Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression

Abstract

The study of T regulatory cells (T reg cells) has been limited by the lack of specific surface markers and an inability to define mechanisms of suppression. We show that the expression of CD39/ENTPD1 in concert with CD73/ecto-5'-nucleotidase distinguishes CD4(+)/CD25(+)/Foxp3(+) T reg cells from other T cells. These ectoenzymes generate pericellular adenosine from extracellular nucleotides. The coordinated expression of CD39/CD73 on T reg cells and the adenosine A2A receptor on activated T effector cells generates immunosuppressive loops, indicating roles in the inhibitory function of T reg cells. Consequently, T reg cells from Cd39-null mice show impaired suppressive properties in vitro and fail to block allograft rejection in vivo. We conclude that CD39 and CD73 are surface markers of T reg cells that impart a specific biochemical signature characterized by adenosine generation that has functional relevance for cellular immunoregulation.

Figure 1.

CD4⁺/CD25⁺ cells express CD39. (A) The expression of CD39 (open profiles) on mouse lymph node cell suspensions is shown in the histograms on the right, contrasted against control IgG (gray). The expression of CD39 on CD4⁺, CD8⁺, and B220⁺ subsets is shown in the density plots. (B) Lymph node cells were stained with CD4-Cy5 and CD25-PE. CD39 expression in gated populations is shown in the histograms (open) with irrelevant control (gray). (C) CD4⁺ cells were stained for CD39 (horizontal axis) and the indicated markers. (D) Real-time PCR analysis in CD4⁺/CD39⁺ and CD4⁺/CD39⁻ cells. (E) 5 × 10⁴ CD4⁺/CD25⁻ T cells were mixed with CD4⁺/CD39⁺ or CD4⁺/CD25⁺ cells. [³H]Thymidine incorporation was measured after 3 d. Data are means with SEM (error bars) of three independent experiments.

Figure 2.

Foxp3⁺ cells express CD39. (A) CD4⁺ T cells from Foxp3⁺ (GFP⁺) knock-in mice stained with CD25-PE or CD39-PE. (B) Real-time PCR analysis of Foxp3⁺/CD39⁺ (black bars), Foxp3⁻/CD39⁺ (shaded bars), and Foxp3⁻/CD39⁻ (white bars) cells. (C) Foxp3⁺/CD39⁺ suppresses T effector (Foxp3⁻/CD39⁻ CD4⁺) proliferation. [³H]Thymidine incorporation was measured after 72 h. Data are representative of two independent experiments, with error bars representing the SD of triplicate wells. (D) Foxp3⁺(GFP⁺) cells stained for CD39-APC and CD73-PE. (E) CD73 mRNA expression in Foxp3⁺/CD39⁺ (black bars), Foxp3⁻/CD39⁺ (shaded bars), Foxp3⁻/CD39⁻ (white bars), and B lymphocytes (striped bars).

Figure 3.

Concomitant expression of CD39/CD73 and generation of adenosine distinguishes T reg cells from other T cells. (A) CD39/NTPDase activity in CD4⁺/CD25⁺ and CD4⁺/CD25⁻ cells. (B) Hydrolysis of extracellular ¹⁴C-radiolabeled ADP to adenosine is catalyzed by WT CD4⁺ (lanes 4–8) but not Cd39-null CD4⁺ (lanes 9–13) mouse T cells. The radiolabeled standards are designated as ADP, AMP, and Adenosine (lanes 1–3). (C) Hydrolysis of extracellular ADP by Foxp3⁺/CD39⁺ (first to fifth lanes), Foxp3⁻/CD39⁺ (6th to 10th lanes), and Foxp3⁻/CD39⁻ (11th to 15th lanes) cells.

Figure 4.

Adenosine generated by CD39 and CD73 suppresses T cell proliferation. (A) mRNA expression of adenosine A2A receptor. Cells were activated for 5 d in vitro. Data are means of three independent experiments. (B) ATL146e inhibits T cell proliferation in response to alloantigens. 5 × 10⁴ CD4⁺/CD25⁻ T cells were cultured alone or in the presence of irradiated allogeneic splenocytes (shaded bar) with ATL146e at indicated concentrations. Data are means with SEM (error bars) of more than five independent experiments (P < 0.05 vs. CD4⁺/CD25⁻ cells alone). (C) Inhibition of T cell proliferation by adenosine receptor agonists. C277, E2387, C7938, and ATL146e (1 μM final) were added at the beginning of the cultures. [³H]Thymidine incorporation was assayed at day 5. Data are representative of five independent experiments. (D) Proliferation of CD4⁺/CD25⁻ T cells purified from A2A-null (black bars) or WT (white bars) mice and stimulated by 2.5 μg/ml of plate-bound anti-CD3 and 2.5 μg/ml of soluble anti-CD28. [³H]Thymidine was added for the last 8 h of culture. Representative data are from three experiments. *, P < 0.002 between WT and A2A null at 6 d of culture.

Figure 5.

T reg cells from Cd39-null mice are constitutively activated and fail to suppress CD4⁺/CD25⁺ cell proliferation. (A) CD25 and CTLA-4 mRNA expression in CD4⁺/CD25⁻ and CD4⁺/CD25⁺ cells isolated from Cd39-null (black bars) or WT mice (white bars). (B) Proliferation assay of CD4⁺/CD25⁺ T cells purified from Cd39-null (black bars) or WT (white bars) mice. Cells were cultured in the presence or absence of 2.5 μg/ml of plate-bound anti-CD3 and 2.5 μg/ml of soluble anti-CD28 for 3 d. (C) Proliferation of CD4⁺/CD25⁻ T cells purified from Cd39-null (black bars) or WT (white bars) mice cultured in the presence of irradiated allogeneic splenocytes. Apyrase reconstitution effects are noted at day 6 (shaded bars). Error bars represent the SEM of three independent experiments. (D) T reg cell function. Cd39-null T reg cell effects of the stimulation of A2A-null Teff (open) and WT T reg cells on WT Teff (closed; 1:1 ratio) are compared at day 5. A represents the nonproliferating cell populations, B represents CD4⁺/CD25⁻ cells that have entered into the cell cycle, and C represents unlabeled T reg cells. The percentage of cells present in A or B was quantified using FlowJo software. Data are representative of three independent experiments. (E) Skin allograft survival. C57BL/6 Rag 1–deficient mice received allogeneic skin grafts 24 h after the passive transfer of CD4⁺/CD25⁺ and/or CD4⁺/CD25⁻ cells from WT or Cd39-null mice in a ratio of 4:1 (32, 33). Mice receiving WT (circles) or Cd39-null (triangles) CD4⁺/CD25⁻ cells rejected the skin graft at a median of 13 d (n = 6). Mice receiving WT CD4⁺/CD25⁺ cells transferred in excess (diamonds; 4:1) with WT CD4⁺/CD25⁻ cells showed long-term graft survival (n = 9). Four out of eight mice receiving Cd39-null CD4⁺/CD25⁺ cells transferred in excess (squares; 4:1) with Cd39-null CD4⁺/CD25⁻ cells rejected the skin allograft at a median of 24.5 d (P = 0.02).