Maintenance of immune tolerance by Foxp3⁺ regulatory T cells requires CD69 expression

Highlights

• CD69 is constitutively expressed by a subset of Tregs in steady state.

• The suppressive function of Tregs is dependent on CD69 expression.

• Inhibition of ERK pathway restores the suppressor potential of Cd69^−/− Tregs.

• Tregs from Cd69^−/− mice have reduced capacity to maintain immune tolerance.

• Immunotherapy with CD69⁺ Tregs, restore immune tolerance and avoid inflammation.

Abstract

Although FoxP3⁺ regulatory T cells are key players in the maintenance of immune tolerance and autoimmunity, the lack of specific markers constitute an obstacle to their use for immunotherapy protocols. In this study, we have investigated the role of the C-type lectin receptor CD69 in the suppressor function of Tregs and maintenance of immune tolerance towards harmless inhaled antigens. We identified a novel FoxP3⁺CD69⁺ Treg subset capable to maintain immune tolerance and protect to developing inflammation. Although CD69⁺ and CD69⁻FoxP3⁺ Tregs exist in homeostasis, only CD69-expressing Tregs express high levels of CTLA-4, ICOS, CD38 and GITR suppression-associated markers, secrete high amounts of TGFβ and have potent suppressor activity. This activity is regulated by STAT5 and ERK signaling pathways and is impaired by antibody-mediated down-regulation of CD69 expression. Moreover, immunotherapy with FoxP3⁺CD69⁺ Tregs restores the homeostasis in Cd69^−/− mice, that fail to induce tolerance, and is also highly proficient in the prevention of inflammation. The identification of the FoxP3⁺CD69⁺ Treg subset paves the way toward the development of new therapeutic strategies to control immune homeostasis and autoimmunity.

Introduction

FoxP3⁺ regulatory T (Treg) cells are a subset of CD4⁺ T lymphocytes with suppressive activity, key mediator of peripheral tolerance and essential for preventing autoimmune and chronic inflammatory diseases. However, the lack of specific markers and insufficient understanding of Tregs biology constitute the two largest obstacles to develop immunotherapy protocols for the treatment of these diseases [1]. Treg cells inhibit proliferation and function of effector T cells through various mechanisms, including cell–cell contact and the production of anti-inflammatory cytokines such as TGF-β or IL-10. Tregs can develop in the thymus, or from naïve CD4⁺ T cells in the periphery [2]. After antigen challenge lymph nodes are the sites of differentiation of effector T cells and/or Tregs, which upregulate specific adhesion and chemokine receptors and migrate to the inflamed tissue. This regulatory mechanism of cell migration is critical for a proper balance between the innate and adaptive immune responses in the inflamed tissue [3]. In this regard, S1P₁ has been described as an important molecule controlling lymphocyte egress from lymphoid organs and leukocyte receptor CD69 negatively regulates its expression [4].

The early leukocyte activation antigen CD69 is a membrane receptor from the family of type II C-type lectins. CD69 is rapidly induced after cell activation in all bone marrow derived cells except erythrocytes [5], [6]. Expression of CD69 in vivo is restricted to positively selected thymocytes and leukocytes undergoing activation, particularly at inflammatory sites. Engagement of CD69 with monoclonal antibodies (mAbs) in the presence of phorbol esters induces a strong Ca²⁺ influx leading to the activation of ERK, induction of IL-2 and IFN-γ genes, and T cell proliferation [7], [8]. However, in vivo studies with CD69-deficient mice revealed an unexpected immunoregulatory role [9]. A mouse model of lymphocyte-dependent collagen-induced arthritis (CIA) suggested that CD69 might act as a regulatory molecule by modulating TGF-β levels at the site of inflammation [10]. Since TGF-β participates in the differentiation both of regulatory T cells and of Th17 cells [11], [12], CD69 might regulate the immune response at the stage of T cell differentiation. The balance between Th17 and Treg cells is critical for the regulation of the immune response by determining the net balance of pro- and anti-inflammatory cytokines at the inflammatory foci.

In this report we have analyzed the role of CD69 in the function of FoxP3⁺ Tregs. We show that around half of the Tregs located in lymphoid organs express CD69 in steady state. CD69⁺ Tregs express higher surface levels of suppression-associated markers than CD69⁻ Tregs cells or Tregs from Cd69^−/− mice, and have enhanced suppressor activity in vitro and in a mouse model of lung tolerance to harmless antigens. Our results strongly support a role for CD69 as a critical receptor for controlling Treg-suppressor function in both physiological and pathological immune responses, including autoimmunity and allergy.