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Article ; Online: Genome-wide CRISPR/Cas9-knockout in human induced Pluripotent Stem Cell (iPSC)-derived macrophages

Elena Navarro-Guerrero / Chwen Tay / Justin P. Whalley / Sally A. Cowley / Ben Davies / Julian C. Knight / Daniel Ebner

Scientific Reports, Vol 11, Iss 1, Pp 1-

2021 Volume 11

Abstract: Abstract Genome engineering using CRISPR/Cas9 technology enables simple, efficient and precise genomic modifications in human cells. Conventional immortalized cell lines can be easily edited or screened using genome-wide libraries with lentiviral ... ...

Abstract	Abstract Genome engineering using CRISPR/Cas9 technology enables simple, efficient and precise genomic modifications in human cells. Conventional immortalized cell lines can be easily edited or screened using genome-wide libraries with lentiviral transduction. However, cell types derived from the differentiation of induced Pluripotent Stem Cells (iPSC), which often represent more relevant, patient-derived models for human pathology, are much more difficult to engineer as CRISPR/Cas9 delivery to these differentiated cells can be inefficient and toxic. Here, we present an efficient, lentiviral transduction protocol for delivery of CRISPR/Cas9 to macrophages derived from human iPSC with efficiencies close to 100%. We demonstrate CRISPR/Cas9 knockouts for three nonessential proof-of-concept genes—HPRT1, PPIB and CDK4. We then scale the protocol and validate for a genome-wide pooled CRISPR/Cas9 loss-of-function screen. This methodology enables, for the first time, systematic exploration of macrophage involvement in immune responses, chronic inflammation, neurodegenerative diseases and cancer progression, using efficient genome editing techniques.
Keywords	Medicine ; R ; Science ; Q
Language	English
Publishing date	2021-02-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Epigenomic analysis reveals a dynamic and context-specific macrophage enhancer landscape associated with innate immune activation and tolerance

Ping Zhang / Harindra E. Amarasinghe / Justin P. Whalley / Chwen Tay / Hai Fang / Gabriele Migliorini / Andrew C. Brown / Alice Allcock / Giuseppe Scozzafava / Phalguni Rath / Benjamin Davies / Julian C. Knight

Genome Biology, Vol 23, Iss 1, Pp 1-

2022 Volume 21

Abstract: Abstract Background Chromatin states and enhancers associate gene expression, cell identity and disease. Here, we systematically delineate the acute innate immune response to endotoxin in terms of human macrophage enhancer activity and contrast with ... ...

Abstract	Abstract Background Chromatin states and enhancers associate gene expression, cell identity and disease. Here, we systematically delineate the acute innate immune response to endotoxin in terms of human macrophage enhancer activity and contrast with endotoxin tolerance, profiling the coding and non-coding transcriptome, chromatin accessibility and epigenetic modifications. Results We describe the spectrum of enhancers under acute and tolerance conditions and the regulatory networks between these enhancers and biological processes including gene expression, splicing regulation, transcription factor binding and enhancer RNA signatures. We demonstrate that the vast majority of differentially regulated enhancers on acute stimulation are subject to tolerance and that expression quantitative trait loci, disease-risk variants and eRNAs are enriched in these regulatory regions and related to context-specific gene expression. We find enrichment for context-specific eQTL involving endotoxin response and specific infections and delineate specific differential regions informative for GWAS variants in inflammatory bowel disease and multiple sclerosis, together with a context-specific enhancer involving a bacterial infection eQTL for KLF4. We show enrichment in differential enhancers for tolerance involving transcription factors NFκB-p65, STATs and IRFs and prioritize putative causal genes directly linking genetic variants and disease risk enhancers. We further delineate similarities and differences in epigenetic landscape between stem cell-derived macrophages and primary cells and characterize the context-specific enhancer activities for key innate immune response genes KLF4, SLAMF1 and IL2RA. Conclusions Our study demonstrates the importance of context-specific macrophage enhancers in gene regulation and utility for interpreting disease associations, providing a roadmap to link genetic variants with molecular and cellular functions.
Keywords	Biology (General) ; QH301-705.5 ; Genetics ; QH426-470
Subject code	610
Language	English
Publishing date	2022-06-01T00:00:00Z
Publisher	BMC
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Use of gene expression studies to investigate the human immunological response to malaria infection

Susanne H. Hodgson / Julius Muller / Helen E. Lockstone / Adrian V. S. Hill / Kevin Marsh / Simon J. Draper / Julian C. Knight

Malaria Journal, Vol 18, Iss 1, Pp 1-

2019 Volume 15

Abstract: Abstract Background Transcriptional profiling of the human immune response to malaria has been used to identify diagnostic markers, understand the pathogenicity of severe disease and dissect the mechanisms of naturally acquired immunity (NAI). However, ... ...

Abstract	Abstract Background Transcriptional profiling of the human immune response to malaria has been used to identify diagnostic markers, understand the pathogenicity of severe disease and dissect the mechanisms of naturally acquired immunity (NAI). However, interpreting this body of work is difficult given considerable variation in study design, definition of disease, patient selection and methodology employed. This work details a comprehensive review of gene expression profiling (GEP) of the human immune response to malaria to determine how this technology has been applied to date, instances where this has advanced understanding of NAI and the extent of variability in methodology between studies to allow informed comparison of data and interpretation of results. Methods Datasets from the gene expression omnibus (GEO) including the search terms; ‘plasmodium’ or ‘malaria’ or ‘sporozoite’ or ‘merozoite’ or ‘gametocyte’ and ‘Homo sapiens’ were identified and publications analysed. Datasets of gene expression changes in relation to malaria vaccines were excluded. Results Twenty-three GEO datasets and 25 related publications were included in the final review. All datasets related to Plasmodium falciparum infection, except two that related to Plasmodium vivax infection. The majority of datasets included samples from individuals infected with malaria ‘naturally’ in the field (n = 13, 57%), however some related to controlled human malaria infection (CHMI) studies (n = 6, 26%), or cells stimulated with Plasmodium in vitro (n = 6, 26%). The majority of studies examined gene expression changes relating to the blood stage of the parasite. Significant heterogeneity between datasets was identified in terms of study design, sample type, platform used and method of analysis. Seven datasets specifically investigated transcriptional changes associated with NAI to malaria, with evidence supporting suppression of the innate pro-inflammatory response as an important mechanism for this in the majority of these studies. However, further ...
Keywords	Plasmodium falciparum ; Gene expression ; Malaria ; Immunity ; Arctic medicine. Tropical medicine ; RC955-962 ; Infectious and parasitic diseases ; RC109-216
Subject code	616
Language	English
Publishing date	2019-12-01T00:00:00Z
Publisher	BMC
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Evaluation of T cell responses to naturally processed variant SARS-CoV-2 spike antigens in individuals following infection or vaccination

Zixi Yin / Ji-Li Chen / Yongxu Lu / Beibei Wang / Leila Godfrey / Alexander J. Mentzer / Xuan Yao / Guihai Liu / Dannielle Wellington / Yiqi Zhao / Peter A.C. Wing / Wanwisa Dejnirattisa / Piyada Supasa / Chang Liu / Philip Hublitz / Ryan Beveridge / Craig Waugh / Sally-Ann Clark / Kevin Clark /

Paul Sopp / Timothy Rostron / Juthathip Mongkolsapaya / Gavin R. Screaton / Graham Ogg / Katie Ewer / Andrew J. Pollard / Sarah Gilbert / Julian C. Knight / Teresa Lambe / Geoffrey L. Smith / Tao Dong / Yanchun Peng

Cell Reports, Vol 42, Iss 5, Pp 112470- (2023)

2023

Abstract: Summary: Most existing studies characterizing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell responses are peptide based. This does not allow evaluation of whether tested peptides are processed and presented canonically. In ... ...

Abstract	Summary: Most existing studies characterizing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell responses are peptide based. This does not allow evaluation of whether tested peptides are processed and presented canonically. In this study, we use recombinant vaccinia virus (rVACV)-mediated expression of SARS-CoV-2 spike protein and SARS-CoV-2 infection of angiotensin-converting enzyme (ACE)-2-transduced B cell lines to evaluate overall T cell responses in a small cohort of recovered COVID-19 patients and uninfected donors vaccinated with ChAdOx1 nCoV-19. We show that rVACV expression of SARS-CoV-2 antigen can be used as an alternative to SARS-CoV-2 infection to evaluate T cell responses to naturally processed spike antigens. In addition, the rVACV system can be used to evaluate the cross-reactivity of memory T cells to variants of concern (VOCs) and to identify epitope escape mutants. Finally, our data show that both natural infection and vaccination could induce multi-functional T cell responses with overall T cell responses remaining despite the identification of escape mutations.
Keywords	CP: Immunology ; Biology (General) ; QH301-705.5
Subject code	616
Language	English
Publishing date	2023-05-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Large clones of pre-existing T cells drive early immunity against SARS-COV-2 and LCMV infection

Martina Milighetti / Yanchun Peng / Cedric Tan / Michal Mark / Gayathri Nageswaran / Suzanne Byrne / Tahel Ronel / Tom Peacock / Andreas Mayer / Aneesh Chandran / Joshua Rosenheim / Matthew Whelan / Xuan Yao / Guihai Liu / Suet Ling Felce / Tao Dong / Alexander J. Mentzer / Julian C. Knight / Francois Balloux /

Erez Greenstein / Shlomit Reich-Zeliger / Corinna Pade / Joseph M. Gibbons / Amanda Semper / Tim Brooks / Ashley Otter / Daniel M. Altmann / Rosemary J. Boyton / Mala K. Maini / Aine McKnight / Charlotte Manisty / Thomas A. Treibel / James C. Moon / Mahdad Noursadeghi / Benny Chain

iScience, Vol 26, Iss 6, Pp 106937- (2023)

2023

Abstract: Summary: T cell responses precede antibody and may provide early control of infection. We analyzed the clonal basis of this rapid response following SARS-COV-2 infection. We applied T cell receptor (TCR) sequencing to define the trajectories of ... ...

Abstract	Summary: T cell responses precede antibody and may provide early control of infection. We analyzed the clonal basis of this rapid response following SARS-COV-2 infection. We applied T cell receptor (TCR) sequencing to define the trajectories of individual T cell clones immediately. In SARS-COV-2 PCR+ individuals, a wave of TCRs strongly but transiently expand, frequently peaking the same week as the first positive PCR test. These expanding TCR CDR3s were enriched for sequences functionally annotated as SARS-COV-2 specific. Epitopes recognized by the expanding TCRs were highly conserved between SARS-COV-2 strains but not with circulating human coronaviruses. Many expanding CDR3s were present at high frequency in pre-pandemic repertoires. Early response TCRs specific for lymphocytic choriomeningitis virus epitopes were also found at high frequency in the preinfection naive repertoire. High-frequency naive precursors may allow the T cell response to respond rapidly during the crucial early phases of acute viral infection.
Keywords	Biological sciences ; Immunology ; Immunity ; Cell biology ; Science ; Q
Language	English
Publishing date	2023-06-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Single cell spatial analysis reveals inflammatory foci of immature neutrophil and CD8 T cells in COVID-19 lungs

Praveen Weeratunga / Laura Denney / Joshua A. Bull / Emmanouela Repapi / Martin Sergeant / Rachel Etherington / Chaitanya Vuppussetty / Gareth D. H. Turner / Colin Clelland / Jeongmin Woo / Amy Cross / Fadi Issa / Carlos Eduardo de Andrea / Ignacio Melero Bermejo / David Sims / Simon McGowan / Yasemin-Xiomara Zurke / David J. Ahern / Eddie C. Gamez /

Justin Whalley / Duncan Richards / Paul Klenerman / Claudia Monaco / Irina A. Udalova / Tao Dong / Agne Antanaviciute / Graham Ogg / Julian C. Knight / Helen M. Byrne / Stephen Taylor / Ling-Pei Ho

Nature Communications, Vol 14, Iss 1, Pp 1-

2023 Volume 20

Abstract: Abstract Single cell spatial interrogation of the immune-structural interactions in COVID −19 lungs is challenging, mainly because of the marked cellular infiltrate and architecturally distorted microstructure. To address this, we develop a suite of ... ...

Abstract	Abstract Single cell spatial interrogation of the immune-structural interactions in COVID −19 lungs is challenging, mainly because of the marked cellular infiltrate and architecturally distorted microstructure. To address this, we develop a suite of mathematical tools to search for statistically significant co-locations amongst immune and structural cells identified using 37-plex imaging mass cytometry. This unbiased method reveals a cellular map interleaved with an inflammatory network of immature neutrophils, cytotoxic CD8 T cells, megakaryocytes and monocytes co-located with regenerating alveolar progenitors and endothelium. Of note, a highly active cluster of immature neutrophils and CD8 T cells, is found spatially linked with alveolar progenitor cells, and temporally with the diffuse alveolar damage stage. These findings offer further insights into how immune cells interact in the lungs of severe COVID-19 disease. We provide our pipeline [Spatial Omics Oxford Pipeline (SpOOx)] and visual-analytical tool, Multi-Dimensional Viewer (MDV) software, as a resource for spatial analysis.
Keywords	Science ; Q
Subject code	610
Language	English
Publishing date	2023-11-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Using de novo assembly to identify structural variation of eight complex immune system gene regions.

Jia-Yuan Zhang / Hannah Roberts / David S C Flores / Antony J Cutler / Andrew C Brown / Justin P Whalley / Olga Mielczarek / David Buck / Helen Lockstone / Barbara Xella / Karen Oliver / Craig Corton / Emma Betteridge / Rachael Bashford-Rogers / Julian C Knight / John A Todd / Gavin Band

PLoS Computational Biology, Vol 17, Iss 8, p e

2021 Volume 1009254

Abstract: Driven by the necessity to survive environmental pathogens, the human immune system has evolved exceptional diversity and plasticity, to which several factors contribute including inheritable structural polymorphism of the underlying genes. ... ...

Abstract	Driven by the necessity to survive environmental pathogens, the human immune system has evolved exceptional diversity and plasticity, to which several factors contribute including inheritable structural polymorphism of the underlying genes. Characterizing this variation is challenging due to the complexity of these loci, which contain extensive regions of paralogy, segmental duplication and high copy-number repeats, but recent progress in long-read sequencing and optical mapping techniques suggests this problem may now be tractable. Here we assess this by using long-read sequencing platforms from PacBio and Oxford Nanopore, supplemented with short-read sequencing and Bionano optical mapping, to sequence DNA extracted from CD14+ monocytes and peripheral blood mononuclear cells from a single European individual identified as HV31. We use this data to build a de novo assembly of eight genomic regions encoding four key components of the immune system, namely the human leukocyte antigen, immunoglobulins, T cell receptors, and killer-cell immunoglobulin-like receptors. Validation of our assembly using k-mer based and alignment approaches suggests that it has high accuracy, with estimated base-level error rates below 1 in 10 kb, although we identify a small number of remaining structural errors. We use the assembly to identify heterozygous and homozygous structural variation in comparison to GRCh38. Despite analyzing only a single individual, we find multiple large structural variants affecting core genes at all three immunoglobulin regions and at two of the three T cell receptor regions. Several of these variants are not accurately callable using current algorithms, implying that further methodological improvements are needed. Our results demonstrate that assessing haplotype variation in these regions is possible given sufficiently accurate long-read and associated data. Continued reductions in the cost of these technologies will enable application of these methods to larger samples and provide a broader catalogue of ...
Keywords	Biology (General) ; QH301-705.5
Subject code	006 ; 612
Language	English
Publishing date	2021-08-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Genomic Response to Vitamin D Supplementation in the Setting of a Randomized, Placebo-Controlled Trial

Antonio J. Berlanga-Taylor / Katharine Plant / Andrew Dahl / Evelyn Lau / Michael Hill / David Sims / Andreas Heger / Jonathan Emberson / Jane Armitage / Robert Clarke / Julian C. Knight

EBioMedicine, Vol 31, Iss , Pp 133-

2018 Volume 142

Abstract: Background: Vitamin D deficiency has been associated with multiple diseases, but the causal relevance and underlying processes are not fully understood. Elucidating the mechanisms of action of drug treatments in humans is challenging, but application of ... ...

Abstract	Background: Vitamin D deficiency has been associated with multiple diseases, but the causal relevance and underlying processes are not fully understood. Elucidating the mechanisms of action of drug treatments in humans is challenging, but application of functional genomic approaches in randomized trials may afford an opportunity to systematically assess molecular responses. Methods: In the Biochemical Efficacy and Safety Trial of Vitamin D (BEST-D), a double-blind, placebo-controlled, dose-finding, randomized clinical trial, 305 community-dwelling individuals aged over 65 years were randomly allocated to treatment with vitamin D3 4000 IU, 2000 IU or placebo daily for 12 months. Genome-wide genotypes at baseline, and transcriptome and plasma levels of cytokines (IFN-γ, IL-10, IL-8, IL-6 and TNF-α) at baseline and after 12 months, were measured. The trial had >90% power to detect 1.2-fold changes in gene expression. Findings: Allocation to vitamin D for 12-months was associated with 2-fold higher plasma levels of 25-hydroxy-vitamin D (25[OH]D, 4000 IU regimen), but had no significant effect on whole-blood gene expression (FDR < 5%) or on plasma levels of cytokines compared with placebo. In pre-specified analysis, rs7041 (intron variant, GC) had a significant effect on circulating levels of 25(OH)D in the low dose, but not in the placebo or high dose vitamin D regimen. A gene expression quantitative trait locus analysis (eQTL) demonstrated evidence of 31,568 cis-eQTLs (unique SNP-probe pairs) among individuals at baseline and 34,254 after supplementation for 12 months (any dose). No significant associations involving vitamin D supplementation response eQTLs were found. Interpretation: We performed a comprehensive functional genomics and molecular analysis of vitamin D supplementation in a randomized, placebo-controlled trial. Although this study was limited to mostly Caucasian individuals aged over 65 years, the results differ from many previous studies and do not support a strong effect of vitamin D on ...
Keywords	Medicine ; R ; Medicine (General) ; R5-920
Subject code	610
Language	English
Publishing date	2018-05-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Context-specific regulation of surface and soluble IL7R expression by an autoimmune risk allele

Hussein Al-Mossawi / Nicole Yager / Chelsea A. Taylor / Evelyn Lau / Sara Danielli / Jelle de Wit / James Gilchrist / Isar Nassiri / Elise A. Mahe / Wanseon Lee / Laila Rizvi / Seiko Makino / Jane Cheeseman / Matt Neville / Julian C. Knight / Paul Bowness / Benjamin P. Fairfax

Nature Communications, Vol 10, Iss 1, Pp 1-

2019 Volume 11

Abstract: Interleukin-7 (IL-7) is a central cytokine in T cell homeostasis. Here the authors show that allelic variation at rs6897932, an autoimmune GWAS risk allele at IL7R, regulates surface and soluble IL-7R in stimulated monocytes, indicating a function of ... ...

Abstract	Interleukin-7 (IL-7) is a central cytokine in T cell homeostasis. Here the authors show that allelic variation at rs6897932, an autoimmune GWAS risk allele at IL7R, regulates surface and soluble IL-7R in stimulated monocytes, indicating a function of monocytes in IL-7-related autoimmunity.
Keywords	Science ; Q
Language	English
Publishing date	2019-10-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Context-specific regulation of surface and soluble IL7R expression by an autoimmune risk allele

Nature Communications, Vol 10, Iss 1, Pp 1-

2019 Volume 11

Abstract	Interleukin-7 (IL-7) is a central cytokine in T cell homeostasis. Here the authors show that allelic variation at rs6897932, an autoimmune GWAS risk allele at IL7R, regulates surface and soluble IL-7R in stimulated monocytes, indicating a function of monocytes in IL-7-related autoimmunity.
Keywords	Science ; Q
Language	English
Publishing date	2019-10-01T00:00:00Z
Publisher	Nature Publishing Group
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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