Biology subjects

Ussher, J. E.

Publications and source records attributed to Ussher, J. E..

8 recordsLinked to original sources

bioRxiv2026

Engineering antigenic breadth against SARS-CoV-2 by pairing divergent RBDs within a single mRNA immunogen

Vaccines capable of eliciting broadly neutralising antibodies (bnAbs) are a major goal for pandemic preparedness. A persistent challenge across vaccine Wields is how to deliberately recruit the rare B cell clones that recognise conserved epitopes shared across diverse viral variants. BnAbs have been known to frequently emerge through extensive somatic hypermutation during afWinity maturation, here we describe an alternative, structure-driven mechanism for bnAb selection. We designed an mRNA vaccine in which two antigenically distinct SARS-CoV-2 variants (Omicron and Delta; O-{Delta}) receptor binding domains (RBDs) are physically fused on a single polypeptide. This design is predicted to favour B cell antigen receptors capable of engaging conserved epitopes on both RBDs with enhanced avidity. A matched non-divergent tandem RBD (Delta-Delta; {Delta}-{Delta}) served as a control. The divergent (O-{Delta}) immunogen was robustly expressed and retained high-afWinity ACE2 binding. In mice, immunisation elicited potent antibody responses and increased the frequency of antigen-speciWic cross-reactive B cells, recognising Delta, Omicron, and the 2002 pandemic strain SARS-CoV RBDs. Using multicolour RBD tetramers and single-cell B cell receptor sequencing, we show that breadth arises via two distinct pathways. The divergent vaccine preferentially enriches clonally distinct cross-reactive B cells (not present within non-cross-reactive B cell pools) with low levels of somatic hypermutation (SHM), consistent with selection of germline-biased precursors. In contrast, the matched control vaccine yields cross-reactivity primarily within existing clonal lineages (clonal overlap between cross-reactive and non-cross-reactive cells) and at higher mutational burdens, consistent with afWinity-maturation-driven acquisition of breadth. Together, these Windings demonstrate that antigen structure can bias B cell selection towards cross-reactive speciWicities without requiring extensive SHM. This work establishes a simple, modular antigen-design principle in which juxtaposing appropriately divergent antigens on a single scaffold promotes the enrichment of bnAb-prone B cells, providing a scalable strategy for vaccine development against rapidly evolving pathogens.

immunology↗

bioRxiv2025

4-1BB expression and signaling regulates MAIT cell activation and effector functions

Mucosal associated invariant T (MAIT) cells are abundant unconventional T cells that recognize microbe-derived riboflavin metabolites presented by MR1. Upon recognition, they become activated, produce proinflammatory cytokines, chemokines and cytotoxic molecules. MAIT cells are also activated by cytokines independently of T cell receptor (TCR) engagement, however these two signals can also act in concert to finetune MAIT cell functions. Additionally, multiple other co-stimulatory signals have also been reported that can boost MAIT cell effector responses to TCR or cytokine stimulation. However, a comprehensive study exploring the role of surface bound TNF receptor superfamily (TNFRSF) molecule 4-1BB, well known for its co-stimulatory function during conventional T cell activation, is lacking in the context of MAIT cells. In this study, we show that 4-1BB is the earliest and most highly expressed TNFRSF co-stimulatory molecule on MAIT cells upon activation by Escherichia coli, with expression seen as early as six hours where it was predominantly MR1 mediated. 4-1BB expression on MAIT cells following late activation was due to both TCR signaling and cytokine signaling. We found marked differences in MAIT cell activation and cytokine expression between 4-1BB+ and 4-1BB- MAIT cells suggesting 4-1BB expression on MAIT cells is associated with functional superiority. By blocking 4-1BB signaling or co-culturing with a 4-1BBL overexpressing cell line we demonstrated an important role of co-stimulation via 4-1BB in MAIT cells during activation. Expression and signaling via 4-1BB also enhanced T-bet and Blimp1 expression. In summary, our study confirms a role for 4-1BB signaling during MAIT cell activation.

immunology↗

bioRxiv2025

IL-21 selectively augments cytotoxic potential of antigen-activated MAIT cells

Mucosal-associated invariant T (MAIT) cells are unconventional cytotoxic T cells restricted by MHC class 1 related molecule, MR1. They are activated through their TCR by derivatives from microbial riboflavin synthesis or independently of TCR signalling via IL-12 and IL-18. Upon activation, MAIT cells upregulate cytotoxic molecules GrzB and perforin and lyse bacterially-infected cells. While cytokines act as co-stimulatory molecules that enhance MAIT cell activation, their specific role in regulating MAIT cell cytotoxicity remains unresolved. We show that the cytokine IL-21 enhances expression of GrzB and perforin on TCR or IL-12/IL-18-activated MAIT cells but has limited effect on MAIT cell cytokine production. Using a flow cytometry-based cytotoxic assay, we show priming MAIT cells with IL-21 enhances their ability to kill 5-OP-RU-treated B cell lines. We demonstrate a previously unexplored co-stimulatory role for IL-21 that selectively augments the cytotoxic potential of MAIT cells.

immunology↗

bioRxiv2022

MR1 dependent MAIT cell activation is regulated by autophagy associated proteins

The antigen presenting molecule, MR1, presents microbial metabolites to MAIT cells, a population of innate-like, anti-microbial T cells. It also presents an unidentified ligand to MR-1 restricted T cells in the setting of cancer. The cellular co-factors that regulate MR1 antigen presentation have yet to be fully defined. We performed a mass spectrometry-based proteomics screen to identify MR1 interacting proteins and discovered the selective autophagy receptor SQSTM1/p62. CRISPR-Cas9-mediated knock out of SQSTM1/p62 increased MAIT cell activation in the presence of E.coli but not the synthetic ligand 5-OP-RU whereas depletion of Atg5 and Atg7, key autophagy proteins, increased MAIT activation irrespective of the ligand used. This regulation appears to occur at an early step in the MR1 trafficking pathway. This data implicates distinct roles for autophagy associated proteins in the regulation of MR1 activity and highlights the contribution of the autophagy pathway to regulation of cellular antigen presentation.

cell biology↗

bioRxiv2019

Neutrophils Suppress Mucosal-Associated Invariant T Cells

Mucosal-associated invariant T (MAIT) cells are innate-like T lymphocytes that are abundant in mucosal tissues and the liver where they can respond rapidly to a broad range of riboflavin producing bacterial and fungal pathogens. Neutrophils, which are recruited early to sites of infection, play a non-redundant role in pathogen clearance and are crucial for controlling infection. The interaction of these two cell types is poorly studied. Here, we investigated both the effect of neutrophils on MAIT cell activation and the effect of activated MAIT cells on neutrophils. We show that neutrophils suppress the activation of MAIT cells by a cell-contact and H2O2 dependent mechanism. Moreover, highly activated MAIT cells were able to produce high levels of TNF that induced neutrophil death. We therefore provide evidence for a negative regulatory feedback mechanism in which neutrophils prevent over-activation of MAIT cells and, in turn, MAIT cells limit neutrophil survival.

immunology↗

bioRxiv2019

Type I interferons are important co-stimulatory signals during T cell receptor mediated MAIT cell activation

Mucosal associated invariant T (MAIT) cells are abundant unconventional T cells which can be stimulated either via their T cell receptor (TCR) or by innate cytokines. The MAIT cell TCR recognises a pyrimidine ligand, derived from riboflavin synthesising bacteria, bound to MR1. In infection, bacteria not only provide the pyrimidine ligand but also co-stimulatory signals, such as Toll-like receptor agonists, that can modulate TCR-mediated activation. Recently, type I interferons (T1-IFNs) have been identified as contributing to cytokine-mediated MAIT cell activation. However, it is unknown whether T1-IFNs also have a role during TCR-mediated MAIT cell activation. In this study, we investigated the co-stimulatory role of T1-IFNs during TCR-mediated activation of MAIT cells by the MR1 ligand 5-amino-6-D-ribitylaminouracil/methylglyoxal (5-A-RU/MG). We found that T1-IFNs were able to boost interferon-{gamma} and granzyme B production in 5-A-RU/MG-stimulated MAIT cells. Similarly, influenza virus-induced T1-IFNs enhanced TCR-mediated MAIT cell activation. An essential role of T1-IFNs in regulating MAIT cell activation by riboflavin synthesising bacteria was also demonstrated. The co-stimulatory role of T1-IFNs was confirmed using liver-derived MAIT cells. T1-IFNs acted directly on MAIT cells to enhance their response to TCR stimulation. Overall, our findings establish an important immunomodulatory role of T1-IFNs during TCR-mediated MAIT cell activation.

immunology↗

bioRxiv2019

Transcriptional analysis defines TCR and cytokine-stimulated MAIT cells as rapid polyfunctional effector T cells that can coordinate the immune response

MAIT cells are an abundant innate-like T cell population which can be activated via either their T cell receptor (TCR), which recognizes MR1-bound pyrimidine antigens derived from microbial riboflavin biosynthesis, or via cytokines, such as IL-12 and IL-18. In vivo, these two modes of activation may act in concert or independently depending upon the nature of the microbial or inflammatory stimuli. It is unknown, however, how the MAIT cell response differs to the different modes of activation. Here, we define the transcriptional and effector responses of human MAIT cells to TCR and cytokine stimulation. We report that MAIT cells rapidly respond to TCR stimulation through the production of multiple effector cytokines and chemokines, alteration of their cytotoxic granule content and transcription factor expression, and upregulation of co-stimulatory proteins CD40L and 4-1BB. In contrast, cytokine-mediated activation is slower and results in more limited production of cytokines, chemokines, and co-stimulatory proteins; differences in granule content and transcription factor expression are also evident. Therefore, we propose that in infections by riboflavin-synthesizing bacteria, MAIT cells play a key early role in effecting and coordinating the immune response, while in the absence of TCR stimulation (e.g. viral infection) their role is likely to differ.

immunology↗

bioRxiv2018

Role of Innate-like Lymphocytes in the Pathogenesis of Community Acquired Pneumonia

BackgroundMucosal-associated invariant T (MAIT) cells and V{delta}2+ {gamma}{delta} T cells are anti-bacterial innate-like lymphocytes (ILLs) that are enriched in blood and mucosa. ILLs have been implicated in control of bacterial infection. However, the role of ILLs in community-acquired pneumonia (CAP) is unknown.\n\nMethodsUsing sputum samples from a well-characterised CAP cohort, MAIT cell (V7.2-J12/20/33) and V{delta}2+ T cell (V{delta}2-J{delta}1/2/3/4) abundance was determined by quantitative PCR. Cytokine and chemokine concentrations in sputum were measured. The capacity of bacteria in sputum to produce activating ligands for MAIT cells and V{delta}2+ T cells was inferred by 16S rRNA sequencing.\n\nResultsMAIT cell abundance in sputum was higher in patients with less severe pneumonia; duration of hospital admission was inversely correlated with both MAIT and V{delta}2+ T cell abundance. The abundance of both ILLs was higher in patients with a confirmed bacterial aetiology, however there was no correlation with total bacterial load or the predicted capacity of bacteria to produce activating ligands. Sputum MAIT cell abundance was associated with interferon- , and interferon-{gamma}, and sputum neutrophil abundance, while V{delta}2+ T cell abundance was associated with CXCL11 and interferon-{gamma}.\n\nConclusionsPulmonary MAIT and V{delta}2+ T cells can be detected in sputum in CAP, where they may contribute to improved clinical outcome.

immunology↗