Biology subjects

Goulding, D. A.

Publications and source records attributed to Goulding, D. A..

3 recordsLinked to original sources

bioRxiv2024

Simulating CD8 T Cell Exhaustion: A Comprehensive Approach

Immunotherapy has revolutionised the treatment of multiple cancer types, however, these treatments only work for a proportion of patients and biomarkers to predict response are lacking. One correlate of response is the reinvigoration of a subset of CD8 T cells that have an exhausted phenotype and impaired functionality. In order to develop new therapies, reproducible models are required to identify candidate target genes that enables the reversal of key hallmarks of T cell exhaustion. Here we describe the development of an in vitro model by chronically stimulating T cells with their cognate antigen and performed an in depth temporal phenotypic characterisation. This model recapitulates many of the critical hallmarks of exhaustion, including increased expression of canonical exhaustion surface markers, impaired proliferation, reduced cytokine production, decreased release of cytotoxic granules, and metabolic alterations, including dysfunctional mitochondria. These exhaustion hallmarks were validated using an in vivo model and a gene signature identified which robustly define the shared in vitro and in vivo exhausted state. Critically, this signature is also observed in tumour infiltrating T cells from multiple human tumour types, validating the translational potential of this model for discovering and triaging new therapies.

immunology↗

bioRxiv2022

Post-vaccine epidemiology of serotype 3 pneumococci identifies transformation inhibition through prophage-driven alteration of a non-coding RNA

The respiratory pathogen Streptococcus pneumoniae (the pneumococcus) is a genetically diverse bacterium associated with over 100 immunologically-distinct polysaccharide capsules (serotypes). Polysaccharide conjugate vaccines (PCVs) have successfully eliminated multiple targeted serotypes, yet the mucoid serotype 3 has persisted despite its inclusion in PCV13. This capsule type is predominantly associated with a single globally-disseminated strain, GPSC12 (CC180), which was split into clades by a genomic analysis. Clade I, the most common, rarely underwent transformation, but was typically infected with the prophage {phi}OXC141. Prior to the introduction of PCV13, this clades composition shifted towards a {phi}OXC141-negative subpopulation in a systematically-sampled UK collection. In the post-PCV era, more rapidly-recombining non-Clade I isolates, also {phi}OXC141-negative, have risen in prevalence. The low in vitro transformation efficiency of a Clade I isolate could not be fully explained by the [~]100-fold reduction attributable to the serotype 3 capsule. Accordingly, prophage {phi}OXC141 was found to modify csRNA3, a non-coding RNA that inhibits the induction of transformation. This alteration was identified in [~]30% of all pneumococci, and was particularly common in the unusually-clonal serotype 1 GPSC2 strain. RNA-seq and quantitative reverse transcriptase PCR data demonstrated the altered csRNA3 was more effective at inhibiting production of the competence stimulating peptide pheromone. This interference with the quorum sensing needed to induce competence lowered the rate of spontaneous transformation, reducing the risk of the prophage being deleted by homologous recombination. Hence the selfish prophage-driven alteration of a regulatory RNA limits cell-cell communication and horizontal gene transfer, complicating the interpretation of post-vaccine population dynamics.

microbiology↗

bioRxiv2020

A novel therapeutic antibody screening method using bacterial high-content imaging reveals functional antibody binding phenotypes of Escherichia coli ST131

The increase of antimicrobial resistance (AMR), and lack of new classes of licensed antimicrobials, have made alternative treatment options for AMR pathogens increasingly attractive. Recent studies have demonstrated anti-bacterial efficacy of a humanised monoclonal antibody (mAb) targeting the O25b O-antigen of Escherichia coli ST131. To evaluate the phenotypic effects of antibody binding to diverse clinical E. coli ST131 O25b bacterial isolates in high-throughput, we designed a novel mAb screening method using high-content imaging (HCI) and image-based morphological profiling to screen a mAb targeting the O25b O-antigen. Screening the antibody against a panel of 86 clinical E. coli ST131 O25:H4 isolates revealed 4 binding phenotypes: no binding (18.60%), weak binding (4.65%), strong binding (69.77%) and strong agglutinating binding (6.98%). Impaired antibody binding could be explained by the presence of insertion sequences or mutations in O-antigen or lipopolysaccharide core biosynthesis genes, affecting the amount, structure or chain length of the O-antigen. The agglutinating binding phenotype was linked with lower O-antigen density, enhanced antibody-mediated phagocytosis and increased serum susceptibly. This study highlights the need to screen candidate mAbs against large panels of clinically relevant isolates, and that HCI can be used to evaluate mAb binding affinity and potential functional efficacy against AMR bacteria.

microbiology↗