MHC Groove Research Articles

Using molecular dynamics simulations to interrogate T cell receptor non-equilibrium kinetics

An atomic-scale mechanism of T Cell Receptor (TCR) mechanosensing of peptides in the binding groove of the peptide-major histocompatibility complex (pMHC) may inform the design of novel TCRs for immunotherapies. Using steered molecular dynamics simulations, our study demonstrates that mutations to peptides in the binding groove of the pMHC – which are known to discretely alter the T cell response to an antigen – alter the MHC conformation at equilibrium. This subsequently impacts the overall strength (duration and length) of the TCR-pMHC bond under constant load. Moreover, physiochemical features of the TCR-pMHC dynamic bond strength, such as hydrogen bonds and Lennard-Jones contacts, correlate with the immunogenic response elicited by the specific peptide in the MHC groove. Thus, formation of transient TCR-pMHC bonds is characteristic of immunogenic peptides, and steered molecular dynamics simulations can be used in the overall design strategy of TCRs for immunotherapies.

Novel approach for the detection of antigen-specific CD8 T cell responses corresponding to treatment outcome in HPV+ cancer patients

Abstract HPV cancer treatment outcome has been positively correlated to the presence of patient immunity against the virus. Six HPV strains account for over 75% of cervical cancer cases worldwide with HPVE6 & E7 protein integration into the host’s DNA being the driving factor in these cancers. However, the sequence of these proteins vary between strains resulting in high diversity in epitope presentation in patients with different HLA haplotypes. Therefore, using one tetramer gives an inaccurate read of a patient’s immunity to the virus. In this study, we set out to develop a method to obtain a more complete read-out of patient immunity to HPV by creating a more comprehensive tetramer staining panel. We first selected 29 in-silico predicted epitopes of both E6 and E7 oncogenic proteins in the 6 most common HPV strains for 3 HLA haplotypes (HLA-A*02:01, HLA-A*11:01 and *24:02). We then utilized a non-UV “peptide exchange MHC tetramer platform” to assess the binding of these peptides to corresponding MHC groove. Since the peptide exchange and affinity of peptides to the cleft of MHC are correlated, we used an algorithm to calculate the measure of the peptide exchange rate. We found that 3 of the 29 peptides showed poor or no binding to the matched MHC, while all others had rates over 81.78%. The MHC tetramers generated by this peptide exchange platform were then used alone or as part of a pool to stain HPV specific CD8+ T cell enriched PBMCs from an HPV positive host. Our results showed no significant difference in the detection of HPV+ CD8 T cells between our generated HPV16 E7 tetramer and the commercially available tetramer. This implies that the use of this peptide exchange tetramer platform can serve as an accurate tool for measuring HPV immunity in cancer patients.

Chaperone-assisted peptide exchange on MHC-I is driven by a negative allostery release cycle: Implications for a role of peptide-editing Molecular Chaperones in scrutinizing the peptide repertoire

Abstract Molecular chaperones TAPBPR (TAP-binding protein related) and tapasin associate with major histocompatibility complex class I (MHC-I) to promote the loading of antigenic peptides through a poorly understood mechanism. Here, we use solution Nuclear Magnetic Resonance (NMR) spectroscopy to probe TAPBPR-induced changes on MHC-I. Dynamic motions present in the empty MHC groove become progressively dampened with increasing peptide occupancy, while allosteric communication between the A- and F-pockets regulates a conformational switch located near the TAPBPR binding site, which is crucial for chaperone release from the complex. Our analysis of NMR data recorded for a range of TAPBPR complexes prepared with both murine H2 and human HLA alleles complements the recent X-ray structures to provide atomic-resolution mechanistic insights into the selection of optimal peptide sequences for the displayed antigen repertoire. In particular, our results show that negative allosteric coupling between the MHC groove and chaperone binding sites allows TAPBPR to proofread MHC molecules containing a range of different peptides. Since the affinity of incoming peptides for the empty groove is greatly reduced in the chaperone complex, (micromolar range, relative to nanomolar for the free MHC), these interactions can provide a mechanism for optimizing the peptide repertoire, where only the highest-affinity peptides can drive chaperone release. Finally, our results suggest that TAPBPR may promote the dissociation of tightly bound peptides from MHC molecules, thereby further scrutinizing the displayed repertoire. These findings imply a similar mechanism for the specificity and editing function of tapasin in the peptide-loading complex.

A Recurrent Mutation in Anaplastic Lymphoma Kinase with Distinct Neoepitope Conformations.

The identification of recurrent human leukocyte antigen (HLA) neoepitopes driving T cell responses against tumors poses a significant bottleneck in the development of approaches for precision cancer therapeutics. Here, we employ a bioinformatics method, Prediction of T Cell Epitopes for Cancer Therapy, to analyze sequencing data from neuroblastoma patients and identify a recurrent anaplastic lymphoma kinase mutation (ALK R1275Q) that leads to two high affinity neoepitopes when expressed in complex with common HLA alleles. Analysis of the X-ray structures of the two peptides bound to HLA-B*15:01 reveals drastically different conformations with measurable changes in the stability of the protein complexes, while the self-epitope is excluded from binding due to steric hindrance in the MHC groove. To evaluate the range of HLA alleles that could display the ALK neoepitopes, we used structure-based Rosetta comparative modeling calculations, which accurately predict several additional high affinity interactions and compare our results with commonly used prediction tools. Subsequent determination of the X-ray structure of an HLA-A*01:01 bound neoepitope validates atomic features seen in our Rosetta models with respect to key residues relevant for MHC stability and T cell receptor recognition. Finally, MHC tetramer staining of peripheral blood mononuclear cells from HLA-matched donors shows that the two neoepitopes are recognized by CD8+ T cells. This work provides a rational approach toward high-throughput identification and further optimization of putative neoantigen/HLA targets with desired recognition features for cancer immunotherapy.

Two snapshots of the TAPBPR-MHC I complex

Immunology Cytotoxic CD8+ T cells recognize infected and cancerous cells by scrutinizing the antigenic peptides presented by the major histocompatibility complex class I (MHC I). Peptide binding and exchange occurs in the endoplasmic reticulum in a sequence of events mediated by the chaperones tapasin and TAPBPR (see the Perspective by Cresswell). Thomas and Tampe resolved the crystal structure of the TAPBPR–MHC I editing complex by using a photocleavable high-affinity peptide to stabilize the MHC molecule. Jiang et al. crystalized MHC I molecules inhabited by truncated disulfide-linked peptides that still permit TAPBPR to bind. These complimentary snapshots elucidate the dynamic process by which chaperones stabilize the groove of peptide-free MHC I molecules. This helps MHC I sample peptide candidates and facilitates the generation of peptide repertoires enriched with high-affinity antigenic peptides. Science , this issue p. [1060][1], p. [1064][2]; see also p. [992][3] [1]: /lookup/doi/10.1126/science.aao6001 [2]: /lookup/doi/10.1126/science.aao5154 [3]: /lookup/doi/10.1126/science.aaq1398

Crystal structure of a TAPBPR-MHC I complex reveals the mechanism of peptide editing in antigen presentation.

Central to CD8+ T cell-mediated immunity is the recognition of peptide-major histocompatibility complex class I (p-MHC I) proteins displayed by antigen-presenting cells. Chaperone-mediated loading of high-affinity peptides onto MHC I is a key step in the MHC I antigen presentation pathway. However, the structure of MHC I with a chaperone that facilitates peptide loading has not been determined. We report the crystal structure of MHC I in complex with the peptide editor TAPBPR (TAP-binding protein-related), a tapasin homolog. TAPBPR remodels the peptide-binding groove of MHC I, resulting in the release of low-affinity peptide. Changes include groove relaxation, modifications of key binding pockets, and domain adjustments. This structure captures a peptide-receptive state of MHC I and provides insights into the mechanism of peptide editing by TAPBPR and, by analogy, tapasin.

Machine learning reveals a non-canonical mode of peptide binding to MHC class II molecules.

MHC class II molecules play a fundamental role in the cellular immune system: they load short peptide fragments derived from extracellular proteins and present them on the cell surface. It is currently thought that the peptide binds lying more or less flat in the MHC groove, with a fixed distance of nine amino acids between the first and last residue in contact with the MHCII. While confirming that the great majority of peptides bind to the MHC using this canonical mode, we report evidence for an alternative, less common mode of interaction. A fraction of observed ligands were shown to have an unconventional spacing of the anchor residues that directly interact with the MHC, which could only be accommodated to the canonical MHC motif either by imposing a more stretched out peptide backbone (an 8mer core) or by the peptide bulging out of the MHC groove (a 10mer core). We estimated that on average 2% of peptides bind with a core deletion, and 0·45% with a core insertion, but the frequency of such non-canonical cores was as high as 10% for certain MHCII molecules. A mutational analysis and experimental validation of a number of these anomalous ligands demonstrated that they could only fit to their MHC binding motif with a non-canonical binding core of length different from nine. This previously undescribed mode of peptide binding to MHCII molecules gives a more complete picture of peptide presentation by MHCII and allows us to model more accurately this event.

Computational Identification and Characterization of a Promiscuous T-Cell Epitope on the Extracellular Protein 85B of Mycobacterium spp. for Peptide-Based Subunit Vaccine Design.

Tuberculosis (TB) is a reemerging disease that remains as a leading cause of morbidity and mortality in humans. To identify and characterize a T-cell epitope suitable for vaccine design, we have utilized the Vaxign server to assess all antigenic proteins of Mycobacterium spp. recorded to date in the Protegen database. We found that the extracellular protein 85B displayed the most robust antigenicity among the proteins identified. Computational tools for identifying T-cell epitopes predicted an epitope, 181-QQFIYAGSLSALLDP-195, that could bind to at least 13 major histocompatibility complexes, revealing the promiscuous nature of the epitope. Molecular docking simulation demonstrated that the epitope could bind to the binding groove of MHC II and MHC I molecules by several hydrogen bonds. Molecular docking analysis further revealed that the epitope had a distinctive binding pattern to all DRB1 and A and B series of MHC molecules and presented almost no polymorphism in its binding site. Moreover, using “Allele Frequency Database,” we checked the frequency of HLA alleles in the worldwide population and found a higher frequency of both class I and II HLA alleles in individuals living in TB-endemic regions. Our results indicate that the identified peptide might be a universal candidate to produce an efficient epitope-based vaccine for TB.

Protein Complexes from MD Simulations: The Rope-Pulling Game of Tapasin and Histocompatibility Molecules

Using multi-microsecond all-atom molecular dynamics (MD) simulations in explicit solvent, we characterized the association between protein tapasin and major histocompatibility complex class I (MHC I) molecules, two key players of the antigen-processing pathway. MHC I molecules are proteins that present peptide epitopes to cytotoxic T cells and hence are crucial to the mammalian adaptive immune response. Due to a lack of structural data on the tapasin / MHC I complex, the molecular events taking place during peptide loading onto MHC I were not well understood. We show that tapasin not only acts as a chaperone that stabilizes the MHC I structure: it also catalyzes the exchange of abundant non-specific peptides against the rare, immunodominant ones. This selection of high-affinity antigens from the vast pool of cytosolic degradation products is the result of a molecular game of rope-pulling: tapasin pulls on a region (α2-1) of the peptide binding groove of MHC I to open it, while the peptide simultaneously tries to close the groove. Low-affinity peptides “lose” this challenge and are exchanged until, eventually, a high-affinity contender binds to and closes the groove, thereby initiating complex break-down. Properly loaded MHC I molecules then migrate to the cell surface, where recognition by T-cell receptors takes place. Our simulations explain experimental kinetics and mutagenesis data, and represent the first in-depth, atomic-level study of the mechanism underlying the peptide-loading complex (PLC), an important step towards a better understanding of adaptive immunity.

T-Cell Receptor Binding Affects the Dynamics of the Peptide/MHC-I Complex.

The recognition of peptide/MHC by T-cell receptors is one of the most important interactions in the adaptive immune system. A large number of computational studies have investigated the structural dynamics of this interaction. However, to date only limited attention has been paid to differences between the dynamics of peptide/MHC with the T-cell receptor bound and unbound. Here we present the first large-scale molecular dynamics simulation study of this type investigating HLA-B*08:01 in complex with the Epstein-Barr virus peptide FLRGRAYGL and all possible single-point mutations (n = 172). All of the simulations were performed with and without the LC 13 T-cell receptor for a simulation time of 100 ns, yielding 344 simulations and a total simulation time of 34 400 ns. Our study is 2 orders of magnitude larger than the average T-cell receptor/peptide/MHC molecular dynamics simulation study. This data set provides reliable insights into alterations of the peptide/MHC-I dynamics caused by the presence of the T-cell receptor. We found that simulations in the presence of T-cell receptors have more hydrogen bonds between the peptide and MHC, altered flexibility patterns in the MHC helices and the peptide, a lower MHC groove width range, and altered solvent-accessible surface areas. This indicates that without a T-cell receptor the MHC binding groove can open and close, while the presence of the T-cell receptor inhibits these breathing-like motions.

Gapped sequence alignment using artificial neural networks: application to the MHC class I system

Many biological processes are guided by receptor interactions with linear ligands of variable length. One such receptor is the MHC class I molecule. The length preferences vary depending on the MHC allele, but are generally limited to peptides of length 8-11 amino acids. On this relatively simple system, we developed a sequence alignment method based on artificial neural networks that allows insertions and deletions in the alignment. We show that prediction methods based on alignments that include insertions and deletions have significantly higher performance than methods trained on peptides of single lengths. Also, we illustrate how the location of deletions can aid the interpretation of the modes of binding of the peptide-MHC, as in the case of long peptides bulging out of the MHC groove or protruding at either terminus. Finally, we demonstrate that the method can learn the length profile of different MHC molecules, and quantified the reduction of the experimental effort required to identify potential epitopes using our prediction algorithm. The NetMHC-4.0 method for the prediction of peptide-MHC class I binding affinity using gapped sequence alignment is publicly available at: http://www.cbs.dtu.dk/services/NetMHC-4.0.

X‐Ray Single Molecule Tracking Reveal Motions of Peptides in the Binding Groove of MHC II Regulated by DM

Class II MHC can take loosely bound peptides conformations, which generated in the recycling endosomes or cell surface, that are recognized by autoimmune T cells. DM converts loosely bound complexes to tightly bound complexes; crystal structure advocated that DM bound form of MHC II generate novel threshold for peptide binding by creating a hydrogen bond inside the binding groove, that form loosely bound intermediate peptides complex. We used soluble I‐Ak and two peptides that differ in their length as a model system to detect such floating peptides. Fluorescein quenching study provided remarkable difference; fluorescein on the peptides gave about twice a strength signal with DM than without DM, suggests peptides floating half way from MHC II. Diffracted X‐ray tracking (DXT) that monitors real‐time motion of individual peptides/MHC in solution at micro‐second level, revealed that motions of peptides inside binding groove divided into two groups without DM. Addition of DM shifted the slow velocity peak to the fast one. Thus, we successfully detected the DM mediated loosely bound peptides intermediate by two distinct methods. We suggest that measuring the motion of peptide at micro‐second level presents new parameters for both peptide/MHC complex and DM function.

Cholesterol lowering drug may influence cellular immune response by altering MHC II function

Major histocompatibility complex class II (MHC II) expressed on the surface of antigen-presenting cells (APCs) displays peptides to CD4⁺ T cells. Depletion of membrane cholesterol from APCs by methyl β-cyclodextrin treatment compromises peptide-MHC II complex formation coupled with impaired binding of conformational antibody, which binds close to the peptide binding groove of MHC II. Interestingly, the total cell surface of MHC II remains unaltered. These defects can be corrected by restoring membrane cholesterol. In silico docking studies with a three-dimensional model showed the presence of a cholesterol binding site in the transmembrane domain of MHC II (TM-MHC-II). From the binding studies it was clear that cholesterol, indeed, interacts with the TM-MHC-II and alters its conformation. Mutation of cholesterol binding residues (F240, L243, and F246) in the TM-MHC-II decreased the affinity for cholesterol. Furthermore, transfection of CHO cells with full-length mutant MHC II, but not wild-type MHC II, failed to activate antigen-specific T cells coupled with decreased binding of conformation-specific antibodies. Thus, cholesterol-induced conformational change of TM-MHC-II may allosterically modulate the peptide binding groove of MHC II leading to T cell activation.

The length of the peptide in the MHC groove compartmentalizes the CD8+ T cell repertoire (P1186)

Abstract TCRs expressed at the CD8+ T-cell surface interact with short peptide fragments bound to MHC class I molecules (pMHCI). The TCR/pMHCI interaction is pivotal in all aspects of CD8+ T-cell immunity. However, the rules that govern the outcome of TCR/pMHCI engagement are not entirely understood, and this is a major barrier to understanding the requirements for both effective immunity and vaccination. Using large combinatorial peptide arrays, each containing between twenty billion to forty quadtrillion distinct peptides, we have discovered an unexpected feature of the TCR/pMHCI interaction by showing that all TCRs exhibit an explicit preference for an MHCI-bound peptide of defined length. Agonists of nonpreferred length were extremely rare, suboptimal, and often entirely distinct in sequence. Structural analysis indicated that alterations in peptide length have a major impact on antigenic complexity, to which individual TCRs are unable to adapt. This surprising finding, that the CD8+ T cell repertoire is rigorously "compartmentalized" by peptide length, has important consequences for cellular immune system research and vaccine development.

Dynamical Peptide Motions in MHC Groove is Crucial for Recognition and Activation of Certain T Cells

We detected dynamic motions of peptides in MHC II with single-molecule technology using X-rays. T cells see peptides in the context of MHC molecules. This recognition should be specific to each antigens to prevent anomalous self-attack by the activated T cells. However, cross-reactivity of T cell is also well known phenomena. Also, some T cells see peptides/MHC in peculiar forms. As a typical of such peptide/MHC, we studied type B form of I-Ak with HEL peptides, as well as I-Ag7 with newly found diabetogenic peptides for molecular flexibility. in order to elucidate the mechanism of the recognition, we used diffracted X-ray tracking method (DXT) that monitors real-time movements of individual proteins in solution at the single-molecular level. We found that peptides have its individual Brownian motions that are different from MHC protein themself, and the extent of motion diminishes by time in order of days, which may mimic the DM function. We also found that the rotational motions of peptides correlate with the type B T cell activation or with diabetogenic I-Ag7 activation. The rotational motion of peptides may create transient conformation of peptide/MHC complex that recognized by a population of T cells.

Characterization of the binding profile of peptide to transporter associated with antigen processing (TAP) using Gaussian process regression

Although MHC–peptide binding is the most selective event in epitope presentation process, the protein fragments generated by proteasomal cleavage require to be recognized by transporter associated with antigen processing (TAP) and translocated from cytosol to endoplasmic reticulum before they can be loaded into the ligand-binding groove of MHC. In this article, we report the use of a new and powerful machine learning tool called Gaussian process (GP) to model the linear and nonlinear relationships between the sequence pattern and binding affinity of peptide to TAP, and to explain the physicochemical properties and structural implications underlying the specific recognition and association of peptide with TAP. The resulting statistics are compared systematically with those obtained by sophisticated PLS, ANN and SVM. Results show that: (i) Nonlinear methods such as the ANN and GP perform much better than the linear PLS. (ii) GP is capable of handling both linearity- and nonlinearity-hybrid relationship and thus exhibits a good performance relative to other two nonlinear methods. (iii) Investigation of the GP model shows that the P1, P2, P3 and P9 of peptide are the most important positions that dominate TAP–peptide recognition, P5 contributes slightly to the peptide binding, whereas P4, P6, P7 and P8 can only exert very limited potency on the binding. (iv) Diverse properties cast remarkable effects on the interaction between TAP and peptide. In particular, hydrophobility, electronic property and hydrogen bond contribute most significantly to the binding affinity of TAP–peptide association.

Molecular un-stability of peptide-MHC complex and activation of T cell

CD4+ T cell responses require the recognition of specific peptide-MHC complexes displayed by APC. It is important to determine how Ag presentations affect the ensuing T cell response. Imunization of B10.BR mice with immunodominant peptide 48-61 of Hen egg lysozyme elicit two different types of T cell responses. First type of T cell (type A termed by Unanue et. al) respond to APC pulsed with either peptide or whole HEL protein. Second type of T cell (termed type B) respond to APC incubated with peptide but showed no response to APC with whole protein. Some of the type B T cell clone exhibit unusual response to the variant of 48-61 peptide and responded better to poor MHC binding peptide that to strong MHC binding peptide. In contrast, reactivity of the type A of T cell clones correlate well with the affinity of the peptide to the MHC molecules. Since weak MHC binding peptides form unstable complex, we hypothesize that T cells, like type B T cell, respond well to unstable MHC peptide complex by interacting with one of multiple transitional conformations. To test this hypothesis, we observed the movement of peptide/MHC complex at the single molecular level by using diffracted X-ray tracking (DXT) method. It was found that movement of the low affinity peptide MHC complex was greater than that of high affinity peptide MHC complex. However, the difference between two complexes was mainly due to the overall movement of the molecule rather than the different movement of the peptides in the MHC groove. Thus, peptide bound to the MHC greatly influence the movement of the whole MHC peptide complex and this movement strongly affect the recognition by T cell.

Promiscuous Interaction between Gold-Specific T Cells and APCs in Gold Allergy

Gold compounds clinically used as immunomodulators have high potential to evoke hypersensitivity reactions as an adverse effect. To explore the mechanism of gold allergy, we immunologically characterized T cells infiltrating skin rashes and generated 44 gold-specific T cell clones and lines from a rheumatoid arthritis patient who developed skin rashes and systemic symptoms after gold treatment. CD4(+) and CD8(+) cells predominantly infiltrating the skin rashes and some of the T cell clones and lines shared common Vbetas. These cells exhibited Th0-like, Th2-like, and Tc1-like cytokine profiles, and showed chemotactic activities for thymus and activation-regulated chemokine and IFN-gamma-inducible protein-10 corresponding to the cytokine profiles. T cell recognition of gold consisted of MHC-restricted and MHC-independent pathways. Blocking studies with anti-MHC Abs indicated that the groove of MHC in APCs, where Ags should ordinarily be settled, did not serve as a conjugating site of gold for these T cells in certain cases. These observations raise the possibility that gold-specific CD4(+) and CD8(+) T cells and APCs promiscuously interact under stimulation with gold, resulting in various clinical manifestations in gold allergy.

De-Immunization of Human Factor VIII: Identification of Epitopes in the C2 Domain

Most immune responses to protein antigens are dependent on T-cell recognition of discrete peptide epitopes presented in an MHC groove. The pattern of peptide recognition can be predicted from the primary structure of a given protein based on residues that bind (anchor) to a given HLA phenotype. Algorithms such as EpiMatrix can be applied to identify such epitopes and measure the potential immunogenicity of proteins based on epitope content. One approach to reduce immunogenicity is to generate recombinant proteins whose constituent epitopes have been modified so as to reduce their HLA binding. Modification of the sequence can be performed in silico using algorithms such as OptiMatrix. This “de-immunization” method has been used effectively with a number of therapeutic proteins already in use in clinical trials. It may therefore also be possible to target those residues of fVIII that, while contributing to HLA binding, can be mutated without altering the functional ability of fVIII to initiate clotting. We have begun this de-immunization process with the fVIII, starting with C2 domain because C2 has been confirmed to be a major target of both T cells and inhibitory antibodies. Using EpiMatrix, we selected 10 peptides in human fVIII that would be predicted to bind to eight class II HLA DR molecules that encompass over 95% of the U.S. population. These epitopes were synthesized and eight of the ten were assayed in vitro and found to bind to HLA at IC50 <100μM. Immunization of fVIII knockout mice with whole fVIII or with constituent fVIII peptide epitopes resulted in significant T cell proliferation to the peptide epitopes as measured by thymidine incorporation assays. By contrast, homologous peptide epitopes modified by 1–2 residues elicited significantly lower levels of proliferation. These preliminary results point to the feasibility of generating a de-immunized fVIII molecule with reduced immunogenicity; validation of these results is planned. If such proteins retain even partial activity to initiate clotting, then they would become useful in treating hemophilia A patients to avoid inhibitor formation. (Supported by NIH R43 HL088834-01)

Self-reporting peptides illuminate the MHC groove

Synthetic oligopeptides chemically modified with environmentally sensitive fluorophores enable real-time visualization of peptide binding to MHC molecules. This technology will expand our understanding of antigen presentation and enable visualization of fluorescent peptide binding to a wide variety of receptors in living cells.