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The intricate interaction between major histocompatibility complexes (MHCs) and antigen peptides with diverse amino acid sequences plays a pivotal role in immune responses and T cell activity. In recent years, deep learning (DL)-based models have emerged as promising tools for accelerating antigen peptide screening. However, most of these models solely rely on one-dimensional amino acid sequences, overlooking crucial information required for the three-dimensional (3-D) space binding process. In this study, we propose TransfIGN, a structure-based DL model that is inspired by our previously developed framework, Interaction Graph Network (IGN), and incorporates sequence information from transformers to predict the interactions between HLA-A*02:01 and antigen peptides. Our model, trained on a comprehensive data set containing 61,816 sequences with 9051 binding affinity labels and 56,848 eluted ligand labels, achieves an area under the curve (AUC) of 0.893 on the binary data set, better than state-of-the-art sequence-based models trained on larger data sets such as NetMHCpan4.1, ANN, and TransPHLA. Furthermore, when evaluated on the IEDB weekly benchmark data sets, our predictions (AUC = 0.816) are better than those of the recommended methods like the IEDB consensus (AUC = 0.795). Notably, the interaction weight matrices generated by our method highlight the strong interactions at specific positions within peptides, emphasizing the model's ability to provide physical interpretability. This capability to unveil binding mechanisms through intricate structural features holds promise for new immunotherapeutic avenues.

The startling diversity in αβ T-cell receptor (TCR) sequences and structures complicates molecular-level analyses of the specificity and sensitivity determining T-cell immunogenicity. A number of three-dimensional (3D) structures are now available of ternary complexes between TCRs and peptides: major histocompatibility complexes (pMHC). Here, to glean molecular-level insights we analyze structures of TCRs bound to human class I nonamer peptide-MHC complexes. Residues at peptide positions 4-8 are found to be particularly important for TCR binding. About 90% of the TCRs hydrogen bond with one or both of the peptide residues at positions 4 and 8 presented by MHC allele HLA-A2, and this number is still ~79% for peptides presented by other MHC alleles. Residue 8, which lies outside the previously-identified central peptide region, is crucial for TCR recognition of class I MHC-presented nonamer peptides. The statistics of the interactions also sheds light on the MHC residues important for TCR binding. The present analysis will aid in the structural modeling of TCR:pMHC complexes and has implications for the rational design of peptide-based vaccines and T-cell-based immunotherapies., (© 2022 Wiley Periodicals LLC.)

Adoptive cell therapy with tumor-specific T cells can mediate durable cancer regression. The prime target of tumor-specific T cells are neoantigens arising from mutations in self-proteins during malignant transformation. To understand T cell recognition of cancer neoantigens at the atomic level, we studied oligoclonal T cell receptors (TCRs) that recognize a neoepitope arising from a driver mutation in the p53 oncogene (p53R175H) presented by the major histocompatibility complex class I molecule HLA-A2. We previously reported the structures of three p53R175H-specific TCRs (38-10, 12-6, and 1a2) bound to p53R175H and HLA-A2. The structures showed that these TCRs discriminate between WT and mutant p53 by forming extensive interactions with the R175H mutation. Here, we report the structure of a fourth p53R175H-specific TCR (6-11) in complex with p53R175H and HLA-A2. In contrast to 38-10, 12-6, and 1a2, TCR 6-11 makes no direct contacts with the R175H mutation, yet is still able to distinguish mutant from WT p53. Structure-based in silico mutagenesis revealed that the 60-fold loss in 6-11 binding affinity for WT p53 compared to p53R175H is mainly due to the higher energetic cost of desolvating R175 in the WT p53 peptide during complex formation than H175 in the mutant. This indirect strategy for preferential neoantigen recognition by 6-11 is fundamentally different from the direct strategies employed by other TCRs and highlights the multiplicity of solutions to recognizing p53R175H with sufficient selectivity to mediate T cell killing of tumor but not normal cells., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

T cells play a vital role in combatting SARS-CoV-2 and forming long-term memory responses. Whereas extensive structural information is available on neutralizing antibodies against SARS-CoV-2, such information on SARS-CoV-2-specific T-cell receptors (TCRs) bound to their peptide-MHC targets is lacking. Here we determine the structures of a public and a private TCR from COVID-19 convalescent patients in complex with HLA-A2 and two SARS-CoV-2 spike protein epitopes (YLQ and RLQ). The structures reveal the basis for selection of particular TRAV and TRBV germline genes by the public but not the private TCR, and for the ability of the TCRs to recognize natural variants of RLQ but not YLQ. Neither TCR recognizes homologous epitopes from human seasonal coronaviruses. By elucidating the mechanism for TCR recognition of an immunodominant yet variable epitope (YLQ) and a conserved but less commonly targeted epitope (RLQ), this study can inform prospective efforts to design vaccines to elicit pan-coronavirus immunity., (© 2022. The Author(s).)

The data currently available on how the immune system recognises the SARS-CoV-2 virus is growing rapidly. While there are structures of some SARS-CoV-2 proteins in complex with antibodies, which helps us understand how the immune system is able to recognise this new virus; however, we lack data on how T cells are able to recognise this virus. T cells, especially the cytotoxic CD8+ T cells, are critical for viral recognition and clearance. Here we report the X-ray crystallography structure of a T cell receptor, shared among unrelated individuals (public TCR) in complex with a dominant spike-derived CD8+ T cell epitope (YLQ peptide). We show that YLQ activates a polyfunctional CD8+ T cell response in COVID-19 recovered patients. We detail the molecular basis for the shared TCR gene usage observed in HLA-A*02:01+ individuals, providing an understanding of TCR recognition towards a SARS-CoV-2 epitope. Interestingly, the YLQ peptide conformation did not change upon TCR binding, facilitating the high-affinity interaction observed.

Understanding T-cell responses requires identifying viral peptides presented by human leukocyte antigens (HLAs). X-ray crystallography can be used to visualize their presentation. This protocol describes the expression, purification, and crystallization of HLA-A∗02:01, one of the most frequent HLA in the global population in complex with peptides derived from the SARS-CoV-2 nucleocapsid protein. This protocol can be applied to different HLA class I molecules bound to other peptides. For complete details on the use and execution of this protocol, please refer to Szeto et al. (2021)., Competing Interests: The authors have no competing interests., (© 2021 The Authors.)

CD8 + T cells express T cell receptors (TCRs) that recognize short peptide antigens in the context of major histocompatibility class I (MHC I) molecules. This recognition process produces an array of cytokine-mediated signals that help to govern immunological responses. Design of biostable MHC I peptide vaccines containing unnatural subunits is desirable, and synthetic antigens in which a native α-amino acid residue is replaced by a homologous β-amino acid residue (native side chain but extended backbone) might be useful in this regard. We have evaluated the impact of α-to-β backbone modification at a single site on T cell-mediated recognition of six clinically important viral and tumor-associated antigens bound to an MHC I. Effects of this modification on MHC I affinity and T cell activation were measured. Many of these modifications diminish or prevent T cell response. However, a number of α/β-peptide antigens were found to mimic the activity of natural antigens or to enhance maximal T cell response, as measured by interferon-γ release. Results from this broad exploratory study advance our understanding of immunological responses to antigens bearing unnatural modifications and suggest that α/β-peptides could be a source of potent and proteolytically stable variants of native antigens.

TP53 (tumor protein p53) is the most commonly mutated cancer driver gene, but drugs that target mutant tumor suppressor genes, such as TP53 , are not yet available. Here, we describe the identification of an antibody highly specific to the most common TP53 mutation (R175H, in which arginine at position 175 is replaced with histidine) in complex with a common human leukocyte antigen-A (HLA-A) allele on the cell surface. We describe the structural basis of this specificity and its conversion into an immunotherapeutic agent: a bispecific single-chain diabody. Despite the extremely low p53 peptide-HLA complex density on the cancer cell surface, the bispecific antibody effectively activated T cells to lyse cancer cells that presented the neoantigen in vitro and in mice. This approach could in theory be used to target cancers containing mutations that are difficult to target in conventional ways., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Melanoma is a type of skin cancer with increasing incidence worldwide and high lethality. Conventional forms of treatment are not effective in advanced cancer stages. Hence, immunotherapeutic approaches have been tested to modulate immune response against tumor cells. Some vaccine models using tumor-associated antigens (TAAs) such as glycoprotein 100 (gp100) have been studied, but their expected effectiveness has not been shown until now. Antigen immunogenicity is a crucial point to improve the immune response, and therefore mutations are inserted in peptide sequences. It is possible to understand the interactions which occur between peptides and immune system molecules through computer simulation, and this is essential in order to guide efficient vaccine models. In this work, we have calculated the interaction binding energies of crystallographic data based on modified gp100 peptides and HLA-A*0201 using density functional theory (DFT) and the molecular fractionation with conjugated caps (MFCC) approach. Our results show the most relevant residue-residue interactions, the impact of three mutations in their binding sites, and the main HLA-A*0201 amino acids for peptide-HLA binding.

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., Competing Interests: The authors declare no competing interest.

SARS-CoV-2, a new world coronavirus belonging to class Nidovirales of Coronaviridae family causes COVID-19 infection which is the leading cause of death worldwide. Currently there are no approved drugs and vaccines available for the prevention of COVID-19 infection, although couples of immunizations are being tested in clinical trials. However, the present efforts are focused on computational vaccination technique for evaluating candidates to design multi-epitope-based vaccine against pathogenic mechanism of novel SARS-COV-2. Based on recent published evidence, we recognized spike glycoprotein and envelope small membrane protein are the potential targets to combat the pathogenic mechanism of SARS-CoV-2. Similarly, in the present study we identified epitope of both B and T cell associated with these proteins. Extremely antigenic, conserve, immunogenic and nontoxic epitope of B and T cell of Spike protein are WPWYVWLGFI, SRVKNLNSSEGVPDLLV whereas the CWCARPTCIK and YCCNIVNVSL are associated with envelope small membrane protein were selected as potential candidate for vaccine designing. These epitopes show virtuous interaction with HLAA0201 during molecular docking analysis. Under simulation protocol the predicted vaccine candidates show stability. Collectively, this work provides novel potential candidates for epitope-based vaccine designing against COVID-19 infection.

Most biomolecular interactions are typically thought to increase the (local) rigidity of a complex, for example, in drug-target binding. However, detailed analysis of specific biomolecular complexes can reveal a more subtle interplay between binding and rigidity. Here, we focussed on the human leucocyte antigen (HLA), which plays a crucial role in the adaptive immune system by presenting peptides for recognition by the αβ T-cell receptor (TCR). The role that the peptide plays in tuning HLA flexibility during TCR recognition is potentially crucial in determining the functional outcome of an immune response, with obvious relevance to the growing list of immunotherapies that target the T-cell compartment. We have applied high-pressure/temperature perturbation experiments, combined with molecular dynamics simulations, to explore the drivers that affect molecular flexibility for a series of different peptide-HLA complexes. We find that different peptide sequences affect peptide-HLA flexibility in different ways, with the peptide cargo tuning a network of correlated motions throughout the pHLA complex, including in areas remote from the peptide-binding interface, in a manner that could influence T-cell antigen discrimination., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The CD8 T cell response to the HLA-A2-restricted epitope LLWNGPMAV (LLW) of the non-structural protein 4b of Yellow Fever Virus (YFV) is remarkably immunodominant, highly prevalent and powerful in YFV-vaccinated humans. Here we used a combinatorial peptide library screening in the context of an A2/LLW-specific CD8 T cell clone to identify a superagonist that features a methionine to isoleucine substitution at position 7. Based on in silico modeling, the functional enhancement of this LLW-7I mutation was associated with alterations in the structural dynamics of the peptide in the major histocompatibility complex (pMHC) binding with the T cell receptor (TCR). While the TCR off-rate of LLW-7I pMHC is comparable to the wild type peptide, the rigidity of the 7I peptide seems to confer less entropy loss upon TCR binding. This LLW-7I superagonist is an example of improved functionality in human CD8 T cells associated with optimized ligand rigidity for TCR binding and not with changes in TCR:pMHC off-rate kinetics., Competing Interests: Declaration of Competing Interest The authors declare no commercial or financial conflict of interest., (Crown Copyright © 2020. Published by Elsevier Ltd. All rights reserved.)

T cell-mediated immunity is governed primarily by T cell receptor (TCR) recognition of peptide-human leukocyte antigen (pHLA) complexes and is essential for immunosurveillance and disease control. This interaction is generally stabilized by interactions between the HLA surface and TCR germline-encoded complementarity-determining region (CDR) loops 1 and 2, whereas peptide selectivity is guided by direct interactions with the TCR CDR3 loops. Here, we solved the structure of a newly identified TCR in complex with a clinically relevant peptide derived from the cancer testis antigen melanoma antigen-A4 (MAGE-A4). The TCR bound pHLA in a position shifted toward the peptide's N terminus. This enabled the TCR to achieve peptide selectivity via an indirect mechanism, whereby the TCR sensed the first residue of the peptide through HLA residue Trp-167, which acted as a tunable gateway. Amino acid substitutions at peptide position 1 predicted to alter the HLA Trp-167 side-chain conformation abrogated TCR binding, indicating that this indirect binding mechanism is essential for peptide recognition. These findings extend our understanding of the molecular rules that underpin antigen recognition by TCRs and have important implications for the development of TCR-based therapies., Competing Interests: Conflict of interest—C. C., C. M., A. L., M. H., L. H., M. C. C. R., C. H., D. K. C., and S. H. are/were employees of Immunocore Ltd. P. L. was an employee of Genentech Inc. The authors declare that the research was conducted in the absence of any other commercial or financial relationships that could be construed as a potential conflict of interest., (© 2020 Coles et al.)

Adoptive cell therapy (ACT) with tumor-specific T cells can mediate cancer regression. The main target of tumor-specific T cells are neoantigens arising from mutations in self-proteins. Although the majority of cancer neoantigens are unique to each patient, and therefore not broadly useful for ACT, some are shared. We studied oligoclonal T-cell receptors (TCRs) that recognize a shared neoepitope arising from a driver mutation in the p53 oncogene (p53R175H) presented by HLA-A2. Here we report structures of wild-type and mutant p53-HLA-A2 ligands, as well as structures of three tumor-specific TCRs bound to p53R175H-HLA-A2. These structures reveal how a driver mutation in p53 rendered a self-peptide visible to T cells. The TCRs employ structurally distinct strategies that are highly focused on the mutation to discriminate between mutant and wild-type p53. The TCR-p53R175H-HLA-A2 complexes provide a framework for designing TCRs to improve potency for ACT without sacrificing specificity.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims antigenic peptide precursors to generate mature antigenic peptides for presentation by major histocompatibility complex class I (MHCI) molecules and regulates adaptive immune responses. ERAP1 has been proposed to trim peptide precursors both in solution and in preformed MHCI-peptide complexes, but which mode is more relevant to its biological function remains controversial. Here, we compared ERAP1-mediated trimming of antigenic peptide precursors in solution or when bound to three MHCI alleles, HLA-B*58, HLA-B*08, and HLA-A*02. For all MHCI-peptide combinations, peptide binding onto MHCI protected against ERAP1-mediated trimming. In only a single MHCI-peptide combination, trimming of an HLA-B*08-bound 12-mer progressed at a considerable rate, albeit still slower than in solution. Results from thermodynamic, kinetic, and computational analyses suggested that this 12-mer is highly labile and that apparent on-MHC trimming rates are always slower than that of MHCI-peptide dissociation. Both ERAP2 and leucine aminopeptidase, an enzyme unrelated to antigen processing, could trim this labile peptide from preformed MHCI complexes as efficiently as ERAP1. A pseudopeptide analogue with high affinity for both HLA-B*08 and the ERAP1 active site could not promote the formation of a ternary ERAP1/MHCI/peptide complex. Similarly, no interactions between ERAP1 and purified peptide-loading complex were detected in the absence or presence of a pseudopeptide trap. We conclude that MHCI binding protects peptides from ERAP1 degradation and that trimming in solution along with the dynamic nature of peptide binding to MHCI are sufficient to explain ERAP1 processing of antigenic peptide precursors., (© 2020 Mavridis et al.)

Histocompatibility Leukocyte Antigens, or HLAs, are one of the most polymorphic molecules in humans. This high degree of polymorphism endows HLA molecules with the ability to present a vast array of peptides, an essential trait for responding to ever-evolving pathogens. Unlike classical HLA molecules (HLA-Ia), some non-classical HLA-Ib molecules, including HLA-E, are almost monomorphic. Several studies show HLA-E can present self-peptides originating from the leader sequence of other HLA molecules, which signals to our immune system that the cell is healthy. Therefore, it was traditionally thought that the chief role of HLA-E in the body was in immune surveillance. However, there is emerging evidence that HLA-E is also able to present pathogen-derived peptides to the adaptive immune system, namely T cells, in a manner that is similar to classical HLA-Ia molecules. Here we describe the early findings of this less conventional role of HLA-E in the adaptive immune system and its importance for immunity., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: The re-emergence of mumps among vaccinated young adults has become a global issue. Besides waning of antibody responses, suboptimal induction of T-cell responses may reduce protection. In a recent study, we observed a dominant polyfunctional CD8+ T-cell response after natural mumps virus (MuV) infection that was not present after vaccination. Unraveling the MuV epitope repertoire can provide insight in the specificity, functionality, and breadth of the T-cell response against MuV., Methods: Peptides were eluted from human leukocyte antigen (HLA) class I molecules of MuV-infected cells and characterized by advanced mass spectrometry. Selected identified MuV peptides were tested for in vitro and ex vivo immunogenicity., Results: In this study, we identified a broad landscape of 83 CD8+ T-cell epitopes of MuV, 41 of which were confirmed based on synthetic peptide standards. For 6 epitopes, we showed induction of an HLA-A*02-restriced CD8+ T-cell response. Moreover, robust T-cell responses against 5 selected MuV epitopes could be detected in all tested mumps patients using peptide/HLA-A*02:01 dextramers., Conclusions: The identified CD8+ T-cell epitopes will help to further characterize MuV-specific T-cell immunity after natural MuV infection or vaccination. These MuV epitopes may provide clues for a better understanding of, and possibly for preventing, mumps vaccine failure.We identified for the first time 41 mumps virus (MuV)-specific HLA-A*02 epitopes. For 6 epitopes, CD8+ T-cell responses were confirmed in T cells derived from several mumps cases, and MuV-specific CD8+ T cells could be identified by peptide/dextramer staining., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.)

Understanding how peptide selection is controlled on different major histocompatibility complex class I (MHC I) molecules is pivotal for determining how variations in these proteins influence our predisposition to infectious diseases, cancer, and autoinflammatory conditions. Although the intracellular chaperone TAPBPR edits MHC I peptides, it is unclear which allotypes are subjected to TAPBPR-mediated peptide editing. Here, we examine the ability of 97 different human leukocyte antigen (HLA) class I allotypes to interact with TAPBPR. We reveal a striking preference of TAPBPR for HLA-A, particularly for supertypes A2 and A24, over HLA-B and -C molecules. We demonstrate that the increased propensity of these HLA-A molecules to undergo TAPBPR-mediated peptide editing is determined by molecular features of the HLA-A F pocket, specifically residues H114 and Y116. This work reveals that specific polymorphisms in MHC I strongly influence their susceptibility to chaperone-mediated peptide editing, which may play a significant role in disease predisposition., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

The T cell receptor (TCR)-peptide-MHC (pMHC) interaction is the only antigen-specific interaction during T lymphocyte activation. Recent work suggests that formation of catch bonds is characteristic of activating TCR-pMHC interactions. However, whether this binding behavior is an intrinsic feature of the molecular bond, or a consequence of more complex multimolecular or cellular responses, remains unclear. We used a laminar flow chamber to measure, first, 2D TCR-pMHC dissociation kinetics of peptides of various activating potency in a cell-free system in the force range (6 to 15 pN) previously associated with catch-slip transitions and, second, 2D TCR-pMHC association kinetics, for which the method is well suited. We did not observe catch bonds in dissociation, and the off-rate measured in the 6- to 15-pN range correlated well with activation potency, suggesting that formation of catch bonds is not an intrinsic feature of the TCR-pMHC interaction. The association kinetics were better explained by a model with a minimal encounter duration rather than a standard on-rate constant, suggesting that membrane fluidity and dynamics may strongly influence bond formation., Competing Interests: The authors declare no conflict of interest.

The HLA-A*02:01-restricted decapeptide EAAGIGILTV, derived from melanoma antigen recognized by T-cells-1 (MART-1) protein, represents one of the best-studied tumor associated T-cell epitopes, but clinical results targeting this peptide have been disappointing. This limitation may reflect the dominance of the nonapeptide, AAGIGILTV, at the melanoma cell surface. The decapeptide and nonapeptide are presented in distinct conformations by HLA-A*02:01 and TCRs from clinically relevant T-cell clones recognize the nonapeptide poorly. Here, we studied the MEL5 TCR that potently recognizes the nonapeptide. The structure of the MEL5-HLA-A*02:01-AAGIGILTV complex revealed an induced fit mechanism of antigen recognition involving altered peptide-MHC anchoring. This "flexing" at the TCR-peptide-MHC interface to accommodate the peptide antigen explains previously observed incongruences in this well-studied system and has important implications for future therapeutic approaches. Finally, this study expands upon the mechanisms by which molecular plasticity can influence antigen recognition by T cells., (© 2019 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

T cell receptor (TCR) recognition of antigenic peptides bound and presented by class I major histocompatibility complex (MHC) proteins underlies the cytotoxic immune response to diseased cells. Crystallographic structures of TCR-peptide/MHC complexes have demonstrated how TCRs simultaneously interact with both the peptide and the MHC protein. However, it is increasingly recognized that, beyond serving as a static platform for peptide presentation, the physical properties of class I MHC proteins are tuned by different peptides in ways that are not always structurally visible. These include MHC protein motions, or dynamics, which are believed to influence interactions with a variety of MHC-binding proteins, including not only TCRs, but other activating and inhibitory receptors as well as components of the peptide loading machinery. Here, we investigated the mechanisms by which peptides tune the dynamics of the common class I MHC protein HLA-A2. By examining more than 50 lengthy molecular dynamics simulations of HLA-A2 presenting different peptides, we identified regions susceptible to dynamic tuning, including regions in the peptide binding domain as well as the distal α3 domain. Further analyses of the simulations illuminated mechanisms by which the influences of different peptides are communicated throughout the protein, and involve regions of the peptide binding groove, the β 2 -microglobulin subunit, and the α3 domain. Overall, our results demonstrate that the class I MHC protein is a highly tunable peptide sensor whose physical properties vary considerably with bound peptide. Our data provides insight into the underlying principles and suggest a role for dynamically driven allostery in the immunological function of MHC proteins.

TCRs recognize cognate pMHCs to initiate T cell signaling and adaptive immunity. Mechanical force strengthens TCR-pMHC interactions to elicit agonist-specific catch bonds to trigger TCR signaling, but the underlying dynamic structural mechanism is unclear. We combined steered molecular dynamics (SMD) simulation, single-molecule biophysical approaches, and functional assays to collectively demonstrate that mechanical force induces conformational changes in pMHCs to enhance pre-existing contacts and activates new interactions at the TCR-pMHC binding interface to resist bond dissociation under force, resulting in TCR-pMHC catch bonds and T cell activation. Intriguingly, cancer-associated somatic mutations in HLA-A2 that may restrict these conformational changes suppressed TCR-pMHC catch bonds. Structural analysis also indicated that HLA polymorphism might alter the equilibrium of these conformational changes. Our findings not only reveal critical roles of force-induced conformational changes in pMHCs for activating TCR-pMHC catch bonds but also have implications for T cell-based immunotherapy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Antigen-specific T cells are found at low frequencies in circulation but carry important diagnostic information as liquid biomarkers in numerous biomedical settings, such as monitoring the efficacy of vaccines and cancer immunotherapies. To enable detection of antigen-specific T cells with high sensitivity, we develop peptide-MHC (pMHC) tetramers labeled with DNA barcodes to detect single T cells by droplet digital PCR (ddPCR). We show that site-specific conjugation of DNA via photocleavable linkers allows barcoded tetramers to stain T cells with similar avidity compared to conventional fluorescent tetramers and efficient recovery of barcodes by light with no loss in cell viability. We design an orthogonal panel of DNA-barcoded tetramers to simultaneously detect multiple antigen-specific T cell populations, including from a mouse model of viral infection, and discriminate single cancer-specific T cells with high diagnostic sensitivity and specificity. This approach of DNA-barcoding can be broadened to encompass additional rare cells for monitoring immunological health at the single cell level.

The m embrane- a ssociated R ING- CH (MARCH) family of membrane-bound E3 ubiquitin ligases regulates the levels of cell-surface membrane proteins, many of which are involved in immune responses. Although their role in ubiquitin-dependent endocytosis and degradation of cell-surface proteins is extensively documented, the features of MARCH proteins and their substrates that drive the molecular recognition events leading to ubiquitin transfer remain poorly defined. In this study, we sought to determine the features of human MARCH9 that are required for regulating the surface levels of its substrate proteins. Consistent with previous studies of other MARCH proteins, we found that susceptibility to MARCH9 activity is encoded in the transmembrane (TM) domains of its substrates. Accordingly, substitutions at specific residues and motifs within MARCH9's TM domains resulted in varying degrees of functional impairment. Most notably, a single serine-to-alanine substitution in the first of its two TM domains rendered MARCH9 completely unable to alter the surface levels of two different substrates: the major histocompatibility class I molecule HLA-A2 and the T-cell co-receptor CD4. Solution NMR analysis of a MARCH9 fragment encompassing the two TM domains and extracellular connecting loop revealed that the residues contributing most to MARCH9 activity are located in the α-helical portions of TM1 and TM2 that are closest to the extracellular face of the lipid bilayer. This observation defines a key region required for substrate regulation. In summary, our biochemical and structural findings demonstrate that specific sequences in the α-helical MARCH9 TM domains make crucial contributions to its ability to down-regulate its protein substrates., (© 2019 Tan et al.)

X-ray crystallographic studies of class I peptide-MHC molecules (pMHC) continue to provide important insights into immune recognition, however their success depends on generation of diffraction-quality crystals, which remains a significant challenge. While protein engineering techniques such as surface-entropy reduction and lysine methylation have proven utility in facilitating and/or improving protein crystallisation, they risk affecting the conformation and biochemistry of the class I MHC antigen binding groove. An attractive alternative is the use of noncovalent crystallisation chaperones, however these have not been developed for pMHC. Here we describe a method for promoting class I pMHC crystallisation, by exploiting its natural ligand interaction with the immunoregulatory receptor LILRB1 as a novel crystallisation chaperone. First, focussing on a model HIV-1-derived HLA-A2-restricted peptide, we determined a 2.4 Å HLA-A2/LILRB1 structure, which validated that co-crystallisation with LILRB1 does not alter conformation of the antigenic peptide. We then demonstrated that addition of LILRB1 enhanced the crystallisation of multiple peptide-HLA-A2 complexes, and identified a generic condition for initial co-crystallisation. LILRB1 chaperone-based crystallisation enabled structure determination for HLA-A2 complexes previously intransigent to crystallisation, including both conventional and post-translationally-modified peptides, of diverse lengths. Since both the LILRB1 recognition interface on the HLA-A2 α3 domain molecule and HLA-A2-mediated crystal contacts are predominantly conserved across class I MHC molecules, the approach we outline could prove applicable to a diverse range of class I pMHC. LILRB1 chaperone-mediated crystallisation should expedite molecular insights into the immunobiology of diverse immune-related diseases and immunotherapeutic strategies, particularly involving class I pMHC complexes that are challenging to crystallise., (Copyright © 2018. Published by Elsevier B.V.)

Mutated peptides (neoantigens) from a patient's cancer genome can serve as targets for T-cell immunity, but identifying which peptides can be presented by an MHC molecule and elicit T cells has been difficult. Although algorithms that predict MHC binding exist, they are not yet able to distinguish experimental differences in half-lives of the complexes (an immunologically relevant parameter, referred to here as kinetic stability). Improvement in determining actual neoantigen peptide/MHC stability could be important, as only a small fraction of peptides in most current vaccines are capable of eliciting CD8 + T-cell responses. Here, we used a rapid, high-throughput method to experimentally determine peptide/HLA thermal stability on a scale that will be necessary for analysis of neoantigens from thousands of patients. The method combined the use of UV-cleavable peptide/HLA class I complexes and differential scanning fluorimetry to determine the T m values of neoantigen complexes. Measured T m values were accurate and reproducible and were directly proportional to the half-lives of the complexes. Analysis of known HLA-A2-restricted immunogenic peptides showed that T m values better correlated with immunogenicity than algorithm-predicted binding affinities. We propose that temperature stability information can be used as a guide for the selection of neoantigens in cancer vaccines in order to focus attention on those mutated peptides with the highest probability of being expressed on the cell surface., (©2018 American Association for Cancer Research.)

T cells generate adaptive immune responses mediated by the T cell receptor (TCR)-CD3 complex comprising an αβ TCR heterodimer noncovalently associated with three CD3 dimers. In early T cell activation, αβ TCR engagement by peptide-major histocompatibility complex (pMHC) is first communicated to the CD3 signaling apparatus of the TCR-CD3 complex, but the underlying mechanism is incompletely understood. It is possible that pMHC binding induces allosteric changes in TCR conformation or dynamics that are then relayed to CD3. Here, we carried out NMR analysis and molecular dynamics (MD) simulations of both the α and β chains of a human antiviral TCR (A6) that recognizes the Tax antigen from human T cell lymphotropic virus-1 bound to the MHC class I molecule HLA-A2. We observed pMHC-induced NMR signal perturbations in the TCR variable (V) domains that propagated to three distinct sites in the constant (C) domains: 1) the Cβ FG loop projecting from the Vβ/Cβ interface; 2) a cluster of Cβ residues near the Cβ αA helix, a region involved in interactions with CD3; and 3) the Cα AB loop at the membrane-proximal base of the TCR. A biological role for each of these allosteric sites is supported by previous mutational and functional studies of TCR signaling. Moreover, the pattern of long-range, ligand-induced changes in TCR A6 revealed by NMR was broadly similar to that predicted by the MD simulations. We propose that the unique structure of the TCR β chain enables allosteric communication between the TCR-binding sites for pMHC and CD3., (© 2018 Rangarajan et al.)

The binding of the adenovirus (Ad) protein E3-19K with the human leukocyte antigen (HLA) plays an important role in Ad infections, which is the causative agent of a series of gastrointestinal, respiratory and ocular diseases. The objective of this research is to evaluate the essential interactions between E3-19K and HLA-A2 using the X-ray crystal structure of the E3-19K/HLA-A2 complex, and to identify small molecules that could potentially disrupt their binding. Computational methods, including molecular dynamic simulations, MM/GBSA calculations, and computational solvent mapping, were implemented to determine potential binding site(s) for small molecules. The previous experimentally determined hot spot residues, Q54 and E177 in HLA-A2, were also predicted to be the dominant residues for binding to E3-19K by our theoretical calculations. Several other residues were also found to play pivotal roles for the binding of E3-19K with HLA-A2. Residues adjacent to E177, including Q54 and several other residues theoretically predicted to be crucial in HLA-A2 were selected as a potential binding pocket to perform virtual screening with 1200 compounds from the Prestwick library. Seven hits were validated by surface plasmon resonance (SPR) as binders to HLA-A2 as a first step in identifying molecules that can perturb its association with the Ad E3-19K protein., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The discovery of heat shock protein 16 kDa antigen protein has deepen the understanding of latent tuberculosis since it was found to be primarily expressed by Mycobacterium tuberculosis during latent phase leading to the rapid optimization and development in terms of diagnosis and therapeutics. Recently, T cell receptor-like antibody has been explored extensively targeting various diseases due to its dual functionality (T cell receptor and antibody). In this study, a TCR-like domain antibody (A2/Ab) with the binding capacity to Mtb heat shock protein (HSP) 16 kDa antigen presented by major histocompatible complex (MHC) HLA-A*02 was successfully generated via biopanning against human domain antibody library. The generated antibody (A2/Ab) exhibited strong functionality and binding capacity against the target assuring the findings of this study to be beneficial for the development of latent tuberculosis diagnosis and immunotherapeutics in future., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A method for calculating the free energy difference between two structurally defined conformational states of a chemical system is developed. A path is defined using a previously reported collective variable that interpolates between two or more conformations, and a restraint is introduced in order to keep the system close to the path. The evolution of the system along the path, which typically presents a high free energy barrier, is generated using enhanced sampling schemes. Although the formulation of the method in terms of a path is quite general, an important advance in this work is the demonstration that prior knowledge of the path is, in fact, not needed and that the free energy difference can be obtained using a simplified definition of the path collective variable that only involves the endpoints. We first validate this method on cyclohexane isomerization. The method is then tested for an extensive conformational change in a realistic molecular system by calculating the free energy difference between the α -helix and β -hairpin conformations of deca-alanine in solution. Finally, the method is applied to a biologically relevant system to calculate the free energy difference of an observed and a hypothetical conformation of an antigenic peptide bound to a major histocompatibility complex.

Using personalized peptide vaccines (PPVs) to target tumor-specific nonself-antigens (neoantigens) is a promising approach to cancer treatment. However, the development of PPVs is hindered by the challenge of identifying tumor-specific neoantigens, in part because current in silico methods for identifying such neoantigens have limited effectiveness. In this article, we report the results of molecular dynamics simulations of 12 oligopeptides bound with an HLA, revealing a previously unrecognized association between the inability of an oligopeptide to elicit a T cell response and the contraction of the peptide-binding groove upon binding of the oligopeptide to the HLA. Our conformational analysis showed that this association was due to incompatibility at the interface between the contracted groove and its αβ-T cell Ag receptor. This structural demonstration that having the capability to bind HLA does not guarantee immunogenicity prompted us to develop an atom-based method (SE FF12MC ) to predict immunogenicity through using the structure and energy of a peptide·HLA complex to assess the propensity of the complex for further complexation with its TCR. In predicting the immunogenicities of the 12 oligopeptides, SE FF12MC achieved a 100% success rate, compared with success rates of 25-50% for 11 publicly available residue-based methods including NetMHC-4.0. Although further validation and refinements of SE FF12MC are required, our results suggest a need to develop in silico methods that assess peptide characteristics beyond their capability to form stable binary complexes with HLAs to help remove hurdles in using the patient tumor DNA information to develop PPVs for personalized cancer immunotherapy., (Copyright © 2018 The Authors.)

Purpose: PR1 is a human leukocyte antigen (HLA)-A2 nonameric peptide derived from neutrophil elastase (NE) and proteinase 3 (P3). We have previously shown that PR1 is cross-presented by solid tumors, leukemia, and antigen-presenting cells, including B cells. We have also shown that cross-presentation of PR1 by solid tumors renders them susceptible to killing by PR1-targeting immunotherapies. As multiple myeloma is derived from B cells, we investigated whether multiple myeloma is also capable of PR1 cross-presentation and subsequently capable of being targeted by using PR1 immunotherapies. Experimental Design: We tested whether multiple myeloma is capable of cross-presenting PR1 and subsequently becomes susceptible to PR1-targeting immunotherapies, using multiple myeloma cell lines, a xenograft mouse model, and primary multiple myeloma patient samples. Results: Here we show that multiple myeloma cells lack endogenous NE and P3, are able to take up exogenous NE and P3, and cross-present PR1 on HLA-A2. Cross-presentation by multiple myeloma utilizes the conventional antigen processing machinery, including the proteasome and Golgi, and is not affected by immunomodulating drugs (IMiD). Following PR1 cross-presentation, we are able to target multiple myeloma with PR1-CTL and anti-PR1/HLA-A2 antibody both in vitro and in vivo Conclusions: Collectively, our data demonstrate that PR1 is a novel tumor-associated antigen target in multiple myeloma and that multiple myeloma is susceptible to immunotherapies that target cross-presented antigens. Clin Cancer Res; 24(14); 3386-96. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Adoptive transfer of T cells engineered with a cancer-specific T cell receptor (TCR) has demonstrated clinical benefit. However, the risk for off-target toxicity of TCRs remains a concern. Here, we examined the cross-reactive profile of T cell clone (7B5) with a high functional sensitivity for the hematopoietic-restricted minor histocompatibility antigen HA-2 in the context of HLA-A*02:01. HA-2 pos Epstein-Barr virus-transformed B lymphoblastic cell lines (EBV-LCLs) and primary acute myeloid leukemia samples, but not hematopoietic HA-2 neg samples, are effectively recognized. However, we found unexpected off-target recognition of human fibroblasts and keratinocytes not expressing the HA-2 antigen. To uncover the origin of this off-target recognition, we performed an alanine scanning approach, identifying six out of nine positions to be important for peptide recognition. This indicates a low risk for broad cross-reactivity. However, using a combinatorial peptide library scanning approach, we identified a CDH13-derived peptide activating the 7B5 T cell clone. This was confirmed by recognition of CDH13-transduced EBV-LCLs and cell subsets endogenously expressing CDH13, such as proximal tubular epithelial cells. As such, we recommend the use of a combinatorial peptide library scan followed by screening against additional cell subsets to validate TCR specificity and detect off-target toxicity due to cross-reactivity directed against unrelated peptides before selecting candidate TCRs for clinical testing., (Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

EBV infection is associated with a number of malignancies of clinical unmet need, including Hodgkin lymphoma, nasopharyngeal carcinoma, gastric cancer, and posttransplant lymphoproliferative disease (PTLD), all of which express the EBV protein latent membrane protein 2A (LMP2A), an antigen that is difficult to target by conventional antibody approaches. To overcome this, we utilized phage display technology and a structure-guided selection strategy to generate human T cell receptor-like (TCR-like) monoclonal antibodies with exquisite specificity for the LMP2A-derived nonamer peptide, C426LGGLLTMV434 (CLG), as presented on HLA-A*02:01. Our lead construct, clone 38, closely mimics the native binding mode of a TCR, recognizing residues at position P3-P8 of the CLG peptide. To enhance antitumor potency, we constructed dimeric T cell engaging bispecific antibodies (DiBsAb) of clone 38 and an affinity-matured version clone 38-2. Both DiBsAb showed potent antitumor properties in vitro and in immunodeficient mice implanted with EBV transformed B lymphoblastoid cell lines and human T cell effectors. Clone 38 DiBsAb showed a stronger safety profile compared with its affinity-matured variant, with no activity against EBV- tumor cell lines and a panel of normal tissues, and was less cross-reactive against HLA-A*02:01 cells pulsed with a panel of CLG-like peptides predicted from a proteomic analysis. Clone 38 was also shown to recognize the CLG peptide on other HLA-A*02 suballeles, including HLA-A*02:02, HLA-A*02:04, and HLA-A*02:06, allowing for its potential use in additional populations. Clone 38 DiBsAb is a lead candidate to treat EBV malignancies with one of the strongest safety profiles documented for TCR-like mAbs.

Most affinity-maturation campaigns for antibodies and T-cell receptors (TCRs) operate on the residues at the binding site, located within the loops known as complementarity-determining regions (CDRs). Accordingly, mutations in contact residues, or so-called "second shell" residues, that increase affinity are typically identified by directed evolution involving combinatorial libraries. To determine the impact of residues located at a distance from the binding site, here we used single-codon libraries of both CDR and non-CDR residues to generate a deep mutational scan of a human TCR against the cancer antigen MART-1·HLA-A2. Non-CDR residues included those at the interface of the TCR variable domains (Vα and Vβ) and surface-exposed framework residues. Mutational analyses showed that both Vα/Vβ interface and CDR residues were important in maintaining binding to MART-1·HLA-A2, probably due to either structural requirements for proper Vα/Vβ association or direct contact with the ligand. More surprisingly, many Vα/Vβ interface substitutions yielded improved binding to MART-1·HLA-A2. To further explore this finding, we constructed interface libraries and selected them for improved stability or affinity. Among the variants identified, one conservative substitution (F45βY) was most prevalent. Further analysis of F45βY showed that it enhanced thermostability and increased affinity by 60-fold. Thus, introducing a single hydroxyl group at the Vα/Vβ interface, at a significant distance from the TCR·peptide·MHC-binding site, remarkably affected ligand binding. The variant retained a high degree of specificity for MART-1·HLA-A2, indicating that our approach provides a general strategy for engineering improvements in either soluble or cell-based TCRs for therapeutic purposes., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Influenza A virus (IAV) causes an acute infection in humans that is normally eliminated by CD8 + cytotoxic T lymphocytes. Individuals expressing the MHC class I molecule HLA-A2 produce cytotoxic T lymphocytes bearing T-cell receptors (TCRs) that recognize the immunodominant IAV epitope GILGFVFTL (GIL). Most GIL-specific TCRs utilize α/β chain pairs encoded by the TRAV27/TRBV19 gene combination to recognize this relatively featureless peptide epitope (canonical TCRs). However, ∼40% of GIL-specific TCRs express a wide variety of other TRAV/TRBV combinations (non-canonical TCRs). To investigate the structural underpinnings of this remarkable diversity, we determined the crystal structure of a non-canonical GIL-specific TCR (F50) expressing the TRAV13-1/TRBV27 gene combination bound to GIL-HLA-A2 to 1.7 Å resolution. Comparison of the F50-GIL-HLA-A2 complex with the previously published complex formed by a canonical TCR (JM22) revealed that F50 and JM22 engage GIL-HLA-A2 in markedly different orientations. These orientations are distinguished by crossing angles of TCR to peptide-MHC of 29° for F50 versus 69° for JM22 and by a focus by F50 on the C terminus rather than the center of the MHC α1 helix for JM22. In addition, F50, unlike JM22, uses a tryptophan instead of an arginine to fill a critical notch between GIL and the HLA-A2 α2 helix. The F50-GIL-HLA-A2 complex shows that there are multiple structurally distinct solutions to recognizing an identical peptide-MHC ligand with sufficient affinity to elicit a broad anti-IAV response that protects against viral escape and T-cell clonal loss.

Extracellular vesicles (EVs) are released by most cell types and have been associated with multiple immunomodulatory functions. MHC class I molecules have crucial roles in antigen presentation and in eliciting immune responses and are known to be incorporated into EVs. However, the MHC class I immunopeptidome of EVs has not been established. Here, using a small-scale immunoisolation of the antigen serotypes HLA-A*02:01 and HLA-B*27:05 expressed on the Epstein-Barr virus-transformed B cell line Jesthom and MS of the eluted peptides from both cells and EVs, we identified 516 peptides that bind either HLA-A*02:01 or HLA-B*27:05. Of importance, the predicted serotype-binding affinities and peptide-anchor motifs did not significantly differ between the peptide pools isolated from cells or EVs, indicating that during EV biogenesis, no obvious editing of the MHC class I immunopeptidome occurs. These results, for the first time, establish EVs as a source of MHC class I peptides that can be used for the study of the immunopeptidome and in the discovery of potential neoantigens for immunotherapies., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

MEX-3 is a K-homology (KH) domain-containing RNA-binding protein first identified as a translational repressor in Caenorhabditis elegans , and its four orthologs (MEX-3A-D) in human and mouse were subsequently found to have E3 ubiquitin ligase activity mediated by a RING domain and critical for RNA degradation. Current evidence implicates human MEX-3C in many essential biological processes and suggests a strong connection with immune diseases and carcinogenesis. The highly conserved dual KH domains in MEX-3 proteins enable RNA binding and are essential for the recognition of the 3'-UTR and post-transcriptional regulation of MEX-3 target transcripts. However, the molecular mechanisms of translational repression and the consensus RNA sequence recognized by the MEX-3C KH domain are unknown. Here, using X-ray crystallography and isothermal titration calorimetry, we investigated the RNA-binding activity and selectivity of human MEX-3C dual KH domains. Our high-resolution crystal structures of individual KH domains complexed with a noncanonical U-rich and a GA-rich RNA sequence revealed that the KH1/2 domains of human MEX-3C bound MRE10, a 10-mer RNA (5'-CAGAGUUUAG-3') consisting of an eight-nucleotide MEX-3-recognition element (MRE) motif, with high affinity. Of note, we also identified a consensus RNA motif recognized by human MEX-3C. The potential RNA-binding sites in the 3'-UTR of the human leukocyte antigen serotype ( HLA-A2 ) mRNA were mapped with this RNA-binding motif and further confirmed by fluorescence polarization. The binding motif identified here will provide valuable information for future investigations of the functional pathways controlled by human MEX-3C and for predicting potential mRNAs regulated by this enzyme., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Although the precise host-defence mechanisms are not completely understood, T-cell-mediated immune responses are believed to play a pivotal role in controlling parasite infection. In this study, the potential HLA*A2 restricted peptides were predicted and the ability of peptides to bind HLA-A*02 was confirmed by a MHC stabilization assay. Two of the peptides tested stabilized HLA-A*02: (a) LLATTVSGL (P1) and (b) LMTNGPLEV (P3). The potential of the peptides to generate protective immune response was evaluated in patients with treated visceral leishmaniasis as well as in healthy control subjects. Our data suggest that CD8 + T-cell proliferation against the selected peptide was significantly higher compared to unstimulated culture conditions. The stimulation of peripheral blood mononuclear cells with epitopes individually or as a cocktail upregulated IFN-γ production, which indicates its pivotal role in protective immune response. The IFN-γ production was mainly in a CD8 + T-cells-dependent manner, which suggested that these epitopes had an immunoprophylactic potential in a MHC class I-dependent manner. Moreover, no role of the CD3 + T cell was observed in the IL-10 production against the selected peptides, and no role was found in disease pathogenesis. Further studies on the role of these synthetic peptides may contribute significantly to developing a polytope vaccine idea towards leishmaniasis., (© 2017 John Wiley & Sons Ltd.)

Epstein-Barr virus (EBV) is a common gammaherpesvirus associated with various human malignancies. Antibodies with T cell receptor-like specificities (TCR-like mAbs) provide a means to target intracellular tumor- or virus-associated antigens by recognising their processed peptides presented on major histocompatibility complex (MHC) class I (pMHC) complexes. These antibodies are however thought to be relevant only for a single HLA allele. Here, we show that HLA-A*02:01-restricted EBV antigenic peptides EBNA1 562-570 , LMP1 125-133 and LMP2A 426-434 display binding degeneracy towards HLA-A*02 allelic microvariants, and that these pMHC complexes are recognised by anti-EBV TCR-like mAbs E1, L1 and L2 raised in the context of HLA-A*02:01. These antibodies bound endogenously derived pMHC targets on EBV-transformed human B lymphoblastoid cell lines expressing A*02:01, A*02:03, A*02:06 and A*02:07 alleles. More importantly, these TCR-like mAbs mediated both complement-dependent and antibody-dependent cellular cytotoxicity of these cell lines in vitro. This finding suggests the utility of TCR-like mAbs against target cells of closely related HLA subtypes, and the potential applicability of similar reagents within populations of diverse HLA-A*02 alleles.

Tumor exomes provide comprehensive information on mutated, overexpressed genes and aberrant splicing, which can be exploited for personalized cancer immunotherapy. Of particular interest are mutated tumor antigen T-cell epitopes, because neoepitope-specific T cells often are tumoricidal. However, identifying tumor-specific T-cell epitopes is a major challenge. A widely used strategy relies on initial prediction of human leukocyte antigen-binding peptides by in silico algorithms, but the predictive power of this approach is unclear. Here, we used the human tumor antigen NY-ESO-1 (ESO) and the human leukocyte antigen variant HLA-A*0201 (A2) as a model and predicted in silico the 41 highest-affinity, A2-binding 8-11-mer peptides and assessed their binding, kinetic complex stability, and immunogenicity in A2-transgenic mice and on peripheral blood mononuclear cells from ESO-vaccinated melanoma patients. We found that 19 of the peptides strongly bound to A2, 10 of which formed stable A2-peptide complexes and induced CD8 + T cells in A2-transgenic mice. However, only 5 of the peptides induced cognate T cells in humans; these peptides exhibited strong binding and complex stability and contained multiple large hydrophobic and aromatic amino acids. These results were not predicted by in silico algorithms and provide new clues to improving T-cell epitope identification. In conclusion, our findings indicate that only a small fraction of in silico -predicted A2-binding ESO peptides are immunogenic in humans, namely those that have high peptide-binding strength and complex stability. This observation highlights the need for improving in silico predictions of peptide immunogenicity., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

T-cell receptor (TCR) allorecognition is often presumed to be relatively nonspecific, attributable to either a TCR focus on exposed major histocompatibility complex (MHC) polymorphisms or the degenerate recognition of allopeptides. However, paradoxically, alloreactivity can proceed with high peptide and MHC specificity. Although the underlying mechanisms remain unclear, the existence of highly specific alloreactive TCRs has led to their use as immunotherapeutics that can circumvent central tolerance and limit graft-versus-host disease. Here, we show how an alloreactive TCR achieves peptide and MHC specificity. The HCV1406 TCR was cloned from T cells that expanded when a hepatitis C virus (HCV)-infected HLA-A2 - individual received an HLA-A2 + liver allograft. HCV1406 was subsequently shown to recognize the HCV nonstructural protein 3 (NS3):1406-1415 epitope with high specificity when presented by HLA-A2. We show that NS3/HLA-A2 recognition by the HCV1406 TCR is critically dependent on features unique to both the allo-MHC and the NS3 epitope. We also find cooperativity between structural mimicry and a crucial peptide "hot spot" and demonstrate its role, along with the MHC, in directing the specificity of allorecognition. Our results help explain the paradox of specificity in alloreactive TCRs and have implications for their use in immunotherapy and related efforts to manipulate TCR recognition, as well as alloreactivity in general., Competing Interests: The authors declare no conflict of interest.

Hepatitis B virus (HBV) infection has persisted as a major public health problem due to the lack of an effective treatment for those chronically infected. Therapeutic vaccination holds promise, and targeting HBV polymerase is pivotal for viral eradication. In this research, a computational approach was employed to predict suitable HBV polymerase targeting multi-peptides for vaccine candidate selection. We then performed in-depth computational analysis to evaluate the predicted epitopes' immunogenicity, conservation, population coverage, and toxicity. Lastly, molecular docking and MHC-peptide complex stabilization assay were utilized to determine the binding energy and affinity of epitopes to the HLA-A0201 molecule. Criteria-based analysis provided four predicted epitopes, RVTGGVFLV, VSIPWTHKV, YMDDVVLGA and HLYSHPIIL. Assay results indicated the lowest binding energy and high affinity to the HLA-A0201 molecule for epitopes VSIPWTHKV and YMDDVVLGA and epitopes RVTGGVFLV and VSIPWTHKV, respectively. Regions 307 to 320 and 377 to 387 were considered to have the highest probability to be involved in B cell epitopes. The T cell and B cell epitopes identified in this study are promising targets for an epitope-focused, peptide-based HBV vaccine, and provide insight into HBV-induced immune response.

The tumor suppressor p53 is widely dysregulated in cancer and represents an attractive target for immunotherapy. Because of its intracellular localization, p53 is inaccessible to classical therapeutic monoclonal antibodies, an increasingly successful class of anticancer drugs. However, peptides derived from intracellular antigens are presented on the cell surface in the context of MHC I and can be bound by T-cell receptors (TCR). Here, we report the development of a novel antibody, T1-116C, that acts as a TCR mimic to recognize an HLA-A*0201-presented wild-type p53 T-cell epitope, p53 65-73 (RMPEAAPPV). The antibody recognizes a wide range of cancers, does not bind normal peripheral blood mononuclear cells, and can activate immune effector functions to kill cancer cells in vitro In vivo , the antibody targets p53 65-73 peptide-expressing breast cancer xenografts, significantly inhibiting tumor growth. This represents a promising new agent for future cancer immunotherapy. Cancer Res; 77(10); 2699-711. ©2017 AACR ., (©2017 American Association for Cancer Research.)

PR1, an HLA-A2-restricted peptide derived from both proteinase 3 and neutrophil elastase, is recognized on myeloid leukemia cells by cytotoxic T lymphocytes (CTLs) that preferentially kill leukemia and contribute to cytogenetic remission. To evaluate safety, immunogenicity and clinical activity of PR1 vaccination, a phase I/II trial was conducted. Sixty-six HLA-A2+ patients with acute myeloid leukemia (AML: 42), chronic myeloid leukemia (CML: 13) or myelodysplastic syndrome (MDS: 11) received three to six PR1 peptide vaccinations, administered subcutaneously every 3 weeks at dose levels of 0.25, 0.5 or 1.0 mg. Patients were randomized to the three dose levels after establishing the safety of the highest dose level. Primary end points were safety and immune response, assessed by doubling of PR1/HLA-A2 tetramer-specific CTL, and the secondary end point was clinical response. Immune responses were noted in 35 of 66 (53%) patients. Of the 53 evaluable patients with active disease, 12 (24%) had objective clinical responses (complete: 8; partial: 1 and hematological improvement: 3). PR1-specific immune response was seen in 9 of 25 clinical responders versus 3 of 28 clinical non-responders (P=0.03). In conclusion, PR1 peptide vaccine induces specific immunity that correlates with clinical responses, including molecular remission, in AML, CML and MDS patients.

Human JC and BK polyomaviruses (JCV/BKV) can establish a latent infection without any clinical symptoms in healthy individuals. In immunocompromised hosts infection or reactivation of JCV and BKV can cause lethal progressive multifocal leukoencephalopathy (PML) and hemorrhagic cystitis, respectively. Vaccination with JCV/BKV derived antigen epitope peptides or adoptive transfer of virus-specific T cells would constitute an elegant approach to clear virus-infected cells. Furthermore, donor leukocyte infusion (DLI) is another therapeutic approach which could be helpful for patients with JCV/BKV infections.So far, only few immunodominant T cell epitopes of JCV and BKV have been described and therefore is a fervent need for the definition of novel epitopes. In this study, we identified novel T cell epitopes by screening libraries of overlapping peptides derived from the major capsid protein VP1 of JCV. Virus like particles (VLPs) were used to confirm naturally processing. Two human leucocyte antigen (HLA)-A*02-restricted epitopes were characterized by fine mapping with overlapping peptides and nonamer peptide sequences were identified. Cytokine release profile of the epitope-specific T cells was analyzed by enzyme-linked immunospot (ELISPOT) assays and by flow cytometry. We demonstrated that T cell responses were of polyfunctional nature with the potential of epitope-specific killing and cross-reactivity between JCV and BKV. These novel epitopes might constitute a new potential tool to design effective diagnostic and therapeutic approaches against both polyomaviruses.

Proteins are often engineered to have higher affinity for their ligands to achieve therapeutic benefit. For example, many studies have used phage or yeast display libraries of mutants within complementarity-determining regions to affinity mature antibodies and T cell receptors (TCRs). However, these approaches do not allow rapid assessment or evolution across the entire interface. By combining directed evolution with deep sequencing, it is now possible to generate sequence fitness landscapes that survey the impact of every amino acid substitution across the entire protein-protein interface. Here we used the results of deep mutational scans of a TCR-peptide-MHC interaction to guide mutational strategies. The approach yielded stable TCRs with affinity increases of >200-fold. The substitutions with the greatest enrichments based on the deep sequencing were validated to have higher affinity and could be combined to yield additional improvements. We also conducted in silico binding analyses for every substitution to compare them with the fitness landscape. Computational modeling did not effectively predict the impacts of mutations distal to the interface and did not account for yeast display results that depended on combinations of affinity and protein stability. However, computation accurately predicted affinity changes for mutations within or near the interface, highlighting the complementary strengths of computational modeling and yeast surface display coupled with deep mutational scanning for engineering high affinity TCRs., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The interaction between the T cell antigen receptor (TCR) and antigenic peptide in complex with major histocompatibility complex (MHC) molecules is a crucial step in T cell activation. The relative contributions of TCR:peptide and TCR:MHC contacts to the overall binding energy remain unclear. This has important implications for our understanding of T cell development and function. In this study we used site directed mutagenesis to estimate the contribution of HLA-A2 side-chains to the binding of four TCRs. Our results show that these TCRs have very different energetic 'footprints' on HLA-A2, with no residues contributing to all TCR interactions. The estimated overall contribution of MHC side-chains to the total interaction energy was variable, with lower limits ranging from 11% to 50%. Kinetic analysis suggested a minor and variable contribution of MHC side-chains to the transition state complex, arguing against a two-step mechanism for TCR binding.

Antibodies and B cell receptors often bind their antigen at cell-cell interface while both molecular species are surface-bound, which impacts bond kinetics and function. Despite the description of complex energy landscapes for dissociation kinetics which may also result in significantly different association kinetics, surface-bound molecule (2D) association kinetics usually remain described by an on-rate due to crossing of a single free energy barrier, and few experimental works have measured association kinetics under conditions implying force and two-dimensional relative ligand-receptor motion. We use a new laminar flow chamber to measure 2D bond formation with systematic variation of the distribution of encounter durations between antigen and antibody, in a range from 0.1 to 10 ms. Under physiologically relevant forces, 2D association is 100-fold slower than 3D association as studied by surface plasmon resonance assays. Supported by brownian dynamics simulations, our results show that a minimal encounter duration is required for 2D association; an energy landscape featuring a rough initial part might be a reasonable way of accounting for this. By systematically varying the temperature of our experiments, we evaluate roughness at 2k B T, in the range of previously proposed rough parts of landscapes models during dissociation.