Maintenance of immune tolerance by Foxp3+ regulatory T cells requires CD69 expression
Graphical abstract
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.
Section snippets
Animals
CD69-deficient mice were generated as described [13]. C57BL/6 Tg(TcraTcrb)425Cbn/J mice expressing a T-cell receptor specific for peptide 323–339 of OVA in the context of I-Ab (OTII mice) were purchased from the Jackson Laboratory (stock number 004194). OTII mice were backcrossed with CD69-deficient mice in the C57BL/6 background (OTKO mice). For in vivo tolerogenic asthma experiments, we used females 10–12 weeks old that were either littermates or age-matched offspring in BALB/c or OTII
CD69 is constitutively expressed by a subset of thymic and peripheral Tregs in steady state
A subpopulation of about fifty percent of total CD4+CD25+FoxP3+ Treg cells in thymus and secondary lymphoid organs express CD69 on their membrane in steady state (Fig. 1A). Further phenotypic analysis of peripheral CD4+FoxP3+ gated CD69+ and CD69− Tregs from Cd69+/+ or Cd69−/− mice in homeostasis (Fig. 1B), revealed that the suppression-associated markers CTLA-4, ICOS, CD38 and GITR are expressed at higher levels in CD69+-Tregs in steady state compared to CD69− Tregs or Tregs from Cd69−/− mice (
Discussion
Here we identify a unique CD69-constitutive expressing subset of Treg cells in homeostatic and inflammatory conditions with higher suppressor potential, than the classical FoxP3+ Treg cell population as a whole. Consistent with previous evidence that a subset of thymocytes with high expression of CD69 is the precursor of human natural Tregs [21], the peripheral subset of CD69+ Treg differs from CD69− Tregs in the basal expression levels of naturally occurring Treg markers, such as CD25, CTLA-4,
Conclusions
Several mechanisms of suppression by Tregs have been proposed; however, Tregs need to express CD69 to sustain immune tolerance. Our results thus show that CD69 is a key molecular target for controlling Treg-suppressive function and development of inflammation. The identification of this new subset of CD69+ Tregs paves the way toward the development of new strategies of Treg cell isolation for therapeutic purposes towards chronic inflammatory and autoimmune diseases.
Author contributions
J.R.C. and R.S-D performed most experiments and analyzed the data; O.B. contributed confocal microscopy images, analyzed the data and reviewed the manuscript; E.R.B., S.L. and A.M-M. performed experiments; M.L.T. designed experiments and revised the manuscript; F.S-M. designed experiments and revised the manuscript; and P.M. conceived and coordinated the study, designed experiments, oversaw the data analysis and wrote the paper.
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgments
The authors thank Dr. R.A. Flavell for kindly provided the Foxp3-RFP reporter mice, S. Bartlett for editorial assistance, R. Strippoli and M.A. del Pozo for kindly providing the Ci1040 ERK inhibitor and Mª Carmen Gómez de Frutos for technical help. This work was supported by funding from the Spanish Ministry of Economy and Competitiveness: SAF2011-27330 to P.M., SAF2010-15106 to M.L.T and SAF2011-25834 to F.S-M.; grant INDISNET (S2010/BMD-2332) from Comunidad de Madrid and RETICS Enfermedades
References (44)
- et al.
Mechanisms of allergen-specific immunotherapy
J Allergy Clin Immunol
(2011) - et al.
The CD69 receptor: a multipurpose cell-surface trigger for hematopoietic cells
Immunol Today
(1994) - et al.
TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells
Immunity
(2006) - et al.
Plasmacytoid dendritic cells resident in human thymus drive natural Treg cell development
Blood
(2010) - et al.
Foxp3 transcription-factor-dependent and -independent regulation of the regulatory T cell transcriptional signature
Immunity
(2007) - et al.
Adaptive Foxp3+ regulatory T cell-dependent and -independent control of allergic inflammation
Immunity
(2008) - et al.
CD69 suppresses sphingosine 1-phosophate receptor-1 (S1P1) function through interaction with membrane helix 4
J Biol Chem
(2010) - et al.
FOXP3+ regulatory T cells in the human immune system
Nat Rev Immunol
(2010) - et al.
Molecular cues guiding inflammatory responses
Cardiovasc Res
(2010) - et al.
CD69 acts downstream of interferon-alpha/beta to inhibit S1P1 and lymphocyte egress from lymphoid organs
Nature
(2006)
Molecular cloning, expression, and chromosomal localization of the human earliest lymphocyte activation antigen AIM/CD69, a new member of the C-type animal lectin superfamily of signal-transmitting receptors
J Exp Med
CD69-triggered ERK activation and functions are negatively regulated by CD94/NKG2-A inhibitory receptor
Eur J Immunol
Triggering of T cell proliferation through AIM, an activation inducer molecule expressed on activated human lymphocytes
J Exp Med
CD69: an unexpected regulator of TH17 cell-driven inflammatory responses
Sci Signal
CD69 downregulates autoimmune reactivity through active transforming growth factor-beta production in collagen-induced arthritis
J Clin Invest
Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells
Nature
CD69 association with Jak3/Stat5 proteins regulates Th17 cell differentiation
Mol Cell Biol
Essential role of lung plasmacytoid dendritic cells in preventing asthmatic reactions to harmless inhaled antigen
J Exp Med
CD69 targeting differentially affects the course of collagen-induced arthritis
J Leukoc Biol
IL-2 receptor beta-dependent STAT5 activation is required for the development of Foxp3+ regulatory T cells
J Immunol
Blockade of the MAP kinase pathway suppresses growth of colon tumors in vivo
Nat Med
Phenotypical and functional specialization of FOXP3+ regulatory T cells
Nat Rev Immunol
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Those authors contributed equally to this work.