Structure-Based Rational and General Strategy for Stabilizing Single-Chain T-Cell Receptors to Enhance Affinity.

The T-cell receptor (TCR) is a crucial molecule in cellular immunity. The single-chain T-cell receptor (scTCR) is a potential format in TCR therapeutics because it eliminates the possibility of αß-TCR mispairing. However, its poor stability and solubility impede the in vitro study and manufacturing of therapeutic applications. In this study, some conserved structural motifs are identified in variable domains regardless of germlines and species. Theoretical analysis helps to identify those unfavored factors and leads to a general strategy for stabilizing scTCRs by substituting residues at exact IMGT positions with beneficial propensities on the consensus sequence of germlines. Several representative scTCRs are displayed to achieve stability optimization and retain comparable binding affinities with the corresponding αß-TCRs in the range of µM to pM. These results demonstrate that our strategies for scTCR engineering are capable of providing the affinity-enhanced and specificity-retained format, which are of great value in facilitating the development of TCR-related therapeutics.

Tumor resident, TRA anti-viral CDR3 chemical sequence motifs are associated with a better breast cancer outcome.

While for certain cancers, such as cervical cancer, the link to viral infections is very strong and very clear, other cancers represent a history of links to viral infections that are either co-morbidities or drive the cancer in ways that are not yet fully understood, for example the "hit and run" possibility. To further understand the connection of viral infections and the progress of breast cancer, we identified the chemical features of known anti-viral, T-cell receptor alpha chain (TRA) complementarity determining region-3 (CDR3) amino acid sequences among the CDR3s of breast cancer patient TRA recombinations and assessed the association of those features with patient outcomes. The application of this novel paradigm indicated consistent associations of tumor-derived, anti-CMV CDR3 chemical sequence motifs with better breast cancer patient outcomes but did not indicate an opportunity to establish risk stratifications for other cancer types. Interestingly, breast cancer samples with no detectable TRA recombinations represented a better outcome than samples with the non-anti-CMV CDR3s, further adding to a rapidly developing series of results allowing a distinction between positive and possibly harmful cancer immune responses.

Delineation of a T-cell receptor CDR3-cancer mutanome aromaticity factor, assessable via blood samples, that facilitates the establishment of survival distinctions in bladder cancer.

PURPOSE: A very large and still expanding collection of adaptive immune receptor (IR) recombination reads, representing many diseases, is becoming available for downstream analyses. Among the most productive approaches has been to establish risk stratification parameters via the chemical features of the IR complementarity determining region-3 (CDR3) amino acid (AA) sequences, particularly for large datasets where clinical information is available. Because the IR CDR3 AA sequences often play a large role in antigen binding, the chemistry of these AAs has the likelihood of representing a disease-related fingerprint as well as providing pre-screening information for candidate antigens. To approach this issue in a novel manner, we developed a bladder cancer, case evaluation approach based on CDR3 aromaticity. METHODS: We developed and applied a simple and efficient algorithm for assessing aromatic, chemical complementarity between T-cell receptor (TCR) CDR3 AA sequences and the cancer specimen mutanome. RESULTS: Results indicated a survival distinction for aromatic CDR3-aromatic mutanome complementary, versus non-complementary, bladder cancer case sets. This result applied to both tumor resident and blood TCR CDR3 AA sequences and was supported by CDR3 AA sequences represented by both exome and RNAseq files. CONCLUSION: The described aromaticity factor algorithm has the potential of assisting in prognostic assessments and guiding immunotherapies for bladder cancer.

Structure of a fully assembled tumor-specific T cell receptor ligated by pMHC.

The T cell receptor (TCR) expressed by T lymphocytes initiates protective immune responses to pathogens and tumors. To explore the structural basis of how TCR signaling is initiated when the receptor binds to peptide-loaded major histocompatibility complex (pMHC) molecules, we used cryogenic electron microscopy to determine the structure of a tumor-reactive TCRαß/CD3δγε2ζ2 complex bound to a melanoma-specific human class I pMHC at 3.08 Å resolution. The antigen-bound complex comprises 11 subunits stabilized by multivalent interactions across three structural layers, with clustered membrane-proximal cystines stabilizing the CD3-εδ and CD3-εγ heterodimers. Extra density sandwiched between transmembrane helices reveals the involvement of sterol lipids in TCR assembly. The geometry of the pMHC/TCR complex suggests that efficient TCR scanning of pMHC requires accurate pre-positioning of T cell and antigen-presenting cell membranes. Comparisons of the ligand-bound and unliganded receptors, along with molecular dynamics simulations, indicate that TCRs can be triggered in the absence of spontaneous structural rearrangements.

Computational discovery of binding mode of anti-TRBC1 antibody and predicted key amino acids of TRBC1.

Peripheral T-cell lymphoma (PTCL) is a type of non-Hodgkin lymphoma that progresses aggressively with poor survival rate. CAR T cell targeting T-cell receptor ß-chain constant domains 1 (TRBC1) of malignant T cells has been developed recently by using JOVI.1 monoclonal antibody as a template. However, the mode of JOVI.1 binding is still unknown. This study aimed to investigate the molecular interaction between JOVI.1 antibody and TRBC1 by using computational methods and molecular docking. Therefore, the TRBC protein crystal structures (TRBC1 and TRBC2) as well as the sequences of JOVI.1 CDR were chosen as the starting materials. TRBC1 and TRBC2 epitopes were predicted, and molecular dynamic (MD) simulation was used to visualize the protein dynamic behavior. The structure of JOVI.1 antibody was also generated before the binding mode was predicted using molecular docking with an antibody mode. Epitope prediction suggested that the N3K4 region of TRBC1 may be a key to distinguish TRBC1 from TCBC2. MD simulation showed the major different surface conformation in this area between two TRBCs. The JOVI.1-TRBC1 structures with three binding modes demonstrated JOVI.1 interacted TRBC1 at N3K4 residues, with the predicted dissociation constant (Kd) ranging from 1.5 × 108 to 1.1 × 1010 M. The analysis demonstrated JOVI.1 needed D1 residues of TRBC1 for the interaction formation to N3K4 in all binding modes. In conclusion, we proposed the three binding modes of the JOVI.1 antibody to TRBC1 with the new key residue (D1) necessary for N3K4 interaction. This data was useful for JOVI.1 redesign to improve the PTCL-targeting CAR T cell.

The Full Model of the pMHC-TCR-CD3 Complex: A Structural and Dynamical Characterization of Bound and Unbound States.

The machinery involved in cytotoxic T-cell activation requires three main characters: the major histocompatibility complex class I (MHC I) bound to the peptide (p), the T-cell receptor (TCR), and the CD3 complex, a multidimer interfaced with the intracellular side. The pMHC:TCR interaction has been largely studied by means of both experimental and computational models, giving a contribution in understanding the complexity of the TCR triggering. Nevertheless, a detailed study of the structural and dynamical characterization of the full complex (pMHC:TCR:CD3 complex) is still missing due to a lack of structural information of the CD3-chains arrangement around the TCR. Very recently, the determination of the TCR:CD3 complex structure by means of Cryo-EM technique has given a chance to build the entire system essential in the activation of T-cells, a fundamental mechanism in the adaptive immune response. Here, we present the first complete model of the pMHC interacting with the TCR:CD3 complex, built in a lipid environment. To describe the conformational behavior associated with the unbound and the bound states, all-atom Molecular Dynamics simulations were performed for the TCR:CD3 complex and for two pMHC:TCR:CD3 complex systems, bound to two different peptides. Our data point out that a conformational change affecting the TCR Constant ß (Cß) region occurs after the binding to the pMHC, revealing a key role of this region in the propagation of the signal. Moreover, we found that TCR reduces the flexibility of the MHC I binding groove, confirming our previous results.

Cholesterol Binds in a Reversed Orientation to TCRß-TM in Which Its OH Group is Localized to the Center of the Lipid Bilayer.

T cell receptor (TCR) signaling in response to antigen recognition is essential for the adaptive immune response. Cholesterol keeps TCRs in the resting conformation and mediates TCR clustering by directly binding to the transmembrane domain of the TCRß subunit (TCRß-TM), while cholesterol sulfate (CS) displaces cholesterol from TCRß. However, the atomic interaction of cholesterol or CS with TCRß remains elusive. Here, we determined the cholesterol and CS binding site of TCRß-TM in phospholipid bilayers using solution nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulation. Cholesterol binds to the transmembrane residues within a CARC-like cholesterol recognition motif. Surprisingly, the polar OH group of cholesterol is placed in the hydrophobic center of the lipid bilayer stabilized by its polar interaction with K154 of TCRß-TM. An aromatic interaction with Y158 and hydrophobic interactions with V160 and L161 stabilize this reverse orientation. CS binds to the same site, explaining how it competes with cholesterol. Site-directed mutagenesis of the CARC-like motif disrupted the cholesterol/CS binding to TCRß-TM, validating the NMR and MD results.

TCRpair: prediction of functional pairing between HLA-A*02:01-restricted T-cell receptor α and ß chains.

SUMMARY: The ability of a T cell to recognize foreign peptides is defined by a single α and a single ß hypervariable complementarity determining region (CDR3), which together form the T-cell receptor (TCR) heterodimer. In ∼30-35% of T cells, two α chains are expressed at the mRNA level but only one α chain is part of the functional TCR. This effect can also be observed for ß chains, although it is less common. The identification of functional α/ß chain pairs is instrumental in high-throughput characterization of therapeutic TCRs. TCRpair is the first method that predicts whether an α and ß chain pair forms a functional, HLA-A*02:01 specific TCR without requiring the sequence of a recognized peptide. By taking additional amino acids flanking the CDR3 regions into account, TCRpair achieves an AUC of 0.71. AVAILABILITY AND IMPLEMENTATION: TCRpair is implemented in Python using TensorFlow 2.0 and is freely available at https://www.github.com/amoesch/TCRpair. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Clonotypic architecture of a Gag-specific CD8+ T-cell response in chronic human HIV-2 infection.

The dynamics of T-cell receptor (TCR)selection in chronic HIV-1 infection, and its association with clinical outcome, is well documented for an array of MHC-peptide complexes and disease stages. However, the factors that may contribute to the selection and expansion of CD8+ T-cells in chronic HIV-2 infection, especially at the clonal level remain unclear. To address this question, we undertook a detailed molecular characterization of the clonotypic architecture of an HLA-B*3501 restricted Gag-specific CD8+ T-cell response in donors chronically infected with HIV-2 using a combination of flow cytometry, tetramer-specific CD8+ TCR clonotyping, and in vitro assays. We show that the response to the NY9 epitope is hierarchical and narrow in terms of T-cell receptor-alpha (TCRA) and -beta (TCRB) gene usage yet clonotypically diverse. Furthermore, clonotypic dominance in shared origin CTL clones was associated with a greater magnitude of cytokine production and antigen sensitivity at limiting antigen dilution as well as enhanced cross-reactivity for known HIV-2 variants. Hence, our data suggest that effector mobilization and expansion in human chronic HIV-2 infection may be linked to the qualitative features of specific CD8+ T-cell clonotypes, which could have implications for viral control and disease outcome.

T Cell Receptor Genotype and Ubash3a Determine Susceptibility to Rat Autoimmune Diabetes.

Genetic analyses of human type 1 diabetes (T1D) have yet to reveal a complete pathophysiologic mechanism. Inbred rats with a high-risk class II major histocompatibility complex (MHC) haplotype (RT1B/Du) can illuminate such mechanisms. Using T1D-susceptible LEW.1WR1 rats that express RT1B/Du and a susceptible allele of the Ubd promoter, we demonstrate that germline knockout of Tcrb-V13S1A1, which encodes the Vß13a T cell receptor ß chain, completely prevents diabetes. Using the RT1B/Du-identical LEW.1W rat, which does not develop T1D despite also having the same Tcrb-V13S1A1 ß chain gene but a different allele at the Ubd locus, we show that knockout of the Ubash3a regulatory gene renders these resistant rats relatively susceptible to diabetes. In silico structural modeling of the susceptible allele of the Vß13a TCR and its class II RT1u ligand suggests a mechanism by which a germline TCR ß chain gene could promote susceptibility to T1D in the absence of downstream immunoregulation like that provided by UBASH3A. Together these data demonstrate the critical contribution of the Vß13a TCR to the autoimmune synapse in T1D and the regulation of the response by UBASH3A. These experiments dissect the mechanisms by which MHC class II heterodimers, TCR and regulatory element interact to induce autoimmunity.

Molecular design of the γδT cell receptor ectodomain encodes biologically fit ligand recognition in the absence of mechanosensing.

High-acuity αßT cell receptor (TCR) recognition of peptides bound to major histocompatibility complex molecules (pMHCs) requires mechanosensing, a process whereby piconewton (pN) bioforces exert physical load on αßTCR-pMHC bonds to dynamically alter their lifetimes and foster digital sensitivity cellular signaling. While mechanotransduction is operative for both αßTCRs and pre-TCRs within the αßT lineage, its role in γδT cells is unknown. Here, we show that the human DP10.7 γδTCR specific for the sulfoglycolipid sulfatide bound to CD1d only sustains a significant load and undergoes force-induced structural transitions when the binding interface-distal γδ constant domain (C) module is replaced with that of αß. The chimeric γδ-αßTCR also signals more robustly than does the wild-type (WT) γδTCR, as revealed by RNA-sequencing (RNA-seq) analysis of TCR-transduced Rag2-/- thymocytes, consistent with structural, single-molecule, and molecular dynamics studies reflective of γδTCRs as mediating recognition via a more canonical immunoglobulin-like receptor interaction. Absence of robust, force-related catch bonds, as well as γδTCR structural transitions, implies that γδT cells do not use mechanosensing for ligand recognition. This distinction is consonant with the fact that their innate-type ligands, including markers of cellular stress, are expressed at a high copy number relative to the sparse pMHC ligands of αßT cells arrayed on activating target cells. We posit that mechanosensing emerged over ∼200 million years of vertebrate evolution to fulfill indispensable adaptive immune recognition requirements for pMHC in the αßT cell lineage that are unnecessary for the γδT cell lineage mechanism of non-pMHC ligand detection.

Canonical T cell receptor docking on peptide-MHC is essential for T cell signaling.

T cell receptor (TCR) recognition of peptide-major histocompatibility complexes (pMHCs) is characterized by a highly conserved docking polarity. Whether this polarity is driven by recognition or signaling constraints remains unclear. Using "reversed-docking" TCRß-variable (TRBV) 17+ TCRs from the naïve mouse CD8+ T cell repertoire that recognizes the H-2Db-NP366 epitope, we demonstrate that their inability to support T cell activation and in vivo recruitment is a direct consequence of reversed docking polarity and not TCR-pMHCI binding or clustering characteristics. Canonical TCR-pMHCI docking optimally localizes CD8/Lck to the CD3 complex, which is prevented by reversed TCR-pMHCI polarity. The requirement for canonical docking was circumvented by dissociating Lck from CD8. Thus, the consensus TCR-pMHC docking topology is mandated by T cell signaling constraints.

CD8+ T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope display high naive precursor frequency and TCR promiscuity.

To better understand primary and recall T cell responses during coronavirus disease 2019 (COVID-19), it is important to examine unmanipulated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells. By using peptide-human leukocyte antigen (HLA) tetramers for direct ex vivo analysis, we characterized CD8+ T cells specific for SARS-CoV-2 epitopes in COVID-19 patients and unexposed individuals. Unlike CD8+ T cells directed toward subdominant epitopes (B7/N257, A2/S269, and A24/S1,208) CD8+ T cells specific for the immunodominant B7/N105 epitope were detected at high frequencies in pre-pandemic samples and at increased frequencies during acute COVID-19 and convalescence. SARS-CoV-2-specific CD8+ T cells in pre-pandemic samples from children, adults, and elderly individuals predominantly displayed a naive phenotype, indicating a lack of previous cross-reactive exposures. T cell receptor (TCR) analyses revealed diverse TCRαß repertoires and promiscuous αß-TCR pairing within B7/N105+CD8+ T cells. Our study demonstrates high naive precursor frequency and TCRαß diversity within immunodominant B7/N105-specific CD8+ T cells and provides insight into SARS-CoV-2-specific T cell origins and subsequent responses.

A general chemical crosslinking strategy for structural analyses of weakly interacting proteins applied to preTCR-pMHC complexes.

T lymphocytes discriminate between healthy and infected or cancerous cells via T-cell receptor-mediated recognition of peptides bound and presented by cell-surface-expressed major histocompatibility complex molecules (MHCs). Pre-T-cell receptors (preTCRs) on thymocytes foster development of αßT lymphocytes through their ß chain interaction with MHC displaying self-peptides on thymic epithelia. The specific binding of a preTCR with a peptide-MHC complex (pMHC) has been identified previously as forming a weak affinity complex with a distinct interface from that of mature αßTCR. However, a lack of appropriate tools has limited prior efforts to investigate this unique interface. Here we designed a small-scale linkage screening protocol using bismaleimide linkers for determining residue-specific distance constraints between transiently interacting protein pairs in solution. Employing linkage distance restraint-guided molecular modeling, we report the oriented solution docking geometry of a preTCRß-pMHC interaction. The linkage model of preTCRß-pMHC complex was independently verified with paramagnetic pseudocontact chemical shift (PCS) NMR of the unlinked protein mixtures. Using linkage screens, we show that the preTCR binds with differing affinities to peptides presented by MHC in solution. Moreover, the C-terminal peptide segment is a key determinant in preTCR-pMHC recognition. We also describe the process for future large-scale production and purification of the linked constructs for NMR, X-ray crystallography, and single-molecule electron microscopy studies.

T Cell Receptor Beta-Chain Profiling of Tumor Tissue, Peripheral Blood and Regional Lymph Nodes From Patients With Papillary Thyroid Carcinoma.

Objective: To study the characteristics of the T cell receptor (TCR) repertoire in cancer tissue, peripheral blood and regional lymph nodes (LNs) from patients with papillary thyroid carcinoma (PTC). Methods: PTC tissue, peripheral blood mononuclear cells (PBMCs) and regional LNs of six patients with papillary thyroid carcinoma were harvested. T cell receptor beta-chain (TCRß) profiling was performed though high-throughput sequencing (HTS), and IMonitor, MiXCR and VDJtools were used to analyze the characteristics of the TCR repertoire. Results: The results of IMonitor and those of MiXCR and VDJtools were very similar. The unique CDR3 of TCRß from LNs was higher than that of PBMCs, and the CDR3 of TCRß from LNs was higher than that of PTC tissue. Shannon's diversity index, D50, inverse Simpson index_mean and normalized Shannon's diversity index_mean of CDR3 from LNs were higher than those of PTCs and PBMCs. The HEC (high expansion clones) rate of CDR3 sequences at the amino acid level in PTC tissue was higher than that of PBMCs, which was higher than that of LNs. The V-J HEC rate of CDR3 was highest in PTC tissue, followed by PBMCs and LNs. Conclusion: TCR CDR3 profiling showed differences among and within the PBMCs, PTC tissues and regional LNs of PTC, including unique CDR3, CDR3 HEC at the amino acid level, CDR3 V-J HEC at the amino acid level, Shannon's diversity index and D50. The TCRß repertoire of PTC tissue, peripheral blood and regional LNs of PTC provide a reference for further study of immunity mechanisms against PTC.

Electrostatic Complementarity of T-Cell Receptor-Alpha CDR3 Domains and Mutant Amino Acids Is Associated with Better Survival Rates for Sarcomas.

While sarcoma immunology has advanced with regard to basic, and even some applied topics, this disease has not been subject to more recent immunogenomics approaches. Thus, we assessed the immune receptor recombinations available from the cancer genome atlas (TCGA) sarcoma database via tumor sample exome and RNASeq files. Results indicated that recovery of T-cell receptor-alpha recombination reads (TRA) correlated with a better survival rate, with the expression of T-cell biomarkers, and with tumor sample apoptosis signatures consistent with the longer patient survival times. Furthermore, samples representing TRA complementarity determining region-3 (CDR3) net charge per residue (NCPR) based complementarity with the corresponding sarcoma mutanome had a better survival rate, and more granzyme expression, than samples lacking such complementarity. By specifically using RNASeq-recovered TRA CDR3s and related NCPR assessments, three genes, TP53, ATRX, and RB1, were identified as being key components of the mutanome-based complementarity. Thus, these genes may represent key immune system targets for soft tissue sarcomas. Also, several key results from above were reproduced with a pediatric osteosarcoma dataset, work that led to identification of MUC6 mutations as potentially linked to a strong immune response. In sum, TRA CDR3s are likely to be important prognostic indicators, and possibly a beginning tool for immunotherapy development strategies, for adult and pediatric sarcomas.

Structurally silent peptide anchor modifications allosterically modulate T cell recognition in a receptor-dependent manner.

Presentation of peptides by class I MHC proteins underlies T cell immune responses to pathogens and cancer. The association between peptide binding affinity and immunogenicity has led to the engineering of modified peptides with improved MHC binding, with the hope that these peptides would be useful for eliciting cross-reactive immune responses directed toward their weak binding, unmodified counterparts. Increasing evidence, however, indicates that T cell receptors (TCRs) can perceive such anchor-modified peptides differently than wild-type (WT) peptides, although the scope of discrimination is unclear. We show here that even modifications at primary anchors that have no discernible structural impact can lead to substantially stronger or weaker T cell recognition depending on the TCR. Surprisingly, the effect of peptide anchor modification can be sensed by a TCR at regions distant from the site of modification, indicating a through-protein mechanism in which the anchor residue serves as an allosteric modulator for TCR binding. Our findings emphasize caution in the use and interpretation of results from anchor-modified peptides and have implications for how anchor modifications are accounted for in other circumstances, such as predicting the immunogenicity of tumor neoantigens. Our data also highlight an important need to better understand the highly tunable dynamic nature of class I MHC proteins and the impact this has on various forms of immune recognition.

Pre-T cell receptors topologically sample self-ligands during thymocyte ß-selection.

Self-discrimination, a critical but ill-defined molecular process programmed during thymocyte development, requires myriad pre-T cell receptors (preTCRs) and αßTCRs. Using x-ray crystallography, we show how a preTCR applies the concave ß-sheet surface of its single variable domain (Vß) to "horizontally" grab the protruding MHC α2-helix. By contrast, αßTCRs purpose all six complementarity-determining region (CDR) loops of their paired VαVß module to recognize peptides bound to major histocompatibility complex molecules (pMHCs) in "vertical" head-to-head binding. The preTCR topological fit ensures that CDR3ß reaches the peptide's featured C-terminal segment for pMHC sampling, establishing the subsequent αßTCR canonical docking mode. "Horizontal" docking precludes germline CDR1ß- and CDR2ß-MHC binding to broaden ß-chain repertoire diversification before αßTCR-mediated selection refinement. Thus, one subunit successively attunes the recognition logic of related multicomponent receptors.

T-Cell Receptor CDR3 Loop Conformations in Solution Shift the Relative Vα-Vß Domain Distributions.

T-cell receptors are an important part in the adaptive immune system as they are responsible for detecting foreign proteins presented by the major histocompatibility complex (MHC). The affinity is predominantly determined by structure and sequence of the complementarity determining regions (CDRs), of which the CDR3 loops are responsible for peptide recognition. We present a kinetic classification of T-cell receptor CDR3 loops with different loop lengths into canonical and non-canonical solution structures. Using molecular dynamics simulations, we do not only sample available X-ray structures, but we also observe a substantially broader CDR3 loop ensemble with various distinct kinetic minima in solution. Our results strongly imply, that for given CDR3 loop sequences several canonical structures have to be considered to characterize the conformational diversity of these loops. Our suggested dominant solution structures could extend the repertoire of available canonical clusters by including kinetic minimum structures present in solution. Thus, the CDR3 loops need to be characterized as conformational ensembles in solution. Furthermore, the conformational changes of the CDR3 loops follow the paradigm of conformational selection, because the experimentally determined binding competent state is present within this ensemble of pre-existing conformations without the presence of the antigen. We also identify strong correlations between the CDR3 loops and include combined state descriptions. Additionally, we observe a strong dependency of the CDR3 loop conformations on the relative Vα-Vß interdomain orientations, revealing that certain CDR3 loop states favor specific interface orientations.

Atypical TRAV1-2- T cell receptor recognition of the antigen-presenting molecule MR1.

MR1 presents vitamin B-related metabolites to mucosal associated invariant T (MAIT) cells, which are characterized, in part, by the TRAV1-2+ αß T cell receptor (TCR). In addition, a more diverse TRAV1-2- MR1-restricted T cell repertoire exists that can possess altered specificity for MR1 antigens. However, the molecular basis of how such TRAV1-2- TCRs interact with MR1-antigen complexes remains unclear. Here, we describe how a TRAV12-2+ TCR (termed D462-E4) recognizes an MR1-antigen complex. We report the crystal structures of the unliganded D462-E4 TCR and its complex with MR1 presenting the riboflavin-based antigen 5-OP-RU. Here, the TRBV29-1 ß-chain of the D462-E4 TCR binds over the F'-pocket of MR1, whereby the complementarity-determining region (CDR) 3ß loop surrounded and projected into the F'-pocket. Nevertheless, the CDR3ß loop anchored proximal to the MR1 A'-pocket and mediated direct contact with the 5-OP-RU antigen. The D462-E4 TCR footprint on MR1 contrasted that of the TRAV1-2+ and TRAV36+ TCRs' docking topologies on MR1. Accordingly, diverse MR1-restricted T cell repertoire reveals differential docking modalities on MR1, thus providing greater scope for differing antigen specificities.