Iron and reactive oxygen in wheat-pathogen interactions

Iron is an essential component of various proteins and pigments for both plants and pathogenic fungi. However, redox cycling between the ferric and ferrous forms of iron can also catalyse the production of dangerous free radicals and iron homeostasis is therefore tightly regulated. During pathogen attack, plants quickly produce large amounts of reactive oxygen species at the site of attempted pathogen ingress. This so-called oxidative burst has received considerable attention, but no single enzyme has been shown to account for the phenomenon. Using inductively coupled plasma mass spectrometry and histochemistry, I show that iron is secreted to the apoplast of the diploid wheat Triticum monococcum during attack by the powdery mildew fungus Blumeria graminis f.sp. tritici. This iron accumulates at cell wall appositions synthesised de novo beneath sites of pathogen attack. I further show, using histochemistry and pharmaceutical inhibitors, that this apoplastic iron accumulation is required for production of H2O2 in the oxidative burst. To understand the impact of this massive change in iron homeostasis on gene transcription, I employ a 187 gene targeted macroarray platform and establish that iron overload induces the expression of iron homeostasis-related genes and defence-related genes through iron itself and iron-mediated H2O2 production, respectively. To illustrate how the plant is able to withstand the negative effects of its own oxidative defences, I characterise a novel quinone redox cycle, and show that simultaneous induction of a protective quinone reductase isoform and downregulation of reactive oxygen-producing quinone reductase isoform prevents the spread of reactive oxygen during pathogen attack. Finally, in an effort to understand the impact of iron on fungal pathogenicity, I investigate iron uptake in the head blight pathogen, Fusarium graminearum. Fungi use at least two separate systems to take up iron, one based on enzymatic iron reduction and the other based on the synthesis and secretion of small iron chelators termed siderophores. Using mutants disrupted in either of two modes of iron uptake, I establish that siderophore production is essential for full F. graminearum virulence on wheat. This thesis exposes iron as an important component of both plant defence and fungal virulence

Phased electromagnetic acoustic transducer array for Rayleigh wave surface defect detection

A phased electromagnetic acoustic transducer (EMAT) array system has been developed for detection and characterisation of surface breaking defects. An array of four linear coils which are individually controlled are used to generate a Rayleigh wave. The high current electronics combined with the coil designs enables the array to generate either narrowband or broadband signals, and controlling the phase delay between the channels makes it possible to change the ultrasound wavelength without requiring the physical separation of the coils to be changed. Experimental results show that the four-coil phased array is able to generate a wavelength range from 3.0 mm to 11.7 mm. Surface breaking defects were characterised using a transmit-receive set-up with a broadband EMAT detector being used to detect the Rayleigh wave. Machined surface slots with different depths were used for technique validation. The results show that the array is sensitive to surface defects and that a wide depth sensitivity range for defect sizing can be easily achieved by applying phasing to tune the wavelength of operation. A large increase in detection flexibility is immediately shown

High power phased EMAT arrays for nondestructive testing of as-cast steel

A new high-power electromagnetic acoustic transducer (EMAT) solid state pulser system has been developed that is capable of driving up to 4 EMAT coils with programmable phase delays, allowing for focusing and steering of the acoustic field. Each channel is capable of supplying an excitation current of up to 1.75 kA for a pulse with a rise time of 1 μs. Finite element and experimental data are presented which demonstrate a signal enhancement by a factor of 3.5 (compared to a single EMAT coil) when using the system to transmit a longitudinal ultrasound pulse through a 22.5 cm thick as-cast steel slab sample. Further signal enhancement is demonstrated through the use of an array of detection EMATs, and a demonstration of artificial internal defect detection is presented on a thick steel sample. The design of this system is such that it has the potential to be employed at elevated temperatures for diagnostic measurements of steel during the continuous casting process

Diversity within the adenovirus fiber knob hypervariable loops influences primary receptor interactions

Adenovirus based vectors are of increasing importance for wide ranging therapeutic applications. As vaccines, vectors derived from human adenovirus species D serotypes 26 and 48 (HAdV-D26/48) are demonstrating promising efficacy as protective platforms against infectious diseases. Significant clinical progress has been made, yet definitive studies underpinning mechanisms of entry, infection, and receptor usage are currently lacking. Here, we perform structural and biological analysis of the receptor binding fiber-knob protein of HAdV-D26/48, reporting crystal structures, and modelling putative interactions with two previously suggested attachment receptors, CD46 and Coxsackie and Adenovirus Receptor (CAR). We provide evidence of a low affinity interaction with CAR, with modelling suggesting affinity is attenuated through extended, semi-flexible loop structures, providing steric hindrance. Conversely, in silico and in vitro experiments are unable to provide evidence of interaction between HAdV-D26/48 fiber-knob with CD46, or with Desmoglein 2. Our findings provide insight into the cell-virus interactions of HAdV-D26/48, with important implications for the design and engineering of optimised Ad-based therapeutics

T-cell libraries allow simple parallel generation of multiple peptide-specific human T-cell clones

Isolation of peptide-specific T-cell clones is highly desirable for determining the role of T-cells in human disease, as well as for the development of therapies and diagnostics. However, generation of monoclonal T-cells with the required specificity is challenging and time-consuming. Here we describe a library-based strategy for the simple parallel detection and isolation of multiple peptide-specific human T-cell clones from CD8+ or CD4+ polyclonal T-cell populations. T-cells were first amplified by CD3/CD28 microbeads in a 96U-well library format, prior to screening for desired peptide recognition. T-cells from peptide-reactive wells were then subjected to cytokine-mediated enrichment followed by single-cell cloning, with the entire process from sample to validated clone taking as little as 6 weeks. Overall, T-cell libraries represent an efficient and relatively rapid tool for the generation of peptide-specific T-cell clones, with applications shown here in infectious disease (Epstein–Barr virus, influenza A, and Ebola virus), autoimmunity (type 1 diabetes) and cancer

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, LAG3 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 inhibitio

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

VDJdb in 2019: database extension, new analysis infrastructure and a T-cell receptor motif compendium

Here, we report an update of the VDJdb database with a substantial increase in the number of T-cell receptor (TCR) sequences and their cognate antigens. The update further provides a new database infrastructure featuring two additional analysis modes that facilitate database querying and real-world data analysis. The increased yield of TCR specificity identification methods and the overall increase in the number of studies in the field has allowed us to expand the database more than 5-fold. Furthermore, several new analysis methods are included. For example, batch annotation of TCR repertoire sequencing samples allows for annotating large datasets on-line. Using recently developed bioinformatic methods for TCR motif mining, we have built a reduced set of high-quality TCR motifs that can be used for both training TCR specificity predictors and matching against TCRs of interest. These additions enhance the versatility of the VDJdb in the task of exploring T-cell antigen specificities. The database is available at https://vdjdb.cdr3.net