Structural definition of HLA class II-presented SARS-CoV-2 epitopes reveals a mechanism to escape pre-existing CD4+ T cell immunity.

CD4+ T cells recognize a broad range of peptide epitopes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which contribute to immune memory and limit COVID-19 disease. We demonstrate that the immunogenicity of SARS-CoV-2 peptides, in the context of the model allotype HLA-DR1, does not correlate with their binding affinity to the HLA heterodimer. Analyzing six epitopes, some with very low binding affinity, we solve X-ray crystallographic structures of each bound to HLA-DR1. Further structural definitions reveal the precise molecular impact of viral variant mutations on epitope presentation. Omicron escaped ancestral SARS-CoV-2 immunity to two epitopes through two distinct mechanisms: (1) mutations to TCR-facing epitope positions and (2) a mechanism whereby a single amino acid substitution caused a register shift within the HLA binding groove, completely altering the peptide-HLA structure. This HLA-II-specific paradigm of immune escape highlights how CD4+ T cell memory is finely poised at the level of peptide-HLA-II presentation.

Seven mysteries of LAG-3: a multi-faceted immune receptor of increasing complexity.

Despite three decades of research to its name and increasing interest in immunotherapies that target it, LAG-3 remains an elusive co-inhibitory receptor in comparison to the well-established PD-1 and CTLA-4. As such, LAG-3 targeting therapies have yet to achieve the clinical success of therapies targeting other checkpoints. This could, in part, be attributed to the many unanswered questions that remain regarding LAG-3 biology. Of these, we address: (i) the function of the many LAG-3-ligand interactions, (ii) the hurdles that remain to acquire a high-resolution structure of LAG-3, (iii) the under-studied LAG-3 signal transduction mechanism, (iv) the elusive soluble form of LAG-3, (v) the implications of the lack of (significant) phenotype of LAG-3 knockout mice, (vi) the reports of LAG-3 expression on the epithelium, and (vii) the conflicting reports of LAG-3 expression (and potential contributions to pathology) in the brain. These mysteries which surround LAG-3 highlight how the ever-evolving study of its biology continues to reveal ever-increasing complexity in its role as an immune receptor. Importantly, answering the questions which shroud LAG-3 in mystery will allow the maximum therapeutic benefit of LAG-3 targeting immunotherapies in cancer, autoimmunity and beyond.

Pouring petrol on the flames: Using oncolytic virotherapies to enhance tumour immunogenicity.

Oncolytic viruses possess the ability to infect, replicate and lyse malignantly transformed tumour cells. This oncolytic activity amplifies the therapeutic advantage and induces a form of immunogenic cell death, characterized by increased CD8 + T-cell infiltration into the tumour microenvironment. This important feature of oncolytic viruses can result in the warming up of immunologically 'cold' tumour types, presenting the enticing possibility that oncolytic virus treatment combined with immunotherapies may enhance efficacy. In this review, we assess some of the most promising candidates that might be used for oncolytic virotherapy: immunotherapy combinations. We assess their potential as separate agents or as agents combined into a single therapy, where the immunotherapy is encoded within the genome of the oncolytic virus. The development of such advanced agents will require increasingly sophisticated model systems for their preclinical assessment and evaluation. In vivo rodent model systems are fraught with limitations in this regard. Oncolytic viruses replicate selectively within human cells and therefore require human xenografts in immune-deficient mice for their evaluation. However, the use of immune-deficient rodent models hinders the ability to study immune responses against any immunomodulatory transgenes engineered within the viral genome and expressed within the tumour microenvironment. There has therefore been a shift towards the use of more sophisticated ex vivo patient-derived model systems based on organoids and explant co-cultures with immune cells, which may be more predictive of efficacy than contrived and artificial animal models. We review the best of those model systems here.

Molecular characterization of HLA class II binding to the LAG-3 T cell co-inhibitory receptor.

Immune checkpoint inhibitors (antibodies that block the T cell co-inhibitory receptors PD-1/PD-L1 or CTLA-4) have revolutionized the treatment of some forms of cancer. Importantly, combination approaches using drugs that target both pathways have been shown to boost the efficacy of such treatments. Subsequently, several other T cell inhibitory receptors have been identified for the development of novel immune checkpoint inhibitors. Included in this list is the co-inhibitory receptor lymphocyte activation gene-3 (LAG-3), which is upregulated on T cells extracted from tumor sites that have suppressive or exhausted phenotypes. However, the molecular rules that govern the function of LAG-3 are still not understood. Using surface plasmon resonance combined with a novel bead-based assay (AlphaScreenTM ), we demonstrate that LAG-3 can directly and specifically interact with intact human leukocyte antigen class II (HLA-II) heterodimers. Unlike the homologue CD4, which has an immeasurably weak affinity using these biophysical approaches, LAG-3 binds with low micromolar affinity. We further validated the interaction at the cell surface by staining LAG-3+ cells with pHLA-II-multimers. These data provide new insights into the mechanism by which LAG-3 initiates T cell inhibition.

CD4+ T Cells Recognize Conserved Influenza A Epitopes through Shared Patterns of V-Gene Usage and Complementary Biochemical Features.

T cell recognition of peptides presented by human leukocyte antigens (HLAs) is mediated by the highly variable T cell receptor (TCR). Despite this built-in TCR variability, individuals can mount immune responses against viral epitopes by using identical or highly related TCRs expressed on CD8+ T cells. Characterization of these TCRs has extended our understanding of the molecular mechanisms that govern the recognition of peptide-HLA. However, few examples exist for CD4+ T cells. Here, we investigate CD4+ T cell responses to the internal proteins of the influenza A virus that correlate with protective immunity. We identify five internal epitopes that are commonly recognized by CD4+ T cells in five HLA-DR1+ subjects and show conservation across viral strains and zoonotic reservoirs. TCR repertoire analysis demonstrates several shared gene usage biases underpinned by complementary biochemical features evident in a structural comparison. These epitopes are attractive targets for vaccination and other T cell therapies.

Human leukocyte antigen (HLA) class II peptide flanking residues tune the immunogenicity of a human tumor-derived epitope.

CD4+ T-cells recognize peptide antigens, in the context of human leukocyte antigen (HLA) class II molecules (HLA-II), which through peptide-flanking residues (PFRs) can extend beyond the limits of the HLA binding. The role of the PFRs during antigen recognition is not fully understood; however, recent studies have indicated that these regions can influence T-cell receptor (TCR) affinity and pHLA-II stability. Here, using various biochemical approaches including peptide sensitivity ELISA and ELISpot assays, peptide-binding assays and HLA-II tetramer staining, we focused on CD4+ T-cell responses against a tumor antigen, 5T4 oncofetal trophoblast glycoprotein (5T4), which have been associated with improved control of colorectal cancer. Despite their weak TCR-binding affinity, we found that anti-5T4 CD4+ T-cells are polyfunctional and that their PFRs are essential for TCR recognition of the core bound nonamer. The high-resolution (1.95 Å) crystal structure of HLA-DR1 presenting the immunodominant 20-mer peptide 5T4111-130, combined with molecular dynamic simulations, revealed how PFRs explore the HLA-proximal space to contribute to antigen reactivity. These findings advance our understanding of what constitutes an HLA-II epitope and indicate that PFRs can tune weak affinity TCR-pHLA-II interactions.

Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens.

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies.