Height,but not binding epitope,affects the potency of synthetic TCR agonists

Under physiological conditions, peptide-major histocompatibility complex (pMHC) molecules can trigger T cell receptors (TCRs) as monovalent ligands that are sparsely distributed on the plasma membrane of an antigen-presenting cell. TCRs can also be triggered by artificial clustering, such as with pMHC tetramers or antibodies; however, these strategies circumvent many of the natural ligand discrimination mechanisms of the T cell and can elicit nonphysiological signaling activity. We have recently introduced a synthetic TCR agonist composed of an anti-TCRβ Fab′ antibody fragment covalently bound to a DNA oligonucleotide, which serves as a membrane anchor. This Fab′-DNA ligand efficiently triggers TCR as a monomer when membrane associated and exhibits a potency and activation profile resembling agonist pMHC. In this report, we explore the geometric requirements for efficient TCR triggering and cellular activation by Fab′-DNA ligands. We find that T cells are insensitive to the ligand binding epitope on the TCR complex but that length of the DNA tether is important. Increasing, the intermembrane distance spanned by Fab′-DNA:TCR complexes decreases TCR triggering efficiency and T cell activation potency, consistent with the kinetic-segregation model of TCR triggering. These results establish design parameters for constructing synthetic TCR agonists that are able to activate polyclonal T cell populations, such as T cells from a human patient, in a similar manner as the native pMHC ligand.     

T-cell Receptor (TCR)-Peptide Specificity Overrides Affinity-enhancing TCR-Major Histocompatibility Complex Interactions

αβ T-cell receptors (TCRs) engage antigens using complementarity-determining region (CDR) loops that are either germ line-encoded (CDR1 and CDR2) or somatically rearranged (CDR3). TCR ligands compose a presentation platform (major histocompatibility complex (MHC)) and a variable antigenic component consisting of a short “foreign” peptide. The sequence of events when the TCR engages its peptide-MHC (pMHC) ligand remains unclear. Some studies suggest that the germ line elements of the TCR engage the MHC prior to peptide scanning, but this order of binding is difficult to reconcile with some TCR-pMHC structures. Here, we used TCRs that exhibited enhanced pMHC binding as a result of mutations in either CDR2 and/or CDR3 loops, that bound to the MHC or peptide, respectively, to dissect the roles of these loops in stabilizing TCR-pMHC interactions. Our data show that TCR-peptide interactions play a strongly dominant energetic role providing a binding mode that is both temporally and energetically complementary with a system requiring positive selection by self-pMHC in the thymus and rapid recognition of non-self-pMHC in the periphery.     

Targeting hepatitis B virus-infected cells with a T-cell receptor-like antibody

Virus-specific CD8 T cells are activated when their T-cell receptors (TCRs) recognize the specific viral peptide/major histocompatibility complex (MHC) class I (pMHC) complexes present on the surface of infected cells. Antibodies able to recognize the specific pMHC can mimic TCR specificity and both represent a valuable biological tool to visualize pMHC complexes on infected cells and serve as a delivery system for highly targeted therapies. To evaluate these possibilities, we created a monoclonal antibody able to specifically recognize a hepatitis B virus (HBV) envelope epitope (Env at positions 183 to 91 [Env183-91]) presented by the HLA-A201 molecule, and we tested its ability to recognize HBV-infected hepatocytes and to deliver a cargo to a specific target. We demonstrate that this antibody detects and visualizes the processed product of HBV proteins produced in naturally HBV-infected cells, is not inhibited by soluble HBV proteins present in patient sera, and mediates the intracellular delivery of a fluorescent molecule to target cells. Additionally, compared to CD8 T cells specific for the same HBV epitope, the TCR-like antibody has both a superior sensitivity and a specificity focused on distinct amino acids within the epitope. These data demonstrate that a T-cell receptor-like antibody can be used to determine the quantitative relationship between HBV replication and specific antigen presentation to CD8 T cells and serves as a novel therapeutic delivery platform for personalized health care for HBV-infected patients.     

Generation of peptide-MHC class I complexes through UV-mediated ligand exchange

Major histocompatibility complex (MHC) class I molecules present peptide ligands on the cell surface for recognition by appropriate cytotoxic T cells. MHC-bound peptides are critical for the stability of the MHC complex, and standard strategies for the production of recombinant MHC complexes are based on in vitro refolding reactions with specific peptides. This strategy is not amenable to high-throughput production of vast collections of MHC molecules. We have developed conditional MHC ligands that form stable complexes with MHC molecules but can be cleaved upon UV irradiation. The resulting empty, peptide-receptive MHC molecules can be charged with epitopes of choice under native conditions. Here we describe in-depth procedures for the high-throughput production of peptide-MHC (pMHC) complexes by MHC exchange, the analysis of peptide exchange efficiency by ELISA and the parallel production of MHC tetramers for T-cell detection. The production of the conditional pMHC complex by an in vitro refolding reaction can be achieved within 2 weeks, and the actual high-throughput MHC peptide exchange and subsequent MHC tetramer formation require less than a day.     

The versatility of the αβ T‐cell antigen receptor

The T‐cell antigen receptor is a heterodimeric αβ protein (TCR) expressed on the surface of T‐lymphocytes, with each chain of the TCR comprising three complementarity‐determining regions (CDRs) that collectively form the antigen‐binding site. Unlike antibodies, which are closely related proteins that recognize intact protein antigens, TCRs classically bind, via their CDR loops, to peptides (p) that are presented by molecules of the major histocompatibility complex (MHC). This TCR‐pMHC interaction is crucially important in cell‐mediated immunity, with the specificity in the cellular immune response being attributable to MHC polymorphism, an extensive TCR repertoire and a variable peptide cargo. The ensuing structural and biophysical studies within the TCR‐pMHC axis have been highly informative in understanding the fundamental events that underpin protective immunity and dysfunctional T‐cell responses that occur during autoimmunity. In addition, TCRs can recognize the CD1 family, a family of MHC‐related molecules that instead of presenting peptides are ideally suited to bind lipid‐based antigens. Structural studies within the CD1‐lipid antigen system are beginning to inform us how lipid antigens are specifically presented by CD1, and how such CD1‐lipid antigen complexes are recognized by the TCR. Moreover, it has recently been shown that certain TCRs can bind to vitamin B based metabolites that are bound to an MHC‐like molecule termed MR1. Thus, TCRs can recognize peptides, lipids, and small molecule metabolites, and here we review the basic principles underpinning this versatile and fascinating receptor recognition system that is vital to a host's survival.     

How a single T cell receptor recognizes both self and foreign MHC

alphabeta T cell receptors (TCRs) can crossreact with both self- and foreign- major histocompatibility complex (MHC) proteins in an enigmatic phenomenon termed alloreactivity. Here we present the 2.35 A structure of the 2C TCR complexed with its foreign ligand H-2L(d)-QL9. Surprisingly, we find that this TCR utilizes a different strategy to engage the foreign pMHC in comparison to the manner in which it recognizes a self ligand H-2K(b)-dEV8. 2C engages both shared and polymorphic residues on L(d) and K(b), as well as the unrelated QL9 and dEV8 peptide antigens, in unique pair-wise contacts, resulting in greater structural complementarity with the L(d)-QL9 complex. In the structure of an engineered, high-affinity 2C TCR variant bound to H-2L(d)-QL9, the "wild-type" TCR-MHC binding orientation persists despite modified TCR-CDR3alpha interactions with peptide. Thus, a single TCR recognizes two globally similar, but distinct ligands by divergent mechanisms, indicating that receptor-ligand crossreactivity can occur in the absence of molecular mimicry.     

Differential peptide dynamics is linked to major histocompatibility complex polymorphism

Peptide presentation by major histocompatibility complex (MHC) molecules is of central importance for immune responses, which are triggered through recognition of peptide-loaded MHC molecules (pMHC) by cellular ligands such as T-cell receptors (TCR). However, a unifying link between structural features of pMHC and cellular responses has not been established. Instead, pMHC/TCR binding studies suggest conformational and/or flexibility changes of the binding partners as a possible cause of differential T-cell stimulation, but information on real-time dynamics is lacking. We therefore probed the real-time dynamics of a MHC-bound nonapeptide (m9), by combining time-resolved fluorescence depolarization and molecular dynamics simulations. Here we show that the nanosecond dynamics of this peptide presented by two human MHC class I subtypes (HLA-B*2705 and HLA-B*2709) with differential autoimmune disease association varies dramatically, despite virtually identical crystal structures. The peptide dynamics is linked to the single, buried polymorphic residue 116 in the peptide binding groove. Pronounced peptide flexibility is seen only for the non-disease-associated subtype HLA-B*2709, suggesting an entropic control of peptide recognition. Thermodynamic data obtained for two additional peptides support this hypothesis.     

T-cell receptor structure and TCR complexes

The first crystal structures of intact T-cell receptors (TCRs) and their complexes with MHC peptide antigens (pMHC) were reported during the past year, along with those of a single-chain TCR Fv fragment and a β-chain complexed with two different bacterial superantigens. These structures have shown the similarities and differences in the architecture of the antigen-binding regions of TCRs and antibodies, and how the TCR interacts with pMHC ligands as well as with superantigens     

A flexible docking approach for prediction of T cell receptor–peptide–MHC complexes

T cell receptors (TCRs) are immune proteins that specifically bind to antigenic molecules, which are often foreign peptides presented by major histocompatibility complex proteins (pMHCs), playing a key role in the cellular immune response. To advance our understanding and modeling of this dynamic immunological event, we assembled a protein–protein docking benchmark consisting of 20 structures of crystallized TCR/pMHC complexes for which unbound structures exist for both TCR and pMHC. We used our benchmark to compare predictive performance using several flexible and rigid backbone TCR/pMHC docking protocols. Our flexible TCR docking algorithm, TCRFlexDock, improved predictive success over the fixed backbone protocol, leading to near‐native predictions for 80% of the TCR/pMHC cases among the top 10 models, and 100% of the cases in the top 30 models. We then applied TCRFlexDock to predict the two distinct docking modes recently described for a single TCR bound to two different antigens, and tested several protein modeling scoring functions for prediction of TCR/pMHC binding affinities. This algorithm and benchmark should enable future efforts to predict, and design of uncharacterized TCR/pMHC complexes.     

ATLAS: A database linking binding affinities with structures for wild‐type and mutant TCR‐pMHC complexes

The ATLAS (Altered TCR Ligand Affinities and Structures) database ( https://zlab.umassmed.edu/atlas/web /) is a manually curated repository containing the binding affinities for wild‐type and mutant T cell receptors (TCRs) and their antigens, peptides presented by the major histocompatibility complex (pMHC). The database links experimentally measured binding affinities with the corresponding three dimensional (3D) structures for TCR‐pMHC complexes. The user can browse and search affinities, structures, and experimental details for TCRs, peptides, and MHCs of interest. We expect this database to facilitate the development of next‐generation protein design algorithms targeting TCR‐pMHC interactions. ATLAS can be easily parsed using modeling software that builds protein structures for training and testing. As an example, we provide structural models for all mutant TCRs in ATLAS, built using the Rosetta program. Utilizing these structures, we report a correlation of 0.63 between experimentally measured changes in binding energies and our predicted changes. Proteins 2017; 85:908–916. © 2016 Wiley Periodicals, Inc.     

Thermodynamics of T-cell receptor-peptide/MHC interactions: progress and opportunities

alphabeta T-cell receptors (TCRs) recognize peptide antigens presented by class I or class II major histocompatibility complex molecules (pMHC). Here we review the use of thermodynamic measurements in the study of TCR-pMHC interactions, with attention to the diversity in binding thermodynamics and how this is related to the variation in TCR-pMHC interfaces. We show that there is no enthalpic or entropic signature for TCR binding; rather, enthalpy and entropy changes vary in a compensatory manner that reflects a narrow free energy window for the interactions that have been characterized. Binding enthalpy and entropy changes do not correlate with structural features such as buried surface area or the number of hydrogen bonds within TCR-pMHC interfaces, possibly reflecting the myriad of contributors to binding thermodynamics, but likely also reflecting a reliance on van't Hoff over calorimetric measurements and the unaccounted influence of equilibria linked to binding. TCR-pMHC binding heat capacity changes likewise vary considerably. In some cases, the heat capacity changes are consistent with conformational differences between bound and free receptors, but there is little data indicating these conformational differences represent the need to organize disordered CDR loops. In this regard, we discuss how thermodynamics may provide additional insight into conformational changes occurring upon TCR binding. Finally, we highlight opportunities for the further use of thermodynamic measurements in the study of TCR-pMHC interactions, not only for understanding TCR binding in general, but also for understanding specifics of individual interactions and the engineering of TCRs with desired molecular recognition properties.     

Spatial coordination of CD8 and TCR molecules controls antigen recognition by CD8+ T-cells

The interactions between the TCR and peptides bound to class I MHC encoded molecules (pMHC) and a mechanism for CD8 cooperation in this process are reviewed. Observation of two TCR/CD8 populations with different lateral diffusion rate constants as well as two distinct association phases of class I MHC tetramers ((pMHC)4) with T-cells suggest that the most efficient pMHC-T-cell association route corresponds to a fast tetramer binding to a colocalized CD8/TCR population, which apparently resides within membrane rafts. Thus, ligand-cell association starts by pMHC binding to the CD8. This rather fast step promotes pMHC association with CD8-proximal TCRs and thereby enhances the overall association process. The model suggests that this raft-associated CD8-TCR subpopulation is responsible for evoking T-cell activation.     

Human TCR-binding affinity is governed by MHC class restriction

T cell recognition is initiated by the binding of TCRs to peptide-MHCs (pMHCs), the interaction being characterized by weak affinity and fast kinetics. Previously, only 16 natural TCR/pMHC interactions have been measured by surface plasmon resonance (SPR). Of these, 5 are murine class I, 5 are murine class II, and 6 are human class I-restricted responses. Therefore, a significant gap exists in our understanding of human TCR/pMHC binding due to the limited SPR data currently available for human class I responses and the absence of SPR data for human class II-restricted responses. We have produced a panel of soluble TCR molecules originating from human T cells that respond to naturally occurring disease epitopes and their cognate pMHCs. In this study, we compare the binding affinity and kinetics of eight class-I-specific TCRs (TCR-Is) to pMHC-I with six class-II-specific TCRs (TCR-IIs) to pMHC-II using SPR. Overall, there is a substantial difference in the TCR-binding equilibrium constants for pMHC-I and pMHC-II, which arises from significantly faster on-rates for TCRs binding to pMHC-I. In contrast, the off-rates for all human TCR/pMHC interactions fall within a narrow window regardless of class restriction, thereby providing experimental support for the notion that binding half-life is the principal kinetic feature controlling T cell activation.     

Attenuated T Cell Responses to a High-Potency Ligand In Vivo

αβ T cell receptor (TCR) recognition of foreign peptides bound to major histocompatibility complex (pMHC) molecules on the surface of antigen presenting cells is a key event in the initiation of adaptive cellular immunity. In vitro, high-affinity binding and/or long-lived interactions between TCRs and pMHC correlate with high-potency T cell activation. However, less is known about the influence of TCR/pMHC interaction parameters on T cell responses in vivo. We studied the influence of TCR/pMHC binding characteristics on in vivo T cell immunity by tracking CD4⁺ T cell activation, effector, and memory responses to immunization with peptides exhibiting a range of TCR/pMHC half-lives and in vitro T cell activation potencies. Contrary to predictions from in vitro studies, we found that optimal in vivo T cell responses occur to ligands with intermediate TCR/pMHC half-lives. The diminished in vivo responses we observed to the ligand exhibiting the longest TCR/pMHC half-life were associated with attenuation of intracellular signaling, expansion, and function over a broad range of time points. Our results reveal a level of control over T cell activation in vivo not recapitulated in in vitro assays and highlight the importance of considering in vivo efficacy of TCR ligands as part of vaccine design.     

Altered recognition of antigen is a mechanism of CD8+ T cell tolerance in cancer

Antigen-specific CD8+ T-cell tolerance, induced by myeloid-derived suppressor cells (MDSCs), is one of the main mechanisms of tumor escape. Using in vivo models, we show here that MDSCs directly disrupt the binding of specific peptide-major histocompatibility complex (pMHC) dimers to CD8-expressing T cells through nitration of tyrosines in a T-cell receptor (TCR)-CD8 complex. This process makes CD8-expressing T cells unable to bind pMHC and to respond to the specific peptide, although they retain their ability to respond to nonspecific stimulation. Nitration of TCR-CD8 is induced by MDSCs through hyperproduction of reactive oxygen species and peroxynitrite during direct cell-cell contact. Molecular modeling suggests specific sites of nitration that might affect the conformational flexibility of TCR-CD8 and its interaction with pMHC. These data identify a previously unknown mechanism of T-cell tolerance in cancer that is also pertinent to many pathological conditions associated with accumulation of MDSCs.     

The molecular basis of alloreactivity in antigen-specific, major histocompatibility complex-restricted T cell clones

We have studied the relationship between major histocompatibility complex (MHC)-restricted antigen recognition and alloreactivity by examining T cell receptor (TCR) alpha and beta gene expression in cytochrome c-specific, Ek alpha:Ek beta (Ek)-restricted helper T cell clones derived from B10.A mice. The clones could be segregated on the basis of four distinct alloreactivity patterns. Clones cross-reactive for three different allogeneic la molecules (As alpha:As beta [As], Ab alpha:Ab beta [Ab], Ek alpha: Eb beta [Eb]) expressed the same V alpha and V beta gene segments, generating the distinct alloreactive specificities via unique V alpha-J alpha and V beta-D beta-J beta joining events. Ek alpha:Es beta (Es)-alloreactive B10.A clones expressed the same V alpha, J alpha, and V beta segments as an Es-restricted, Ek-alloreactive, cytochrome c-specific, H-2-congenic B10.S(9R) clone. This homology between TCRs mediating allorecognition of la molecules and recognition of the same la molecules as restriction elements associated with nominal antigen suggests that MHC-restricted recognition and allorecognition represent differences in the affinity of the TCR-MHC molecule interaction.     

Directed evolution of human T cell receptor CDR2 residues by phage display dramatically enhances affinity for cognate peptide-MHC without increasing apparent cross-reactivity

The mammalian alpha/beta T cell receptor (TCR) repertoire plays a pivotal role in adaptive immunity by recognizing short, processed, peptide antigens bound in the context of a highly diverse family of cell-surface major histocompatibility complexes (pMHCs). Despite the extensive TCR-MHC interaction surface, peptide-independent cross-reactivity of native TCRs is generally avoided through cell-mediated selection of molecules with low inherent affinity for MHC. Here we show that, contrary to expectations, the germ line-encoded complementarity determining regions (CDRs) of human TCRs, namely the CDR2s, which appear to contact only the MHC surface and not the bound peptide, can be engineered to yield soluble low nanomolar affinity ligands that retain a surprisingly high degree of specificity for the cognate pMHC target. Structural investigation of one such CDR2 mutant implicates shape complementarity of the mutant CDR2 contact interfaces as being a key determinant of the increased affinity. Our results suggest that manipulation of germ line CDR2 loops may provide a useful route to the production of high-affinity TCRs with therapeutic and diagnostic potential.     

Different T cell receptor affinity thresholds and CD8 coreceptor dependence govern cytotoxic T lymphocyte activation and tetramer binding properties

T cells have evolved a unique system of ligand recognition involving an antigen T cell receptor (TCR) and a coreceptor that integrate stimuli provided by the engagement of peptide-major histocompatibility complex (pMHC) antigens. Here, we use altered pMHC class I (pMHCI) molecules with impaired CD8 binding (CD8-null) to quantify the contribution of coreceptor extracellular binding to (i) the engagement of soluble tetrameric pMHCI molecules, (ii) the kinetics of TCR/pMHCI interactions on live cytotoxic T lymphocytes (CTLs), and (iii) the activation of CTLs by cell-surface antigenic determinants. Our data indicate that the CD8 coreceptor substantially enhances binding efficiency at suboptimal TCR/pMHCI affinities through effects on both association and dissociation rates. Interestingly, coreceptor requirements for efficient tetramer labeling of CTLs or for CTL activation by determinants displayed on the cell surface operated in different TCR/pMHCI affinity ranges. Wild-type and CD8-null pMHCI tetramers required monomeric affinities for cognate TCRs of KD < approximately 80 microM and approximately 35 microM, respectively, to label human CTLs at 37 degrees C. In contrast, activation by cellular pMHCI molecules was strictly dependent on CD8 binding only for TCR/pMHCI interactions with KD values >200 microM. Altogether, our data provide information on the binding interplay between CD8 and the TCR and support a model of CTL activation in which the extent of coreceptor dependence is inversely correlated to TCR/pMHCI affinity. In addition, the results reported here define the range of TCR/pMHCI affinities required for the detection of antigen-specific CTLs by flow cytometry.