Richard Berry

Molecular architecture of the αβ T cell receptor–CD3 complex

Significance The T-cell receptor (TCR) does not signal on its own. Instead, it is constitutively associated with the CD3 coreceptors, which contain intracellular signaling motifs. Although antigen (Ag) recognition by the TCR and the activation of T cells after CD3 activation have been extensively studied, there is far less known about how the TCR relays the binding of Ag to initiate intracellular signaling. Detailed study of the eight-chain TCR–CD3 complex is hampered by limited structural information for how the components of the complex interact. We use complementary structural approaches to examine where the TCR–CD3 extracellular domains are placed relative to each other and the overall organization of the complex. αβ T-cell receptor (TCR) activation plays a crucial role for T-cell function. However, the TCR itself does not possess signaling domains. Instead, the TCR is noncovalently coupled to a conserved multisubunit signaling apparatus, the CD3 complex, that comprises the CD3εγ, CD3εδ, and CD3ζζ dimers. How antigen ligation by the TCR triggers CD3 activation and what structural role the CD3 extracellular domains (ECDs) play in the assembled TCR–CD3 complex remain unclear. Here, we use two complementary structural approaches to gain insight into the overall organization of the TCR–CD3 complex. Small-angle X-ray scattering of the soluble TCR–CD3εδ complex reveals the CD3εδ ECDs to sit underneath the TCR α-chain. The observed arrangement is consistent with EM images of the entire TCR–CD3 integral membrane complex, in which the CD3εδ and CD3εγ subunits were situated underneath the TCR α-chain and TCR β-chain, respectively. Interestingly, the TCR–CD3 transmembrane complex bound to peptide–MHC is a dimer in which two TCRs project outward from a central core composed of the CD3 ECDs and the TCR and CD3 transmembrane domains. This arrangement suggests a potential ligand-dependent dimerization mechanism for TCR signaling. Collectively, our data advance our understanding of the molecular organization of the TCR–CD3 complex, and provides a conceptual framework for the TCR activation mechanism.

Effective functional maturation of invariant natural killer T cells is constrained by negative selection and T-cell antigen receptor affinity

Significance Several different populations of T lymphocytes develop in the thymus from a common precursor. Each population plays a unique and critical role in the mounting and resolution of an immune response. The mechanisms responsible for the emergence of these different populations remain incompletely understood. We demonstrate that strict “Goldilocks” conditions of affinity for self-lipids by the T-cell antigen receptor expressed on T-cell precursors are necessary for imprinting the proper developmental program toward the invariant NK T-cell lineage. Our results establish a direct link between the affinity of the T-cell receptor for self-antigens and the proper development of a unique population of lymphocytes that has been implicated in the modulation of a multitude of immune responses in mice and humans. The self-reactivity of their T-cell antigen receptor (TCR) is thought to contribute to the development of immune regulatory cells, such as invariant NK T cells (iNKT). In the mouse, iNKT cells express TCRs composed of a unique Vα14-Jα18 rearrangement and recognize lipid antigens presented by CD1d molecules. We created mice expressing a transgenic TCR-β chain that confers high affinity for self-lipid/CD1d complexes when randomly paired with the mouse iNKT Vα14-Jα18 rearrangement to study their development. We show that although iNKT cells undergo agonist selection, their development is also shaped by negative selection in vivo. In addition, iNKT cells that avoid negative selection in these mice express natural sequence variants of the canonical TCR-α and decreased affinity for self/CD1d. However, limiting the affinity of the iNKT TCRs for “self” leads to inefficient Egr2 induction, poor expression of the iNKT lineage-specific zinc-finger transcription factor PLZF, inadequate proliferation of iNKT cell precursors, defects in trafficking, and impaired effector functions. Thus, proper development of fully functional iNKT cells is constrained by a limited range of TCR affinity that plays a key role in triggering the iNKT cell-differentiation pathway. These results provide a direct link between the affinity of the TCR expressed by T-cell precursors for self-antigens and the proper development of a unique population of lymphocytes essential to immune responses.

The structure of the atypical killer cell immunoglobulin-like receptor, KIR2DL4

Background: KIR2DL4 is an important natural killer cell receptor with properties distinct from other KIRs. Results: The D0 domain of KIR2DL4 drove self-association of the receptor. Conclusion: Among KIRs, the self-association of KIR2DL4 is unique and a result of discrete differences in its D0 domain. Significance: The self-association of KIR2DL4 has implications for its unique signaling and function. The engagement of natural killer cell immunoglobulin-like receptors (KIRs) with their target ligands, human leukocyte antigen (HLA) molecules, is a critical component of innate immunity. Structurally, KIRs typically have either two (D1-D2) or three (D0-D1-D2) extracellular immunoglobulin domains, with the D1 and D2 domain recognizing the α1 and α2 helices of HLA, respectively, whereas the D0 domain of the KIR3DLs binds a loop region flanking the α1 helix of the HLA molecule. KIR2DL4 is distinct from other KIRs (except KIR2DL5) in that it does not contain a D1 domain and instead has a D0-D2 arrangement. Functionally, KIR2DL4 is also atypical in that, unlike all other KIRs, KIR2DL4 has both activating and inhibitory signaling domains. Here, we determined the 2.8 Å crystal structure of the extracellular domains of KIR2DL4. Structurally, KIR2DL4 is reminiscent of other KIR2DL receptors, with the D0 and D2 adopting the C2-type immunoglobulin fold arranged with an acute elbow angle. However, KIR2DL4 self-associated via the D0 domain in a concentration-dependent manner and was observed as a tetramer in the crystal lattice by size exclusion chromatography, dynamic light scattering, analytical ultracentrifugation, and small angle x-ray scattering experiments. The assignment of residues in the D0 domain to forming the KIR2DL4 tetramer precludes an interaction with HLA akin to that observed for KIR3DL1. Accordingly, no interaction was observed to HLA by direct binding studies. Our data suggest that the unique functional properties of KIR2DL4 may be mediated by self-association of the receptor.

An Extensive Antigenic Footprint Underpins Immunodominant TCR Adaptability against a Hypervariable Viral Determinant

Mutations in T cell epitopes are implicated in hepatitis C virus (HCV) persistence and can impinge on vaccine development. We recently demonstrated a narrow bias in the human TCR repertoire targeted at an immunodominant, but highly mutable, HLA-B*0801–restricted epitope (1395HSKKKCDEL1403 [HSK]). To investigate if the narrow TCR repertoire facilitates CTL escape, structural and biophysical studies were undertaken, alongside comprehensive functional analysis of T cells targeted at the natural variants of HLA-B*0801–HSK in different HCV genotypes and quasispecies. Interestingly, within the TCR–HLA-B*0801–HSK complex, the TCR contacts all available surface-exposed residues of the HSK determinant. This broad epitope coverage facilitates cross-genotypic reactivity and recognition of common mutations reported in HCV quasispecies, albeit to a varying degree. Certain mutations did abrogate T cell reactivity; however, natural variants comprising these mutations are reportedly rare and transient in nature, presumably due to fitness costs. Overall, despite a narrow bias, the TCR accommodated frequent mutations by acting like a blanket over the hypervariable epitope, thereby providing effective viral immunity. Our findings simultaneously advance the understanding of anti-HCV immunity and indicate the potential for cross-genotype HCV vaccines.

Structure of the chicken CD3εδ/γ heterodimer and its assembly with the αβT cell receptor

Background: Chickens possess a CD3δ/γ chain that assembles with T cell receptor to mediate immune signaling. Results: Chicken CD3ϵδ/γ has an atypical heterodimer interface and surface but associates with TCRαβ. Conclusion: Chicken CD3δ/γ represents a hybrid chain possessing features in common with human CD3δ and CD3γ. Significance: Understanding the ancestral TCR signaling complex provides insights into the evolution of this signaling apparatus. In mammals, the αβT cell receptor (TCR) signaling complex is composed of a TCRαβ heterodimer that is noncovalently coupled to three dimeric signaling molecules, CD3ϵδ, CD3ϵγ, and CD3ζζ. The nature of the TCR signaling complex and subunit arrangement in different species remains unclear however. Here we present a structural and biochemical analysis of the more primitive ancestral form of the TCR signaling complex found in chickens. In contrast to mammals, chickens do not express separate CD3δ and CD3γ chains but instead encode a single hybrid chain, termed CD3δ/γ, that is capable of pairing with CD3ϵ. The NMR structure of the chicken CD3ϵδ/γ heterodimer revealed a unique dimer interface that results in a heterodimer with considerable deviation from the distinct side-by-side architecture found in human and murine CD3ϵδ and CD3ϵγ. The chicken CD3ϵδ/γ heterodimer also contains a unique molecular surface, with the vast majority of surface-exposed, nonconserved residues being clustered to a single face of the heterodimer. Using an in vitro biochemical assay, we demonstrate that CD3ϵδ/γ can assemble with both chicken TCRα and TCRβ via conserved polar transmembrane sites. Moreover, analogous to the human TCR signaling complex, the presence of two copies of CD3ϵδ/γ is required for ζζ assembly. These data provide insight into the evolution of this critical receptor signaling apparatus.

Recognition of the Major Histocompatibility Complex (MHC) Class Ib Molecule H2-Q10 by the Natural Killer Cell Receptor Ly49C

Murine natural killer (NK) cells are regulated by the interaction of Ly49 receptors with major histocompatibility complex class I molecules (MHC-I). Although the ligands for inhibitory Ly49 were considered to be restricted to classical MHC (MHC-Ia), we have shown that the non-classical MHC molecule (MHC-Ib) H2-M3 was a ligand for the inhibitory Ly49A. Here we establish that another MHC-Ib, H2-Q10, is a bona fide ligand for the inhibitory Ly49C receptor. H2-Q10 bound to Ly49C with a marginally lower affinity (∼5 μm) than that observed between Ly49C and MHC-Ia (H-2Kb/H-2Dd, both ∼1 μm), and this recognition could be prevented by cis interactions with H-2K in situ. To understand the molecular details underpinning Ly49·MHC-Ib recognition, we determined the crystal structures of H2-Q10 and Ly49C bound H2-Q10. Unliganded H2-Q10 adopted a classical MHC-I fold and possessed a peptide-binding groove that exhibited features similar to those found in MHC-Ia, explaining the diverse peptide binding repertoire of H2-Q10. Ly49C bound to H2-Q10 underneath the peptide binding platform to a region that encompassed residues from the α1, α2, and α3 domains, as well as the associated β2-microglobulin subunit. This docking mode was conserved with that previously observed for Ly49C·H-2Kb. Indeed, structure-guided mutation of Ly49C indicated that Ly49C·H2-Q10 and Ly49C·H-2Kb possess similar energetic footprints focused around residues located within the Ly49C β4-stand and L5 loop, which contact the underside of the peptide-binding platform floor. Our data provide a structural basis for Ly49·MHC-Ib recognition and demonstrate that MHC-Ib represent an extended family of ligands for Ly49 molecules.

Recognition of nectin-2 by the natural killer cell receptor T cell immunoglobulin and ITIM domain (TIGIT)

T cell immunoglobulin and ITIM domain (TIGIT) is an inhibitory receptor expressed on the surface of natural killer (NK) cells. TIGIT recognizes nectin and nectin-like adhesion molecules and thus plays a critical role in the innate immune response to malignant transformation. Although the TIGIT nectin-like protein-5 (necl-5) interaction is well understood, how TIGIT engages nectin-2, a receptor that is broadly over-expressed in breast and ovarian cancer, remains unknown. Here, we show that TIGIT bound to the immunoglobulin domain of nectin-2 that is most distal from the membrane with an affinity of 6 μm, which was moderately lower than the affinity observed for the TIGIT/necl-5 interaction (3.2 μm). The TIGIT/nectin-2 binding disrupted pre-assembled nectin-2 oligomers, suggesting that receptor-ligand and ligand-ligand associations are mutually exclusive events. Indeed, the crystal structure of TIGIT bound to the first immunoglobulin domain of nectin-2 indicated that the receptor and ligand dock using the same molecular surface and a conserved “lock and key” binding motifs previously observed to mediate nectin/nectin homotypic interactions as well as TIGIT/necl-5 recognition. Using a mutagenesis approach, we dissected the energetic basis for the TIGIT/nectin-2 interaction and revealed that an “aromatic key” of nectin-2 is critical for this interaction, whereas variations in the lock were tolerated. Moreover, we found that the C-C′ loop of the ligand dictates the TIGIT binding hierarchy. Altogether, these findings broaden our understanding of nectin/nectin receptor interactions and have implications for better understanding the molecular basis for autoimmune disease and cancer.

Insight into the basis of autonomous immunoreceptor activation

Expression of the pre-T cell receptor (pTCR) by immature thymocytes is crucial for T cell development. The pTCR comprises an invariant pre-Tα chain that pairs with a newly rearranged TCRβ chain and CD3 signaling components. Despite its similarity to the mature αβTCR, which binds to specific peptide-loaded major histocompatibility molecules, the pTCR functions in a ligand-independent manner. Precisely how pTCR functions autonomously has remained a source of intense debate. Recently, the structure of the extracellular domain of the pTCR has been determined, providing insight into the mechanism of pTCR autonomous signaling. In this review, we reflect on the current understanding of pTCR function and draw comparisons to the mechanisms employed by the mature αβTCR and the related pre-B cell receptor.

A highly conserved sequence of the viral TAP inhibitor ICP47 is required for freezing of the peptide transport cycle.

The transporter associated with antigen processing (TAP) translocates antigenic peptides into the endoplasmic reticulum (ER) lumen for loading onto MHC class I molecules. This is a key step in the control of viral infections through CD8+ T-cells. The herpes simplex virus type-1 encodes an 88 amino acid long species-specific TAP inhibitor, ICP47, that functions as a high affinity competitor for the peptide binding site on TAP. It has previously been suggested that the inhibitory function of ICP47 resides within the N-terminal region (residues 1–35). Here we show that mutation of the highly conserved 50PLL52 motif within the central region of ICP47 attenuates its inhibitory capacity. Taking advantage of the human cytomegalovirus-encoded TAP inhibitor US6 as a luminal sensor for conformational changes of TAP, we demonstrated that the 50PLL52 motif is essential for freezing of the TAP conformation. Moreover, hierarchical functional interaction sites on TAP dependent on 50PLL52 could be defined using a comprehensive set of human-rat TAP chimeras. This data broadens our understanding of the molecular mechanism underpinning TAP inhibition by ICP47, to include the 50PLL52 sequence as a stabilizer that tethers the TAP-ICP47 complex in an inward-facing conformation.

The Role of L(u)ck in T Cell Triggering

The Src family kinase Lck is activated prior to T cell receptor engagement and poised for action upon antigen binding. T cells use a highly complex signaling apparatus to discriminate between a diverse array of foreign antigens and a myriad of self molecules. The precise mechanism whereby signals are communicated from the antigen-binding T cell receptor to the intracellular signal transduction machinery remains a source of intense debate. This Journal Club article highlights recent research elucidating the role of the Src family kinase Lck in T cell triggering. Resting T cells contain a newly described form of preactivated Lck that is both necessary and sufficient for T cell activation but remains uncoupled from the T cell receptor in the absence of antigen. This research allows a reappraisal of the mechanisms underlying T cell triggering.