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Students presented their research at a one day symposium open to all staff at the Institute
hadge: a comprehensive pipeline for donor deconvolution in single-cell studies.
Single-cell multiplexing techniques (cell hashing and genetic multiplexing) combine multiple samples, optimizing sample processing and reducing costs. Cell hashing conjugates antibody-tags or chemical-oligonucleotides to cell membranes, while genetic multiplexing allows to mix genetically diverse samples and relies on aggregation of RNA reads at known genomic coordinates. We develop hadge (hashing deconvolution combined with genotype information), a Nextflow pipeline that combines 12 methods to perform both hashing- and genotype-based deconvolution. We propose a joint deconvolution strategy combining best-performing methods and demonstrate how this approach leads to the recovery of previously discarded cells in a nuclei hashing of fresh-frozen brain tissue.
Blimp-1 and c-Maf regulate immune gene networks to protect against distinct pathways of pathobiont-induced colitis.
Intestinal immune responses to microbes are controlled by the cytokine IL-10 to avoid immune pathology. Here, we use single-cell RNA sequencing of colon lamina propria leukocytes (LPLs) along with RNA-seq and ATAC-seq of purified CD4+ T cells to show that the transcription factors Blimp-1 (encoded by Prdm1) and c-Maf co-dominantly regulate Il10 while negatively regulating proinflammatory cytokines in effector T cells. Double-deficient Prdm1fl/flMaffl/flCd4Cre mice infected with Helicobacter hepaticus developed severe colitis with an increase in TH1/NK/ILC1 effector genes in LPLs, while Prdm1fl/flCd4Cre and Maffl/flCd4Cre mice exhibited moderate pathology and a less-marked type 1 effector response. LPLs from infected Maffl/flCd4Cre mice had increased type 17 responses with increased Il17a and Il22 expression and an increase in granulocytes and myeloid cell numbers, resulting in increased T cell-myeloid-neutrophil interactions. Genes over-expressed in human inflammatory bowel disease showed differential expression in LPLs from infected mice in the absence of Prdm1 or Maf, revealing potential mechanisms of human disease.
Beyond bNAbs: Uses, Risks, and Opportunities for Therapeutic Application of Non-Neutralising Antibodies in Viral Infection.
The vast majority of antibodies generated against a virus will be non-neutralising. However, this does not denote an absence of protective capacity. Yet, within the field, there is typically a large focus on antibodies capable of directly blocking infection (neutralising antibodies, NAbs) of either specific viral strains or multiple viral strains (broadly-neutralising antibodies, bNAbs). More recently, a focus on non-neutralising antibodies (nNAbs), or neutralisation-independent effects of NAbs, has emerged. These can have additive effects on protection or, in some cases, be a major correlate of protection. As their name suggests, nNAbs do not directly neutralise infection but instead, through their Fc domains, may mediate interaction with other immune effectors to induce clearance of viral particles or virally infected cells. nNAbs may also interrupt viral replication within infected cells. Developing technologies of antibody modification and functionalisation may lead to innovative biologics that harness the activities of nNAbs for antiviral prophylaxis and therapeutics. In this review, we discuss specific examples of nNAb actions in viral infections where they have known importance. We also discuss the potential detrimental effects of such responses. Finally, we explore new technologies for nNAb functionalisation to increase efficacy or introduce favourable characteristics for their therapeutic applications.
Immune microniches shape intestinal Treg function.
The intestinal immune system is highly adapted to maintaining tolerance to the commensal microbiota and self-antigens while defending against invading pathogens1,2. Recognizing how the diverse network of local cells establish homeostasis and maintains it in the complex immune environment of the gut is critical to understanding how tolerance can be re-established following dysfunction, such as in inflammatory disorders. Although cell and molecular interactions that control T regulatory (Treg) cell development and function have been identified3,4, less is known about the cellular neighbourhoods and spatial compartmentalization that shapes microorganism-reactive Treg cell function. Here we used in vivo live imaging, photo-activation-guided single-cell RNA sequencing5-7 and spatial transcriptomics to follow the natural history of T cells that are reactive towards Helicobacter hepaticus through space and time in the settings of tolerance and inflammation. Although antigen stimulation can occur anywhere in the tissue, the lamina propria-but not embedded lymphoid aggregates-is the key microniche that supports effector Treg (eTreg) cell function. eTreg cells are stable once their niche is established; however, unleashing inflammation breaks down compartmentalization, leading to dominance of CD103+SIRPα+ dendritic cells in the lamina propria. We identify and validate the putative tolerogenic interaction between CD206+ macrophages and eTreg cells in the lamina propria and identify receptor-ligand pairs that are likely to govern the interaction. Our results reveal a spatial mechanism of tolerance in the lamina propria and demonstrate how knowledge of local interactions may contribute to the next generation of tolerance-inducing therapies.
Obesity Differs from Diabetes Mellitus in Antibody and T Cell Responses Post COVID-19 Recovery.
Obesity and type 2 diabetes (DM) are risk factors for severe COVID-19 outcomes, which disproportionately affect South Asian populations. This study aims to investigate the humoral and cellular immune responses to SARS-CoV-2 in adult COVID-19 survivors with obesity and DM in Bangladesh. In this cross-sectional study, SARS-CoV-2-specific antibody and T cell responses were investigated in 63 healthy and 75 PCR-confirmed COVID-19 recovered individuals in Bangladesh, during the pre-vaccination first wave of the COVID-19 pandemic in 2020. In COVID-19 survivors, SARS-CoV-2 infection induced robust antibody and T cell responses, which correlated with disease severity. After adjusting for age, sex, DM status, disease severity, and time since onset of symptoms, obesity was associated with decreased neutralising antibody titers, and increased SARS-CoV-2 spike-specific IFN-γ response along with increased proliferation and IL-2 production by CD8+ T cells. In contrast, DM was not associated with SARS-CoV-2-specific antibody and T cell responses after adjustment for obesity and other confounders. Obesity is associated with lower neutralising antibody levels and higher T cell responses to SARS-CoV-2 post COVID-19 recovery, while antibody or T cell responses remain unaltered in DM.
Semmaphorin 3 A causes immune suppression by inducing cytoskeletal paralysis in tumour-specific CD8+ T cells.
Semaphorin-3A (SEMA3A) functions as a chemorepulsive signal during development and can affect T cells by altering their filamentous actin (F-actin) cytoskeleton. The exact extent of these effects on tumour-specific T cells are not completely understood. Here we demonstrate that Neuropilin-1 (NRP1) and Plexin-A1 and Plexin-A4 are upregulated on stimulated CD8+ T cells, allowing tumour-derived SEMA3A to inhibit T cell migration and assembly of the immunological synapse. Deletion of NRP1 in both CD4+ and CD8+ T cells enhance CD8+ T-cell infiltration into tumours and restricted tumour growth in animal models. Conversely, over-expression of SEMA3A inhibit CD8+ T-cell infiltration. We further show that SEMA3A affects CD8+ T cell F-actin, leading to inhibition of immune synapse formation and motility. Examining a clear cell renal cell carcinoma patient cohort, we find that SEMA3A expression is associated with reduced survival, and that T-cells appear trapped in SEMA3A rich regions. Our study establishes SEMA3A as an inhibitor of effector CD8+ T cell tumour infiltration, suggesting that blocking NRP1 could improve T cell function in tumours.
Study protocol for a Randomised controlled trial of EArly transjugular intrahepatiC porTosystemic stent-shunt in Acute Variceal Bleeding (REACT-AVB trial).
INTRODUCTION: In liver cirrhosis, acute variceal bleeding (AVB) is associated with a 1-year mortality rate of up to 40%. Data on early or pre-emptive transjugular intrahepatic portosystemic stent-shunt (TIPSS) in AVB is inconclusive and may not reflect current management strategies. Randomised controlled trial of EArly transjugular intrahepatiC porTosystemic stent-shunt in AVB (REACT-AVB) aims to investigate the clinical and cost-effectiveness of early TIPSS in patients with cirrhosis and AVB after initial bleeding control. METHODS AND ANALYSIS: REACT-AVB is a multicentre, randomised controlled, open-label, superiority, two-arm, parallel-group trial with an internal pilot. The two interventions allocated randomly 1:1 are early TIPSS within 4 days of diagnostic endoscopy or secondary prophylaxis with endoscopic therapy in combination with non-selective beta blockers. Patients aged ≥18 years with cirrhosis and Child-Pugh Score 7-13 presenting with AVB with endoscopic haemostasis are eligible for inclusion. The primary outcome is transplant-free survival at 1 year post randomisation. Secondary endpoints include transplant-free survival at 6 weeks, rebleeding, serious adverse events, other complications of cirrhosis, Child-Pugh and Model For End-Stage Liver Disease (MELD) scores at 6 and 12 months, health-related quality of life, use of healthcare resources, cost-effectiveness and use of cross-over therapies. The sample size is 294 patients over a 4-year recruitment period, across 30 hospitals in the UK. ETHICS AND DISSEMINATION: Research ethics committee of National Health Service has approved REACT-AVB (reference number: 23/WM/0085). The results will be submitted for publication in a peer-reviewed journal. A lay summary will also be emailed or posted to participants before publication. TRIAL REGISTRATION NUMBER: ISRCTN85274829; protocol version 3.0, 1 July 2023.
Exclusive Enteral Nutrition Mediates Beneficial Gut Microbiome Enrichment in Acute Severe Colitis.
BACKGROUND: Exclusive enteral nutrition (EEN) supplementation of the standard of care (SOC) augments steroid responsiveness in patients with acute severe ulcerative colitis (ASUC). EEN is known to alter gut microbial composition. The present study investigates EEN-driven gut microbial alterations in patients with ASUC and examines their correlations with clinical parameters. METHODS: Stool samples from patients with ASUC (n = 44) who received either EEN-supplemented SOC (EEN group; n = 20) or SOC alone (SOC group; n = 24) for 7 days were collected at baseline (day 0) and postintervention (day 7). Microbiome analysis was carried out using 16S ribosomal RNA gene sequencing followed by data processing using QIIME2 and R packages. RESULTS: Seven-day EEN-conjugated corticosteroid therapy in patients with ASUC enhanced the abundances of beneficial bacterial genera Faecalibacterium and Veillonella and reduced the abundance of Sphingomonas (generalized linear model fitted with Lasso regularization with robustness of 100%), while no such improvements in gut microbiota were observed in the SOC group. The EEN-associated taxa correlated with the patient's clinical parameters (serum albumin and C-reactive protein levels). Unlike the SOC group, which retained its preintervention core microbiota, EEN contributed Faecalibacterium prausnitzii, a beneficial gut bacterial taxon, to the gut microbial core. EEN responders showed enhancement of Ligilactobacillus and Veillonella and reduction in Prevotella and Granulicatella. Analysis of baseline gut microbiota showed relative enhancement of certain microbial genera being associated with corticosteroid response and baseline clinical parameters and that this signature could conceivably be used as a predictive tool. CONCLUSIONS: Augmentation of clinical response by EEN-conjugated corticosteroid therapy is accompanied by beneficial gut microbial changes in patients with ASUC.
Mitochondrial control of lymphocyte homeostasis.
Mitochondria play a multitude of essential roles within mammalian cells, and understanding how they control immunity is an emerging area of study. Lymphocytes, as integral cellular components of the adaptive immune system, rely on mitochondria for their function, and mitochondria can dynamically instruct their differentiation and activation by undergoing rapid and profound remodelling. Energy homeostasis and ATP production are often considered the primary functions of mitochondria in immune cells; however, their importance extends across a spectrum of other molecular processes, including regulation of redox balance, signalling pathways, and biosynthesis. In this review, we explore the dynamic landscape of mitochondrial homeostasis in T and B cells, and discuss how mitochondrial disorders compromise adaptive immunity.
Surgical Treatment of Lymphedema in the Upper Extremity.
The advent of supermicrosurgery has led to an increasing interest in the surgical management of lymphedema through the reconstruction of the lymphatic network, that is, the physiologic approach. Broadly, this can be divided into 2 main techniques: lymphaticovenous anastomosis and lymph node transfer. In the United Kingdom, the British Lymphology Society does not provide any recommendations on surgical management. Moreover, surgical treatment of lymphedema is not widely practiced within the National Health Service due to low-certainty evidence. Herein, we discuss our experience in physiologic reconstruction for lymphedema.
Short-chain fatty acids: linking diet, the microbiome and immunity.
The short-chain fatty acids (SCFAs) butyrate, propionate and acetate are microbial metabolites and their availability in the gut and other organs is determined by environmental factors, such as diet and use of antibiotics, that shape the diversity and metabolism of the microbiota. SCFAs regulate epithelial barrier function as well as mucosal and systemic immunity via evolutionary conserved processes that involve G protein-coupled receptor signalling or histone deacetylase activity. Indicatively, the anti-inflammatory role of butyrate is mediated through direct effects on the differentiation of intestinal epithelial cells, phagocytes, B cells and plasma cells, and regulatory and effector T cells. Intestinally derived SCFAs also directly and indirectly affect immunity at extra-intestinal sites, such as the liver, the lungs, the reproductive tract and the brain, and have been implicated in a range of disorders, including infections, intestinal inflammation, autoimmunity, food allergies, asthma and responses to cancer therapies. An ecological understanding of microbial communities and their interrelated metabolic states, as well as the engineering of butyrogenic bacteria may support SCFA-focused interventions for the prevention and treatment of immune-mediated diseases.
Prevalence of resistance-associated viral variants to the HIV-specific broadly neutralising antibody 10-1074 in a UK bNAb-naïve population.
Broadly neutralising antibodies (bNAbs) targeting HIV show promise for both prevention of infection and treatment. Among these, 10-1074 has shown potential in neutralising a wide range of HIV strains. However, resistant viruses may limit the clinical efficacy of 10-1074. The prevalence of both de novo and emergent 10-1074 resistance will determine its use at a population level both to protect against HIV transmission and as an option for treatment. To help understand this further, we report the prevalence of pre-existing mutations associated with 10-1074 resistance in a bNAb-naive population of 157 individuals presenting to UK HIV centres with primary HIV infection, predominantly B clade, receiving antiretroviral treatment. Single genome analysis of HIV proviral envelope sequences showed that 29% of participants' viruses tested had at least one sequence with 10-1074 resistance-associated mutations. Mutations interfering with the glycan binding site at HIV Env position 332 accounted for 95% of all observed mutations. Subsequent analysis of a larger historic dataset of 2425 B-clade envelope sequences sampled from 1983 to 2019 revealed an increase of these mutations within the population over time. Clinical studies have shown that the presence of pre-existing bNAb mutations may predict diminished therapeutic effectiveness of 10-1074. Therefore, we emphasise the importance of screening for these mutations before initiating 10-1074 therapy, and to consider the implications of pre-existing resistance when designing prevention strategies.