CD69: from activation marker to metabolic gatekeeper

Introduction

CD69 is a disulfide-linked homodimer protein with two differentially glycosylated subunits (28-32 kDa). Each subunit consists of an extracellular C-type lectin domain (CTLD) connected with a single-spanning transmembrane region followed by a short cytoplasmic tail (Fig. 1). The CD69 gene is located in the natural killer (NK) gene cluster, chromosome 6 in mouse and 12 in human [1, 2]. Binding sites for several inducible transcriptional factors such as nuclear factor (NF)-κB, erythroblast transformation-specific related gene-1 (ERG-1) and activator protein- (AP-) 1 are located within the CD69 gene promoter [3, 4]. CD69 expression is readily upregulated upon activation in most leukocytes, which underlies its widespread use as a marker of activated lymphocytes and NK cells, mainly [5]. In addition to its intrinsic value as an activation marker, recent evidence suggests that CD69 is also an important regulator of immune responses. Although the precise role of CD69 expression on immune cells function is yet to be elucidated, accumulating experimental evidence has revealed that CD69 may determine patterns of cytokine release as well as homing and migration of activated lymphocytes. In this review, we aim to update the state of the art regarding the functional role of CD69 in the regulation of the immune responses. We offer an integrated perspective of the mechanisms that drive the immune effects mediated by CD69 as well as potential synergies and antagonisms with other signaling routes involved in the immune response.

CD69 is an early activation marker

CD69 expression is rapidly induced on the surface of T lymphocytes after TCR/CD3 engagement, activating cytokines and polyclonal, mitogenic stimulation. Transcriptional expression of the CD69 gene is detected early after activation (30-60 min); however, it declines rapidly after 4-6 h. CD69 protein expression can be detected as early as 2-3 h after stimulation. The appearance of CD69 on the plasma membrane of activated cells is faster than that of CD25, underlying its widespread use as a very early marker of lymphocyte activation [5].

CD69 is expressed on infiltrated leukocytes at inflammatory sites in several human chronic inflammatory conditions, for example rheumatoid arthritis [6], systemic lupus erythematosus [7], systemic sclerosis [8], allergic asthma [9] and atopic dermatitis [10]. Early studies with CD69-deficient cells were unable to definitely prove the function of CD69 in T lymphocyte proliferation and priming [11, 12]. However, in vivo strategies using CD69-deficient mice and blocking antibodies, showed that CD69 expression modulates the severity of different murine inflammation models, including arthritis [13]; asthma and contact hypersensitivity [14]; myocarditis [15]; pathogen clearance [16]; tumor immunity [17]; and inflammatory bowel disease [18–20].

CD69 ligands

The identification of specific ligands for CD69 is critical to understand its roles in physiology and pathology. Based on the identification of a CTLD region within its structure, early studies postulated that CD69 could bind to carbohydrate moieties. However, the results of those early experiments were not conclusive, likely due to the fact that CD69 interacts with both, carbohydrate and protein residues (reviewed in [5]). These early inquiries planted the seed for a later study in which the protein Gal-1 (Fig. 1) was identified as a specific ligand of CD69 [21]. Gal-1 is a carbohydrate-binding protein expressed in dendritic cells and macrophages. Its systemic deficiency exacerbates TH1 and TH17 responses [22], similar to the phenotype observed in CD69-deficient mice [23]. The binding of CD69 to Gal-1 on DCs negatively modulates the in vitro differentiation of TH17 cells [21], which could control inflammatory responses in vivo. Gal-1 also enhances IL-10 secretion in T cells through the activation of the aryl hydrocarbon receptor (AHR) pathway, although the mechanism is not fully elucidated [24]. Another putative ligand of CD69 is the S100A8/S100A9 complex, which binds to CD69 in a glycosylation-dependent manner, and regulates Treg-cell differentiation [25]. In addition to these ligands, which bind CD69 in a trans manner, CD69 establishes lateral (cis) interactions with the sphingosine 1-phosphate receptor 1 (S1P1) [26], that regulates lymph nodes T-cell egress (Fig. 1), and the amino acid transporter complex LAT1-CD98 [27] (Fig. 2). These data suggest that, in addition to its possible role as a signal transducer of its ligands, CD69 may independently modulate the function of at least these two receptors, S1P1 and LAT1, which are involved in migration and cell metabolism, respectively.

CD69 controls TH-cell differentiation

T-cell differentiation is mainly driven by polarizing cytokines that activate JAK/STAT routes, leading to the expression of lineage-specific transcription factors and effector cytokines. IL-12 (which signals through STAT4), IL-4 (which signals through STAT6) and TGF-β + IL-2 (which signals through STAT5) all drive the differentiation of TH1, TH2 and inducible Treg cells respectively. On the other hand, TGF-β stimulation in the presence of IL-6 or IL-21 (which signal through STAT3) promotes the development of TH17 cells [28]. The cytoplasmic tail of CD69 triggers intracellular signals through JAK3, which then phosphorylates and activates STAT5 (Fig. 1) [23]. IL-17 production is inhibited by the direct competition of STAT5 with IL-6-dependent binding of STAT3 to transcriptional enhancers within the Il17 gene [29]. In vitro and in vivo evidence demonstrated that the TH1 and TH17 responses of TCR-activated CD4⁺ T cells were exacerbated when CD69 was deleted, and that this effect was linked to decreased STAT5 phosphorylation [23]. Interestingly, the role played by CD69 as a negative regulator of TH17-mediated immune responses has been observed mainly in models of adaptive immune response, which employ cognate antigens such as OVA or specific peptides [23].

The balance of STAT5 and STAT3 signaling is a key conductor of the development of inducible Treg cells. CD4⁺ T cells deficient for CD69 were less capable of differentiating into Foxp3⁺ Treg cells in vitro and in vivo in a cell transfer-induced colitis model [18]. In the steady state, about 50% of Treg cells residing in lymphoid organs express CD69. Moreover, FOXP3⁺CD69⁺ Treg cells expressed higher surface levels of suppression-associated markers and displayed enhanced suppressor activity compared to FOXP3⁺CD69^- Treg cells in a mouse model of lung tolerance induced by harmless inhaled antigens [30]. In addition, a recent report showed that CD69 expression is required for thymus Treg-cell development. A possible mechanism relies in the interaction of CD69 with JAK3/STAT5, which increases miR-155 expression and controls its downstream target molecule signaling (SOCS-1) [31].

Besides polarizing cytokines, other molecules can influence the outcome of Treg and Th17 cells. These include mediators involved in metabolic control, such as the mechanistic target of rapamycin (mTOR) pathway and hypoxia induced factor (HIF) 1α, as well as the ligand-induced transcriptional factor AHR [28, 32]. Other controllers of Treg and TH17 responses are CD69 [23], Gal-1 [22], S1P1 [33] and LAT1 [34]. The interactions between some of these molecules in a cis or a trans fashion (see previous section) delineates a more complete picture of the mechanisms that control the outcome of TH17 and Treg cells. Such a picture depicts a tightly regulated, intertwined network that involves cytokines, soluble factors, membrane receptors, signaling molecules and metabolic mediators (Fig. 1 and 2).

CD69-S1P1 balance in the plasma membrane of T cells determines tissue egress or retention

Immune surveillance and antigen patrolling involves the continuous egress and return of naïve T cells from the lymphoid organs to the circulation. Murine T-cell egress from lymphoid organs is mediated by S1P1 expression and its interaction with sphingosine-1 phosphate (S1P) from the bloodstream. S1P is a sphingolipid metabolite that stimulates diverse signaling pathways, including calcium mobilization, actin polymerization, chemotaxis/migration and survival. S1P can bind to five related G-protein-coupled receptors (S1P1-5) although the S1P1 isoform is the most represented in murine T lymphocytes [35].

Antigen recognition by the TCR as well as certain pro-inflammatory stimuli such as IFN-α/β and TNF-α can temporarily impair lymphocyte egress from the lymph nodes [36]. These stimuli can also directly induce CD69 expression in lymphocytes [5]. Although the mechanism is not completely established, IFN-AR signaling promotes the formation of a complex that includes CD69 and S1P1 on the plasma membrane (Fig. 1). This complex negatively regulates the egress function of S1P1, thus promoting lymphocyte retention in secondary lymphoid organs [26]. This interaction is independent of the CTLD region of CD69 and involves the transmembrane and membrane proximal regions of CD69, and helix 4 of S1P1 [37]. CD69 and S1P1 association can be detected by co-immunoprecipitation, whereas no association is detected between CD69 and the related receptor S1P3. Increased CD69 expression on the surface of activated T cells promotes S1P1 receptor internalization and degradation with no effect at a transcriptional level [37]. These biochemical studies placed CD69 as a regulator of lymphocyte migration and promoter of T-cell retention in the lymph nodes and throughout the body. Upon activation, T lymphocytes express CD69 and are transiently retained in the lymph nodes, likely to favor their full activation. For this reason, CD69⁺ T cells are seldom found in the circulation compared to lymph nodes or inflamed tissues, although they appear increased in some chronic inflammatory conditions [8, 38]. Whether the proposed IFN-AR/CD69/S1P1 axis of egress shutdown underlies the actual role of CD69 in immune responses in vivo has not been fully assessed. Two independent studies showed that transgenic overexpression of CD69 impaired egress of mature single-positive T cells from the thymus [39, 40]. However, CD69-deficient mice display normal hematopoietic cell development and T-cell subpopulations in the thymus and periphery [11]. A recent report showed that Salmonella infection, or LPS stimulation, block lymphocyte egress from Peyer’s patches independently of the IFN-AR/CD69/S1P1 axis. This study also showed that CD69-mediated retention was only effective in the case of TCR-activated CD4⁺ lymphocytes [41]. In support of these results, another study demonstrated that CD4⁺ T-cell accumulation in inflamed colon requires CD69 expression, which also determines the pattern of chemokine expression [19]. In addition, CD69 is required for the trafficking of effector CD4⁺ T cells to the bone marrow, particularly for their relocation and the persistence of their interaction with stromal cells as memory T helper cells [42]. A role for CD69 regulation of migration through the control of S1P1 expression has been also reported in skin dendritic cells [43]. Further studies are required in order to explore the role of CD69 in the control of inflammatory cell migration; whether it always depends on the function of the S1P1 receptor; and if the egress-limiting role of CD69 is tissue-, cell- and/or stimulus-dependent.

CD69 as a marker for tissue resident cells

Once infection has been resolved, the number of effector activated T cells decreases, with a fraction of effector T cells becoming memory T cells. Current classification of memory T cells distinguishes between central or effector memory cells, which recirculate through the blood, secondary lymphoid organs and peripheral tissues; and tissue-resident memory T cells (TRM), which reside permanently in the peripheral tissues [44]. TRM cells have been identified in diverse peripheral organs like the gut and skin, in mice and in humans, where they confer long-lived protection against local infection. They also participate in the development of autoimmune and allergic diseases [44]. CD69 is a typical marker of peripheral TRM cells, especially in the skin, together with CD103. Furthermore, CD69-deficient mice display a marked reduction in the number of skin TRM [45]. CD69 expression is upregulated in TRM precursor cells after tissue entry into peripheral tissues, where it blocks S1P1 function, promoting cell retention into the tissue [46]. This idea is also supported by experiments in which forced expression of S1P1 prevented the appearance of TRM cells [47]. Recent observations classify TRM cells according to their expression of CD69. However, CD69-CD103⁺TRM cells are also present in peripheral organs, and recent works emphasized the independence of CD69 expression of the inability of TRM cells to recirculate [48, 49]. Nevertheless, resident CD8⁺, CD4⁺, Treg and γδ T cells, as well as innate lymphocytes and NKT cells express CD69 in all the tissues explored so far, regardless of their cell origin (embryonic or bone marrow circulating cells) and the presence of antigen [44]. This likely indicates that tissue-specific environment cues may control CD69 expression to promote the retention and/or survival of tissue resident immune cells. In addition, expression of AHR is required for maintaining the TRM population in the skin [50]. It would therefore be of interest to assess whether the expression of CD69 in TRM cells contributes to the regulation of the transcriptional activity of AHR through the control of tryptophan (Trp) uptake by LAT1, as demonstrated for skin resident γδ T cells [27].

CD69-LAT1 cooperative alliance on the lymphocyte membrane

Recently, we have reported that CD69 associates with the amino acid transporter complex LAT1-CD98 (SLC7A5-4F2) on the plasma membrane of T cells (Fig. 2) [27]. This interaction was first identified by differential proteomic analysis using activated CD4⁺ T lymphocytes from CD69-deficient and wild type mice. Further biochemical and duo-link assays confirmed the interaction of CD69 with LAT1 on the membrane of activated T cells. Through this interaction, CD69 contributes to the stability and/or transport of LAT1 to the plasma membrane; thus regulating amino acid uptake in activated cells [27]. LAT1 mediates entrance of leucine and aromatic amino acids such as tryptophan (Trp) into the cytoplasm of activated lymphocytes (Fig. 2). Oxidative metabolism of Trp produces several metabolites that bind and activate AHR, for example 6-formylindolo [3,2-b] carbazole (FICZ), which bears high affinity for AHR (Fig. 2) [51]. LAT1-mediated amino acid uptake has been previously associated to the control of the mTOR signaling pathway [34]; but it also regulates AHR activation [27]. Accordingly, the transcriptional activity of AHR is reduced in CD69-deficient cells as well as the maintenance of mTOR signaling after TCR/CD3 engagement. Importantly, these effects have been observed in TCRα/β and γδ T cells [27]. Further studies involving direct mutagenesis and silencing strategies will be required to identify the molecular basis of the association of LAT1 with CD69. It will be also interesting to explore whether the CD69-LAT1 pair could act as an independent and functional channel unit, even in the absence of CD98.

CD69 as a metabolic gatekeeper

T-cell activation and differentiation requires a metabolic reprogramming to meet new demands, including the increased synthesis of several inflammatory mediators. Glucose, amino acid, and iron uptake transporters are up-regulated in response to TCR stimulation and are necessary for the differentiation of effector T cells [34, 52, 53]. Hence, the T-cell immune responses can be potentially shaped by the T-cell nutrient microenvironment [54]. In this regard, CD69 expression is not affected when TCR-dependent T-cell activation takes place in medium depleted of glutamine or glucose [55]. However, earlier studies reported that anti-CD69 antibodies triggered an increase of glucose uptake in activated T cells. This could be due to function mimicking of some ligands, or potential crosslinking of associated molecules on the membrane [56]. Very recently, ChIP experiments have revealed that CD69 is a direct target gene of HIF-1α in hypoxia [57]. A newly hypoxia response element (HRE) was identified in the human CD69 locus. These results explain the high expression of CD69 by tumor-infiltrating T-lymphocytes that reside in the hypoxic tumor microenvironment [57]. In addition, these findings suggest that tissue O₂ levels could play a role in the regulation of CD69 expression in different tissues, as well as their role in global immune responses.

The mTOR signaling cascade integrates activation signals and nutrient-sensing pathways to control the metabolism of T cells, their survival and proliferation, as well as their terminal differentiation [58]. TCR engagement induces a modest activation of mTOR in a PI3K-dependent manner, but this pathway is further amplified by co-stimulatory signals, including CD28 and IL-2. Intense signaling through mTOR is required for T-cell commitment to TH1, TH2 and TH17 effector lineages. Conversely, Treg-cell development is associated with low levels of mTOR activation [32]. HIF-1α, which is activated downstream of mTOR, enhances TH17 development by direct transcriptional activation of RORγt and IL-17. At the same time, HIF-1α attenuates Treg-cell development by promoting proteosomal degradation of FOXP3 [59] (Fig. 1). However, proper mTOR activation is entirely dependent on environmental amino acid in the extracellular medium. Therefore, low amino acid availability potently inhibits mTOR activity. Likewise, the immunosuppressive drug rapamycin has a similar effect to that of amino acid starvation [58]. Although mTOR inhibition with rapamycin favors Treg-cell development, recent evidence supports that mTOR activity is still required for the development and function of Treg cells [60]. Hence, fine tuning mTOR signaling through amino acid uptake and intracellular signaling pathways can determine the phenotypic outcome and plasticity of activated T cells.

It is established that increased amino acid uptake through LAT1-CD98 complex is required for mTOR signaling after TCR activation as well as for TH1 and TH17 differentiation responses, although no effect was observed in Treg-cell differentiation [34]. Our group has also provided evidence that CD69 deficiency attenuates activation of mTOR signaling at late stages of in vitro TH1 and TH17 differentiation, and that CD69 promotes amino acid transport through LAT1-CD98 complex, which increases mTOR signaling [27]. Hence, together with cytokine signaling, the CD69/LAT1 pair controls the metabolism and differentiation of activated T cells. However, S1P1 expression on the membrane sustains mTOR activation in T cells [33]. This fact may explain the lack of functional differences between CD69-deficient and wild type cells in mTOR signaling at early times points after activation. In addition, S1P1 expression inhibits the differentiation of Foxp3⁺Treg cells by interfering with TGF-β-induced SMAD3 signaling, while it promotes the development of TH1 cells [33]. Interestingly, blocking S1P1 internalization increased the JAK2-STAT3 signal-transduction pathway in the presence of IL-6, thus promoting TH17 polarization and exacerbated autoimmune neuroinflammation [61]. It is thus conceivable that, in addition to increased STAT5 phosphorylation, CD69 may control the T effector/ Treg balance through the negative regulation of S1P1 expression on the plasma membrane [23]. However, this potential regulatory effect of the CD69-S1P1 pair in the balance of the different T effector and memory subsets has not been fully explored yet. In summary, the lateral associations of CD69 with S1P1 (Fig. 1) and the amino acid transporter LAT1 (SLC7A5) (Fig. 2) exert opposite effects on the functions of these plasma membrane-associated proteins. Because both molecules promote mTOR activation, CD69 fine-tunes these signals after TCR stimulation, thereby determining the metabolism of the cell and the pattern of released cytokines. Further studies are required to explore the role of CD69 and the mTOR signaling in vivo and in vitro, evaluating the participation of S1P1 and LAT1 function.

AHR is a ligand-activated transcriptional factor that also regulates Treg and TH17 cell differentiation in a ligand-specific manner [62]. AHR activation by FICZ, a metabolic derivative from Trp oxidation, attenuated Treg-cell differentiation and boosted TH17-cell differentiation, worsening EAE development [62]. The interaction of CD69 with LAT1-CD98 regulates Trp uptake and AHR activation (Fig. 2) [27]. Expression of IL-22 is directly associated with the transcriptional activity of AHR, which was upregulated in TH17 and γδ T cells expressing CD69, thus increasing the severity of psoriasis in a murine model [27]. HIF1α and AHR share the ability to form a complex with the AHR nuclear translocator (ARNT), also known as HIF-1β, during the transcription of their target genes. The expression of AHR limits the cellular content of HIF-1α, while the stabilized form of HIF-1α promotes the proteosomal degradation of AHR in activated T cells [63]. Thus, AHR and HIF-1α cross-mutually regulate and through their balance can determine the metabolic fate and differentiation of T cells. In this manner, CD69 could also modulate the immune response due to its ability to regulate AHR activation in different cell types, and under specific environmental cues, such as amino acid abundance, and the presence of specific Trp-associated metabolites (Fig. 2).

Conclusion

The expression of CD69 in infiltrating lymphocytes in inflamed tissues is a marker of different signaling pathways, which potentially regulate tissue retention, metabolism and their activated phenotype. Increased STAT5 signaling, prevention of S1P1 expression in the membrane, and the control of mTOR and AHR activation through the regulation of amino acid uptake by LAT1 are crucial pathways regulated by CD69. The metabolic composition of the tissue environment together with cytokines could differentially regulate the role of CD69 in the development of immune responses. In addition, the expression of ligands in inflamed tissues as well as in dendritic cells is an alternative mechanism to regulate CD69-mediated signaling during the development of the inflammatory responses. CD69 upregulation is observed after activation in most of the cells of the hematopoietic system, indicating a general requirement for its regulatory effects. Further studies will likely push the state of the art regarding the function of CD69 to other immune cell types and contexts.