CD69 Protein Structure
The product of the CD69 coding gene is a polypeptide of 199 animo acids with a size of 22.5 kDa. The N-terminal region of this polypeptide forms a short cytoplasmatic tail (amino acids 1 to 40, Fig. 1) that is able to generate and transduce intracellular signals. There is a single hydrophobic transmembrane α-helix region present in the CD69 polypeptide (amino acids 41-61, Fig. 1), which classifies CD69 as a member of type II transmembrane proteins. The C-terminal 138 amino acids form the extracellular CTLD domain (Hamann et al. 1993) (Fig. 1). There are 3 consensus N-glycosylation motifs in the mouse CD69 polypeptide, all located in the CTLD domain. On the other hand, the human CD69 polypeptide has only one consensus N-glycosylation motif (amino acids 166-168; Fig. 1), while the other N-glycosylation site (amino acids 111-113; Fig. 1) is the atypical one (Vance et al. 1997). Depending on the number of N-glycan chains, the CD69 polypeptide forms subunits of the size 28 kDa or 32 kDa. If one N-glycan chain is present either on the typical or on the atypical N-glycosylation site, the unit size will be 28 kDa. Both N-glycosylation sites are occupied when subunits of 32 kDa are formed (Vance et al. 1997). These subunits can then randomly be combined to form different types of CD69 homodimer proteins with the size of 28–28 kDa, 32–32 kDa, and 28–32 kDa where the last combination is the most common combination. The two subunits are connected with the disulfide bridge located in the short neck region of the extracellular domain (Testi et al. 1994) (Fig. 1).
CD69 Expression and Signaling Pathways
Resting circulating leukocytes do not express CD69, but CD69 is induced very early after activation of T and B lymphocytes, NK cells, neutrophiles, macrophages, and eosinophiles. CD69 is hence considered the earliest activation marker of lymphocytes as it is detected within 2 h after the stimulation and its expression declines after 24 h (Testi et al. 1994). This discovery led to the wide usage of CD69 as a marker of recently in vivo activated leukocytes. This rapid induction of CD69 upon activation is independent of RNA or protein synthesis. Instead it relies on the CD69 protein depot present in the cytoplasm of the resting cells, which is shuttled to the cell surface after activation (Risso et al. 1991). The binding of a putative ligand to CD69 triggers different signaling pathways in different cell types. In NK cells induction of CD69 expression is associated with activation of extracellular signal-regulated kinases (ERK) to induce degranulation and cytotoxicity (Zingoni et al. 2000) (Fig. 2). In activated T lymphocytes CD69 associates with Janus kinase (Jak) 3 to mediate phosphorylation and activation of signal transducer and activator of transcription (STAT) 5 leading to the blockade of retinoic acid-related orphan receptor (ROR)γt gene transcription. This inhibits cell differentiation into T helper (Th) 17 cells (Martin et al. 2010a) (Fig. 2).
CD69 is expressed by half of the CD4 lymphocytes present in the intestinal lamina propria. In the spleen much less T cells express CD69. The expression of CD69 in the intestinal lamina propria is driven by the intestinal microbiota, as CD69 cannot be detected on T cells isolated from grem-free mice (Radulovic et al. 2012). The presence of a high antigen burden in the intestine (derived from food and intestinal microbiota) initiates a state of “physiological inflammation” keeping some intestinal leukocytes in the activated state even in the absence of pathogens. Therefore, the detection of CD69 on the cell surface of intestinal lymphocytes does not indicate constitutive expression, but rather an activated state of the leukocytes.
For a long time it was speculated that the ligand of CD69 should be a carbohydrate as for many other CLEC family members. Crystallographic studies of the CD69 extracellular domain showed the absence of Ca2+-binding amino acid residues that create the classical C-type lectin sugar-binding site (Llera et al. 2001). Furthermore, molecular crosslinking is necessary for the generation of CD69-dependent signal transduction, suggesting that the ligand is a cell-associated protein.
Sphingosine-1 phosphate-1 (S1P1) receptor is another binding partner expressed by the same cell as CD69. Lymphocytes egress from the secondary lymphoid organs (SLO) into the circulation thanks to the S1P1 receptor expressed on their surface. CD69 induced on activated lymphocytes directly associates with the S1P1 receptor on the lymphocyte surface. This leads to the internalization of S1P1 (Fig. 4). By removing the S1P1 receptor from the cell surface, CD69 induces retention of activated lymphocytes in the SLO (Shiow et al. 2006). CD69 affects lymphocyte migration also by regulating their response to chemokines in a yet unknown manner (Radulovic et al. 2013).
Galectin-1 is another potential ligand of CD69 as identified in studies with human monocyte-derived antigen presenting cells (APCs) (Fig. 4). The interaction between CD69 on activated T cells and Galectin-1 expressed on APCs is carbohydrate-dependent. Binding of Galectin-1 to CD69 downregulates the expression of RORC2, the human equivalent of RORγt, a master regulator for the differentiation of T cells into Th17 cells. Furthermore, this interaction inhibits the production of IFN-γ and IL-17 by T cells even in Th17-polarization conditions in vitro (de la Fuente et al. 2014). Possibly, the binding of Galectin-1 to CD69 activates Jak3/STAT5 cascade to suppress the Th17 cell differentiation.
CD69 expressed on the surface of CD4 lymphocytes binds also to calprotectin (S100A8/A9) expressed on the cell surface of human PBMCs. (Fig. 4). Binding of these two molecules required for the CD-69-dependent induction of Treg cells is carbohydrate-dependent. This interaction upregulates the expression of suppressor of cytokine signaling 3 (SOCS3) resulting in inhibited phosphorylation of STAT3 (Lin et al. 2015) (Fig. 4). Inactive STAT3 might support the secretion of transforming growth factor (TGF)-β, which is a key regulator of Treg cell differentiation.
The heavy and light chain of the heterodimeric amino acid transporter “L-type amino acid transporter 1” (LAT1) – CD98 can bind CD69 (Fig. 4). With this interaction CD69 controls the uptake of L-tryptophan by the lymphocyte. L-tryptophan metabolites are then activating the aryl hydrocrbon receptor (AhR), which promotes the transcription of IL-22 cytokine without altering the transcription of IL-17 (Cibrian et al. 2016).
CD69 in Inflammation and Infection
Because CD69 is induced after cell activation and expressed on different cell types (lymphocytes, macrophages, NK cells, eosinophiles, neutrophiles) present in the cellular infiltrates of diseased human tissues (e.g., rheumatoid arthritis, asthma, skin diseases, gastric inflammation, diabetes mellitus), CD69 was initially believed to help the cell activation and proliferation processes enabling their differentiation into effector cell types. Since that time CD69 expression is used as a marker of activated cells. However, future studies have shown that CD69 is not simply an activation marker, but rather has a complex role in inflammation regulating some of the key processes that enable efficient resolution of inflammation and tolerance to harmless antigens.
The establishment of animal models facilitated the investigation of CD69 role in disease processes. Experiments with CD69-deficient mice suggest a protective role of CD69 in inflammation.
For example, CD69-deficient mice showed higher disease severity in an experimental model of autoimmune rheumatoid arthritis (Sancho et al. 2003). In this model CD69 downregulates the autoimmune responses of T and B lymphocytes that infiltrate the inflamed joints. This is accomplished by the induction of TGF-β synthesis, a major cytokine that limits the extent of inflammation and reduces the levels of pro-inflammatory cytokines, like IL-1β (Sancho et al. 2003). Indeed, direct engagement of CD69 on activated spleen cells resulted in TGF-β production (Sancho et al. 2003). This suggests that CD69 signaling contributes to the regulation of joint inflammation by induction of TGF-β-dependent Treg differentiation.
In an allergic asthma model, CD69-deficient mice showed enhanced inflammatory responses with augmented Th2 and Th17 cytokines in the lung (Martin et al. 2010b). In the skin contact hypersensitivity model, the absence of CD69 leads to increased inflammation in skin and enhanced Th1 and Th17 inflammatory responses (Martin et al. 2010b). These results suggest that allergic reactions are controlled by CD69, probably by the signaling pathway that inhibits the differentiation of Th17 pro-inflammatory cells.
Interestingly, in a model of psoriasis CD69 promotes the accumulation of granulocytes in the skin and also the proliferation of keratinocytes. This is achieved by the induction of IL-22 after CD69-dependent activation of LAT1 – CD98 amino acid transporter and binding of tryptophan metabolites to the AhR receptor (Cibrian et al. 2016).
CD69–/– mice showed enhanced disease severity accompanied with increased IL-17 production and Th17 cell infiltration in peritoneum in a mouse model of peritoneal fibrosis (Liappas et al. 2016). This effect seems to be entirely mediated by the CD69-dependent inhibition of Th17 cell differentiation as the blockade of IL-17 reduced the disease severity to the level of control wild-type mice (Liappas et al. 2016).
During infection with the food-derived intracellular pathogen Listeria monocytogenes, CD69-deficient mice showed increased susceptibility to listeriosis (Vega-Ramos et al. 2010). Spleen and liver damage observed in infected CD69−/− mice is associated with elevated levels of type I and II IFN (Vega-Ramos et al. 2010). IFN type I induce CD69 expression on lymphocytes (Shiow et al. 2006; Radulovic et al. 2012). CD69 internalize S1P1 receptor trapping the lymphocytes in local lymph nodes (Shiow et al. 2006). Likely, CD69-deficient lymphocytes do not retain long enough in the local lymph nodes to fully mature which is required for local resolution of the L. monocytogenes infection.
For a long time it has been thought that the expression of CD69 in tumors is associated with a better prognostic outcome. Surprisingly, CD69-deficient mice are protected from the formation of MHC class I-dependent tumors (Esplugues et al. 2003). Absence of CD69 in this experimental model leads to a reduced tumor growth and prolonged survival, proving that CD69 suppresses immune responses. The effects observed in CD69−/− mice with tumors are lymphocyte-dependent as CD69 regulates the migration of lymphocytes to the tumors and TGF-β production required for the escape of tumors from the immune system (Esplugues et al. 2003). Blockade of either TGF-β or CD69 by antibodies in control wild type mice is able to increase the antitumor responses (Esplugues et al. 2003). These results suggest that CD69 could be considered as a target molecule for potential future therapies in malignancies.
CD69 also regulates intestinal immune responses. CD69 is required for the development of oral tolerance to food-derived proteins (Radulovic et al. 2012). The absence of CD69 leads to a more severe colitis in different colitis models (Radulovic et al. 2012; Radulovic et al. 2013). This effect correlates with the increased levels of pro-inflammatory cytokines, such as IFN-γ and IL-17. CD69 cross-linking on CD4 T cells increases TGF-β secretion and decreases the secretion of pro-inflammatory mediators (Radulovic et al. 2012). Consequently CD69-deficient lymphocytes are not able to differentiate into Treg cells explaining the decreased number of Treg cells observed in the intestine, mesenteric lymph nodes and spleen of CD69−/− mice (Radulovic et al. 2012). As in other disease models, the inability of CD69-deficient mice to generate Treg cells contributes to the pathogenesis of colitis and might explain why CD69-deficient mice do not develop oral tolerance.
- de la Fuente H, Cruz-Adalia A, Martinez Del Hoyo G, Cibrian-Vera D, Bonay P, Perez-Hernandez D, et al. The leukocyte activation receptor CD69 controls T cell differentiation through its interaction with galectin-1. Mol Cell Biol. 2014;34:2479–87. doi: 10.1128/MCB.00348-14. MCB.00348-14 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
- Lopez-Cabrera M, Munoz E, Blazquez MV, Ursa MA, Santis AG, Sanchez-Madrid F. Transcriptional regulation of the gene encoding the human C-type lectin leukocyte receptor AIM/CD69 and functional characterization of its tumor necrosis factor-alpha-responsive elements. J Biol Chem. 1995;270:21545–51.CrossRefPubMedGoogle Scholar
- Martin P, Gomez M, Lamana A, Matesanz Marin A, Cortes JR, Ramirez-Huesca M, et al. The leukocyte activation antigen CD69 limits allergic asthma and skin contact hypersensitivity. J Allergy Clin Immunol. 2010b;126:355–65, 65 e1–3. doi: 10.1016/j.jaci.2010.05.010 S0091-6749(10)00811-0 [pii].
- Radulovic K, Manta C, Rossini V, Holzmann K, Kestler HA, Wegenka UM, et al. CD69 regulates type I IFN-induced tolerogenic signals to mucosal CD4 T cells that attenuate their colitogenic potential. J Immunol. 2012;188:2001–13. doi: 10.4049/jimmunol.1100765. jimmunol.1100765 [pii]CrossRefPubMedGoogle Scholar
- Radulovic K, Rossini V, Manta C, Holzmann K, Kestler HA, Niess JH. The early activation marker CD69 regulates the expression of chemokines and CD4 T cell accumulation in intestine. PLoS One. 2013;8:e65413. doi: 10.1371/journal.pone.0065413. PONE-D-13-03295 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
- Sancho D, Gomez M, Viedma F, Esplugues E, Gordon-Alonso M, Garcia-Lopez MA, et al. CD69 downregulates autoimmune reactivity through active transforming growth factor-beta production in collagen-induced arthritis. J Clin Invest. 2003;112:872–882. 10.1172/JCI19112 112/6/872 [pii].Google Scholar
- Vega-Ramos J, Alari-Pahissa E, Valle JD, Carrasco-Marin E, Esplugues E, Borras M, et al. CD69 limits early inflammatory diseases associated with immune response to Listeria monocytogenes infection. Immunol Cell Biol. 2010;88:707–15. doi: 10.1038/icb.2010.62. icb201062 [pii]CrossRefPubMedGoogle Scholar
- Zingoni A, Palmieri G, Morrone S, Carretero M, Lopez-Botel M, Piccoli M, et al. CD69-triggered ERK activation and functions are negatively regulated by CD94 / NKG2-A inhibitory receptor. Eur J Immunol. 2000;30:644–51. doi: 10.1002/1521–4141(200002)30:2<644::AID-IMMU644>3.0.CO;2-H.CrossRefPubMedGoogle Scholar