Human CD8+ T cell cross-reactivity across influenza A, B and C viruses.

Influenza A, B and C viruses (IAV, IBV and ICV, respectively) circulate globally and infect humans, with IAV and IBV causing the most severe disease. CD8+ T cells confer cross-protection against IAV strains, however the responses of CD8+ T cells to IBV and ICV are understudied. We investigated the breadth of CD8+ T cell cross-recognition and provide evidence of CD8+ T cell cross-reactivity across IAV, IBV and ICV. We identified immunodominant CD8+ T cell epitopes from IBVs that were protective in mice and found memory CD8+ T cells directed against universal and influenza-virus-type-specific epitopes in the blood and lungs of healthy humans. Lung-derived CD8+ T cells displayed tissue-resident memory phenotypes. Notably, CD38+Ki67+CD8+ effector T cells directed against novel epitopes were readily detected in IAV- or IBV-infected pediatric and adult subjects. Our study introduces a new paradigm whereby CD8+ T cells confer unprecedented cross-reactivity across all influenza viruses, a key finding for the design of universal vaccines.

MHCpLogics: an interactive machine learning-based tool for unsupervised data visualization and cluster analysis of immunopeptidomes.

The major histocompatibility complex (MHC) encodes a range of immune response genes, including the human leukocyte antigens (HLAs) in humans. These molecules bind peptide antigens and present them on the cell surface for T cell recognition. The repertoires of peptides presented by HLA molecules are termed immunopeptidomes. The highly polymorphic nature of the genres that encode the HLA molecules confers allotype-specific differences in the sequences of bound ligands. Allotype-specific ligand preferences are often defined by peptide-binding motifs. Individuals express up to six classical class I HLA allotypes, which likely present peptides displaying different binding motifs. Such complex datasets make the deconvolution of immunopeptidomic data into allotype-specific contributions and further dissection of binding-specificities challenging. Herein, we developed MHCpLogics as an interactive machine learning-based tool for mining peptide-binding sequence motifs and visualization of immunopeptidome data across complex datasets. We showcase the functionalities of MHCpLogics by analyzing both in-house and published mono- and multi-allelic immunopeptidomics data. The visualization modalities of MHCpLogics allow users to inspect clustered sequences down to individual peptide components and to examine broader sequence patterns within multiple immunopeptidome datasets. MHCpLogics can deconvolute large immunopeptidome datasets enabling the interrogation of clusters for the segregation of allotype-specific peptide sequence motifs, identification of sub-peptidome motifs, and the exportation of clustered peptide sequence lists. The tool facilitates rapid inspection of immunopeptidomes as a resource for the immunology and vaccine communities. MHCpLogics is a standalone application available via an executable installation at: https://github.com/PurcellLab/MHCpLogics.

Benchmarking Bioinformatics Pipelines in Data-Independent Acquisition Mass Spectrometry for Immunopeptidomics.

Immunopeptidomes are the peptide repertoires bound by the molecules encoded by the major histocompatibility complex [human leukocyte antigen (HLA) in humans]. These HLA-peptide complexes are presented on the cell surface for immune T-cell recognition. Immunopeptidomics denotes the utilization of tandem mass spectrometry to identify and quantify peptides bound to HLA molecules. Data-independent acquisition (DIA) has emerged as a powerful strategy for quantitative proteomics and deep proteome-wide identification; however, DIA application to immunopeptidomics analyses has so far seen limited use. Further, of the many DIA data processing tools currently available, there is no consensus in the immunopeptidomics community on the most appropriate pipeline(s) for in-depth and accurate HLA peptide identification. Herein, we benchmarked four commonly used spectral library-based DIA pipelines developed for proteomics applications (Skyline, Spectronaut, DIA-NN, and PEAKS) for their ability to perform immunopeptidome quantification. We validated and assessed the capability of each tool to identify and quantify HLA-bound peptides. Generally, DIA-NN and PEAKS provided higher immunopeptidome coverage with more reproducible results. Skyline and Spectronaut conferred more accurate peptide identification with lower experimental false-positive rates. All tools demonstrated reasonable correlations in quantifying precursors of HLA-bound peptides. Our benchmarking study suggests a combined strategy of applying at least two complementary DIA software tools to achieve the greatest degree of confidence and in-depth coverage of immunopeptidome data.

HLA-A*11:01-restricted CD8+ T cell immunity against influenza A and influenza B viruses in Indigenous and non-Indigenous people.

HLA-A*11:01 is one of the most prevalent human leukocyte antigens (HLAs), especially in East Asian and Oceanian populations. It is also highly expressed in Indigenous people who are at high risk of severe influenza disease. As CD8+ T cells can provide broadly cross-reactive immunity to distinct influenza strains and subtypes, including influenza A, B and C viruses, understanding CD8+ T cell immunity to influenza viruses across prominent HLA types is needed to rationally design a universal influenza vaccine and generate protective immunity especially for high-risk populations. As only a handful of HLA-A*11:01-restricted CD8+ T cell epitopes have been described for influenza A viruses (IAVs) and epitopes for influenza B viruses (IBVs) were still unknown, we embarked on an epitope discovery study to define a CD8+ T cell landscape for HLA-A*11:01-expressing Indigenous and non-Indigenous Australian people. Using mass-spectrometry, we identified IAV- and IBV-derived peptides presented by HLA-A*11:01 during infection. 79 IAV and 57 IBV peptides were subsequently screened for immunogenicity in vitro with peripheral blood mononuclear cells from HLA-A*11:01-expressing Indigenous and non-Indigenous Australian donors. CD8+ T cell immunogenicity screening revealed two immunogenic IAV epitopes (A11/PB2320-331 and A11/PB2323-331) and the first HLA-A*11:01-restricted IBV epitopes (A11/M41-49, A11/NS1186-195 and A11/NP511-520). The immunogenic IAV- and IBV-derived peptides were >90% conserved among their respective influenza viruses. Identification of novel immunogenic HLA-A*11:01-restricted CD8+ T cell epitopes has implications for understanding how CD8+ T cell immunity is generated towards IAVs and IBVs. These findings can inform the development of rationally designed, broadly cross-reactive influenza vaccines to ensure protection from severe influenza disease in HLA-A*11:01-expressing individuals.

Quantitative affinity measurement of small molecule ligand binding to major histocompatibility complex class-I-related protein 1 MR1.

The Major Histocompatibility Complex class I-related protein 1 (MR1) presents small molecule metabolites, drugs, and drug-like molecules that are recognized by MR1-reactive T cells. While we have an understanding of how antigens bind to MR1 and upregulate MR1 cell surface expression, a quantitative, cell-free, assessment of MR1 ligand-binding affinity was lacking. Here, we developed a fluorescence polarization-based assay in which fluorescent MR1 ligand was loaded into MR1 protein in vitro and competitively displaced by candidate ligands over a range of concentrations. Using this assay, ligand affinity for MR1 could be differentiated as strong (IC50 < 1 µM), moderate (1 µM < IC50 < 100 µM), and weak (IC50 > 100 µM). We demonstrated a clear correlation between ligand-binding affinity for MR1, the presence of a covalent bond between MR1 and ligand, and the number of salt bridge and hydrogen bonds formed between MR1 and ligand. Using this newly developed fluorescence polarization-based assay to screen for candidate ligands, we identified the dietary molecules vanillin and ethylvanillin as weak bona fide MR1 ligands. Both upregulated MR1 on the surface of C1R.MR1 cells and the crystal structure of a MAIT cell T cell receptor-MR1-ethylvanillin complex revealed that ethylvanillin formed a Schiff base with K43 of MR1 and was buried within the A'-pocket. Collectively, we developed and validated a method to quantitate the binding affinities of ligands for MR1 that will enable an efficient and rapid screening of candidate MR1 ligands.

Anthem: a user customised tool for fast and accurate prediction of binding between peptides and HLA class I molecules.

Neopeptide-based immunotherapy has been recognised as a promising approach for the treatment of cancers. For neopeptides to be recognised by CD8+ T cells and induce an immune response, their binding to human leukocyte antigen class I (HLA-I) molecules is a necessary first step. Most epitope prediction tools thus rely on the prediction of such binding. With the use of mass spectrometry, the scale of naturally presented HLA ligands that could be used to develop such predictors has been expanded. However, there are rarely efforts that focus on the integration of these experimental data with computational algorithms to efficiently develop up-to-date predictors. Here, we present Anthem for accurate HLA-I binding prediction. In particular, we have developed a user-friendly framework to support the development of customisable HLA-I binding prediction models to meet challenges associated with the rapidly increasing availability of large amounts of immunopeptidomic data. Our extensive evaluation, using both independent and experimental datasets shows that Anthem achieves an overall similar or higher area under curve value compared with other contemporary tools. It is anticipated that Anthem will provide a unique opportunity for the non-expert user to analyse and interpret their own in-house or publicly deposited datasets.

The allopurinol metabolite, oxypurinol, drives oligoclonal expansions of drug-reactive T cells in resolved hypersensitivity cases and drug-naïve healthy donors.

Allopurinol (ALP) is a successful drug used in the treatment of gout. However, this drug has been implicated in hypersensitivity reactions that can cause severe to life-threatening reactions such as Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). Individuals who carry the human leukocyte antigen (HLA)-B*58:01 allotype are at higher risk of experiencing a hypersensitivity reaction (odds ratios ranging from 5.62 to 580.3 for mild to severe reactions, respectively). In addition to the parent drug, the metabolite oxypurinol (OXP) is implicated in triggering T cell-mediated immunopathology via a labile interaction with HLA-B*58:01. To date, there has been limited information regarding the T-cell receptor (TCR) repertoire usage of reactive T cells in patients with ALP-induced SJS or TEN and, in particular, there are no reports examining paired αßTCRs. Here, using in vitro drug-treated PBMCs isolated from both resolved ALP-induced SJS/TEN cases and drug-naïve healthy donors, we show that OXP is the driver of CD8+ T cell-mediated responses and that drug-exposed memory T cells can exhibit a proinflammatory immunophenotype similar to T cells described during active disease. Furthermore, this response supported the pharmacological interaction with immune receptors (p-i) concept by showcasing (i) the labile metabolite interaction with peptide/HLA complexes, (ii) immunogenic complex formation at the cell surface, and (iii) lack of requirement for antigen processing to elicit drug-induced T cell responsiveness. Examination of paired OXP-induced αßTCR repertoires highlighted an oligoclonal and private clonotypic profile in both resolved ALP-induced SJS/TEN cases and drug-naïve healthy donors.

The molecular basis of how buried human leukocyte antigen polymorphism modulates natural killer cell function.

Micropolymorphisms within human leukocyte antigen (HLA) class I molecules can change the architecture of the peptide-binding cleft, leading to differences in peptide presentation and T cell recognition. The impact of such HLA variation on natural killer (NK) cell recognition remains unclear. Given the differential association of HLA-B*57:01 and HLA-B*57:03 with the control of HIV, recognition of these HLA-B57 allomorphs by the killer cell immunoglobulin-like receptor (KIR) 3DL1 was compared. Despite differing by only two polymorphic residues, both buried within the peptide-binding cleft, HLA-B*57:01 more potently inhibited NK cell activation. Direct-binding studies showed KIR3DL1 to preferentially recognize HLA-B*57:01, particularly when presenting peptides with positively charged position (P)Ω-2 residues. In HLA-B*57:01, charged PΩ-2 residues were oriented toward the peptide-binding cleft and away from KIR3DL1. In HLA-B*57:03, the charged PΩ-2 residues protruded out from the cleft and directly impacted KIR3DL1 engagement. Accordingly, KIR3DL1 recognition of HLA class I ligands is modulated by both the peptide sequence and conformation, as determined by the HLA polymorphic framework, providing a rationale for understanding differences in clinical associations.

A natural product compound inhibits coronaviral replication in vitro by binding to the conserved Nsp9 SARS-CoV-2 protein.

The Nsp9 replicase is a conserved coronaviral protein that acts as an essential accessory component of the multi-subunit viral replication/transcription complex. Nsp9 is the predominant substrate for the essential nucleotidylation activity of Nsp12. Compounds specifically interfering with this viral activity would facilitate its study. Using a native mass-spectrometry-based approach to screen a natural product library for Nsp9 binders, we identified an ent-kaurane natural product, oridonin, capable of binding to purified SARS-CoV-2 Nsp9 with micromolar affinities. By determining the crystal structure of the Nsp9-oridonin complex, we showed that oridonin binds through a conserved site near Nsp9's C-terminal GxxxG-helix. In enzymatic assays, oridonin's binding to Nsp9 reduces its potential to act as substrate for Nsp12's Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain. We also showed using in vitro cellular assays oridonin, while cytotoxic at higher doses has broad antiviral activity, reducing viral titer following infection with either SARS-CoV-2 or, to a lesser extent, MERS-CoV. Accordingly, these preliminary findings suggest that the oridonin molecular scaffold may have the potential to be developed into an antiviral compound to inhibit the function of Nsp9 during coronaviral replication.

Immunopeptidomic Analysis Reveals That Deamidated HLA-bound Peptides Arise Predominantly from Deglycosylated Precursors.

The presentation of post-translationally modified (PTM) peptides by cell surface HLA molecules has the potential to increase the diversity of targets for surveilling T cells. Although immunopeptidomics studies routinely identify thousands of HLA-bound peptides from cell lines and tissue samples, in-depth analyses of the proportion and nature of peptides bearing one or more PTMs remains challenging. Here we have analyzed HLA-bound peptides from a variety of allotypes and assessed the distribution of mass spectrometry-detected PTMs, finding deamidation of asparagine or glutamine to be highly prevalent. Given that asparagine deamidation may arise either spontaneously or through enzymatic reaction, we assessed allele-specific and global motifs flanking the modified residues. Notably, we found that the N-linked glycosylation motif NX(S/T) was highly abundant across asparagine-deamidated HLA-bound peptides. This finding, demonstrated previously for a handful of deamidated T cell epitopes, implicates a more global role for the retrograde transport of nascently N-glycosylated polypeptides from the ER and their subsequent degradation within the cytosol to form HLA-ligand precursors. Chemical inhibition of Peptide:N-Glycanase (PNGase), the endoglycosidase responsible for the removal of glycans from misfolded and retrotranslocated glycoproteins, greatly reduced presentation of this subset of deamidated HLA-bound peptides. Importantly, there was no impact of PNGase inhibition on peptides not containing a consensus NX(S/T) motif. This indicates that a large proportion of HLA-I bound asparagine deamidated peptides are generated from formerly glycosylated proteins that have undergone deglycosylation via the ER-associated protein degradation (ERAD) pathway. The information herein will help train deamidation prediction models for HLA-peptide repertoires and aid in the design of novel T cell therapeutic targets derived from glycoprotein antigens.

Resourcing, annotating, and analysing synthetic peptides of SARS-CoV-2 for immunopeptidomics and other immunological studies.

SARS-CoV-2 has caused a significant ongoing pandemic worldwide. A number of studies have examined the T cell mediated immune responses against SARS-CoV-2, identifying potential T cell epitopes derived from the SARS-CoV-2 proteome. Such studies will aid in identifying targets for vaccination and immune monitoring. In this study, we applied tandem mass spectrometry and proteomic techniques to a library of ∼40,000 synthetic peptides, in order to generate a large dataset of SARS-CoV-2 derived peptide MS/MS spectra. On this basis, we built an online knowledgebase, termed virusMS (https://virusms.erc.monash.edu/), to document, annotate and analyse these synthetic peptides and their spectral information. VirusMS incorporates a user-friendly interface to facilitate searching, browsing and downloading the database content. Detailed annotations of the peptides, including experimental information, peptide modifications, predicted peptide-HLA (human leukocyte antigen) binding affinities, and peptide MS/MS spectral data, are provided in virusMS.

The complexity of T cell-mediated penicillin hypersensitivity reactions.

Penicillin refers to a group of beta-lactam antibiotics that are the first-line treatment for a range of infections. However, they also possess the ability to form novel antigens, or neoantigens, through haptenation of proteins and can stimulate a range of immune-mediated adverse reactions-collectively known as drug hypersensitivity reactions (DHRs). IgE-mediated reactions towards these neoantigens are well studied; however, IgE-independent reactions are less well understood. These reactions usually manifest in a delayed manner as different forms of cutaneous eruptions or liver injury consistent with priming of an immune response. Ex vivo studies have confirmed the infiltration of T cells into the site of inflammation, and the subsets of T cells involved appear dependent on the nature of the reaction. Here, we review the evidence that has led to our current understanding of these immune-mediated reactions, discussing the nature of the lesional T cells, the characterization of drug-responsive T cells isolated from patient blood, and the potential mechanisms by which penicillins enter the antigen processing and presentation pathway to stimulate these deleterious responses. Thus, we highlight the need for a more comprehensive understanding of the underlying genetic and molecular basis of penicillin-induced DHRs.

Modification of the cyclopropyl moiety of abacavir provides insight into the structure activity relationship between HLA-B*57:01 binding and T-cell activation.

BACKGROUND: Abacavir is associated with hypersensitivity reactions in individuals positive for the HLA-B*57:01 allele. The drug binds within the peptide binding groove of HLA-B*57:01 altering peptides displayed on the cell surface. Presentation of these HLA-abacavir-peptide complexes to T-cells is hypothesized to trigger a CD8+ T-cell response underpinning the hypersensitivity. Thus, the aim of this study was to explore the relationship between the structure of abacavir with HLA-B*57:01 binding and the CD8+ T-cell activation. METHODS: Seventeen abacavir analogues were synthesized and cytokine secretion from abacavir/abacavir analogue-responsive CD8+ T-cell clones was measured using IFN-γ ELIspot. In silico docking studies were undertaken to assess the predicted binding poses of the abacavir analogues within the HLA-B*57:01 peptide binding groove. In parallel, the effect of selected abacavir analogues on the repertoire of self-peptides presented by cellular HLA-B*57:01 was characterized using mass spectrometry. RESULTS: Abacavir and ten analogues stimulated CD8+ T-cell IFN-γ release. Molecular docking of analogues that retained antiviral activity demonstrated a relationship between predicted HLA-B*57:01 binding orientations and the ability to induce a T-cell response. Analogues that stimulated T-cells displayed a perturbation of the natural peptides displayed by HLA-B*57:01. The antigen-specific CD8+ T-cell response was dependent on the enantiomeric form of abacavir at both cyclopropyl and cyclopentyl regions. CONCLUSION: Alteration of the chemical constitution of abacavir generates analogues that retain a degree of pharmacological activity, but have variable ability to activate T-cells. Modelling and immunopeptidome analysis delineate how drug HLA-B*57:01 binding and peptide display by antigen presenting cells relate to the activation of CD8+ T-cells.

Identification of Native and Posttranslationally Modified HLA-B*57:01-Restricted HIV Envelope Derived Epitopes Using Immunoproteomics.

The recognition of pathogen-derived peptides by T lymphocytes is the cornerstone of adaptive immunity, whereby intracellular antigens are degraded in the cytosol and short peptides assemble with class I human leukocyte antigen (HLA) molecules in the ER. These peptide-HLA complexes egress to the cell surface and are scrutinized by cytotoxic CD8+ T-cells leading to the eradication of the infected cell. Here, naturally presented HLA-B*57:01 bound peptides derived from the envelope protein of the human immunodeficiency virus (HIVenv) are identified. HIVenv peptides are present at a very small percentage of the overall HLA-B*57:01 peptidome (<0.1%) and both native and posttranslationally modified forms of two distinct HIV peptides are identified. Notably, a peptide bearing a natively encoded C-terminal tryptophan residue is also present in a modified form containing a kynurenine residue. Kynurenine is a major product of tryptophan catabolism and is abundant during inflammation and infection. Binding of these peptides at a molecular level and their immunogenicity in preliminary functional studies are examined. Modest immune responses are observed to the modified HIVenv peptide, highlighting a potential role for kynurenine-modified peptides in the immune response to HIV and other viral infections.

The molecular basis for peptide repertoire selection in the human leucocyte antigen (HLA) C*06:02 molecule.

Human leukocyte antigen (HLA)-C*06:02 is identified as the allele associated with the highest risk for the development of the autoimmune skin disease psoriasis. However, the diversity and mode of peptide presentation by the HLA-C*06:02 molecule remains unclear. Here, we describe the endogenous peptide repertoire of ∼3,000 sequences for HLA-C*06:02 that defines the peptide-binding motif for this HLA allomorph. We found that HLA-C*06:02 predominantly presents nonamer peptides with dominant arginine anchors at the P2 and P7 positions and a preference for small hydrophobic residues at the C terminus (PΩ). To determine the structural basis of this selectivity, we determined crystal structures of HLA-C*06:02 in complex with two self-peptides (ARTELYRSL and ARFNDLRFV) and an analogue of a melanocyte autoantigen (ADAMTSL5, VRSRR-abu-LRL) implicated in psoriasis. These structures revealed that HLA-C*06:02 possesses a deep peptide-binding groove comprising two electronegative B- and E-pockets that coincide with the preference for P2 and P7 arginine anchors. The ADAMTSL5 autoantigen possessed a P7-Leu instead of the P7-Arg residue, but nevertheless was accommodated within the HLA-C*06:02 antigen-binding cleft. Collectively, our results provide the structural basis for understanding peptide repertoire selection in HLA-C*06:02.

Employing proteomics in the study of antigen presentation: an update.

INTRODUCTION: Our immune system discriminates self from non-self by examining the peptide cargo of human leukocyte antigen (HLA) molecules displayed on the cell surface. Successful recognition of HLA-bound non-self peptides can induce T cell responses leading to, for example, the destruction of infected cells. Today, largely due to advances in technology, we have an unprecedented capability to identify the nature of these presented peptides and unravel the true complexity of antigen presentation. Areas covered: In addition to conventional linear peptides, HLA molecules also present post-translationally modified sequences comprising a wealth of chemical and structural modifications, including a novel class of noncontiguous spliced peptides. This review focuses on these emerging themes in antigen presentation and how mass spectrometry in particular has contributed to a new view of the antigenic landscape that is presented to the immune system. Expert Commentary: Advances in the sensitivity of mass spectrometers and use of hybrid fragmentation technologies will provide more information-rich spectra of HLA bound peptides leading to more definitive identification of T cell epitopes. Coupled with improvements in sample preparation and new informatics workflows, studies will access novel classes of peptide antigen and allow interrogation of rare and clinically relevant samples.

Immune self-reactivity triggered by drug-modified HLA-peptide repertoire.

Human leukocyte antigens (HLAs) are highly polymorphic proteins that initiate immunity by presenting pathogen-derived peptides to T cells. HLA polymorphisms mostly map to the antigen-binding cleft, thereby diversifying the repertoire of self-derived and pathogen-derived peptide antigens selected by different HLA allotypes. A growing number of immunologically based drug reactions, including abacavir hypersensitivity syndrome (AHS) and carbamazepine-induced Stevens-Johnson syndrome (SJS), are associated with specific HLA alleles. However, little is known about the underlying mechanisms of these associations, including AHS, a prototypical HLA-associated drug reaction occurring exclusively in individuals with the common histocompatibility allele HLA-B*57:01, and with a relative risk of more than 1,000 (refs 6, 7). We show that unmodified abacavir binds non-covalently to HLA-B*57:01, lying across the bottom of the antigen-binding cleft and reaching into the F-pocket, where a carboxy-terminal tryptophan typically anchors peptides bound to HLA-B*57:01. Abacavir binds with exquisite specificity to HLA-B*57:01, changing the shape and chemistry of the antigen-binding cleft, thereby altering the repertoire of endogenous peptides that can bind HLA-B*57:01. In this way, abacavir guides the selection of new endogenous peptides, inducing a marked alteration in 'immunological self'. The resultant peptide-centric 'altered self' activates abacavir-specific T-cells, thereby driving polyclonal CD8 T-cell activation and a systemic reaction manifesting as AHS. We also show that carbamazepine, a widely used anti-epileptic drug associated with hypersensitivity reactions in HLA-B*15:02 individuals, binds to this allotype, producing alterations in the repertoire of presented self peptides. Our findings simultaneously highlight the importance of HLA polymorphism in the evolution of pharmacogenomics and provide a general mechanism for some of the growing number of HLA-linked hypersensitivities that involve small-molecule drugs.

The role of HLA genes in pharmacogenomics: unravelling HLA associated adverse drug reactions.

Genetic polymorphism in the genes encoding the human leukocyte antigen (HLA) molecules enables presentation of a wide range peptide ligands thus maximising immune surveillance of pathogens. A consequence of the diversification of the HLA Ag-binding pocket is the enhanced opportunity for off-target binding of small drugs by HLA molecules, with subsequent immune reactivity. These potential off-target interactions are 'set up' to generate T cell-mediated adverse drug reactions even though the precise mechanisms of most HLA-drug interactions are still poorly understood. The association between abacavir hypersensitivity syndrome and HLA-B*57:01 is one exception that has been resolved at a molecular and mechanistic level. Here, we explore the road to understanding the interaction between abacavir and the HLA-B*57:01 molecule and review the current state of understanding of interactions between other drugs and HLA molecules implicated in adverse drug reactions, which appear to involve multiple mechanisms. The continued expansion of the pharmacopoeia generates an imperative to understand these interactions at the molecular level in order to prevent the continued burden on individuals and the health care system.

The cellular redox environment alters antigen presentation.

Cysteine-containing peptides represent an important class of T cell epitopes, yet their prevalence remains underestimated. We have established and interrogated a database of around 70,000 naturally processed MHC-bound peptides and demonstrate that cysteine-containing peptides are presented on the surface of cells in an MHC allomorph-dependent manner and comprise on average 5-10% of the immunopeptidome. A significant proportion of these peptides are oxidatively modified, most commonly through covalent linkage with the antioxidant glutathione. Unlike some of the previously reported cysteine-based modifications, this represents a true physiological alteration of cysteine residues. Furthermore, our results suggest that alterations in the cellular redox state induced by viral infection are communicated to the immune system through the presentation of S-glutathionylated viral peptides, resulting in altered T cell recognition. Our data provide a structural basis for how the glutathione modification alters recognition by virus-specific T cells. Collectively, these results suggest that oxidative stress represents a mechanism for modulating the virus-specific T cell response.

CD8+ T-cell responses towards conserved influenza B virus epitopes across anatomical sites and age.

Influenza B viruses (IBVs) cause substantive morbidity and mortality, and yet immunity towards IBVs remains understudied. CD8+ T-cells provide broadly cross-reactive immunity and alleviate disease severity by recognizing conserved epitopes. Despite the IBV burden, only 18 IBV-specific T-cell epitopes restricted by 5 HLAs have been identified currently. A broader array of conserved IBV T-cell epitopes is needed to develop effective cross-reactive T-cell based IBV vaccines. Here we identify 9 highly conserved IBV CD8+ T-cell epitopes restricted to HLA-B*07:02, HLA-B*08:01 and HLA-B*35:01. Memory IBV-specific tetramer+CD8+ T-cells are present within blood and tissues. Frequencies of IBV-specific CD8+ T-cells decline with age, but maintain a central memory phenotype. HLA-B*07:02 and HLA-B*08:01-restricted NP30-38 epitope-specific T-cells have distinct T-cell receptor repertoires. We provide structural basis for the IBV HLA-B*07:02-restricted NS1196-206 (11-mer) and HLA-B*07:02-restricted NP30-38 epitope presentation. Our study increases the number of IBV CD8+ T-cell epitopes, and defines IBV-specific CD8+ T-cells at cellular and molecular levels, across tissues and age.