Biology subjects

Leslie, A.

Publications and source records attributed to Leslie, A..

12 recordsLinked to original sources

bioRxiv2026

Human CD1c-autoreactive T cells recognise Mycobacterium tuberculosis-infected antigen-presenting cells and display cytotoxic effector programmes

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains the leading cause of death from infection globally, yet the contribution of non-classical T-cell pathways to human immunity remains poorly defined. CD1c-autoreactive T-cells, which recognise self-lipids presented by the antigen-presenting molecule CD1c, are frequent in human blood, but their role during infection is unclear. Here, we investigate how CD1c-expressing antigen-presenting cells (APCs) and Mtb infection shape CD1c-autoreactive T-cell responses using engineered human APC systems, complemented by single-cell transcriptomic profiling to define the ex vivo phenotypic landscape of these T-cells. CD1c is present within human TB granulomas, whereas Mtb down-modulates CD1c expression on infected APCs, consistent with an immune evasion strategy. CD1c-autoreactive T-cells respond more strongly to Mtb-infected CD1c+ APCs than to uninfected cells, exhibiting enhanced activation, cytotoxicity, and diverse cytokine secretion via CD1c-dependent recognition. Under in vitro conditions, these T-cells reduce relative Mtb burden in infected phagocytes. Single-cell RNA-sequencing reveals cytotoxic effector-memory programmes and expression of antimicrobial molecules, providing a mechanistic basis for these responses. Together, these findings define a human CD1c-restricted T-cell response to Mtb-infected APCs and identify autoreactive CD1c-restricted T-cells as a candidate cellular axis for lipid-directed immunity in TB.

immunology↗

bioRxiv2025

Inflammatory Biomarkers of Asymptomatic and Symptomatic Tuberculosis

A large proportion of individuals with tuberculosis (TB) are asymptomatic. The biological and inflammatory underpinnings of asymptomatic TB are unknown and may differ from symptomatic TB. We characterised blood transcriptomic and proteomic profiles in South African community screening vs. health facility-based triage cohorts. Asymptomatic TB shared core transcriptomic and proteomic features with symptomatic TB, including upregulation of innate, interferon and inflammatory pathways and downregulation of T and B cell pathways. Integration of transcriptomic and proteomic data from asymptomatic TB individuals identified two distinct sub-clusters characterized by higher or lower bacterial burden, blood IFN-{gamma} responses, BMI, and chest radiographic abnormalities, suggesting different disease severity. We identified a new blood transcriptomic signature of asymptomatic TB. However, diagnostic performance of transcriptomic and proteomic markers was weaker for asymptomatic TB than symptomatic TB, suggesting that policy development for community-based, asymptomatic TB screening should not adopt biomarkers developed for symptomatic TB triage without further optimization.

immunology↗

bioRxiv2025

MegaTrack: a framework for the anatomically accurate and time-efficient virtual dissection and analysis of large-scale tractography datasets

The increasing prevalence of large-scale neuroimaging initiatives has created a need for efficient and anatomically accurate methods for analysing tractography data. Here, we present MegaTrack, a framework designed to combine the anatomical precision of manual dissections with the efficiency required for big data analysis. At its core, MegaTrack operates by normalising individual tractography datasets to a standard space, concatenating them into a single "mega" tractography dataset the user manually dissects before automatically extracting subject-specific tracts and metrics in native space. This approach permits identifying and extracting tracts not yet available in existing templates or automatic dissection tools. We validated this approach using multiple datasets and applications. Comparison with individual manual dissections showed high spatial agreement (weighted dice coefficient > 0.95) and strong correlations in tract-specific metrics (R2 > 0.9). In a longitudinal dataset, MegaTrack demonstrated improved reproducibility across time points and higher inter-rater reliability compared to manual dissections. In a motor neuron disease cohort, MegaTrack replicated known group differences in corticospinal tract microstructure with comparable sensitivity to manual dissections (p = 0.001) but with significantly reduced processing time. Finally, we showcase MegaTracks scalability by creating a comprehensive white matter atlas from 140 healthy adults and demonstrate its application for lesion analysis in stroke patients. This framework is accompanied by an interactive online tool that allows users to explore these atlases and perform customised lesion analyses. The MegaTrack framework is a time-efficient solution for the anatomically precise analysis of large tractography datasets, enabling the rapid simultaneous dissection of multiple subjects while preserving individual anatomical features. The frameworks ability to efficiently process large datasets makes it an excellent tool for generating training data for machine learning and deep learning algorithms in tractography analysis. This approach has broad applications in both research and clinical settings, from group comparisons to personalised lesion mapping, and can significantly contribute to the development of advanced automated tractography methods.

neuroscience↗

bioRxiv2025

Evolution of T cell responses in the tuberculin skin test reveals generalisable Mtb-reactive T cell metaclones.

T cells contribute to immune protection and pathogenesis in tuberculosis, but measurements of polyclonal responses have failed to resolve correlates of outcome. We report the first temporal evaluation of the human in vivo clonal repertoire of Mtb-reactive T cell responses, by T cell receptor (TCR) sequencing at the site of a standardised antigenic challenge. Initial recruitment of non-Mtb reactive T cells is followed by enrichment of Mtb-reactive clones arising from oligoclonal T cell proliferation. We introduce a modular computational pipeline, Metaclonotypist, to sensitively cluster distinct TCRs with shared epitope specificity, which we apply here to establish a catalogue of public Mtb-reactive HLA-restricted T cell metaclones. Although most in vivo Mtb-reactive T cells are private, 10 metaclones were sufficient to identify Mtb-T cell reactivity across our study population (N[≥]128), indicating striking population level immunodominance of specific TCR-peptide interactions that may offer novel approaches to patient stratification and vaccine development.

immunology↗

bioRxiv2025

Targeting ALOX5/LTA4H driven granuloma caseation as a host-directed strategy for control of TB associated lung damage

Tuberculosis remains one of the major global health challenges with those previously exposed presenting persisting pulmonary dysfunction. Therefore, improved control measures are still required to prevent or reduce the TB-induced immunopathology in affected individuals. Host directed therapies that target host factors, have been suggested to improve on current treatments against TB. We previously reported a potential role for proteins which metabolize the arachidonic acids during TB immunopathogenesis. The study, therefore, sought to demonstrate the potential role of these proteins in TB-induced immunopathology. Using immune-histopathological assays, the association of macrophage driven ALOX5 signaling with severe pulmonary immunopathology during TB was demonstrated. Furthermore, using both in vitro and in vivo assays, the data demonstrated the contribution of ALOX5 signaling to TB-induced granulomatous inflammation. Interception of the signaling pathway through clinically approved pharmaceutical inhibitors, resulted in reduced lung damage during the disease. The resolution of TB-induced lung pathology further contributed to increased bactericidal activity. Taken together, our data strongly suggest a role for macrophage driven inflammation via ALOX5 signaling which can be targeted for the development of therapeutic treatment to prevent exacerbated TB-induced pulmonary immunopathology.

immunology↗

bioRxiv2024

Guidelines for evaluating the success of large carnivore reintroductions

Anthropogenic impacts have led to widespread species decline and extirpation, thereby compelling a global movement to protect and regenerate biodiversity through holistic ecosystem restoration including reintroductions. Yet, despite the increasing practice of conservation-driven reintroduction efforts over the past century, peer-reviewed literature and policy providing criteria with which to evaluate reintroduction stages and efficacy remain limited. Without these comprehensive and quantifiable metrics of relative success, such drastic conservation intervention strategies cannot be objectively evaluated nor compared, hindering the advancement of restoration as a discipline. Herein, we systematically reviewed 227 large carnivore reintroductions of 14 terrestrial mammal species across 23 countries since 1930 to contextualize global efforts to date, and from these, have developed a standardized framework to evaluate reintroduction success. We further retrospectively determined the extent to which existing studies met these criteria towards identifying current knowledge gaps and guide future reintroduction efforts. Most large carnivore records were of Felidae (70%) reintroduced into closed systems (69%) across southern Africa (70%). Our proposed framework provides a full suite of stages, indicators, and targets for reintroduction evaluation, which, when retrospectively applied to reviewed studies, indicated that at least one-third lacked sufficient information to effectively evaluate and compare reintroduction outcomes. This comprehensive and prioritized framework provides novel transparency and scalability to large carnivore reintroduction programs, which is increasingly required to secure the sustained support of impacted communities and stakeholder networks. Moreover, the incorporation of this framework into future practice and policy as an applied tool may directly benefit the recovery of at least 30 large carnivore species, while its principles may be applied more broadly across taxonomic groups for faunal rewilding and global ecosystem restoration.

ecology↗

bioRxiv2023

A targeted isotope dilution mass spectrometry assay for Osteopontin quantification in plasma of metastatic breast cancer patients

Osteopontin (OPN) is a secreted glycophosphoprotein that derives its name from its high abundance in bone and secretion by osteoblasts. It is also secreted by a number of immune cells and, therefore, is present in human plasma at nanogram per millilitre levels where it affects cell adhesion and motility. These properties implicate OPN as a promotor of tumor progression. Plasma OPN is primarily measured by enzyme-linked immunosorbent assay (ELISA). However, due to the complexity of the various OPN isoforms, conflicting results have been obtained on the use of OPN as a biomarker even in the same disease condition. These discrepant results may result from the difficulty in comparing ELISA results obtained with different antibodies that target unique OPN epitopes. Mass spectrometry can be used to quantify proteins in plasma and, by targeting OPN regions that do not bear post-translational modifications, may provide more consistent quantification. However, the low (ng/mL) levels in plasma present a significant analytical challenge. In order to develop a sensitive assay for plasma OPN, we explored a single-step precipitation method using a recently developed spin-tube format. Quantification was performed using isotope-dilution mass spectrometry. The concentration detection limit of this assay was 39 {+/-} 15 ng/mL. The assay was applied to the analysis of plasma OPN in metastatic breast cancer patients.

biochemistry↗

bioRxiv2022

HIV skews the SARS-CoV-2 B cell response toward an extrafollicular maturation pathway

BackgroundHIV infection dysregulates the B cell compartment, affecting memory B cell formation and the antibody response to infection and vaccination. Understanding the B cell response to SARS-CoV-2 in people living with HIV (PLWH) may explain the increased morbidity, reduced vaccine efficacy, reduced clearance, and intra-host evolution of SARS-CoV-2 observed in some HIV-1 coinfections. MethodsWe compared B cell responses to COVID-19 in PLWH and HIV negative (HIV-ve) patients in a cohort recruited in Durban, South Africa, during the first pandemic wave in July 2020 using detailed flow cytometry phenotyping of longitudinal samples with markers of B cell maturation, homing and regulatory features. ResultsThis revealed a coordinated B cell response to COVID-19 that differed significantly between HIV-ve and PLWH. Memory B cells in PLWH displayed evidence of reduced germinal center (GC) activity, homing capacity and class-switching responses, with increased PD-L1 expression, and decreased Tfh frequency. This was mirrored by increased extrafollicular (EF) activity, with dynamic changes in activated double negative (DN2) and activated naive B cells, which correlated with anti-RBD-titres in these individuals. An elevated SARS-CoV-2 specific EF response in PLWH was confirmed using viral spike and RBD bait proteins. ConclusionsDespite similar disease severity, these trends were highest in participants with uncontrolled HIV, implicating HIV in driving these changes. EF B cell responses are rapid but give rise to lower affinity antibodies, less durable long-term memory, and reduced capacity to adapt to new variants. Further work is needed to determine the long-term effects of HIV on SARS-CoV-2 immunity, particularly as new variants emerge. FundingThis work was supported by a grant from the Wellcome Trust to the Africa Health Research Institute (Wellcome Trust Strategic Core Award [grant number 201433/Z/16/Z]). Additional funding was received from the South African Department of Science and Innovation through the National Research Foundation (South African Research Chairs Initiative, [grant number 64809]), and the Victor Daitz Foundation.

immunology↗

bioRxiv2021

Mycobacterium tuberculosis senses host Interferon- gamma via the membrane protein MmpL10

Mycobacterium tuberculosis (Mtb) is one of the most successful human pathogens and remains a leading cause of death from infectious disease. Interferon-{gamma} (IFN-{gamma}) is a central regulator of the immune defense against Mtb. Several cytokines have been shown to increase virulence of other bacterial pathogens, leading us to investigate whether IFN-{gamma} has a direct effect on Mtb. We found that both recombinant and T-cell derived IFN-{gamma} rapidly induced a dose-dependent increase in the oxygen consumption rate (OCR) of Mtb, consistent with increased bacterial respiration. This was also observed in clinical strains, but not in the vaccine strain Bacillus Calmette-Guerin (BCG), and did not occur for other cytokines tested, including TNF-. IFN-{gamma} binds to the cell surface of intact Mtb, but not BCG, whilst TNF- binds to neither. Mass spectrometry analysis identified mycobacterial membrane protein large 10 (MmpL10) as the transmembrane binding partner. Consistent with this, IFN-{gamma} binding and the OCR response was absent in a Mtb {Delta}mmpl10 strain and restored by complementation of the mutant strain. RNA-sequencing of IFN-{gamma} exposed Mtb revealed a distinct transcriptional profile, including genes involved in virulence and cholesterol catabolism. Finally, exposure of Mtb cells to IFN-{gamma} resulted in sterilization of bacilli treated with isoniazid (INH), indicating clearance of phenotypically resistant bacteria that persist in the presence of INH alone. Our data suggest a novel mechanism allowing Mtb to respond to host immune activation that may be important in the immunopathogenesis of TB and have use in novel eradication strategies. One-Sentence SummaryIFN-{gamma} is a critical component of effective immune defense in human tuberculosis yet its causative agent, Mycobacterium tuberculosis it able to sense this cytokine and increase virulence and respiration in response.

microbiology↗

bioRxiv2021

HIV-specific CD8+ T-cells in tonsils express exhaustive TRM-like signatures

Lymphoid tissues are an important HIV reservoir site that persists in the face of antiretroviral therapy and natural immunity. Targeting these reservoirs by harnessing the antiviral activity of local tissue resident memory ( TRM) CD8+ T-cells is of great interest, but limited data exist on TRMs within lymph nodes of people living with HIV (PLWH). Here, we studied tonsil CD8+ T-cells obtained from PLWH and uninfected controls from South Africa. We show that these cells are preferentially located outside the germinal centers (GCs), the main reservoir site for HIV, and display a low cytolytic and transcriptionally TRM-like profile that is distinct from blood. In PLWH, CD8+ TRM-like cells are highly expanded and adopt a more cytolytic, activated and exhausted phenotype characterized by increased expression of CD69, PD-1 and perforin, but reduced CD127. This phenotype was enhanced in HIV-specific CD8+ T-cells from tonsils compared to matched blood. Single-cell profiling of these cells revealed a clear transcriptional signature of T-cell activation, clonal expansion and exhaustion ex-vivo. In contrast, this signature was absent from HIV-specific CD8+ T-cells in tonsils isolated from a natural HIV controller, who expressed lower levels of cell surface PD-1 and CXCR5, and reduced transcriptional evidence of T-cell activation, exhaustion and cytolytic activity. Thus, we show that HIV-specific TRM-like CD8+ T-cells in tonsils from non-HIV controllers are enriched for activation and exhaustion profiles compared to those in blood, suggesting that lymphoid HIV-specific CD8+ TRM cells are potentially ideal candidates for immunotherapy to modulate their ability to targeting the HIV reservoirs.

immunology↗

bioRxiv2020

Single-cell profiling of tuberculosis lung granulomas reveals functional lymphocyte signatures of bacterial control

Mycobacterium tuberculosis lung infection results in a complex multicellular structure, the granuloma. In some granulomas, immune activity promotes bacterial clearance; in others, bacteria persist and grow. We identified correlates of bacterial control in cynomolgus macaque lung granulomas by co-registering longitudinal PET-CT imaging, single-cell RNA-sequencing, and measures of bacterial clearance. We find that bacterial persistence occurs in granulomas enriched for mast, endothelial, fibroblast and plasma cells, signaling amongst themselves via Type II immunity and wound healing pathways. In contrast, these interactions are largely absent in granulomas that drive bacterial control, which are often those that form later in the course of infection; these restrictive lesions are characterized by cellular ecosystems enriched for Type1-Type17, stem-like, and cytotoxic T cells engaged in pro-inflammatory signaling networks that involve diverse myeloid and non-immune cell populations. There is also a temporal aspect to bacterial control, in that granulomas that arise later in infection (in the context of an established immune response) share the functional characteristics of restrictive granulomas and are more capable of killing Mtb. Taken together, our results define the complex multicellular ecosystems underlying (lack of) granuloma resolution and highlight host immune targets that can be leveraged to develop new vaccine and therapeutic strategies for TB. One-Sentence SummaryBacterial control in TB lung granulomas correlates with distinct cellular immune microenvironments and time of formation after infection.

immunology↗

bioRxiv2020

Molecular determinants of β-arrestin coupling to formoterol-bound β1-adrenoceptor

The {beta}1-adrenoceptor ({beta}1AR) is a G protein-coupled receptor (GPCR) activated by the hormone noradrenaline, resulting in the coupling of the heterotrimeric G protein Gs1. G protein-mediated signalling is terminated by phosphorylation of the receptor C-terminus and coupling of {beta}-arrestin 1 ({beta}arr1, also known as arrestin-2), which displaces Gs and induces signalling through the MAP kinase pathway2. The ability of synthetic agonists to induce signalling preferentially through either G proteins or arrestins (biased agonism)3 is important in drug development, as the therapeutic effect may arise from only one signalling cascade, whilst the other pathway may mediate undesirable side effects4. To understand the molecular basis for arrestin coupling, we determined the electron cryo-microscopy (cryo-EM) structure of the {beta}1AR-{beta}arr1 complex in lipid nanodiscs bound to the biased agonist formoterol5, and the crystal structure of formoterol-bound {beta}1AR coupled to the G protein mimetic nanobody Nb806. {beta}arr1 couples to {beta}1AR in a distinct manner to how Gs couples to {beta}2AR7, with the finger loop of {beta}arr1 occupying a narrower cleft on the intracellular surface closer to transmembrane helix H7 than the C-terminal 5 helix of Gs. The conformation of the finger loop in {beta}arr1 is different from that adopted by the finger loop in visual arrestin when it couples to rhodopsin8, and its {beta}-turn configuration is reminiscent of the loop in Nb80 that inserts at the same position. {beta}1AR coupled to {beta}arr1 showed significant differences in structure compared to {beta}1AR coupled to Nb80, including an inward movement of extracellular loop 3 (ECL3) and the cytoplasmic ends of H5 and H6. In the orthosteric binding site there was also weakening of interactions between formoterol and the residues Ser2115.42 and Ser2155.46, and a reduction in affinity of formoterol for the {beta}1AR-{beta}arr1 complex compared to {beta}1AR coupled to mini-Gs. These differences provide a foundation for the development of small molecules that could bias signalling in the {beta}-adrenoceptors.

molecular biology↗