustained functional impairment after A2AR-mediated T-cell inhibition, the A2AR-independent inhibition under hypoxia was short lived, as evidenced by the quick recovery of IFN-γ-producing activity upon re-stimulation. These data support the view that T-cell inhibition by hypoxia can be mediated by multiple mechanisms and that both A2AR and key molecules in the A2AR-independent T-cell inhibition should be targeted to overcome the hypoxia-related immunosuppression in infected tissues and tumors.

Keywords: IFN-γ; immunosuppression; inflammation; oxygen; tumor microenvironment.

Fig. 1.

Suppression of T-cell functions by hypoxia did not require involvement of A2AR. Spleen cells from WT and A2AR^−/− mice were stimulated with anti-CD3 mAb for 24h in the presence of various concentrations of CGS. Cell proliferative activity was monitored by [³H] thymidine uptake after 24h (A). IFN-γ levels in the supernatant were also determined after 24h (B). Hypoxia significantly inhibited cell proliferation and IFN-γ production in both WT and A2AR^−/− T cells. Inhibition by CGS was observed only in WT cells. Data represent average ± SD of triplicate samples. a, P < 0.05; b, P < 0.01; c, P < 0.001 versus no CGS. d, P < 0.05; e, P < 0.01; f, P < 0.001; 21 versus 1% oxygen (no CGS).

Fig. 2.

Hypoxia strongly suppressed T-cell proliferation as compared with the effect of A2AR stimulation by CGS. Proliferation of CFSE-labeled CD4⁺ and CD8⁺ cells after 36h was monitored separately for WT (A) and A2AR^−/− (B) T cells. Peaks with diminished fluorescence intensity (shown by numbers) indicate dilution of CFSE in proliferated cells. Results for normoxic culture, hypoxic culture and CGS (10⁻⁵ M) treatment shown here are representative of four separate experiments with similar results. (C) Proliferation index calculated as in Methods. The index represents average times of cell division in cells that divided at least once. Therefore, the minimum number of proliferation index is one. WT, WT mice; A2A, A2AR^−/− mice; N, normoxic atmosphere (21% oxygen); H, hypoxic atmosphere (1% oxygen); C, CGS (10⁻⁵ M); CH, CGS + 1% oxygen. Data represent average ± SD of triplicate samples. a, P < 0.05; c, P < 0.001 versus normoxia.

Fig. 3.

Hypoxia inhibited early activation of T cells in vivo even in the absence of A2AR. WT and A2AR^−/− mice received injection of anti-CD3 mAb while breathing a normoxic or hypoxic (8% oxygen) atmosphere. Changes in CD69 (A) and CD25 (B) expression on T cells in the spleen were analyzed 2h after the injection of anti-CD3 mAb. Percentages of CD69-positive or CD25-positive cells in the CD4⁺ or CD8⁺ population are shown. Data represent average ± SD of 3–5 mice. a, P < 0.05; b, P < 0.01; c, P < 0.001 versus anti-CD3 mAb + normoxia.

Fig. 4.

A2AR-independent inhibition of T-cell proliferation in mice exposed to a hypoxic atmosphere. After the transfer of CFSE-labeled spleen cells from WT and A2AR^−/− (A2A) mice, the recipient RAG1^−/− mice received an injection of anti-CD3 mAb and were maintained under an either normoxic (N) or hypoxic (H; 8% oxygen) atmosphere. After 40h, the CFSE pattern in the spleen cells was analyzed by flow cytometry. T cells derived from WT and A2AR^−/− mice were discriminated by the expression of Thy1.1 and Thy1.2 markers. (A) Representative pattern of cell proliferation. (B) Proliferation index calculated as in Methods. Data represent average ± SD of three mice. c, P < 0.001 versus normoxia.

Fig. 5.

Decrease in cytotoxicity of effector T cells induced by mixed lymphocyte culture under hypoxic atmosphere. C57BL/6 WT (A) and A2AR^−/− (B) spleen cells (H-2^b) were cultured with BALB/c spleen cells (H-2^d) for 5 days under 21 or 1% oxygen. Cytotoxicity against allogenic target cells was determined by a ⁵¹Cr release assay using P815 cells (H-2^d). Data represent average ± SD of triplicate samples. a, P < 0.05; b, P < 0.01; c, P < 0.001 versus 21% oxygen.

Fig. 6.

Activated T cells developed in the presence of CGS retain an immunosuppressed phenotype, but the inhibitory effect of hypoxia was temporary and T cells recovered effector function. T-cell activation was induced by anti-CD3 mAb with CGS (10⁻⁶, 10⁻⁵ M) or hypoxia (1% oxygen) for 40h. After washing, the cells were re-stimulated for 24h with immobilized anti-CD3 mAb for the measurement of IFN-γ levels. (A) IFN-γ levels after 40-h primary stimulation. (B) IFN-γ levels after re-stimulation. Data represent average ± SD of triplicate samples. a, P < 0.05; b, P < 0.01; c, P < 0.001 versus control.