There are 2 populations of T lymphocytes, αβ T and γδ T cells, that can be distinguished by the expression of either an αβ TCR or a γδ TCR, respectively. Pairing of the Ag binding heterodimer, which consists of TCR-α/TCR-β (TCRαβ) or TCR-γ/TCR-δ (TCRγδ), with proteins of the CD3 complex forms the complete αβ or γδ TCR. Despite some similarities in the structure of TCRαβ and TCRγδ and the shared subunits of the CD3 complex, the 2 receptors differ in important aspects. These include the assembly geometry of the complex, the glycosylation pattern, the plasma membrane organization, as well as the accessibility of signaling motifs in the CD3 intracellular tails. These differences are reflected in the different demands and outcomes of ligand-induced signaling. It was shown that exposure of the proline-rich sequence (PRS) in CD3ε occurs with all activating αβ TCR ligands and is required to induce αβ TCR signaling. In sharp contrast, CD3ε PRS exposure was not induced by binding of those ligands to the γδ TCR that have been studied. Further, signaling by the γδ TCR occurs independently of CD3ε PRS exposure. Interestingly, it can be enhanced by anti-CD3ε Ab-induced enforcement of CD3ε PRS exposure. This review contrasts these two similar, but different immune receptors.
- cluster of differentiation 3
- connecting peptide
- dendritic epidermal T cell
- Fc receptor γ chain
- hypervariable region 4
- iNKT cells
- invariant natural killer T cells
- lymphocyte-specific protein-tyrosine kinase
- non-catalytic region of tyrosine kinase adaptor protein
- proline rich sequence
- selection and upkeep of intraepithelial T cells 1
- transmembrane region
- γδ NKT cells
- γδ natural killer T cells
The adaptive immune system is characterized by receptors that arise in a process called somatic recombination. These receptors can recognize a nearly infinite variety of structures and are present on T and B lymphocytes. Among T cells, αβ and γδ T cells can be distinguished based on the expression of either an αβ or a γδ TCR. Both αβ and γδ TCRs consist of 2 Ag binding subunits that either form the TCR-α/TCR-β (TCRαβ) heterodimer or the TCR-γ/TCR-δ (TCRγδ) heterodimer, and proteins of the cluster of differentiation 3 (CD3) complex that mediate signal transduction into the cell. In contrast to TCRαβ and TCRγδ, which only have a short cytoplasmic portion, the CD3 subunits have longer cytoplasmic tails that contain signaling motifs such as ITAMs in all CD3 subunits and a proline-rich sequence (PRS) in CD3ε (Fig. 1). Genes encoding for TCR-α, TCR-β, TCR-δ, and TCR-γ are present in all jawed vertebrates and most likely arose by gene duplication that occurred at least 450 million years ago.1 The fact that the presence of αβ and γδ TCRs in jawed vertebrates has persisted over millions of years suggest that both receptors must bear specialized features specific to each receptor.
In this review the terms “TCRαβ” or “TCRγδ” are used for the ligand binding TCR-α/TCR-β or the TCR-γ/TCR-δ heterodimers, and the terms “αβ TCR” and “γδ TCR” depict the complete receptors including the CD3 subunits.
2 STRUCTURE OF THE Ag BINDING TCR SUBUNITS
TCR-α, TCR-β, TCR-γ and TCR-δ chains can be divided into a variable (V) and a constant (C) region (Fig. 1). During thymic development of T cells, the genes coding for the V regions are assembled from gene segments through the activity of RAG1/2 recombinases in a progress called somatic recombination. The V region comprises the membrane-distal immunoglobulin-like domain with the Ag binding site. The C region of the TCR chains, which is present as a complete gene segment, includes the membrane-proximal immunoglobulin-like domain, the connecting peptide (CP), the transmembrane (TM) region, and the cytoplasmic tail.
Although the V regions of TCRαβ and TCRγδ exhibit a similar overall structure, the C regions greatly differ.2 The distance between the immunoglobulin-like domains and the disulfide-bond in the CP is longer in TCRγδ compared to TCRαβ, and the conserved FG loop in the C region of TCR-β is much shorter in TCR-γ (Fig. 1). The FG loop might be involved in the association of TCRαβ with CD3 and is supposed to play a role in αβ TCR-induced signaling.3, 4 Furthermore, the surfaces of the C TCRαβ and C TCRγδ regions do not show clear similarities, neither in their shape nor in their charge distribution.2 Since CD3 interacts with the C regions, the differences in the C regions between the αβ and γδ TCR might be the reason for the experimentally observed biochemical differences between the αβ TCR and the γδ TCR that are discussed in this review.
3 CD3 COMPOSITION UPON ASSEMBLY WITH TCRαβ OR TCRγδ
The CD3 complex comprises of CD3ε, CD3γ, CD3δ, and CD3ζ (Fig. 1). When incorporated into human αβ and γδ TCRs the CD3 complex exhibits the same composition resulting in a TCRαβCD3ε2γδζ2 or TCRγδCD3ε2γδζ2 stoichiometry.5, 6 However, murine αβ and γδ TCRs differ in their stoichiometry. While murine αβ TCRs contain the same CD3 subunits as human αβ TCRs, murine γδ TCRs lack CD3δ and instead incorporate two CD3γ subunits leading to a TCRγδCD3ε2γ2ζ2 stoichiometry.6-8
The different stoichiometries of human and murine γδ TCRs are reflected in the different outcomes of human and murine CD3δ deficiencies. Signals via the TCR are essential for the development of both αβ and γδ T cells. Thus, precursor T cells that cannot assemble a TCR on the cell surface exhibit a block in T cell development. While γδ T cells do not develop in human patients lacking CD3δ,9, 10 since a human γδ TCR cannot form due to the lack of its subunit CD3δ, mice deficient for CD3δ have normal numbers of γδ T cells,11 since CD3δ is not part of the murine γδ TCR und thus a functional γδ TCR can be expressed. In contrast, αβ T cells fail to develop in human and mice deficient for CD3δ, since CD3δ is integrated in both human and murine αβ TCRs.9-11 Interestingly, decreased TCR levels on the surface of γδ T cells in mice lacking simultaneously one allele of CD3γ and CD3δ have been reported that result in reduced γδ TCR signaling.12 This suggests that although murine CD3δ is not integrated into γδ TCRs, it might play a role during the assembly of the γδ TCR in the endoplasmatic reticulum. In contrast, αβ T cells exhibit normal levels of cell surface αβ TCRs in CD3γ and CD3δ-haplodeficient mice and were not affected in their development.12 This might indicate that in addition to the differences in the composition of murine αβ and γδ TCRs, the two TCRs might differ in their assembly process.
A feature of both αβ and γδ TCRs is their ability to exchange one or both CD3ζ chains with the Fc receptor γ chain (FcR-γ) to incorporate an FcR-γ/CD3ζ heterodimer or FcR-γ homodimer.13-18, 8, 19 Since CD3ζ contains three ITAMs and FcR-γ only one ITAM, the replacement of CD3ζ by FcR-γ could reduce the signaling capacity of the TCR. Accordingly, the accumulation of inositol phosphates and interleukin 2 (IL2) secretion in a murine CD3ζ and FcR-γ-deficient αβ T cell line, which was resubstituted with FcR-γ, was reduced as compared to CD3ζ-resubstituted cells.20
4 CD3 GLYCOSYLATION UPON ASSEMBLY WITH TCRαβ OR TCRγδ
Although the same subunits of the CD3 complex are incorporated into human αβ and γδ TCRs, CD3δ and CD3γ exhibit a different glycosylation pattern (Fig. 1). CD3δ incorporated into the human γδ TCR carries mainly complex N-linked oligosaccharides, while CD3δ incorporated in the human αβ TCR contains both complex and high mannose carbohydrates.21, 22 The γδ-type glycosylation pattern of CD3δ is also seen when a human γδ TCR is expressed in a human αβ T cellular background and thus is an intrinsic feature of the γδ TCR.6 Most likely CD3δ is located differently within the αβ compared to the γδ TCR complex, and thus its carbohydrate chains when present in the αβ TCR cannot be reached well by the glycosyltransferase enzymes in the Golgi and thus stay in the high mannose pattern. In contrast, in the γδ TCR the enzymes can access the CD3δ carbohydrates well and complex N-linked oligosaccharides are produced. Therefore, we think that the different CD3δ glycosylations in human αβ versus γδ TCRs are an indication for a different arrangement of these two receptors. To our knowledge, CD3δ glycosylations in murine αβ and γδ TCRs have not been studied so far and thus it remains unknown whether this difference is conserved in evolution.
CD3γ most likely exhibits a higher degree of sialylation when incorporated into a human αβ TCR than when incorporated into a human γδ TCR.21 Whether this reflects different accessibilities of the CD3γ carbohydrate chains or different expression of carbohydrate modifying enzymes remains unexplored. Further, the glycosylation pattern of CD3γ assembled into a murine γδ TCR changes upon the activation and expansion of the γδ T cells.23 A similar modification of the CD3γ glycosylation pattern also takes place upon the activation of murine αβ T cells. However, only minimal amounts of the modified CD3γ are incorporated into αβ TCRs.23
5 CD3 GEOMETRY UPON ASSEMBLY WITH TCRαβ OR TCRγδ
As mentioned briefly, the orientation of CD3 toward the ligand binding chains may differ when assembled into an αβ or a γδ TCR (Fig. 1). Recently, a first structure of the ecto- and TM-regions of the complete detergent extracted human αβ TCR was solved, demonstrating that the ectodomains of CD3εγ and CD3εδ are located on one side of the TCRαβ heterodimer in the order CD3δ:CD3ε:CD3γ:CD3ε.24 While CD3εδ forms mainly contacts with TCR-α, CD3εγ docks on TCR-β.24 Accordingly, in human αβ TCRs CD3γ can be biochemically cross-linked to TCR-β.25, 26 In contrast, CD3γ cannot be cross-linked to TCR-γ, which is the TCR-β homologous TCR subunit.25 Instead CD3γ is cross-linked to TCR-δ, which is homologous to TCR-α.25 This might result in a different overall rearrangement of the ectodomains of αβ and γδ TCRs and might also be true for the TM regions.
The polar amino acids in the TM regions of TCRαβ involved in key interactions between TCRαβ and CD3 are also present in TCRγδ (Fig. 2). However, the sequence of the other amino acids in the TCRαβ and TCRγδ TM regions greatly differ (Fig. 2). K258 of human TCR-α possibly forms a salt bridge with D137 from CD3ε and D111 from CD3δ.24 The corresponding K of TCR-δ might establish a similar interaction with D137 from CD3ε and E122 in CD3γ. Similarly, the K of human TCR-γ that corresponds to K288 of human TCR-β might interact with D137 from CD3ε and D111 from CD3δ. Since murine γδ TCRs do not integrate CD3δ8, 6 the corresponding K of murine TCR-γ might interact with CD3ε and CD3γ like K288 in human TCR-β.24 N243 of human TCR-α contacts Q94 and C96 in the CP of CD3δ.24 The corresponding N of human TCR-δ might interact with Q105 and C107 of CD3γ. The R of TCR-δ and Y of TCR-γ that correspond to R253 of human TCR-α and Y282 of human TCR-β, might establish similar contacts to CD3ζ as described for human TCRαβ.24 However, it is still unclear whether the side chains of these amino acids are located in a way that allows establishment of similar contacts to CD3. Most of the CD3-contacting amino acids located in the CP region of TCRαβ (as identified by Dong et al.24) cannot be found in TCRγδ (Fig. 2). This is the case for E233 and T238 of TCR-α that form contacts with CD3ζ, as well as W259 of TCR-β that interact with CD3ε.24 This further pinpoints to the idea that CD3 is oriented in a different position toward TCRγδ than toward TCRαβ.
6 LIGAND RECOGNITION BY αβ AND γδ TCRS
Ligand recognition by both αβ and γδ TCRs involves the three CDRs of the TCR-α, TCR-β, TCR-γ, and TCR-δ chains (Fig. 1). While CDR1 and 2 are germline encoded, the coding sequence of CDR3 arises by somatic recombination. Thus, CDR3 is the most variable region within the ligand binding TCR chains.
Agonistic ligands for αβ TCRs are foreign or mutated peptides presented on MHC molecules. CD4+ T cells recognize peptides presented on MHC class II, whereas CD8+ T cells recognize peptides on MHC class I. An exception is rare αβ TCR subsets, such as invariant NK T (iNKT) cells that recognize lipids presented on the MHC-related CD1 molecule. In the conventional αβ T cells, CD4 binds to MHC class II and CD8 to MHC class I. The conserved CDR1 and 2 regions in individual TCRα and β chains seem to exhibit a general specificity for MHC molecules, while CDR3 contacts the peptide (Fig. 3B).27, 28 Moreover, the binding geometry of αβ TCRs toward peptide-MHC (pMHC) seems to be conserved, in that the N-terminus of the peptide binds to TCR-α and the C-terminus to TCR-β.29 Since deviant binding geometries do not induce αβ TCR signaling,30, 31 a common mode of activation for αβ TCRs might exist.
In contrast, the majority of γδ TCRs do not recognize MHC molecules, which is in line with a lack of CD4 and CD8 expression by most γδ T cells. While the extracellular domains of CD4 and CD8 bind to MHC class II or class I in αβ T cells,32, 33 the intracellular tails bind to Lck,34 thus decreasing the pool of free Lck. Indeed, it has been shown that the expression of CD4 but not a modified CD4 without the Lck-binding properties decreases αβ TCR signaling in response to anti-TCR-β Abs.35 Thus, the recognition of pMHC by αβ TCRs might be necessary to bring CD4/CD8-coupled Lck in close proximity to the αβ TCR, in order to optimally initiate signaling. Due to the lack of CD4 and CD8 in γδ T cells, γδ TCRs can serve form the large pool of free Lck to induce signaling in response to any Ag. In line with this, αβ T cells that develop in CD4, CD8, MHC class I and II quadruple deficient mice react to ligands that are not MHC.35 This shows that αβ T cells that recognize Ags apart from pMHC can be positively selected if Lck is not sequestrated by CD4 or CD8. Importantly the numbers of thymic and peripheral γδ T cells are unaffected in CD4 and CD8 double KO mice.36 The independence of γδ T cells from CD4 and CD8 might be reflected in the different architecture of the γδ TCR compared to the αβ TCR. It has been shown that the conserved TCR-α CP is important for the interaction of murine αβ TCRs with CD8.37 An αβ TCR with the TM and cytoplasmic regions of TCRγδ and the CP of TCR-δ (Fig. 4, column 8) did not associate with CD8. This might be due to the lack of CD3δ being part of the TCR complex, since CD3δ can interact with CD8.37, 38 Although human γδ TCRs incorporate CD3δ, the orientation or structure of CD3δ might differ compared to αβ TCRs so that CD3δ might couple inefficiently to CD8 in γδ TCRs.
Ligands for γδ TCRs include lipid-presenting or stress-induced MHC-like molecules, other non-MHC cell surface molecules as well as soluble proteins and small peptides.39-41 The main subset of γδ T cells in the human blood, which carry Vγ9Vδ2 TCRs, is known to respond to phosphoantigens, which are intermediates of microbial and eukaryotic metabolic pathways for isoprenoid synthesis.42, 43 Butyrophilins, which are members of the B7 family of TM proteins, are involved in the recognition of phosphoantigens by human Vγ9Vδ2 TCRs.44-47 However, it is debated whether Butyrophilins serve as phosphoantigen-presenting molecules48 or undergo a conformational change upon intracellular binding to phosphoantigens, which would then allow them to bind to the TCR.49, 50 Interestingly, Melandri et al.51 have identified a critical role of the hypervariable region 4 (HV4) in TCR-γ in the recognition of Butyrophilin-like (BTNL) molecules by both murine and human γδ TCRs. HV4 in TCR-β does not contact pMHC.52 While TCR-γ HV4 is the solely determinant for BNTL recognition by certain γδ TCRs those TCRs might still be able to bind a second Ag by the CDR regions of TCRγδ, thus exhibiting a dual Ag specificity.51
A comparison between the CDR3s of TCR-α, TCR-β, TCR-γ, TCR-δ, and Igs showed that the CDR3s of TCR-γ and TCR-δ are more similar in length to those of Igs than to those of TCR-α and TCR-β.53, 54 The long CDR3 loop of the TCRδ chain plays a key role in Ag recognition of the MHC-like molecule T22 by the murine G8 γδ TCR.55 It was shown that CDR3δ contacts T22 in the groove that structurally resembles the peptide binding groove of MHC class I molecules (Fig. 3E).
In summary, the ligands for the γδ TCRs are diverse and do not follow a general principle as the ones for αβ TCRs. The lack of common properties of Ags for γδ TCRs complicates the establishment of the molecular mechanism that lead to γδ TCR activation.
7 EXPOSURE OF THE PROLINE-RICH SEQUENCE (PRS) IN CD3ε BY αβ AND γδ TCRS
Binding of pMHC to the αβ TCR leads to the exposure of the proline-rich sequence (PRS) in CD3ε, which serves as a docking site for the SH3 domain-containing adaptor protein Nck (Fig. 3, upper panels).56, 57 Exposure of the CD3ε PRS and Nck recruitment are critical determinants of αβ T cell development and activation.58, 59 Mice lacking the two Nck isoforms (Nck-KO),60, 61 harboring a mutation in the CD3ε PRS (CD3ε-PRS-KI)62 or in the CD3ε stalk region (CD3ε-C80G) preventing exposure of the CD3ε PRS63 exhibited a block in αβ T cell development. This indicates that the ligand-stabilized exposure of the CD3ε PRS and the subsequent recruitment of Nck are crucial for αβ TCR (and pre-αβ TCR) functioning.
In sharp contrast to αβ T cells, not all murine γδ T cell subsets depend on the exposure of the CD3ε PRS during development. γδ T cells can be divided into different subsets according to their usage of particular V fragments in the recombined TCR-γ and TCR-δ chains, their appearance during ontogeny and their homing to different tissues as well as their cytokine profile.64, 65 The first γδ T cells that leave the thymus exhibit an invariant Vγ5+ TCR and locate to the skin epidermis.66 These cells are known as dendritic epidermal T cells (DETC) and are potent IFN-γ producers. A fraction of Vγ5+ T cells that leave the thymus at later stages of development display polyclonal γδ TCRs and do not become DETC.67 The second wave of murine γδ T cells that leave the thymus are Vγ6+ and mainly localize in uterus, lung, and tongue, while the third and fourth wave express Vγ4+ and Vγ1+ TCRs, respectively, and mainly localize in blood, spleen, and lymph nodes. A fraction of Vγ1+ T cells that express the transcription factor promyelocytic leukemia zinc finger (PLZF) and the NK cell marker NK1.1 are known as γδ NK T (NKT) cells and mainly localize in the spleen and liver.68, 69 Vγ7+ T cells, some of which are supposed to develop extrathymically,70 are mainly found in the intestinal mucosa.
Exposure of the CD3ε PRS was not required for the development of Vγ1+, Vγ7+, and Vγ5+ (non-DETC) γδ T cells as shown in CD3ε-C80G and CD3ε-PRS-KI mice.63 In fact, the CD3ε PRS was not exposed upon engagement of a human Vγ9Vδ2 TCR and the murine G8 γδ TCR with their natural ligands, phosphoantigens, and T22, respectively (Fig. 3E).71 The inability of the G8 γδ TCR to expose the CD3ε PRS was mapped to the C region of TCRγδ, since a murine αβ TCR with the two C regions of the G8 TCRγδ lost its ability to expose the CD3ε PRS upon binding to pMHC tetramers (Fig. 4, column 3). Further, a murine αβ TCR containing the CDR3 loop of the G8 TCR-δ chain responded to T22 tetramers with an exposure of the CD3ε PRS (Fig. 4, column 4). This indicates that differences between the γδ TCR-T22 and αβ TCR-pMHC interactions, such as binding geometry or affinity, are not the cause of the lack of CD3ε PRS exposure at the G8 γδTCR. Consequently, the communication of TCRαβ or TCRγδ with CD3 and thus the 3D arrangement of αβ and γδ TCRs determined whether the CD3ε PRS was exposed or not.
Importantly, binding of the natural ligands to human Vγ9Vδ2 TCRs or the G8 γδ TCR led to the induction of TCR signaling72, 71 although the CD3ε PRS was not exposed. This suggests that in sharp contrast to the αβ TCR, γδ TCR signaling can be induced without the exposure of the CD3ε PRS. However, human αβ T cells transduced with a human αβ TCR containing the C regions of the human TCRγδ (Fig. 4, column 3) failed to secrete IFN-γ upon contact with cognate tumor cells.73 Since in a very similar chimera the CD3ε PRS was not exposed (see above,71), the data by Tao et al.73 suggest that also in their case the CD3ε PRS was not exposed and that this was the cause of impaired signaling. Whether the lack of IFN-γ secretion was due to failure of TCR signaling induction was not tested. However, IFN-γ secretion could be induced by another chimeric αβ TCR in which only the C immunoglobulin-like domains were swapped with the one of TCRγδ (Fig. 4, column 5). This indicates that other parts of the TCRαβ C region besides the immunoglobulin-like domain might have been responsible for PRS exposure. Accordingly, a chimeric αβ TCR with the CP, the TM region, and the cytoplasmic tail of TCRγδ failed to induce IFN-γ secretion (Fig. 4, column 6). This could indicate that these regions in the αβ TCR interact differently with CD3 than in the γδ TCR and that thus the ligand-induced αβ TCR rearrangements leading to the PRS exposure might not occur in the same way in the γδ TCR. As a consequence, the PRS would not be exposed in a TCR with the CP, TM region, and cytoplasmic tail of TCRγδ.
In murine TCRs, it might also be the case that the C regions determine whether PRS becomes accessible upon ligand binding. A chimeric αβ TCR with the TM and cytoplasmic regions of TCRγδ and the CP of TCRδ (Fig. 4, column 8) exhibited a defective signaling to Fyn, Lck, ZAP70, and LAT, an impaired activation of Erk, as well as a defective calcium response and induced no IL-2 secretion.74, 75, 37, 76 However, ITAM phosphorylation was induced,76 which is in line with the observed internalization of an αβ TCR with the C regions of TCRγδ.71 Thus, only partial signaling might be initiated by these chimeric αβ/γδ TCRs not leading to T cell activation. When certain parts of the C regions of TCRγδ are combined with the V regions of TCRαβ, binding of the αβ TCR ligand does not induce T cell activation (Fig. 4, columns 3, 6, and 8). However, the same regions in the wild-type TCRγδ allow TCR signaling and activation upon binding of the γδ TCR ligand.
Although Vγ1+, Vγ7+, and Vγ5+ (non-DETC) cells develop normally in CD3ε-C80G and CD3ε-PRS-KI mice, the number of Vγ4+, Vγ5+ (DETC), and γδ NKT cells are strongly reduced, demonstrating an inhomogeneity in the signaling requirements between different γδ T cell subsets during development.63 A similar albeit milder phenotype was observed in CD3ε-PRS-KI and Nck-KO mice63 indicating that CD3ε PRS exposure and Nck recruitment are at least partially required for the development of certain γδ T cell subsets. Although the CD3ε PRS in the G8 γδ TCR is not exposed in the resting or T22-bound state,71 the CD3ε PRS of Vγ4+, Vγ5+ (DETC), and γδ NKT cells might be accessible at some point during the development of the cells—either in the resting or the ligand-bound state. The development of DETCs depends on the presence of the butyrophilin-like protein Skint1 indicating that these cells require positive selection by a thymic ligand.77 This indicates that γδ TCRs with different Vγ/Vδ usage and/or different γδ TCR ligands have a different potential to expose the CD3ε PRS. The reason why Vγ1+, Vγ7+, and Vγ5+ (non-DETC) cells develop in the C80G mice might be due to a lack of ligand-induced positive selection and thus no need for CD3ε PRS exposure. Alternatively, these cells might encounter a thymic ligand for positive selection, but without inducing CD3ε PRS exposure.
8 ANTI-CD3ε Ab-INDUCED REARRANGEMENTS AT THE γδ TCR AND TRANSLATIONAL APPLICATION IN CANCER IMMUNOTHERAPY
Unlike the natural ligands of human Vγ9Vδ2 TCRs and the murine G8 γδ TCR, Abs that directly bind to CD3ε (UCHT-1 in case of human and 145-2C11 in case of murine γδ TCRs) induce the exposure of the CD3ε PRS in these γδ TCRs (Fig. 3F).71 This artificial exposure of the CD3ε PRS by Abs increase the signaling strength, such as seen in Ca2+ influx and phosphorylation of Erk, Akt, and IκB-α, and enhance tumor cell killing by Vγ9Vδ2 T cells. Importantly, the tumor killing was not enhanced by anti-CD3ε Abs in γδ T cells expressing a mutant CD3ε (CD3ε-K76T) that is not able to expose the CD3ε PRS. This indicates that the enhancing effect on tumor killing is dependent on CD3ε PRS exposure. A similar enhancing effect on tumor killing can be induced by Fab fragments of the anti-CD3ε Abs.78 This has the advantage that anti-CD3ε Fab fragments alone do not activate the Vγ9Vδ2 T cells but increase the effector functions in the presence of the target cells. In fact, exposure of the CD3ε PRS is not sufficient to induce γδ TCR signaling, which is in line with the αβ TCR that requires an additional cross-linking event to induce signaling.58 Interestingly, the enhancing effect on tumor killing is independent of the recruitment of Nck to the Vγ9Vδ2 TCR. This suggests that other proteins than Nck bind to the CD3ε PRS in the Vγ9Vδ2 TCR or that other motifs are exposed by the anti-CD3ε Fab fragment.
9 LIPID INTERACTION AND PLASMA MEMBRANE ORGANIZATION OF αβ AND γδ TCRS
The plasma membrane organization of the αβ TCR is greatly impacted by cholesterol. Cholesterol can bind to the TM region of the TCR-β chain and together with sphingomyelin induces the formation of αβ TCR nanoclusters.79, 80 An increase in the cholesterol level of Ag-experienced αβ T cells results in enhanced nanoclustering of αβ TCRs and this increases the avidity toward pMCH.80-82, 79 The increase in αβ TCR:pMHC avidity is reflected by the enhanced sensitivity of activated and memory αβ T cells compared to naïve αβ T cells.83 Interestingly, cholesterol binding could not be detected at a Vγ9Vδ2 TCR expressed in αβ TCR-deficient Jurkat cells,84 indicating that it is the intrinsic property of the Vγ9Vδ2 TCR to not bind to cholesterol. The TM regions of TCRαβ and TCRγδ differ (Fig. 2) suggesting that this is the cause for the lack of cholesterol binding to the γδ TCR. In fact a chimeric αβ TCR in which the TM region and intracellular tail of TCR-β was exchanged with that of TCR-γ (Fig. 4, column 7) failed to bind to cholesterol.84 In line with this difference in cholesterol binding, a different membrane organization of αβ and Vγ9Vδ2 TCRs was observed by quantum dot-based nanoscale immunofluorescence imaging of macaque T cells.85 While 50% of αβ TCRs could be detected as nanoclusters comprising 2 or more TCRs, 80% of the γδ TCRs were detected as monomers (Fig. 5). However, after in vivo stimulation of Vγ9Vδ2 T cells by treatment with phosphoantigen and IL2, γδ TCR nanoclusters appeared and were sustained for up to 2 weeks. Interestingly, the in vivo expanded Vγ9Vδ2 T cells bearing a high degree of TCR nanoclusters exhibited an increased responsiveness to phosphoantigen stimulation compared to naïve Vγ9Vδ2 T cells. This suggests a similar role for TCR nanoclusters in the regulation of αβ and γδ T cell sensitivity toward Ag. However, the lack of detectable interaction between γδ TCRs and cholesterol suggests that TCR nanoclustering of αβ and γδ TCRs might be regulated by different mechanisms or require different concentrations of cholesterol.
To overcome a potential decreased affinity of γδ TCRs toward cholesterol, a higher concentration of cholesterol would be required to induce nanoclustering. In fact, it has been shown that murine γδ T cells exhibit an increased expression of genes involved in cholesterol metabolism as well as higher levels of cholesterol compared to αβ T cells.86 Furthermore, it has been described that cholesterol sulfate, a naturally occurring derivative of cholesterol, suppresses TCR signaling in αβ and γδ T cells as measured by the phosphorylation of the S6 ribosomal subunit.87 Cholesterol sulfate can displace cholesterol from the αβ TCR, thereby reducing αβ TCR nanoclustering and reducing the avidity for Ags. The inhibition of γδ TCR signaling by cholesterol sulfate might indicate that cholesterol is also involved in γδ TCR nanoclustering and suggests that γδ TCR nanoclusters modulate signaling strength.
10 ALLOSTERIC REGULATION OF THE TCR'S CONFORMATION
In addition to the role in αβ TCR nanoclustering, cholesterol regulates the conformational state of the αβ TCR.84 According to this allosteric model, the αβ TCR resides in an equilibrium between a resting, cholesterol-bound conformation (Fig. 3A) and an active conformation, that is not bound to cholesterol (Fig. 3B and C). Ligand binding can only occur to the active state and thus stabilizes the active αβ TCR conformation, shifting the equilibrium to the active side. Importantly, only in the active αβ TCR conformation, the CD3ε PRS is exposed and the CD3 ITAMs are accessible for phosphorylation by Lck (Fig. 3B and C). Cholesterol binding could not be detected in a chimeric human αβ TCR in which the TCR-β TM and intracellular regions were exchanged with that of TCR-γ (Fig. 4, column 7).84 In this TCR, even in the absence of ligand, the equilibrium is shifted to the active conformation, in which the CD3ε PRS is exposed. Upon stimulation with anti-CD3ε Abs exposure of the PRS in this TCR is also enhanced compared to wild-type αβ TCRs, leading to an increased ITAM phosphorylation. Accordingly, T cells expressing the chimeric TCR exhibit increased levels of phosphorylated Erk in the unstimulated state and upon anti-CD3ε stimulation. Intriguingly, another report in which the TM and intracellular regions of murine TCR-β were exchanged with that of TCR-γ (Fig. 4, column 7) supports this finding.88 The authors used transgenic mice for the wild-type and the chimeric TCR and detected a higher secretion of IL-2 upon antigenic stimulation. This finding suggests that the differences in cholesterol binding between the αβ and γδ TCR and its effect might be conserved in evolution.
Less efficient cholesterol binding by the γδ TCR and thus a higher percentage of γδ TCRs in the active conformation compared to αβ TCRs might be the basis for the higher basal activation and stronger TCR-induced signaling observed in γδ T cells. Increased levels of phosphorylated Erk and ZAP70 were observed in thymic murine γδ T cells compared to pre-αβ TCR expressing thymocytes.89 In addition, murine γδ T cells exhibit an increased Ca2+ and Erk signaling upon anti-CD3ε stimulation compared to murine αβ T cells resulting in an increased proliferation and higher magnitude of the up-regulation of effector genes.8, 90 However, additional differences between αβ and γδ TCRs (as, e.g., the accessibility of motifs in the CD3 intracellular tails) as well as differences in the cellular background between αβ and γδ T cells (as, e.g., expression levels of signaling proteins91), might account for the different signaling strength as well.
It has been shown that the cytoplasmic tails of CD3ε and CD3ζ interact with lipids of the plasma membrane.92-94 The lipid environment of the αβ TCR changes upon engagement with pMHC, which might induce the release of the CD3 cytoplasmic tails from the plasma membrane resulting in the exposure of the ITAMs.94 Furthermore, the influx of Ca2+ ions into the cytosol might help to stabilize the tails in the released conformation by neutralizing the charge of the inner leaflet of the plasma membrane and thus inducing signal amplification.95 Possible differences in the lipid environment between αβ and γδ TCRs might lead to a different orientation of the cytoplasmic CD3 tails. The lipid environment of the γδ TCR has not been studied so far. However, phosphoantigens induce the clustering and co-localization of the human Vγ9Vδ2 TCR and the lipid raft associated lipid GM1,96 a feature that has been reported for the activated αβ TCR as well.97 Thus, activation induced changes in the lipid environment of the γδ TCR can be assumed as well and might be involved in the regulation of ITAM accessibility.
11 CONCLUDING REMARKS
αβ and γδ TCRs share many similarities, however, accumulating reports point to important differences at the molecular level of TCR organization and conformation, most of which are not fully understood. These differences could directly impact the activation mechanism of the 2 TCRs. While the architecture of αβ TCRs is specialized for the recognition of pMHC, γδ TCRs are able to binds to many different ligands. The distinct architecture of the γδ TCR might allow for a stronger coupling to the same signaling molecules used by the αβ TCR or result in the engagement of different pathways. This might enable the strong proliferative response of γδ T cells to TCR stimulation in the absence of CD28 costimulation8 and a fast onset of effector functions that might be linked to their contributing functions to initiate an immune response. A detailed knowledge of the molecular basis of γδ TCR activation would help to improve clinical application of γδ T cells as well as the design of modified γδ TCRs. In addition, this information could help to understand general aspects of γδ T cell biology such as αβ versus γδ fate decision and the requirements for γδ TCR ligands to trigger signaling. Comparative analyses between αβ and γδ TCRs are useful, because they can reveal specific features of both TCRs, which could be used to control the cellular response of αβ and γδ T cells in diseases. We hope that with this review, we can encourage scientists to include both αβ and γδ TCRs in future studies to perform a side-by-side comparison.
A.M. and W.W.S. wrote the manuscript and designed the figures.
This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) through EXC-294 (Center for Biological Signalling Studies, BIOSS), EXC-2189 (Center of Integrative Biological Signaling Studies, CIBSS, Project-ID 390939984), SCHA976/7-1, SCHA976/8-1, Project-ID 403222702 - SFB 1381, and GSC-4 (Spemann Graduate School of Biology and Medicine). Gefördert durch die Deutsche Forschungsgemeinschaft (DFG) im Rahmen der Exzellenzstrategie des Bundes und der Länder – EXC-2189 – Projektnummer 390939984. We thank the members of FOR2799 and the invited guests for the nice and stimulation atmosphere at the γδ meeting “Receiving and Translating Signals via the Gamma Delta T Cell Receptor” in June 2019 in Erlangen, Germany. We thank Susana Minguet, Gina J. Fiala, and O. Sascha Yousefi for discussions.
The authors declare no conflicts of interest.
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