CD69: An Unexpected Regulator of TH17 Cell–Driven Inflammatory Responses

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Science Signaling  22 Mar 2011:
Vol. 4, Issue 165, pp. pe14
DOI: 10.1126/scisignal.2001825


Mice lacking the C-type lectin receptor CD69 develop exacerbated forms of arthritis, contact dermatitis, allergic asthma, and autoimmune myocarditis. Because the immune responses in these diseases are largely mediated by a balance between proinflammatory subsets of T effector cells called T helper (TH) 17 cells and regulatory T cells, these findings indicate a previously unappreciated regulatory role for CD69 in modulating T lymphocyte differentiation toward the TH17 lineage and suggest a role in regulatory T cell function. CD69 promotes activation of the Jak3−signal transducer and activator of transcription 5 (Stat5) signaling pathway, which inhibits TH17 cell differentiation, thus providing a mechanistic link between CD69 and the regulation of TH17 responses. This evidence underscores the potential of CD69 as target in the treatment of autoimmune and allergic diseases and is consistent with mounting evidence linking CD69 to regulatory T cell subsets.

T cells can differentiate, among other types, into CD4+CD25+FoxP3+ cells known as reg­ulatory T cells, which suppress inflammatory responses, and into a subset of effector T cells known as T helper (TH) 17 cells, which secrete interleukin (IL)-17 and promote inflammatory responses. In response to cytokines, the transcription factors retinoic acid receptor–related orphan receptor γt (RORγt) and signal transducer and activator of transcription (Stat) 3 mediate the generation of TH17 cells (13). Thus, signaling pathways that promote the nuclear translocation of phosphorylated and activated Stat3 favor TH17 cell differentiation. In contrast, TH17 differentiation is blocked by signaling pathways that promote phosphorylation and activation of the transcription factor Stat5 (4, 5), in particular those initiated by IL-2 (4). The balance between TH17 and regulatory T cells regulates the immune response through the net balance between pro- and anti-inflammatory cytokines at inflammatory foci. Thus, factors that regulate the differentiation or function of TH17 and regulatory T cells can alter inflammatory responses.

Accumulating evidence implicates the early leukocyte activation antigen CD69, a member of the type II C-lectin natural killer membrane receptor family, as a previously unappreciated regulator of immune responses. The abundance of CD69 rapidly increases after cell activation in all bone marrow–derived cells except erythrocytes (6, 7). Distribution in vivo is restricted to positively selected thymocytes and leukocytes undergoing activation, particularly at inflammatory sites. Engagement of CD69 with monoclonal antibodies in the presence of activators of protein kinase C (phorbol esters) triggers Ca2+ influx that leads to activation of extracellular signal–regulated kinase 1 and 2 (ERK1/2), induction of genes encoding IL-2 and interferon γ, and proliferation of  T cells (8, 9).

These observations provided an initial view of CD69 as a proinflammatory receptor that induced leukocyte activation (9, 10); however, full elucidation of the role of CD69 has been hampered by the limitations of the experimental models used and the unknown identity of CD69 ligand (or ligands). Some in vivo studies with CD69-deficient mice have suggested an immunoregulatory role for CD69. Experiments in a murine model of lymphocyte-dependent collagen-induced arthritis (CIA), a condition characterized by chronic inflammation, suggested that CD69 might regulate transforming growth factor–β (TGF-β) abundance at inflammation sites (11), where it appears to act as a local anti-inflammatory cytokine (12). TGF-β concentrations are low in joint tissue of CD69-deficient mice, and TGF-β participates in the differentiation of both regulatory T cells (13) and TH17 cells (14, 15), suggesting that CD69 might regulate the immune response through TGF-β (11) and by acting at the differentiation stage of T cells. In a model of granulocyte-mediated acute arthritis induced by administration of anti-type II collagen antibodies and endotoxin, one group reported that CD69-deficient mice were resistant to developing the disease (16). However, an independent study found that lack of CD69 had no effect on joint inflammation (17). To resolve this issue, wild-type mice were injected with monoclonal antibodies directed against CD69 that induce CD69 internalization, an approach that avoids possible undesired genetic alterations produced during the recombination process in the generation of the CD69-deficient mice. Antibody-induced CD69 blockade exacerbated CIA, but seemed not to have a major effect on acute inflammatory responses mediated by granulocytes (17, 18).

Several lines of evidence indicate that CD69 regulates the generation and function of TH17 cells (19). First, CD69 deficiency enhances the differentiation of IL-17–secreting T cells in vitro and is associated with higher abundance of TH17-associated molecules, such as IL-23R and the transcription factors required for the generation of TH17 cells, RORγt and Stat3 (13) (Fig. 1). Second, activation of Stat5 is impaired in CD69-deficient TH17 cells, leading to activation of Stat3, an event that favors TH17 cell differentiation (4, 5). Third, compared with wild-type cells, CD69-deficient TH17 cells produce lower amounts of IL-2 and show diminished activation of Stat5 and increased differentiation (19). Addition of exogenous IL-2 to CD69-deficient cells restores Stat5 activation, thereby inhibiting differentiation into TH17 cells. Blockade of IL-2 signaling in CD69+ cells partially inhibits Stat5 activation and stimulates differentiation into TH17 cells. In contrast, IL-2 blockade in CD69-deficient cells had no effect, indicating that CD69 promotes Stat5-mediated inhibition of TH17 cell differentiation independently of the IL-2 pathway (19) (Fig. 1). The cytoplasmic tail of CD69 is associated with Jak3 and Stat5 and increases their activity, thereby decreasing transcription of RORγt in human and mouse TH17 cells (19). These findings thus establish a mechanistic link between CD69 and the regulation of TH17 cell differentiation (Fig. 1).

Fig. 1

CD69 receptors are present on the membrane of T cells after activation. The cytoplasmic tail of CD69 associates with Jak3 and Stat5, triggering phosphorylation of Stat5 and its translocation to the nucleus, where it can activate the transcription factor FoxP3, which in turn stimulates the differentiation of regulatory T cells. CD69 engagement can also induce the expression of genes encoding IL-2 and TGF-β. These cytokines may act in an autocrine manner to induce the differentiation of regulatory T cells. CD69 can inhibit the TH17 differentiation pathway through at least two mechanisms: CD69-activated Stat5 inhibits the translocation of Stat3 to the nucleus and antagonizes Stat3-mediated RORγt activation through FoxP3 activation. MHC II, major histocompatibility complex II; P, phosphorylation. Dashed lines indicate indirect effects or interactions, and multiple arrows indicate multistep processes.


Other evidence supporting a role for CD69 in regulating TH17 cell function comes from the development of exacerbated forms of contact dermatitis, allergic asthma, and autoimmune myocarditis in CD69-deficient mice (20, 21). Miki-Hosokawa et al. reported impaired function and migration of effector T cells to lungs and resistance to induced asthma (22) in CD69-deficient mice. However, Martín et al., using independently generated CD69-deficient mice, found enhanced responses of effector T cells in lung and high susceptibility for developing induced asthma (20). Administration of monoclonal antibodies against CD69 to nondeficient mice increases the inflammatory response in lungs after asthma induction and in skin in a model of oxazolone-induced contact hypersensitivity, supporting the idea that CD69 modulates immune allergic processes by inhibiting T cell effector responses (20). Accordingly, in a model of experimental autoimmune myocarditis induced by immunization with cardiac α-myosin heavy chain–derived peptides, CD69 deficiency enhances responses of TH17 cells in heart, myocardial inflammation, and the progression to heart failure (21).

The diseases studied in these models are mediated, entirely or in part, by TH17 cells, thus indicating that lack of CD69 enhances TH17 cell differentiation in vitro. One way in which CD69 might determine the outcome of TH17 responses in vivo is by regulating the function or differentiation of regulatory T cells. Evidence supporting this hypothesis comes from several reports. NZBxNZW mice, which are a model of T cell–dependent systemic lupus erythematosus, have increased numbers of CD4+ T cells that are also positive for CD69; these cells can suppress the synthesis of IL-2 by CD4+CD69 T cells (23). As mentioned above, diminished production of IL-2 favors the differentiation of TH17 cells and inhibits the generation of regulatory T cells (23). In another study, Han et al. identified a tumor-induced CD69+CD4+CD25 T cell subset with a high abundance of IL-2 receptor β chain (CD122) and of surface TGF-β1, which suppressed T cell proliferation (24). CD69 engagement in these cells through ERK1/2 activation maintains TGF-β1 on the membrane, an effect that is responsible for their immunosuppressive activity (24). In addition, the defective function of regulatory T cells observed in individuals with systemic sclerosis is associated with diminished abundance of CD69 and TGF-β. Plasma from these individuals can also reduce the abundance of CD69 in regulatory T cells and impair their suppressive capacity (25). Finally, the development of natural regulatory T cells in human thymus has been linked to CD69. Activated dendritic cells induce the differentiation of CD4+CD8+ thymocytes with high abundance of CD69 into CD4+CD25+FoxP3+ regulatory T cells (26). Thus, these data indicate that the abundance of CD69 in T cells might regulate the balance between TH17 and regulatory T cells in both homeostasis and inflam­mation.

On the molecular level, CD69 affects the function of the sphingosine 1-phosphate receptor 1 (S1P1), which controls the egress of lymphocytes from lymphoid tissues (27). The absence of CD69 is associated with increased abundance of S1P1 and, therefore, with enhanced migration of T lymphocytes to lymph nodes or inflamed tissues (28). Constitutive expression of S1P1 in CD4+ T cells induces the secretion of high concentrations of IL-17 and differentiation into TH17 cells (29), whereas S1P1 deficiency stimulates the differentiation of CD4+CD25+FoxP3+ regulatory T cells through activation of the mammalian target of rapamycin–AKT signaling pathway (30). CD69 can associate with S1P1 in the cell membrane through a mechanism dependent on membrane helix four and induce a conformation of S1P1 that favors its internalization and degradation (31). Accordingly, S1P1 surface abundance is increased on CD69−/− TH17 cells in vitro and after the adoptive transfer of TH2 and TH17 cells in ovoalbumin-induced allergic asthma (20). High surface abundance of S1P1 promotes the migration of leukocytes to inflamed tissues, thereby supporting the idea that CD69 deficiency indirectly facilitates tissue infiltration by inflammatory cells. For example, in a mouse model of contact sensitization, S1P1 promotes migration of dendritic cells (32), and CD69 deficiency increases the abundance of S1P1 and enhances the migratory behavior of these cells in vitro and in vivo (33). Thus, these studies suggest that CD69 contributes to the resolution of inflammation by modulating the balance between TH17 and T regulatory cells and migration of dendritic cells and that this effect is mediated, at least in part, by decreased S1P1 abundance.

Understanding the mechanisms that control the immune response is an active research area aimed at generating novel therapies for diverse autoimmune and inflammatory diseases. Several inhibitors of  T cell function that regulate the inflammatory cascade are now important targets for the treatment of autoimmune diseases (34, 35). CD69 inhibits TH17 cell differentiation and function; regulates local TGF-β production and, presumably, the function of regulatory T cells; and affects the ability of leukocytes to migrate to inflamed tissues. CD69 can thus potentially join this group of inhibitors as a target molecule for the treatment of autoimmune diseases.

References and Notes

  1. Acknowledgments: We thank R. González-Amaro, D. Sancho, J. L. Rodríguez, and S. Bartlett for comments and critical reading of the manuscript. Funding: This work was supported by grant SAF2008-02719 from the Spanish Ministry of Science and Innovation (MICINN) to P.M. and grants SAF2008-02635 (MICINN) and INSINET 01592006 [Comunidad de Madrid (FONCICYT-C002-2009-1ALA/127249)] and a MEICA award (Genoma España) to F.S.-M. P.M. holds a MICINN “Ramón y Cajal” contract (RYC-2006). The CNIC is supported by the MICINN and the Pro CNIC Foundation.

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