CD3ζ was first discovered in T cells as a component of the TCR in 1985 (Samelson et al. 1985) and as a part of the pre-TCR complex where it was shown to play an important role in T cell development. Later, it was also found to be a component of the activating receptors NK-cell protein 46 ( NKp46), NKp30, and the low affinity Fc receptor for IgG (FcγRIII), which are expressed by NK cells (Lanier et al. 1989). It was also found to be expressed in retinal ganglion cells, where it regulates neuronal development (Xu et al. 2010).
Evolution, Genomic Organization, and Protein Structure
The CD3ζ gene is evolutionarily related to other ITAM-containing proteins as highlighted by analysis of its exon–intron organization. The nucleotide sequence corresponding to each of the three CD3ζ ITAMs is encoded by two exons that are interrupted by a phase 0 intron at the same position (one amino acid after the first tyrosine of the ITAM). This indicates that these repeated motifs probably derive from triplication of an ancestral pair of exons, the product of which participates in the intracellular signaling by binding to tandem SH2 domain–containing proteins. Such exon–intron structure is also present in most of the other sequences coding for ITAMs. Therefore, a primitive two-exon set has probably undergone multiple rounds of duplication and transposition through evolution to give rise to the gene for CD3ζ by triplication and to various Ig-like (Ig-α, Ig-β, CD3γ, CD3δ, and CD3ε) or non-Ig-like (FcεRI-β, FcεRI-γ, DAP-12/KARAP) ectodomain-containing proteins through exon shuffling. It is speculated that the CD3ζ, FcεRI-γ, and DAP-12/KARAP polypeptide set might have branched off from the ancestor of the CD3εγδ set and lost the exon corresponding to the extracellular Ig domain. Therefore, CD3ζ probably shares both a common origin (an ancestral two-exon set) and a common function (recruiting SH2-containing signaling proteins) with the other ITAM-containing proteins found associated with immunoreceptors (Malissen 2003).
Assembly and Membrane Organization
Only a fully assembled TCR complex is transported to the T cell plasma membrane, while individual subunits are most likely degraded in the endoplasmic reticulum. Thus, in T cells lacking CD3ζ, a TCRαβCD3εγεδ complex is only expressed at low levels on cell surface. CD3ζ is the last subunit to be associated with TCR complex and may only assemble to the other subunits in the Golgi, where it shields the lysosomal targeting sequence in CD3γ. Each of the CD3ζ, CD3ε, and CD3δ molecules possess an ionizable aspartic acid in their TM region, while CD3γ possesses a glutamic acid residue. Together, this gives six acidic residues in the TM region of the TCR complex. TCRβ and TCRα possess one and two basic residues, respectively, in their TM region. This gave rise to the speculation that each TCR complex might contain two TCRαβ dimers to balance the six acidic residues in the CD3εγεδζζ. However, several studies have suggested a stoichiometry of TCRαβCD3εγεδζζ (Schamel et al. 2005). Indeed, assembly of each of the three CD3 dimers with TCRαβ involves a trimeric interface between one basic and two acidic TM residues where CD3ζ associates with TCRα. So the basic TCR complex is monovalent, which then associates with other TCR complexes to give rise to TCR nanoclusters. TCR nanoclusters increase the avidity towards multimeric pMHC and thus provide the T cell with high sensitivity to detect minute numbers of antigenic peptides (Schamel et al. 2005, Molnár et al. 2012, Schamel and Alarcón 2013).
Phosphorylation and Internalization of CD3ζ
CD3ζ has not been reported to undergo serine or threonine phosphorylation.
How the binding of pMHC ligand to the ectodomains of TCRαβ transmits the signal to the cytoplasmic tail of CD3ζ has not been resolved yet. In aqueous solution, the cytoplasmic tail of CD3ζ is unstructured, whereas in the presence of liposomes it exhibits a helical secondary structure. This lipid binding-dependent conformational change in CD3ζ could be one activation mechanism. In the preactivation state when CD3ζ is bound to the inner leaflet of the plasma membrane, it is resistant to phosphorylation by the Src family tyrosine kinases. TCR engagement by pMHC ligands might force cytoplasmic tails to be released from the membrane, which now are accessible for phosphorylation by the kinases (Aivazian and Stern 2000; Gagnon et al. 2012). Another report proposes a change in the proximity of the CD3ζ juxtamembrane regions upon TCR triggering (Lee et al. 2015). In this model the CD3ζ dimers are separated in the resting TCR by their interaction with TCRα. Ligand binding to TCRαβ would induce a loosening of this interaction so that the CD3ζ dimers can come together.
Another possibility is that the TCR is in equilibrium between a resting and an active conformation (Swamy et al. 2016). CD3ζ tyrosines would only be accessible for phosphorylation in the active conformation. In resting state, cholesterol-binding to TCRβ favors the resting, inactive conformation. pMHC binding stabilizes the active conformation, hence makes the tyrosines accessible for phosphorylation (Minguet and Schamel 2008). Once signaling by the TCR is initiated, the influx of Ca2+ ions into the cytoplasm facilitates the dissociation of the CD3ζ cytoplasmic tail from the plasma membrane and sustains ITAM phosphorylation (Shi et al. 2013).
The TCR is constitutively internalized and recycled to the plasma membrane in naïve and activated T cells. Phosphorylation of the TCR upon activation by antigen binding leads to enhanced TCR downregulation from the cell surface. Cell surface TCRs lacking CD3ζ are endocytosed more rapidly than completely assembled receptors. CD3ζ may stabilize TCR expression on cell surface by blocking access to the internalization motifs on other CD3 subunits (D’Oro et al. 2002). On the other hand phosphorylated CD3ζ targets internalized TCR for ubiquitin-dependent degradation. Src-like adapter protein (SLAP) binds to internalized and phosphorylated CD3ζ via its SH2 domain in the endosomal compartment and mediates recruitment of the E3 ubiquitin ligase CBL (Casitas B-lineage lymphoma; also known as c-CBL), resulting in ubiquitylation of CD3ζ, thereby targeting the TCR for degradation. Another study suggests that phosphorylated CD3ζ accumulates in endosomes (Yudushkin and Vale 2010). This endosomal CD3ζ remained signaling competent and could possibly help to sustain long-term signaling in T cells.
The TCR complex plays a critical role in the immune response by activating the T cells which then help in the activation of B cells by releasing helper cytokines (helper T cells) or killing the target cell directly by inducing apoptosis. CD3ζ has two important functions: (1) assembly of the TCR complex in the ER/Golgi and transport to the cell surface, as a TCR complex lacking CD3ζ is mostly subjected to lysosomal degradation and (2) signaling, as the TCRαβ or TCRγδ dimer itself lacks the signaling motifs and relies on the CD3ε, CD3γ, CD3δ, and CD3ζ components for intracellular signaling. Each CD3ζ contains six tyrosines, helping in signal amplification. The tyrosines of the ITAM are phosphorylated by the Src family kinases Lck and Fyn. This leads to the recruitment of the tyrosine kinase ZAP70 and Syk to the phospho-tyrosines via its tandem SH2 domains. In addition, the adaptor protein SAP and several other signaling proteins are recruited to phosphorylated CD3ζ ITAMs (Proust et al. 2012; Borroto et al. 2014).This initiates further downstream signaling and ultimately activation of the T cell.
A similar sequence of events takes place in the signal transduction downstream of pre-TCR in the developing pre-T cells. The signals are required for TCRα gene arrangement and further development of these cells. In addition, CD3ζ is expressed on pro-T cells. In pro-T cells CD3ζ was phosphorylated upon anti-CD3ε antibody stimulation, although no direct association between CD3ζ and other CD3 subunits was observed. CD3ζ-deficient mice have a decreased number of peripheral T cells, as will be discussed later.
Further, the small GTPase TC21, the microtubule end-binding protein EB1, and the GPCR-interacting protein β-arrestin 1 can bind to the nonphosphorylated ITAMs of CD3ζ (Delgado et al. 2009; Martín-Cófreces et al. 2012; Fernández-Arenas et al. 2014). These atypical interaction partners provide cell viability, control dynamics of the immune synapse, and play a role in TCR internalization.
Several studies have been performed to explore the role of the individual ITAMs in the different CD3 proteins in T cell development and function. According to one study, together the ITAMs in CD3ε, CD3γ, and CD3δ can provide normal TCR signal transmission in mature, peripheral T cells, and CD3ζ ITAMs play mainly a role in positive selection in the thymus (Pitcher et al. 2005b). However, in another study, expression of the mutant CD3ζ, which lacked Y1 and Y2, so that the 23 kDa form of CD3ζ could not be generated (Fig. 5), partially impaired negative selection and promoted the emergence of potentially autoreactive T cells (Pitcher et al. 2005a). Furthermore, contradicting results were obtained in a comprehensive study of all CD3 ITAMs (Holst et al. 2008). Mice with only six to two wild-type ITAMs developed a lethal, multiorgan autoimmune disease due to breakdown in central tolerance. The proliferation potential of cells was directly proportional to the number of ITAMs, whereas cytokine production was independent of the ITAM number. Thus, a high number of ITAMs in TCR amplifies the signaling that effects proliferation and ensures negative selection to prevent autoimmunity.
In resting human T cells, a portion of CD3ζ associates with the actin cytoskeleton. This interaction, mediated by a sequence in the C-terminus of CD3ζ, may be involved in the localization of the TCR into lipid raft structures and/or in TCR recycling. Some viral proteins, such as simian immunodeficiency virus Nef, bind the CD3ζ and downmodulate TCR. Such interaction seems to have evolved as an immune escape strategy for the virus. CD3ζ is also shown to interact with the transferrin receptor (TfR) and plays a role in T-cell activation via TfR stimulation. The TfR/CD3ζ complex is expressed on the cell surface independent of the expression of the other subunits of the TCR, and activation of this complex might be a signal-amplifying mechanism for T cells. Phosphorylated CD3ζ can bind to several SH2 domain–containing proteins which include adaptor proteins Shc and Grb2 and the p85 subunit of PI3K. SLAP-2, an SH2 domain–containing protein related to SLAP, binds to CD3ζ upon ligand binding and is a negative regulator of downstream signaling. CTLA-4, another negative regulator of the T-cell activation, binds to phospho-CD3ζ and prevents accumulation of the TCR in lipid rafts upon antigen binding.
In addition, CD3ζ is also expressed in cells other than T cells, for example, NK cells and neurons. In NK cells, CD3ζ is associated with NK FcγRIII (CD16) and may be necessary for efficient cell surface expression of this receptor complex. Activation of NK cells with an anti-FcγRIII antibody induces tyrosine phosphorylation of CD3ζ, and FcγRIII-associated CD3ζ might be downregulated in patients with cancer due to chronic inflammation (Eleftheriadis et al. 2008). CD3ζ is also associated with NKp46 and NKp30 receptors on NK cells, and its phosphorylation is required for transmission of activating signals upon antigen binding to these receptors. CD3ζ is also expressed in retinal ganglion cells and brain neurons, where it regulates neuronal development by reducing the size of the dendritic arbor. As a result, CD3ζ knock-out mice show an impaired learning behavior and memory due to altered NMDA and AMPA receptor signaling (Louveau et al. 2013).
Pathophysiological and Clinical Roles
Immunological disorders associated with mutations in CD3ζ
Chimeric antigen receptors (CARs) used in cancer immunotherapy make use of the ITAM-containing and thus signaling-competent CD3ζ cytoplasmic domain (Dai et al. 2016). In these chimeric receptors an extracellular antibody single chain variable fragment (scFv) specific for a tumor-expressed antigen is coupled to the CD3ζ cytoplasmic domain by a transmembrane domain. These constructs are then expressed in T cells. Antigen binding to the scFv activates the CAR which leads to the phosphorylation of the CD3ζ cytoplasmic domain and subsequent activation of the T cell to kill the tumor cells.
CD3ζ is a type 1 TM protein and a subunit of the TCR and pre-TCR complexes. It forms a dimer and is necessary for assembly and expression of these receptors on the cell surface. The TCR exists in two different conformations, a resting state and an active state that is stabilized upon ligand-binding to the TCR. CD3ζ is part of this structural change. CD3ζ possesses three ITAMs with six tyrosines, which are phosphorylated only in the active conformation. Once phosphorylated, these tyrosines recruit SH2 domain–containing proteins for downstream signaling which ultimately leads to the development of pre-T cells in the thymus and activation of mature T cells in the periphery, which then initiate an immune response. CD3ζ is also part of several other receptors on T cells and NK cells and is expressed and has a function in neurons. Downregulation of CD3ζ has been noticed in patients with tumors and autoimmune and infectious diseases. Lastly, the cytosolic signaling tail of CD3ζ is employed in chimeric antigen receptors to be used in immunotherapy against tumors.
- Dai H, Wang Y, et al. Chimeric antigen receptors modified T-cells for cancer therapy. J Natl Cancer Inst. 2016;108(7). doi: 10.1093/jnci/djv439.
- Fernández-Arenas E, Calleja E, Martínez-Martín N, Gharbi SI, Navajas R, García-Medel N, Penela P, Alcamí A, Mayor F Jr, Albar JP, Alarcón B. β-Arrestin-1 mediates the TCR-triggered re-routing of distal receptors to the immunological synapse by a PKC-mediated mechanism. EMBO J. 2014 Mar;33(6):559–77.CrossRefPubMedPubMedCentralGoogle Scholar
- Nambiar MP, Enyedy EJ, et al. Polymorphisms/mutations of TCR-zeta-chain promoter and 3′ untranslated region and selective expression of TCR zeta-chain with an alternatively spliced 3′ untranslated region in patients with systemic lupus erythematosus. J Autoimmun. 2001;16(2):133–42.CrossRefPubMedGoogle Scholar