Key Residues at Third CDR3? Position Impact Structure and Antigen Recognition of Human Invariant NK TCRs.
ABSTRACT: The human invariant NK (iNK) TCR is largely composed of the invariant TCR V?24-J?18 chain and semivariant TCR V?11 chains with variable CDR3? sequences. The direct role of CDR3? in Ag recognition has been studied extensively. Although it was noted that CDR3? can interact with CDR3?, how this interaction might indirectly influence Ag recognition is not fully elucidated. We observed that the third position of V?11 CDR3 can encode an Arg or Ser residue as a result of somatic rearrangement. Clonotypic analysis of the two iNK TCR types with a single amino acid substitution revealed that the staining intensity by anti-V?24 Abs depends on whether Ser or Arg is encoded. When stained with an anti-V?24-J?18 Ab, human primary invariant NKT cells could be divided into V?24 low- and high-intensity subsets, and Arg-encoding TCR V?11 chains were more frequently isolated from the V?24 low-intensity subpopulation compared with the V?24 high-intensity subpopulation. The Arg/Ser substitution also influenced Ag recognition as determined by CD1d multimer staining and CD1d-restricted functional responses. Importantly, in silico modeling validated that this Ser-to-Arg mutation could alter the structure of the CDR3? loop, as well as the CDR3? loop. Collectively, these results indicate that the Arg/Ser encoded at the third CDR3? residue can effectively modulate the overall structure of, and Ag recognition by, human iNK TCRs.
Project description:Mucosal-associated invariant T (MAIT) cells express an invariant T cell receptor (TCR) ?-chain (TRAV1-2 joined to TRAJ33, TRAJ20, or TRAJ12 in humans), which pairs with an array of TCR ?-chains. MAIT TCRs can bind folate- and riboflavin-based metabolites restricted by the major histocompatibility complex (MHC)-related class I-like molecule, MR1. However, the impact of MAIT TCR and MR1-ligand heterogeneity on MAIT cell biology is unclear. We show how a previously uncharacterized MR1 ligand, acetyl-6-formylpterin (Ac-6-FP), markedly stabilized MR1, potently up-regulated MR1 cell surface expression, and inhibited MAIT cell activation. These enhanced properties of Ac-6-FP were attributable to structural alterations in MR1 that subsequently affected MAIT TCR recognition via conformational changes within the complementarity-determining region (CDR) 3? loop. Analysis of seven TRBV6-1(+) MAIT TCRs demonstrated how CDR3? hypervariability impacted on MAIT TCR recognition by altering TCR flexibility and contacts with MR1 and the Ag itself. Ternary structures of TRBV6-1, TRBV6-4, and TRBV20(+) MAIT TCRs in complex with MR1 bound to a potent riboflavin-based antigen (Ag) showed how variations in TRBV gene usage exclusively impacted on MR1 contacts within a consensus MAIT TCR-MR1 footprint. Moreover, differential TRAJ gene usage was readily accommodated within a conserved MAIT TCR-MR1-Ag docking mode. Collectively, MAIT TCR heterogeneity can fine-tune MR1 recognition in an Ag-dependent manner, thereby modulating MAIT cell recognition.
Project description:Ag processing in the endoplasmic reticulum (ER) by the ER aminopeptidase associated with Ag processing (ERAAP) is central to presentation of a normal peptide-MHC class I (MHC I) repertoire. Alternations in ERAAP function cause dramatic changes in the MHC I-presented peptides, which elicit potent immune responses. An unusual subset of CD8+ T cells monitor normal Ag processing by responding to a highly conserved FL9 peptide that is presented by Qa-1b, a nonclassical MHC Ib molecule (QFL) in ERAAP-deficient cells. To understand the structural basis for recognition of the conserved ligand, we analyzed the ?? TCRs of QFL-specific T cells. Individual cells in normal wild-type and TCR?-transgenic mice were assessed for QFL-specific TCR ?- and ?-chains. The QFL-specific cells expressed a predominant semi-invariant TCR generated by DNA rearrangement of TRAV9d-3-TRAJ21 ?-chain and TRBV5-TRBD1-TRBJ2-7 ?-chain gene segments. Furthermore, the CDR3 regions of the ?- as well as ?-chains were required for QFL ligand recognition. Thus, the ?? TCRs used to recognize the peptide-Qa-1 ligand presented by ERAAP-deficient cells are semi-invariant and likely reflect a conserved mechanism for monitoring the fidelity of Ag processing in the ER.
Project description:V?9V?2 T cells respond in a TCR-dependent fashion to both microbial and host-derived pyrophosphate compounds (phosphoantigens, or P-Ag). Butyrophilin-3A1 (BTN3A1), a protein structurally related to the B7 family of costimulatory molecules, is necessary but insufficient for this process. We performed radiation hybrid screens to uncover direct TCR ligands and cofactors that potentiate BTN3A1's P-Ag sensing function. These experiments identified butyrophilin-2A1 (BTN2A1) as essential to V?9V?2 T cell recognition. BTN2A1 synergised with BTN3A1 in sensitizing P-Ag-exposed cells for V?9V?2 TCR-mediated responses. Surface plasmon resonance experiments established V?9V?2 TCRs used germline-encoded V?9 regions to directly bind the BTN2A1 CFG-IgV domain surface. Notably, somatically recombined CDR3 loops implicated in P-Ag recognition were uninvolved. Immunoprecipitations demonstrated close cell-surface BTN2A1-BTN3A1 association independent of P-Ag stimulation. Thus, BTN2A1 is a BTN3A1-linked co-factor critical to V?9V?2 TCR recognition. Furthermore, these results suggest a composite-ligand model of P-Ag sensing wherein the V?9V?2 TCR directly interacts with both BTN2A1 and an additional ligand recognized in a CDR3-dependent manner.
Project description:We describe a simple iterative approach to augment TCR affinity, which we studied using a myelin oligodendrocyte glycoprotein-specific TCR. We hypothesized that single amino acid modifications in TCR CDR3 could enhance TCR sensitivity through focal interactions with antigenic peptide while minimizing the risk of cross-reactivity observed previously in TCR more broadly mutagenized using in vitro evolution techniques. We show that this iterative method can indeed generate TCR with Ag sensitivity 100-fold greater than the parental receptor and can endow TCR with coreceptor independence. However, we also find that single amino acid mutations in the CDR3 can alter TCR fine specificity, affecting recognition requirements for Ag residues over most of the length of the MHC binding groove. Furthermore, minimal changes in surface-exposed CDR3 amino acids, even the addition of a single hydroxyl group or conversion of a methyl or sulfhydryl moiety to a hydroxyl, can confer modified Ag-specific TCR with new self-reactivity. In vivo modeling of modified TCR through retroviral TCR gene transfer into Rag(-/-) mice confirmed the biological significance of these altered reactivities, although it also demonstrated the feasibility of producing Ag-specific, positively selecting, coreceptor-independent receptors with markedly increased Ag sensitivity. These results affirm the possibility of readily generating affinity-enhanced TCR for therapeutic purposes but demonstrate that minimal changes in TCR CDR3 structure can promote self reactivity and thereby emphasize the importance of caution in validating receptors with even subtle alterations before clinical application.
Project description:MR1-restricted mucosal-associated invariant T (MAIT) cells represent a subpopulation of ?? T cells with innate-like properties and limited TCR diversity. MAIT cells are of interest because of their reactivity against bacterial and yeast species, suggesting that they play a role in defense against pathogenic microbes. Despite the advances in understanding MAIT cell biology, the molecular and structural basis behind their ability to detect MR1-Ag complexes is unclear. In this study, we present our structural and biochemical characterization of MAIT TCR engagement of MR1 presenting an Escherichia coli-derived stimulatory ligand, rRL-6-CH2OH, previously found in Salmonella typhimurium. We show a clear enhancement of MAIT TCR binding to MR1 due to the presentation of this ligand. Our structure of a MAIT TCR/MR1/rRL-6-CH2OH complex shows an evolutionarily conserved binding orientation, with a clear role for both the CDR3? and CDR3? loops in recognizing the rRL-6-CH2OH stimulatory ligand. We also present two additional xenoreactive MAIT TCR/MR1 complexes that recapitulate the docking orientation documented previously, despite having variation in the CDR2? and CDR3? loop sequences. Our data support a model by which MAIT TCRs engage MR1 in a conserved fashion, with their binding affinities modulated by the nature of the MR1-presented Ag or diversity introduced by alternate V? usage or CDR3? sequences.
Project description:The majority of human CD8 cytotoxic T lymphocytes express ?? T-cell receptors that recognize peptide-MHC class I complexes. Considerable attention has been devoted to TCR ? repertoires, but study of TCR ? chains has been limited. To gain a better understanding of the features of CDR3? and CDR3? in paired samples, we comprehensively analyzed 776 unique paired ?? TCR CDR3 regions in this study. We found that (I) the CDR3 length among paired ?? TCRs had a fairly narrow distribution due to random assortment of CDR3 length in alpha and beta chains; (II) nucleotide deletions among CDR3 regions were positively correlated with insertions in both ? and ? TCRs; (III) the CDR3 loops of both ? and ? chains contained an abundance of charged/polar residues and the CDR3 base regions contained a conserved motif; and (IV) the occurrence of Gly was CDR3 length- and position-dependent in both chains, whereas the frequency of Ser at positions 106 and 107 was positively correlated with CDR3 length in TCR ?. Overall, the amino acids in CDR3 loop regions were significantly different between TCR ? and ?, which suggests a distinct role for each chain in the recognition of antigen-MHC complexes. Here, we have provided detailed information on CDR3 in paired TCRs expressed on human CD8+ T cells and established the basis of a reference set for ?? TCR repertoires in healthy humans.
Project description:Mucosal-associated invariant T (MAIT) cells are an evolutionarily conserved ?? T-cell lineage that express a semi-invariant T-cell receptor (TCR) restricted to the MHC related-1 (MR1) protein. MAIT cells are dependent upon MR1 expression and exposure to microbes for their development and stimulation, yet these cells can exhibit microbial-independent stimulation when responding to MR1 from different species. We have used this microbial-independent, cross-species reactivity of MAIT cells to define the molecular basis of MAIT-TCR/MR1 engagement and present here a 2.85 Å complex structure of a human MAIT-TCR bound to bovine MR1. The MR1 binding groove is similar in backbone structure to classical peptide-presenting MHC class I molecules (MHCp), yet is partially occluded by large aromatic residues that form cavities suitable for small ligand presentation. The docking of the MAIT-TCR on MR1 is perpendicular to the MR1 surface and straddles the MR1 ?1 and ?2 helices, similar to classical ?? TCR engagement of MHCp. However, the MAIT-TCR contacts are dominated by the ?-chain, focused on the MR1 ?2 helix. TCR ?-chain contacts are mostly through the variable CDR3? loop that is positioned proximal to the CDR3? loop directly over the MR1 open groove. The elucidation of the MAIT TCR/MR1 complex structure explains how the semi-invariant MAIT-TCR engages the nonpolymorphic MR1 protein, and sheds light onto ligand discrimination by this cell type. Importantly, this structure also provides a critical link in our understanding of the evolution of ?? T-cell recognition of MHC and MHC-like ligands.
Project description:Invariant natural killer T (iNKT) cells recognize self-lipids presented by CD1d through characteristic TCRs, which mainly consist of the invariant V?14-J?18 TCR? chain and V?8.2, 7 or 2 TCR? chains with hypervariable CDR3? sequences in mice. The iNKT cell-CD1d axis is conserved between humans and mice, and human CD1d reactivity of murine iNKT cells have been described. However, the detailed differences between the recognition of human and mouse CD1d bound to various self-lipids by mouse iNKT TCRs are largely unknown. In this study, we generated a de novo murine iNKT TCR repertoire with a wider range of autoreactivity compared with that of naturally occurring peripheral iNKT TCRs. V?8.2 mouse iNKT TCRs capable of recognizing the human CD1d-self-lipid tetramer were identified, although such clones were not detectable in the V?7 or V?2 iNKT TCR repertoire. In line with previously reports, clonotypic V?8.2 iNKT TCRs with unique CDR3? loops did not discriminate among lipids presented by mouse CD1d. Unexpectedly, however, these iNKT TCRs showed greater ligand selectivity toward human CD1d presenting the same lipids. Our findings demonstrated that the recognition of mouse and human CD1d-self-lipid complexes by murine iNKT TCRs is not conserved, thereby further elucidating the differences between cognate and cross-species reactivity of self-antigens by mouse iNKT TCRs.
Project description:High-throughput TCR sequencing allows interrogation of the human TCR repertoire, potentially connecting TCR sequences to antigenic targets. Unlike the highly polymorphic MHC proteins, monomorphic Ag-presenting molecules such as MR1, CD1d, and CD1b present Ags to T cells with species-wide TCR motifs. CD1b tetramer studies and a survey of the 27 published CD1b-restricted TCRs demonstrated a TCR motif in humans defined by the TCR ?-chain variable gene 4-1 (TRBV4-1) region. Unexpectedly, TRBV4-1 was involved in recognition of CD1b regardless of the chemical class of the carried lipid. Crystal structures of two CD1b-specific TRBV4-1+ TCRs show that germline-encoded residues in CDR1 and CDR3 regions of TRBV4-1-encoded sequences interact with each other and consolidate the surface of the TCR. Mutational studies identified a key positively charged residue in TRBV4-1 and a key negatively charged residue in CD1b that is shared with CD1c, which is also recognized by TRBV4-1 TCRs. These data show that one TCR V region can mediate a mechanism of recognition of two related monomorphic Ag-presenting molecules that does not rely on a defined lipid Ag.
Project description:Natural killer T cell antigen receptors (NKT TCRs) recognize lipid-based antigens (Ags) presented by CD1d. Although the TCR ?-chain is invariant, NKT TCR V? exhibits greater diversity, with one (V?11) and three (V?8, V?7, and V?2) V? chains in humans and mice, respectively. With the exception of the V?2 NKT TCR, NKT TCRs possess canonical tyrosine residues within complementarity determining region (CDR) 2? that are critical for CD1d binding. Thus, how V?2 NKT TCR docks with CD1d-Ag was unclear. Despite the absence of the CDR2?-encoded tyrosine residues, we show that the V?2 NKT TCR engaged CD1d-Ag in a similar manner and with a comparable affinity and energetic footprint to the manner observed for the V?8.2 and V?7 NKT TCRs. Accordingly, the germline-encoded regions of the TCR ?-chain do not exclusively dictate the innate NKT TCR-CD1d-Ag docking mode. Nevertheless, clear fine specificity differences for the CD1d-Ag existed between the V?2 NKT TCR and the V?8.2 and V?7 NKT TCRs, with the V?2 NKT TCR exhibiting greater sensitivity to modifications to the glycolipid Ag. Furthermore, within the V?2 NKT TCR-CD1d-?GalCer complex, the CDR2? loop mediated fewer contacts with CD1d, whereas the CDR1? and CDR3? loops contacted CD1d to a much greater extent compared with most V?11, V?8.2, and V?7 NKT TCRs. Accordingly, there is a greater interplay between the germline- and nongermline-encoded loops within the TCR ?-chain of the V?2 NKT TCR that enables CD1d-Ag ligation.