Adenosine A_2B receptor antagonist suppresses differentiation to regulatory T cells without suppressing activation of T cells

Abstract

Extracellular adenosine activates P1 receptors (A₁, A_2A, A_2B, A₃) on cellular membranes. Here, we investigated the involvement of P1 receptor-mediated signaling in differentiation to regulatory T cells (Treg). Treg were induced in vitro by incubating isolated CD4⁺CD62L⁺ naïve murine T cells under Treg-skewing conditions. Antagonists of A₁ and A_2B receptors suppressed the expression of Foxp3, a specific marker of Treg, and the production of IL-10, suggesting the involvement of A₁ and A_2B receptors in differentiation to Treg. We also investigated the effect of these antagonists on T cell activation, which is essential for differentiation to Treg, and found that A₁ antagonist, but not A_2B antagonist, suppressed T cell activation. We conclude that A₁ and A_2B receptors are both involved in differentiation to Treg, but through different mechanisms. Since A_2B antagonist blocked differentiation to Treg without suppressing T cell activation, it is possible that blockade of A_2B receptor would facilitate tumor immunity.

Introduction

Nucleotides and nucleosides are physiologically released into extracellular space from various types of cells at sites of inflammation and are thought to modulate immune response [1], [2], [3]. Adenosine exerts its actions by activating P1 receptors, while nucleotides bind to P2X_1–7 and P2Y_1–14 receptors. As regards the process of T cell activation, it has been reported that ATP is released through a gap junction hemichannel, pannexin-1, and maxi-anion channel after stimulation of T cell receptor (TCR) [4], [5]. We have recently shown that vesicular exocytosis of ATP and activation of P2X₇ and P2Y₆ receptors are involved in T cell activation via TCR [6], [7]. Extracellular ATP is immediately degraded to adenosine by extracellular ectonucleotidases, including CD39 and CD73 [8], and activates P1 receptors [9], [10]. Extracellular adenosine binds to P1 receptor, which belongs to the G protein-coupled receptor family. There are four different types of P1 receptors, i.e., adenosine A₁, A_2A, A_2B, and A₃ receptors. Little is known about the functional significance of the A_2B receptor because of its lower affinity for the endogenous ligand, adenosine, and because no specific agonist for this receptor is available, but recent studies have revealed the importance of A_2B receptor in regulating immunity and inflammation [11]. However, the involvement of P1 receptors, especially A_2B receptors, in differentiation of T cells into T-cell subsets has not been reported.

Upon interaction with self- or cross-reactive antigen, naïve CD4⁺ T helper cells are activated, then expand and differentiate into various effector T-cell subsets. Depending on the cytokines they produce, these T-cell subsets have very different properties. Thus, T helper cells include not only the well-defined effector subsets Th1 cells and Th2 cells [12], as well as the more recently described Th17 [13], [14] and Th9 [15], [16] cells, but also regulatory subsets such as induced regulatory T cells (Treg) [17], [18] and Tr1 cells [19]. A CD4⁺ T cell subset, IL-17-producing Th17 cells, has been described and shown to have a critical role in the induction of autoimmune diseases [13], [14]. On the other hand, Treg plays a part in the maintenance of peripheral immune tolerance and prevention of immune-mediated diseases by suppressing immune responses [20]. Regulatory T cells express CD4, IL-2 receptor α-chain (CD25), and transcriptional factor Forkhead box P3 (Foxp3), which appears to function as a master regulator in the development and function of Treg [21]. Although the mechanisms of Treg action remain poorly understood and contentious, secretion of inhibitory cytokines TGF-β and IL-10 has been implicated in the effector function of Treg [22], [23]. Both Th17 cells and Treg are derived from naïve CD4⁺ T cells; the naïve cells differentiate to Th17 in the presence of TGF-β and IL-6 [9], [10], and to Treg in the presence of TGF-β and IL-2 [24], [25]. It has also been reported that extracellular ATP is involved in Th17 cell differentiation in lamina propria [26].

As mentioned above, ATP is released during T cell activation via TCR. Considering that extracellular ATP is immediately degraded to adenosine, we hypothesized that adenosine is involved in differentiation of naïve CD4⁺ T cells into Th cell subsets, such as Treg. Since in vitro studies have shown that conversion of naïve peripheral CD4⁺ T cells to functional Foxp3⁺ Treg could be achieved through ligation of TCR in the presence of IL-2 and TGF-β [25], we utilized this Treg induction system to investigate the involvement of P1 receptors in Treg differentiation. Our data indicate that A₁ receptor is involved in both differentiation of Treg (expression of Foxp3 and production of IL-10) and TCR-dependent T cell activation (expression of CD25 and production of IL-2). On the other hand, A_2B receptor plays an important role in the process of differentiation to Treg, but not in T cell activation. Thus, we suggest that A₁ and A_2B receptors are both involved in the induction of Treg, but through different mechanisms.