CD69 is an immunoregulatory moleculeinduced following activationDavid Sancho, Manuel Gomez and Francisco Sanchez-Madrid

Servicio de Inmunologıa, Hospital de la Princesa, Universidad Autonoma de Madrid, Diego de Leon, 62. E-28006. Madrid, Spain

CD69 is an early leukocyte activation molecule

expressed at sites of chronic inflammation. The precise

role of CD69 in immunity has not been elucidated owing

to the absence of a known ligand and adequate in vivo

models to study its physiological function. Although

previous in vitro studies suggest that CD69 is an

activatory molecule in different leukocyte subsets,

recent studies in CD69-deficient mice have revealed a

non-redundant role for this receptor in downregulation

of the immune response through the production of the

pleiotropic cytokine transforming growth factor-b

(TGF-b). The possible cellular and molecular mechan-

isms of action of this molecule are discussed herein.

Introduction

Self-limitation of the immune response is crucial to itscontrol and molecules induced during lymphocyte acti-vation might act as negative regulators. In this Opinion,we discuss recent results that identify CD69 as a potentialnegative regulator. CD69 is an early membrane receptortransiently expressed on lymphocyte activation, notdetected in resting lymphocytes, and selectively expressedin chronic inflammatory infiltrates and at the sites ofactive immune responses in vivo. Although early in vitrodata suggested that CD69 exerts a proinflammatoryfunction, recent in vivo results indicate that this receptormight act as a regulatory molecule, modulating theinflammatory response. In addition, CD69 might actspecifically on an as yet uncharacterized T-cell regulatorysubset. These recent insights provide a novel view of thefunction of this receptor, even though a full picture of thespatial and temporal regulation of the immune response byCD69 will require detailed characterization of its ligand(s).

Early data: CD69 exerts a co-stimulatory effect in vitro

The CD69 gene is located within the natural killer (NK)gene complex on mouse chromosome 6 and humanchromosome 12 [1,2] and codes for a type II C-type lectinascribed to the family of NK receptors. CD69 is expressedfollowing activation in all bone marrow-derived cellsexcept erythrocytes (reviewed in Ref. [3]). Most NK lectinreceptors directly mediate their activatory or inhibitoryeffects through their cytoplasmic domains [4]. However,the cytoplasmic domain of CD69 is short and lacks anyidentifiable function-associated motifs. Thus, no

Corresponding author: Sanchez-Madrid, F. (fsanchez.hlpr@salud.madrid.org).Available online 5 January 2005

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signalling proteins have been described that directlyassociate with its cytoplasmic domain, although recentresults show that CD69 activates Syk in a Src-dependentmanner in NK cells [5]. These tyrosine kinases controldownstream activation of phospholipase Cg2 (PLCg2) andVav1 that, in turn, activate the Rac–ERK (extracellularsignal-regulated protein kinase) pathway [6], which isimplicated in NK-cell activation. In addition, some studieshave reported the involvement of a CD69-coupled hetero-trimeric G protein in its intracellular signalling path-way [7–9].

The rapid and transient induction of CD69 expressionon Tcells suggests that it might enhance activation and/ordifferentiation, as occurs with CD40L (CD154) or CD25. Inthe absence of a known ligand, in vitro studies to dissectthe possible function of CD69 were based on the use ofspecific monoclonal antibodies (mAbs) (reviewed in Ref.[3]). In the presence of phorbol esters, anti-CD69 mAbsstimulate the production of interleukin-2 (IL-2), whichincreases T-cell proliferation [10,11], and tumour necrosisfactor-a (TNF-a) synthesis [9,12], whereas they inducenitric oxide (NO) secretion by monocytes [13] andactivation of arachidonic acid metabolism and degranula-tion in platelets [14], suggesting that CD69 could act as aproinflammatory receptor. In addition, cross-linking ofCD69 with secondary antibodies mediates early signallingevents, such as extracellular Ca2C influx [9–11], relievingthe blockade in capacitative calcium entry in antigen-primed T cells [8]. Moreover, antibodies against CD69significantly inhibit the ability of T cells to activatemacrophages by cell contact [15], suggesting that aputative CD69 counter-receptor expressed by macro-phages is involved in the production of proinflammatorycytokines. Therefore, CD69 can apparently mediateimmune cell activation and exert proinflammatory effectsin vitro either directly or indirectly. However, CD69engagement also triggers apoptosis in different celltypes, such as monocytes or eosinophils [16,17], andmight mediate inhibitory signals on IL-1 receptor(IL-1R)- or CD3-mediated T-cell proliferation [18]. Allthese data indicate that CD69 behaves in vitro more as aco-stimulatory receptor than as a net inhibitory oractivatory molecule, although the fate of this co-stimu-lation could vary depending on the cellular context.

Recent insights: immunoregulatory role of CD69

The in vivo models initially chosen for the study of CD69function were based on its pattern of expression. Studies

Opinion TRENDS in Immunology Vol.26 No.3 March 2005

. doi:10.1016/j.it.2004.12.006

Opinion TRENDS in Immunology Vol.26 No.3 March 2005 137

in CD69-transgenic mice focused on thymic selection[19,20], a process in which CD69 expression is transientlyinduced (Box 1). Despite the in vitro evidence suggesting apossible proinflammatory role for CD69, constitutiveexpression of CD69 by T cells in transgenic mice is notassociated with inflammatory conditions [19,20]. Further-more, analysis of antigen-specific responses in mice hasnot revealed reduced T-cell activation in the absence ofCD69 [21], suggesting that this receptor does not exert anet positive co-stimulatory effect in T cells in vivo,although a redundant role as a positive co-stimulus forT cells cannot be ruled out.

Given the somewhat contradictory in vitro and in vivoresults, it became appropriate to study the role of CD69 inan in vivo model of chronic inflammation. This analysiswas based on two lines of evidence. First, CD69 ispersistently expressed at inflammatory foci [22]. Second,the CD69 gene is located at the Cia3 trait loci on ratchromosome 4 and mouse chromosome 6 [1], syntenic tohuman 12p12–p13 [2], a region that contains suscepti-bility loci for several autoimmune diseases, includingcollagen induced arthritis (CIA) [23,24]. Remarkably, thestudy of CIA in CD69-deficient mice unveiled a newregulatory role for CD69 (Figure 1). CD69-deficient micedevelop an exacerbated form of CIA with higher T- andB-cell responses against collagen [25]. This hyper-respon-siveness correlates with reduced levels of TGF-b ininflamed joints (Figure 1). TGF-b acts as an anti-inflammatory cytokine in CIA [26], and treatment withblocking anti-TGF-b antibodies exacerbates arthritisseverity, increasing proinflammatory cytokines and che-mokines, in wildtype but not in CD69-deficient mice [25].The reduced levels of TGF-b and the absence of CD69could be causally associated. In this regard, CD69 cross-linking in vitro promotes TGF-b synthesis [25,27]. Inaddition, TGF-b synthesis is dependent on ERK activation[28] and CD69 cross-linking mediates ERK activation [6].Hence, the regulatory effects of CD69 in vivo appear to bemediated through the synthesis of a pleiotropic cytokine,which might be finely tuned by the controlled expressionof CD69 ligand(s).

CD69 cross-linking induces TGF-b production in CD4C

and CD8C T cells as well as in NK cells and macrophages[25,27], suggesting that this receptor exerts a wideimmunoregulatory action, and that other cells, expressing

Box 1. CD69 and thymocyte physiology in vivo

CD69 is transiently expressed in thymocytes that are undergoing

positive selection or that have just completed this process [47–49].

Positive selection is a multi-stage process involving a first step in

which CD69 is induced in double positive thymocytes, a process

dependent on MHC molecules expressed by thymic epithelial cells,

and a second step to maturation to single positive thymocytes that is

MHC-independent [50]. These data indicate that CD69 is a marker of a

thymocyte subset that differentiates and proliferates in an MHC-

independent fashion and suggest that this molecule mediates this

process. However, CD69 deficiency does not affect thymic develop-

ment and positive or negative selection of thymocytes [21]. By

contrast, the constitutive expression of CD69 during T-cell develop-

ment induces an increase in both CD8 and CD4 single positive

thymocytes in thymus medulla [19,20]. These data suggest that the

constitutive expression of CD69 does not interfere with thymocyte

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the corresponding counter-receptor, might also participatein this phenomenon. Thus, CD69 could influence not onlyadaptive but also innate immunity. Accordingly, in anNK-sensitive tumourmodel inmice, CD69 deficiency leadsto reduced TGF-b synthesis by immune cells that resultsin a high production of chemokines, with decreasedlymphocyte apoptosis, accumulation of NK cells andenhanced tumour lysis [27]. Supporting these data,blockade of TGF-b signalling in T cells enhances anti-tumour immunity by facilitating the expansion of tumour-specific CD8C T cells [29].

Both the NK-sensitive tumour model and the CIAmodel demonstrate that CD69 deficiency leads to dimin-ished TGF-b levels that support an enhanced immuneresponse, resulting in a more efficient depletion oftumours or increased inflammation in the CIA model[25,27]. The use of an antibody that downregulates CD69expression in vivo reproduced in wildtype mice thephenotype found in CD69-deficient mice [27], furthersupporting the proposed immunoregulatory role of CD69.

However, as mentioned earlier, CD69 cross-linkingin vitro also mediates production of proinflammatorymediators [9,12–14], thus suggesting that CD69 couldhave a dual role, mediating the synthesis of differentcytokines, depending on the particular cellular context. Ithas been reported that CD69-deficient mice are resistantto the induction of granulocyte-mediated acute arthritis,which is initiated by the administration of exogenous anti-collagen II antibodies and endotoxin [30], an inflamma-tory condition in which the regulatory mechanismsexerted by lymphocytes are not involved. It is feasiblethat although TGF-b has a predominant inhibitory effecton T and B cells [31,32], this cytokine might act as achemotactic and activating agent on granulocytes [33],which are the main mediators of this acute model ofarthritis. Therefore, reduced synthesis of TGF-b mightstill account for the attenuated inflammatory responseseen in this model. Alternatively, CD69 might affect thesynthesis of proinflammatory molecules in a non-redun-dant way in this particular model, whereas in the CIAmodel any effect of CD69 on the synthesis of proinflam-matory molecules [15] could be compensated by otherreceptors. The possible dual role of CD69 makes it difficultto predict the final outcome of engagement by its ligand(s),

development but inhibits the export of mature single positive

thymocytes to the periphery [19]. By crossing CD69 transgenic mice

with different TCR transgenic mice, Nakayama et al. showed enhanced

negative selection that caused a reduction in the number of T cells in

peripheral lymphoid organs [20]. Interestingly, CD69Dcyt transgenic

mice, constitutively expressing CD69 without the cytoplasmic domain,

show a phenotype similar to CD69 transgenic mice [20], suggesting

that the putative CD69 ligand expressed in the thymus is responsible

for this phenotype when CD69 is overexpressed. Because CD69-

deficient mice show normal thymocyte selection [21], either the

putative CD69 thymic ligand acts just as a mechanism of retention of

CD69C thymocytes or the signal for thymocyte selection induced

through CD69L is triggered by molecules other than CD69 that are

expressed by thymocytes.

TRENDS in Immunology

CII

TNF, IL-1β, IL-15IL-18, RANTES, MIP-1α Proinflammatory

cytokines

MΦ

T cell

Synoviocytes Cartilage Bone

CD69CD69L ?

TGF-β

Anti-inflammatorycytokine

Figure 1. CD69 acts as an immunoregulatory molecule through the production of

TGF-b. In collagen-induced arthritis, T cells are activated by collagen-derived

peptides (CII) presented by macrophages (Mf), which induce CD69 expression and

the release of proinflammatory cytokines. These cytokines and co-stimulatory

molecules contribute to the persistent expression of CD69 in the inflammatory foci.

Under such circumstances, the interaction of CD69 with its putative ligand(s)

(CD69L) would induce the synthesis of the anti-inflammatory cytokine TGF-b, which

reduces the secretion of proinflammatory cytokines and the activation of immune

cells, thereby ameliorating tissue damage.

Opinion TRENDS in Immunology Vol.26 No.3 March 2005138

which might result in a pro- or anti-inflammatory state,conditioned by the particular environment.

Possible regulatory steps affected by CD69

Recent results indicate that CD69 modulates the syn-thesis of immunoregulatory molecules. Initial T-cellactivation and antigen-driven T-cell proliferation are notaffected by the absence of CD69 [21]. However, CD69might affect the immune response during T-cell differen-tiation (Figure 2), involving immunoregulatory cytokinesthat include, but might not be limited to, TGF-b, whichcontrols T-cell differentiation [31] and that, depending onthe stimulation provided, could also regulate proinflam-matory molecules.

CD69 is persistently expressed in vivo by T cells undercertain conditions characterized by chronic inflammation[22], and in vitro on constant stimulation with pro-inflammatory cytokines or through certain adhesionreceptors [15,34]. As stated earlier, the CIA model inCD69-deficient mice shows that local TGF-b levels in thejoint are reduced [25], suggesting that in wildtype micethis receptor would interact with its putative ligand(s),inducing TGF-b, and thus dampen the local immuneresponse (Figure 1). In this regard, TGF-b is found in thesynovial fluid from rheumatoid arthritis (RA) patients[35], where it might counterbalance the activity ofproinflammatory cytokines. In addition, the presence ofIgG anti-CD69 autoantibodies, detected in the serum of asubset of RA patients, correlates with disease severity[36]. Therefore, we can hypothesize that these autoanti-bodies are able to block the interaction of CD69 with itsputative ligands, decreasing TGF-b production and result-ing in more severe disease. Alternatively, these autoanti-bodies could enhance signalling through CD69,generating proinflammatory mediators.

The induction of TGF-b synthesis through CD69ligation might also take place in lymph nodes during the

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antigen-induced T-cell differentiation (Figure 2). In thisregard, it is well known that CD69 is transientlyexpressed during T-cell activation and differentiationfollowing antigen presentation by dendritic cells (DCs),and it is feasible that CD69 could exert a negativeregulatory activity through TGF-b production. TGF-bdownregulates antigen-presenting cell (APC) function[37,38] and limits activation and polarization of T cells toa Th1 or Th2 phenotype [31] (Figure 2). Therefore,enhanced T-cell differentiation in the absence of CD69could explain the stronger effector response [25]. Thisnegative regulatory role for every CD69-expressing T cellwould normally lead to a non-specific limitation of theimmune response. However, CD69 might mediate suchrole only in specific circumstances, for example, CD69might be upregulated under tolerogenic conditions in theabsence of other activating molecules that might counter-act the negative effect of CD69. Alternatively, theexpression of CD69 ligands under tolerogenic but notimmunogenic conditions could limit the regulatory func-tion exerted through CD69. Finally, other cell types with avariety of functional subsets, including monocytes andDCs, might express CD69, leading to distinct outcomesdepending on the specific cellular and pathophysiologicalsetting in which CD69 is expressed.

CD69 and regulatory T cells

T regulatory (Treg) cells have an impaired capacity torespond to proliferative signals and are able to inhibitother immune cell functions through cell–cell contact orthrough the production of anti-inflammatory cytokines,such as TGF-b, IL-10 or IL-4 [39,40]. Natural Treg cellsare generated in the thymus and are characterized bytheir high expression of CD25, which suppresses effectorresponses through cell–cell contact in a cytokine-indepen-dent manner. However, adaptive Treg cells are generatedfrom mature T lymphocytes after antigenic stimulation inthe periphery, show a variable expression of CD25, andtheir mechanism of suppression of effector T-cell responsesis cytokine-dependent [41]. It is feasible that CD69 could bepersistently expressed by a subset of these cells (Figure 2).

In a murine lupus model, a subset of CD4CCD69C cellshas been detected in peripheral lymphoid tissues andinflammatory infiltrates. These cells are anergic andunable to synthesize proinflammatory cytokines [42].Moreover, these CD4CCD69C cells inhibit cytokine syn-thesis by CD4CCD69K cells in a process that seems to bedependent on TGF-b because it is inhibited by anti-TGF-bantibodies [42]. Interestingly, peripheral blood mono-nuclear cells from lupus patients show an increasedexpression of CD69 [43] and the poor in vitro response ofthese cells to different stimuli is well known. Likewise,freshly isolated human synovial fluid T cells display aprofound state of hypo-responsiveness that correlateswith the expression of CD69 [44]. Therefore, some T cellsbearing CD69 appear to possess the two main character-istics of Treg cells, namely their anergic behavior and theirregulatory role. However, it is not currently knownwhether only a subset of the entire population of CD69-expressing cells is able to synthesize TGF-b in vivo andacts as a regulatory cell subset in different chronic

TRENDS in Immunology

DC

T cell

Proliferation

CD69

CD69L ?

TGF-β

Antigen presentation

Activation

Differentiation

Effector phase

TCR

MHC-II

Treg

EffectorT cell

Th1/Th2polarization

Inflammation

TolerogenicDC

(a)

(b)(e)

(f)

(g)

(d)

(c)

peptide

ANERGY

Figure 2. CD69 might be involved in multiple regulatory steps during the immune response. The antigen-specific signal activates T cells through the TCR and induces CD69.

(a) However, incomplete activation might lead to anergy. (b) Some of these anergic lymphocytes might act as Treg cells and CD69 could act through TGF-b production as a

survival factor for this cell subset. Alternatively, if the balance of the second signal is positive, T lymphocytes will proceed to cell proliferation. In this case, CD69 might exert

its immunoregulatory effect at two different levels. First, if CD69L were expressed by DCs at lymph nodes, (c) CD69C T cells might produce TGF-b, a cytokine that inhibits both

(d) T-cell differentiation (Th1 and Th2 polarization) and (e) APC function. (f) Second, if CD69L is expressed at inflammatory cell infiltrates, leukocytes persistently expressing

high levels of CD69 would produce TGF-b, which would dampen inflammation. (g) Finally, CD69 expression might define a subset of tolerogenic DCs.

Opinion TRENDS in Immunology Vol.26 No.3 March 2005 139

inflammatory conditions [42–44]. It is expected that CD69should have an essential role in the proper function of thesecells. TGF-b induces FoxP3 and a regulatory phenotype inTCR-challenged CD4CCD25K naıve T cells [45] and thismight also explain that, under certain circumstances, theCD69-dependent induction of TGF-b could influence thedevelopment of a subset of adaptive Treg cells.

Concluding remarks

Previous results in vitro pointed to CD69 as a stimulatoryreceptor, however, recent results in vivo have shown thatthe behaviour of CD69 is more complex. The absence ofCD69 leads to an enhanced immune response in twoindependent models: increased severity of a T-cell drivenanimal arthritis model [25] and augmented rejection ofNK-sensitive tumours [27]. CD69 mediates TGF-b pro-duction and the effect of this pleiotropic cytokine mightaccount for the regulatory effect of CD69, although othermediators could be involved. CD69 could affect differentsteps in the distinct mechanisms responsible for thelimitation of immune responses. First, CD69 might havea role in the deletion of lymphocytes by apoptosis followingactivation [27]. Second, during antigen presentation,partial activation signals might be able to simultaneouslyinduce CD69 expression [15,34] and an anergic state,

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which is a characteristic of CD69-expressing lymphocytesin chronic inflammatory diseases [42,44]. Conceivably,these anergic lymphocytes could correspond to an as yetpoorly defined adaptive Treg-cell subset that could act as abystander suppressor lymphocyte population through theproduction of TGF-b or other immunoregulatory cyto-kines, mediating localized or systemic immune deviation[42,46]. Third, CD69 engagement might regulate the finalbalance of Th1/Th2 differentiation. The characterizationof CD69 ligand(s), and the knowledge of their spatial andtemporal expression, will shed further light on the preciseimmunoregulatory functions of CD69.

AcknowledgementsWe apologize to many colleagues whose important contributions have notbeenquoted due to space constraints.Wewant to thankR.Gonzalez-Amaro,R.R.LobbandM.Vicente-Manzanares forhelpfuldiscussion.Thisworkwassupported by grant BMC02–00563 from the SpanishMinistry of EducationandScience, and theAyudaa la InvestigacionBasica 2002 fromJuanMarchFoundation. D.S. is supported by BEFI 01/9191 from the Instituto de SaludCarlos III (Ministerio de Sanidad y Consumo).

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