Figure 7.
Hypoxia increases the number of Tregs with suppressive characteristics in an A2A-dependent manner. (A) Cumulative data of the percentage of CD4+/CD25high/CD127− cells evaluated in 7 randomly selected CLL patients after 3 days of culture under normoxia or hypoxia. Where indicated, the A2A antagonist SCH58261 (10 µM) was added for the entire experiment. (B) RT-PCR data showing an increased expression of FoxP3 (FOXP3, n = 8) in T lymphocytes purified from CLL PBMCs cultured for 5 days in the presence of anti-CD3 (2 µg/mL) and anti-CD28 (1 µg/mL) antibodies and IL-2 (100 U/mL). Cultures were maintained at 21% or 1% O2. The A2A antagonist SCH58261 (10 μM) was added where indicated. (C) Representative immunohistochemical FoxP3 staining of CLL (left panel) and reactive (middle panel) LN sections indicating a greater number of FoxP3+ cells in the CLL LN compartment than in the reactive counterpart. Cumulative data on the percentage of FoxP3+ area confirmed statistical significant differences (n = 20, right panel). Original magnification ×40. (D-F) RT-PCR data on the expression of TGF-β1 (TGFB1, n = 10; D), interferon γ (IFNG, n = 10; E), and the immunoinhibitory molecule PD-1 (PDCD1, n = 10; F, left panel) in sorted T cells after 5 days of normoxic and hypoxic culture under activated conditions. Antagonist SCH58261 (10 µM) was added where indicated. (F) Representative image showing PD-1 and CD4 co-localization in a typical CLL LN section. Original magnification ×63. Scale bar, 25 µm. (G) RT-PCR analysis of the IL-10 cytokine (IL10, n = 10) in T cells sorted from CLL patients after 5 days of normoxic and hypoxic culture with or without the A2A antagonist (left panel). Secretion of IL-10 was confirmed by ELISA of T-cell culture supernatants (right panel). SCH58261 (10 µM) was added for the entire experiment where indicated. Statistical differences were analyzed with the Wilcoxon signed rank and Mann-Whitney U tests followed by the Tukey test for panels B and E-G. H, hypoxia (gray boxes); N, normoxia (open boxes); SCH, SCH58261.

Hypoxia increases the number of Tregs with suppressive characteristics in an A2A-dependent manner. (A) Cumulative data of the percentage of CD4+/CD25high/CD127− cells evaluated in 7 randomly selected CLL patients after 3 days of culture under normoxia or hypoxia. Where indicated, the A2A antagonist SCH58261 (10 µM) was added for the entire experiment. (B) RT-PCR data showing an increased expression of FoxP3 (FOXP3, n = 8) in T lymphocytes purified from CLL PBMCs cultured for 5 days in the presence of anti-CD3 (2 µg/mL) and anti-CD28 (1 µg/mL) antibodies and IL-2 (100 U/mL). Cultures were maintained at 21% or 1% O2. The A2A antagonist SCH58261 (10 μM) was added where indicated. (C) Representative immunohistochemical FoxP3 staining of CLL (left panel) and reactive (middle panel) LN sections indicating a greater number of FoxP3+ cells in the CLL LN compartment than in the reactive counterpart. Cumulative data on the percentage of FoxP3+ area confirmed statistical significant differences (n = 20, right panel). Original magnification ×40. (D-F) RT-PCR data on the expression of TGF-β1 (TGFB1, n = 10; D), interferon γ (IFNG, n = 10; E), and the immunoinhibitory molecule PD-1 (PDCD1, n = 10; F, left panel) in sorted T cells after 5 days of normoxic and hypoxic culture under activated conditions. Antagonist SCH58261 (10 µM) was added where indicated. (F) Representative image showing PD-1 and CD4 co-localization in a typical CLL LN section. Original magnification ×63. Scale bar, 25 µm. (G) RT-PCR analysis of the IL-10 cytokine (IL10, n = 10) in T cells sorted from CLL patients after 5 days of normoxic and hypoxic culture with or without the A2A antagonist (left panel). Secretion of IL-10 was confirmed by ELISA of T-cell culture supernatants (right panel). SCH58261 (10 µM) was added for the entire experiment where indicated. Statistical differences were analyzed with the Wilcoxon signed rank and Mann-Whitney U tests followed by the Tukey test for panels B and E-G. H, hypoxia (gray boxes); N, normoxia (open boxes); SCH, SCH58261.

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