Publications by authors named "Vincenzo Cerundolo"

210 Publications

HLA-E-restricted, Gag-specific CD8 T cells can suppress HIV-1 infection, offering vaccine opportunities.

Sci Immunol 2021 Mar;6(57)

NDM Research Building, Nuffield Department of Medicine, Oxford University, Oxford OX3 7FZ, UK.

Human leukocyte antigen-E (HLA-E) normally presents an HLA class Ia signal peptide to the NKG2A/C-CD94 regulatory receptors on natural killer (NK) cells and T cell subsets. Rhesus macaques immunized with a cytomegalovirus-vectored simian immunodeficiency virus (SIV) vaccine generated Mamu-E (HLA-E homolog)-restricted T cell responses that mediated post-challenge SIV replication arrest in >50% of animals. However, HIV-1-specific, HLA-E-restricted T cells have not been observed in HIV-1-infected individuals. Here, HLA-E-restricted, HIV-1-specific CD8 T cells were primed in vitro. These T cell clones and allogeneic CD8 T cells transduced with their T cell receptors suppressed HIV-1 replication in CD4 T cells in vitro. Vaccine induction of efficacious HLA-E-restricted HIV-1-specific T cells should therefore be possible.
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http://dx.doi.org/10.1126/sciimmunol.abg1703DOI Listing
March 2021

PLGA Nanoparticles Co-encapsulating NY-ESO-1 Peptides and IMM60 Induce Robust CD8 and CD4 T Cell and B Cell Responses.

Front Immunol 2021 25;12:641703. Epub 2021 Feb 25.

Department of Tumor Immunology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, Nijmegen, Netherlands.

Tumor-specific neoantigens can be highly immunogenic, but their identification for each patient and the production of personalized cancer vaccines can be time-consuming and prohibitively expensive. In contrast, tumor-associated antigens are widely expressed and suitable as an off the shelf immunotherapy. Here, we developed a PLGA-based nanoparticle vaccine that contains both the immunogenic cancer germline antigen NY-ESO-1 and an α-GalCer analog IMM60, as a novel iNKT cell agonist and dendritic cell transactivator. Three peptide sequences (85-111, 117-143, and 157-165) derived from immunodominant regions of NY-ESO-1 were selected. These peptides have a wide HLA coverage and were efficiently processed and presented by dendritic cells various HLA subtypes. Co-delivery of IMM60 enhanced CD4 and CD8 T cell responses and antibody levels against NY-ESO-1 . Moreover, the nanoparticles have negligible systemic toxicity in high doses, and they could be produced according to GMP guidelines. Together, we demonstrated the feasibility of producing a PLGA-based nanovaccine containing immunogenic peptides and an iNKT cell agonist, that is activating DCs to induce antigen-specific T cell responses.
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http://dx.doi.org/10.3389/fimmu.2021.641703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7947615PMC
February 2021

Hepcidin-Mediated Hypoferremia Disrupts Immune Responses to Vaccination and Infection.

Med (N Y) 2021 Feb;2(2):164-179.e12

MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK.

Background: How specific nutrients influence adaptive immunity is of broad interest. Iron deficiency is the most common micronutrient deficiency worldwide and imparts a significant burden of global disease; however, its effects on immunity remain unclear.

Methods: We used a hepcidin mimetic and several genetic models to examine the effect of low iron availability on T cells and on immune responses to vaccines and viral infection in mice. We examined humoral immunity in human patients with raised hepcidin and low serum iron caused by mutant . We tested the effect of iron supplementation on vaccination-induced humoral immunity in piglets, a natural model of iron deficiency.

Findings: We show that low serum iron (hypoferremia), caused by increased hepcidin, severely impairs effector and memory responses to immunizations. The intensified metabolism of activated lymphocytes requires the support of enhanced iron acquisition, which is facilitated by IRP1/2 and TFRC. Accordingly, providing extra iron improved the response to vaccination in hypoferremic mice and piglets, while conversely, hypoferremic humans with chronically increased hepcidin have reduced concentrations of antibodies specific for certain pathogens. Imposing hypoferremia blunted the T cell, B cell, and neutralizing antibody responses to influenza virus infection in mice, allowing the virus to persist and exacerbating lung inflammation and morbidity.

Conclusions: Hypoferremia, a well-conserved physiological innate response to infection, can counteract the development of adaptive immunity. This nutrient trade-off is relevant for understanding and improving immune responses to infections and vaccines in the globally common contexts of iron deficiency and inflammatory disorders.

Funding: Medical Research Council, UK.
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http://dx.doi.org/10.1016/j.medj.2020.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7895906PMC
February 2021

Nanovaccine administration route is critical to obtain pertinent iNKt cell help for robust anti-tumor T and B cell responses.

Oncoimmunology 2020 03 17;9(1):1738813. Epub 2020 Mar 17.

Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center & Oncode Institute, Nijmegen, The Netherlands.

Nanovaccines, co-delivering antigen and invariant natural killer T (iNKT) cell agonists, proved to be very effective in inducing anti-tumor T cell responses due to their exceptional helper function. However, it is known that iNKT cells are not equally present in all lymphoid organs and nanoparticles do not get evenly distributed to all immune compartments. In this study, we evaluated the effect of the vaccination route on iNKT cell help to T and B cell responses for the first time in an antigen and agonist co-delivery setting. Intravenous administration of PLGA nanoparticles was mainly targeting liver and spleen where iNKT1 cells are abundant and induced the highest serum IFN-y levels, T cell cytotoxicity, and Th-1 type antibody responses. In comparison, after subcutaneous or intranodal injections, nanoparticles mostly drained or remained in regional lymph nodes where iNKT17 cells were abundant. After subcutaneous and intranodal injections, antigen-specific IgG2 c production was hampered and IFN-y production, as well as cytotoxic T cell responses, depended on sporadic systemic drainage. Therapeutic anti-tumor experiments also demonstrated a clear advantage of intravenous injection over intranodal or subcutaneous vaccinations. Moreover, tumor control could be further improved by PD-1 immune checkpoint blockade after intravenous vaccination, but not by intranodal vaccination. Anti PD-1 antibody combination mainly exerts its effect by prolonging the cytotoxicity of T cells. Nanovaccines also demonstrated synergism with anti-4-1BB agonistic antibody treatment in controlling tumor growth. We conclude that nanovaccines containing iNKT cell agonists shall be preferentially administered intravenously, to optimally reach cellular partners for inducing effective anti-tumor immune responses.
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http://dx.doi.org/10.1080/2162402X.2020.1738813DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790498PMC
March 2020

Deletion of the deISGylating enzyme USP18 enhances tumour cell antigenicity and radiosensitivity.

Br J Cancer 2021 Feb 20;124(4):817-830. Epub 2020 Nov 20.

TDI Mass Spectrometry Laboratory, Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, Roosevelt Drive, Oxford, OX3 7FZ, UK.

Background: Interferon (IFN) signalling pathways, a key element of the innate immune response, contribute to resistance to conventional chemotherapy, radiotherapy, and immunotherapy, and are often deregulated in cancer. The deubiquitylating enzyme USP18 is a major negative regulator of the IFN signalling cascade and is the predominant human protease that cleaves ISG15, a ubiquitin-like protein tightly regulated in the context of innate immunity, from its modified substrate proteins in vivo.

Methods: In this study, using advanced proteomic techniques, we have significantly expanded the USP18-dependent ISGylome and proteome in a chronic myeloid leukaemia (CML)-derived cell line. USP18-dependent effects were explored further in CML and colorectal carcinoma cellular models.

Results: Novel ISGylation targets were characterised that modulate the sensing of innate ligands, antigen presentation and secretion of cytokines. Consequently, CML USP18-deficient cells are more antigenic, driving increased activation of cytotoxic T lymphocytes (CTLs) and are more susceptible to irradiation.

Conclusions: Our results provide strong evidence for USP18 in regulating antigenicity and radiosensitivity, highlighting its potential as a cancer target.
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http://dx.doi.org/10.1038/s41416-020-01167-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7884788PMC
February 2021

The Immune Modulating Properties of Mucosal-Associated Invariant T Cells.

Front Immunol 2020 13;11:1556. Epub 2020 Aug 13.

Medical Research Council Human Immunology Unit, Medical Research Council Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom.

Mucosal-associated invariant T (MAIT) cells are unconventional T lymphocytes that express a semi-invariant T cell receptor (TCR) recognizing microbial vitamin B metabolites presented by the highly conserved major histocompatibility complex (MHC) class I like molecule, MR1. The vitamin B metabolites are produced by several commensal and pathogenic bacteria and yeast, but not viruses. Nevertheless, viral infections can trigger MAIT cell activation in a TCR-independent manner, through the release of pro-inflammatory cytokines by antigen-presenting cells (APCs). MAIT cells belong to the innate like T family of cells with a memory phenotype, which allows them to rapidly release Interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and in some circumstances Interleukin (IL)-17 and IL-10, exerting an immunomodulatory role on the ensuing immune response, akin to iNKT cells and γδ T cells. Recent studies implicate MAIT cells in a variety of inflammatory, autoimmune diseases, and in cancer. In addition, through the analysis of the transcriptome of MAIT cells activated in different experimental conditions, an important function in tissue repair and control of immune homeostasis has emerged, shared with other innate-like T cells. In this review, we discuss these recent findings, focussing on the understanding of the molecular mechanisms underpinning MAIT cell activation and effector function in health and disease, which ultimately will aid in clinically harnessing this unique, not donor-restricted cell subtype.
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http://dx.doi.org/10.3389/fimmu.2020.01556DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7438542PMC
August 2020

Structural and functional characterization of C0021158, a high-affinity monoclonal antibody that inhibits Arginase 2 function via a novel non-competitive mechanism of action.

MAbs 2020 Jan-Dec;12(1):1801230

Cancer Research UK AstraZeneca Antibody Alliance Laboratory , Cambridge, UK.

Arginase 2 (ARG2) is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine. The dysregulated expression of ARG2 within specific tumor microenvironments generates an immunosuppressive niche that effectively renders the tumor 'invisible' to the host's immune system. Increased ARG2 expression leads to a concomitant depletion of local L-arginine levels, which in turn leads to suppression of anti-tumor T-cell-mediated immune responses. Here we describe the isolation and characterization of a high affinity antibody (C0021158) that inhibits ARG2 enzymatic function completely, effectively restoring T-cell proliferation . Enzyme kinetic studies confirmed that C0021158 exhibits a noncompetitive mechanism of action, inhibiting ARG2 independently of L-arginine concentrations. To elucidate C0021158's inhibitory mechanism at a structural level, the co-crystal structure of the Fab in complex with trimeric ARG2 was solved. C0021158's epitope was consequently mapped to an area some distance from the enzyme's substrate binding cleft, indicating an allosteric mechanism was being employed. Following C0021158 binding, distinct regions of ARG2 undergo major conformational changes. Notably, the backbone structure of a surface-exposed loop is completely rearranged, leading to the formation of a new short helix structure at the Fab-ARG2 interface. Moreover, this large-scale structural remodeling at ARG2's epitope translates into more subtle changes within the enzyme's active site. An arginine residue at position 39 is reoriented inwards, sterically impeding the binding of L-arginine. Arg39 is also predicted to alter the p of a key catalytic histidine residue at position 160, further attenuating ARG2's enzymatic function. molecular docking simulations predict that L-arginine is unable to bind effectively when antibody is bound, a prediction supported by isothermal calorimetry experiments using an L-arginine mimetic. Specifically, targeting ARG2 in the tumor microenvironment through the application of C0021158, potentially in combination with standard chemotherapy regimens or alternate immunotherapies, represents a potential new strategy to target immune cold tumors.
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http://dx.doi.org/10.1080/19420862.2020.1801230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7531564PMC
September 2020

Cell identity and nucleo-mitochondrial genetic context modulate OXPHOS performance and determine somatic heteroplasmy dynamics.

Sci Adv 2020 Jul 29;6(31):eaba5345. Epub 2020 Jul 29.

Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain.

Heteroplasmy, multiple variants of mitochondrial DNA (mtDNA) in the same cytoplasm, may be naturally generated by mutations but is counteracted by a genetic mtDNA bottleneck during oocyte development. Engineered heteroplasmic mice with nonpathological mtDNA variants reveal a nonrandom tissue-specific mtDNA segregation pattern, with few tissues that do not show segregation. The driving force for this dynamic complex pattern has remained unexplained for decades, challenging our understanding of this fundamental biological problem and hindering clinical planning for inherited diseases. Here, we demonstrate that the nonrandom mtDNA segregation is an intracellular process based on organelle selection. This cell type-specific decision arises jointly from the impact of mtDNA haplotypes on the oxidative phosphorylation (OXPHOS) system and the cell metabolic requirements and is strongly sensitive to the nuclear context and to environmental cues.
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http://dx.doi.org/10.1126/sciadv.aba5345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7439646PMC
July 2020

Impacts of combining anti-PD-L1 immunotherapy and radiotherapy on the tumour immune microenvironment in a murine prostate cancer model.

Br J Cancer 2020 09 9;123(7):1089-1100. Epub 2020 Jul 9.

Department of Oncology, University of Oxford, Oxford, UK.

Background: Radiotherapy enhances innate and adaptive anti-tumour immunity. It is unclear whether this effect may be harnessed by combining immunotherapy with radiotherapy fractions used to treat prostate cancer. We investigated tumour immune microenvironment responses of pre-clinical prostate cancer models to radiotherapy. Having defined this landscape, we tested whether radiotherapy-induced tumour growth delay could be enhanced with anti-PD-L1.

Methods: Hypofractionated radiotherapy was delivered to TRAMP-C1 and MyC-CaP flank allografts. Tumour growth delay, tumour immune microenvironment flow-cytometry, and immune gene expression were analysed. TRAMP-C1 allografts were then treated with 3 × 5 Gy ± anti-PD-L1.

Results: 3 × 5 Gy caused tumour growth delay in TRAMP-C1 and MyC-CaP. Tumour immune microenvironment changes in TRAMP-C1 at 7 days post-radiotherapy included increased tumour-associated macrophages and dendritic cells and upregulation of PD-1/PD-L1, CD8 T-cell, dendritic cell, and regulatory T-cell genes. At tumour regrowth post-3 × 5 Gy the tumour immune microenvironment flow-cytometry was similar to control tumours, however CD8, natural killer and dendritic cell gene transcripts were reduced. PD-L1 inhibition plus 3 × 5 Gy in TRAMP-C1 did not enhance tumour growth delay versus monotherapy.

Conclusion: 3 × 5 Gy hypofractionated radiotherapy can result in tumour growth delay and immune cell changes in allograft prostate cancer models. Adjuncts beyond immunomodulation may be necessary to improve the radiotherapy-induced anti-tumour response.
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http://dx.doi.org/10.1038/s41416-020-0956-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7525450PMC
September 2020

Extensive sequence and structural evolution of Arginase 2 inhibitory antibodies enabled by an unbiased approach to affinity maturation.

Proc Natl Acad Sci U S A 2020 07 2;117(29):16949-16960. Epub 2020 Jul 2.

Cancer Research UK-AstraZeneca Antibody Alliance Laboratory, CB21 6GP Cambridge, United Kingdom;

Affinity maturation is a powerful technique in antibody engineering for the in vitro evolution of antigen binding interactions. Key to the success of this process is the expansion of sequence and combinatorial diversity to increase the structural repertoire from which superior binding variants may be selected. However, conventional strategies are often restrictive and only focus on small regions of the antibody at a time. In this study, we used a method that combined antibody chain shuffling and a staggered-extension process to produce unbiased libraries, which recombined beneficial mutations from all six complementarity-determining regions (CDRs) in the affinity maturation of an inhibitory antibody to Arginase 2 (ARG2). We made use of the vast display capacity of ribosome display to accommodate the sequence space required for the diverse library builds. Further diversity was introduced through pool maturation to optimize seven leads of interest simultaneously. This resulted in antibodies with substantial improvements in binding properties and inhibition potency. The extensive sequence changes resulting from this approach were translated into striking structural changes for parent and affinity-matured antibodies bound to ARG2, with a large reorientation of the binding paratope facilitating increases in contact surface and shape complementarity to the antigen. The considerable gains in therapeutic properties seen from extensive sequence and structural evolution of the parent ARG2 inhibitory antibody clearly illustrate the advantages of the unbiased approach developed, which was key to the identification of high-affinity antibodies with the desired inhibitory potency and specificity.
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http://dx.doi.org/10.1073/pnas.1919565117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7382286PMC
July 2020

Ligand-dependent downregulation of MR1 cell surface expression.

Proc Natl Acad Sci U S A 2020 05 27;117(19):10465-10475. Epub 2020 Apr 27.

MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom.

The antigen-presenting molecule MR1 presents riboflavin-based metabolites to Mucosal-Associated Invariant T (MAIT) cells. While MR1 egress to the cell surface is ligand-dependent, the ability of small-molecule ligands to impact on MR1 cellular trafficking remains unknown. Arising from an in silico screen of the MR1 ligand-binding pocket, we identify one ligand, 3-([2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]formamido)propanoic acid, DB28, as well as an analog, methyl 3-([2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]formamido)propanoate, NV18.1, that down-regulate MR1 from the cell surface and retain MR1 molecules in the endoplasmic reticulum (ER) in an immature form. DB28 and NV18.1 compete with the known MR1 ligands, 5-OP-RU and acetyl-6-FP, for MR1 binding and inhibit MR1-dependent MAIT cell activation. Crystal structures of the MAIT T cell receptor (TCR) complexed with MR1-DB28 and MR1-NV18.1, show that these two ligands reside within the A'-pocket of MR1. Neither ligand forms a Schiff base with MR1 molecules; both are nevertheless sequestered by a network of hydrophobic and polar contacts. Accordingly, we define a class of compounds that inhibits MR1 cellular trafficking.
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http://dx.doi.org/10.1073/pnas.2003136117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229755PMC
May 2020

Results of a randomized, double-blind phase II clinical trial of NY-ESO-1 vaccine with ISCOMATRIX adjuvant versus ISCOMATRIX alone in participants with high-risk resected melanoma.

J Immunother Cancer 2020 04;8(1)

Ludwig Institute for Cancer Research Austin Branch, Heidelberg, Victoria, Australia.

Background: To compare the clinical efficacy of New York Esophageal squamous cell carcinoma-1 (NY-ESO-1) vaccine with ISCOMATRIX adjuvant versus ISCOMATRIX alone in a randomized, double-blind phase II study in participants with fully resected melanoma at high risk of recurrence.

Methods: Participants with resected stage IIc, IIIb, IIIc and IV melanoma expressing NY-ESO-1 were randomized to treatment with three doses of NY-ESO-1/ISCOMATRIX or ISCOMATRIX adjuvant administered intramuscularly at 4-week intervals, followed by a further dose at 6 months. Primary endpoint was the proportion free of relapse at 18 months in the intention-to-treat (ITT) population and two per-protocol populations. Secondary endpoints included relapse-free survival (RFS) and overall survival (OS), safety and NY-ESO-1 immunity.

Results: The ITT population comprised 110 participants, with 56 randomized to NY-ESO-1/ISCOMATRIX and 54 to ISCOMATRIX alone. No significant toxicities were observed. There were no differences between the study arms in relapses at 18 months or for median time to relapse; 139 vs 176 days (p=0.296), or relapse rate, 27 (48.2%) vs 26 (48.1%) (HR 0.913; 95% CI 0.402 to 2.231), respectively. RFS and OS were similar between the study arms. Vaccine recipients developed strong positive antibody responses to NY-ESO-1 (p≤0.0001) and NY-ESO-1-specific CD4 and CD8 responses. Biopsies following relapse did not demonstrate differences in NY-ESO-1 expression between the study populations although an exploratory study demonstrated reduced (NY-ESO-1)/Human Leukocyte Antigen (HLA) class I double-positive cells in biopsies from vaccine recipients performed on relapse in 19 participants.

Conclusions: The vaccine was well tolerated, however, despite inducing antigen-specific immunity, it did not affect survival endpoints. Immune escape through the downregulation of NY-ESO-1 and/or HLA class I molecules on tumor may have contributed to relapse.
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http://dx.doi.org/10.1136/jitc-2019-000410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7204806PMC
April 2020

Enhanced Immunogenicity of Mitochondrial-Localized Proteins in Cancer Cells.

Cancer Immunol Res 2020 05 23;8(5):685-697. Epub 2020 Mar 23.

MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom.

Epitopes derived from mutated cancer proteins elicit strong antitumor T-cell responses that correlate with clinical efficacy in a proportion of patients. However, it remains unclear whether the subcellular localization of mutated proteins influences the efficiency of T-cell priming. To address this question, we compared the immunogenicity of NY-ESO-1 and OVA localized either in the cytosol or in mitochondria. We showed that tumors expressing mitochondrial-localized NY-ESO-1 and OVA proteins elicit significantdly higher frequencies of antigen-specific CD8 T cells . We also demonstrated that this stronger immune response is dependent on the mitochondrial location of the antigenic proteins, which contributes to their higher steady-state amount, compared with cytosolic localized proteins. Consistent with these findings, we showed that injection of mitochondria purified from B16 melanoma cells can protect mice from a challenge with B16 cells, but not with irrelevant tumors. Finally, we extended these findings to cancer patients by demonstrating the presence of T-cell responses specific for mutated mitochondrial-localized proteins. These findings highlight the utility of prioritizing epitopes derived from mitochondrial-localized mutated proteins as targets for cancer vaccination strategies.
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http://dx.doi.org/10.1158/2326-6066.CIR-19-0467DOI Listing
May 2020

The Repertoire of Serous Ovarian Cancer Non-genetic Heterogeneity Revealed by Single-Cell Sequencing of Normal Fallopian Tube Epithelial Cells.

Cancer Cell 2020 02;37(2):226-242.e7

Ovarian Cancer Cell Laboratory, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK; Nuffield Department of Women's & Reproductive Health, University of Oxford, Oxford OX3 9DU, UK; Department of Gynecological Oncology, Churchill Hospital, Oxford University Hospitals, Oxford OX3 7LE, UK. Electronic address:

The inter-differentiation between cell states promotes cancer cell survival under stress and fosters non-genetic heterogeneity (NGH). NGH is, therefore, a surrogate of tumor resilience but its quantification is confounded by genetic heterogeneity. Here we show that NGH in serous ovarian cancer (SOC) can be accurately measured when informed by the molecular signatures of the normal fallopian tube epithelium (FTE) cells, the cells of origin of SOC. Surveying the transcriptomes of ∼6,000 FTE cells, predominantly from non-ovarian cancer patients, identified 6 FTE subtypes. We used subtype signatures to deconvolute SOC expression data and found substantial intra-tumor NGH. Importantly, NGH-based stratification of ∼1,700 tumors robustly correlated with survival. Our findings lay the foundation for accurate prognostic and therapeutic stratification of SOC.
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http://dx.doi.org/10.1016/j.ccell.2020.01.003DOI Listing
February 2020

The Chemical Synthesis, Stability, and Activity of MAIT Cell Prodrug Agonists That Access MR1 in Recycling Endosomes.

ACS Chem Biol 2020 02 14;15(2):437-445. Epub 2020 Jan 14.

The Ferrier Research Institute , Victoria University of Wellington , Wellington , New Zealand.

Mucosal-associated invariant T (MAIT) cells are antibacterial effector T cells that react to pyrimidines derived from bacterial riboflavin synthesis presented by the monomorphic molecule MR1. A major challenge in MAIT cell research is that the commonly used MAIT agonist precursor, 5-amino-6-d-ribitylaminouracil (5-A-RU), is labile to autoxidation, resulting in a loss of biological activity. Here, we characterize two independent autoxidation processes by LCMS. To overcome the marked instability, we report the synthesis of a 5-A-RU prodrug generated by modification of the 5-amino group with a cleavable valine-citrulline--aminobenzyl carbamate. The compound is stable in prodrug form, with the parent amine (i.e., 5-A-RU) released only after enzymatic cleavage. Analysis of the prodrug and showed an enhanced MAIT cell activation profile compared to 5-A-RU, which was associated with preferential loading within recycling endosomes, a route used by some natural agonists. This prodrug design therefore overcomes the difficulties associated with 5-A-RU in biological studies and provides an opportunity to explore different presentation pathways.
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http://dx.doi.org/10.1021/acschembio.9b00902DOI Listing
February 2020

Re-evaluation of human BDCA-2+ DC during acute sterile skin inflammation.

J Exp Med 2020 03;217(3)

Medical Research Council Human Immunology Unit, Radcliffe Department of Medicine, Oxford National Institute for Health Research Biomedical Research Centre, Medical Research Council Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

Plasmacytoid dendritic cells (pDCs) produce type I interferon (IFN-I) and are traditionally defined as being BDCA-2+CD123+. pDCs are not readily detectable in healthy human skin, but have been suggested to accumulate in wounds. Here, we describe a CD1a-bearing BDCA-2+CD123int DC subset that rapidly infiltrates human skin wounds and comprises a major DC population. Using single-cell RNA sequencing, we show that these cells are largely activated DCs acquiring features compatible with lymph node homing and antigen presentation, but unexpectedly express both BDCA-2 and CD123, potentially mimicking pDCs. Furthermore, a third BDCA-2-expressing population, Axl+Siglec-6+ DCs (ASDC), was also found to infiltrate human skin during wounding. These data demonstrate early skin infiltration of a previously unrecognized CD123intBDCA-2+CD1a+ DC subset during acute sterile inflammation, and prompt a re-evaluation of previously ascribed pDC involvement in skin disease.
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http://dx.doi.org/10.1084/jem.20190811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7062525PMC
March 2020

Interactions Between MAIT Cells and Dendritic Cells.

Methods Mol Biol 2020 ;2098:125-139

MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK.

Mucosal-associated invariant T (MAIT) cells recognize intermediates of the vitamin B2 biosynthetic pathway present in a variety of bacteria, presented by the monomorphic MR1 molecules. Because of their central role in shaping adaptive immunity through interaction with dendritic cells (DCs) and B cells, their manipulation can be of translational relevance. We describe a method to routinely isolate and maintain MAIT cells from peripheral blood and to investigate their activity using DC as targets.
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http://dx.doi.org/10.1007/978-1-0716-0207-2_8DOI Listing
January 2021

Self-Maintaining CD103 Cancer-Specific T Cells Are Highly Energetic with Rapid Cytotoxic and Effector Responses.

Cancer Immunol Res 2020 02 26;8(2):203-216. Epub 2019 Nov 26.

Chinese Academy of Medical Sciences (CAMS) Oxford Institute, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.

Enrichment of CD103 tumor-infiltrating T lymphocytes (TIL) is associated with improved outcomes in patients. However, the characteristics of human CD103 cytotoxic CD8 T cells (CTL) and their role in tumor control remain unclear. We investigated the features and antitumor mechanisms of CD103 CTLs by assessing T-cell receptor (TCR)-matched CD103 and CD103 cancer-specific CTL immunity and its immunophenotype Interestingly, we found that differentiated CD103 cancer-specific CTLs expressed the active form of TGFβ1 to continually self-regulate CD103 expression, without relying on external TGFβ1-producing cells. The presence of CD103 on CTLs improved TCR antigen sensitivity, which enabled faster cancer recognition and rapid antitumor cytotoxicity. These CD103 CTLs had elevated energetic potential and faster migration capacity. However, they had increased inhibitory receptor coexpression and elevated T-cell apoptosis following prolonged cancer exposure. Our data provide fundamental insights into the properties of matured human CD103 cancer-specific CTLs, which could have important implications for future designs of tissue-localized cancer immunotherapy strategies.
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http://dx.doi.org/10.1158/2326-6066.CIR-19-0554DOI Listing
February 2020

A Comprehensive Analysis of Key Immune Checkpoint Receptors on Tumor-Infiltrating T Cells From Multiple Types of Cancer.

Front Oncol 2019 25;9:1066. Epub 2019 Oct 25.

Nuffield Department of Medicine (NDM), Chinese Academy of Medical Sciences Oxford Institute (CAMS Oxford Institute), University of Oxford, Oxford, United Kingdom.

Cancer patients often display dysfunctional antitumor T-cell responses. Because noteworthy benefits of immune checkpoint pathway blockade, such as programmed cell death protein 1 (PD-1) inhibitors, have been achieved in multiple advanced cancers, the next critical question is which mono-blockade or combinatorial blockade regimens may reinvigorate antitumor T-cell immunity in those cancer patients while limiting immune-related adverse effects. This study recruited, in total, 172 primary cancer patients (131 were blood-tumor-matched patients) who were treatment-naïve prior to the surgeries or biopsies covering the eight most prevalent types of cancer. With access to fresh surgical samples, this study simultaneously investigated the expression level of eight known immune checkpoint receptors [PD-1, cytotoxic T-lymphocyte antigen-4 [CTLA-4], T-cell immunoglobulin and mucin-domain containing-3 [Tim-3], 2B4, killer cell lectin like receptor G1 [KLRG-1], TIGIT, B- and T-lymphocyte attenuator [BTLA], and CD160] on tumor-infiltrating T cells (TILs) and paired circulating T cells in blood from a 131-patient cohort. We found increased an expression of PD-1 and Tim-3 but a decreased expression of BTLA on TILs when compared with peripheral blood from multiple types of cancer. Moreover, our co-expression analysis of key immune checkpoint receptors delineates "shared" subsets as PD-1+Tim-3+TIGIT+2B4+KLRG-1-CTLA-4- and PD-1+TIGIT+2B4+Tim-3-KLRG-1-CTLA-4- from bulk CD8 TILs. Furthermore, we found that a higher frequency of advanced differentiation stage T cells (CD27-CCR7-CD45RA-) among the "shared" subset (PD-1+Tim-3+TIGIT+2B4+KLRG-1-CTLA-4-) in bulk CD8 TILs was associated with poorly differentiated cancer type in cervical cancer patients. To our knowledge, our study is the first comprehensive analysis of key immune checkpoint receptors on T cells in treatment-naïve, primary cancer patients from the eight most prevalent types of cancer. These findings might provide useful information for future design of mono-blockade/combinatorial blockades and/or genetically modified T-cell immunotherapy.
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http://dx.doi.org/10.3389/fonc.2019.01066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6823747PMC
October 2019

Sterile activation of invariant natural killer T cells by ER-stressed antigen-presenting cells.

Proc Natl Acad Sci U S A 2019 11 5;116(47):23671-23681. Epub 2019 Nov 5.

Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, OX3 9DS Oxford, United Kingdom;

Invariant NKT (iNKT) cells have the unique ability to shape immunity during antitumor immune responses and other forms of sterile and nonsterile inflammation. Recent studies have highlighted a variety of classes of endogenous and pathogen-derived lipid antigens that can trigger iNKT cell activation under sterile and nonsterile conditions. However, the context and mechanisms that drive the presentation of self-lipid antigens in sterile inflammation remain unclear. Here we report that endoplasmic reticulum (ER)-stressed myeloid cells, via signaling events modulated by the protein kinase RNA-like ER kinase (PERK) pathway, increase CD1d-mediated presentation of immunogenic endogenous lipid species, which results in enhanced iNKT cell activation both in vitro and in vivo. In addition, we demonstrate that actin cytoskeletal reorganization during ER stress results in an altered distribution of CD1d on the cell surface, which contributes to enhanced iNKT cell activation. These results define a previously unidentified mechanism that controls iNKT cell activation during sterile inflammation.
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http://dx.doi.org/10.1073/pnas.1910097116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6876220PMC
November 2019

Capturing the antigen landscape: HLA-E, CD1 and MR1.

Curr Opin Immunol 2019 08 21;59:121-129. Epub 2019 Aug 21.

Nuffield Department of Medicine Research Building, University of Oxford, UK. Electronic address:

T cell receptor (TCR) recognition of antigens presented by relatively non-polymorphic MHC-like molecules is emerging as a significant contributor to health and disease. These evolutionarily ancient pathways have been inappropriately labelled 'non-conventional' because their roles were discovered after viral-specific peptide presentation by polymorphic MHC class I molecules. We suggest that these pathways are complementary to mainstream peptide presentation. HLA-E, CD1 and MR1 can present diverse self and foreign antigens to TCRs and therefore contribute to tissue homeostasis, pathogen defence, inflammation and immune responses to cancer. Despite presenting different classes of antigens, they share many features and are under common selective pressures. Through understanding their roles in disease, therapeutic manipulation for disease prevention and treatment should become possible.
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http://dx.doi.org/10.1016/j.coi.2019.07.006DOI Listing
August 2019

Enriched HLA-E and CD94/NKG2A Interaction Limits Antitumor CD8 Tumor-Infiltrating T Lymphocyte Responses.

Cancer Immunol Res 2019 08 18;7(8):1293-1306. Epub 2019 Jun 18.

CAMS-Oxford International Centre for Translational Immunology, CAMS Oxford Institute, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.

Immunotherapy treatments with anti-PD-1 boost recovery in less than 30% of treated cancer patients, indicating the complexity of the tumor microenvironment. Expression of HLA-E is linked to poor clinical outcomes in mice and human patients. However, the contributions to immune evasion of HLA-E, a ligand for the inhibitory CD94/NKG2A receptor, when expressed on tumors, compared with adjacent tissue and peripheral blood mononuclear cells, remains unclear. In this study, we report that epithelial-derived cancer cells, tumor macrophages, and CD141 conventional dendritic cells (cDC) contributed to HLA-E enrichment in carcinomas. Different cancer types showed a similar pattern of enrichment. Enrichment correlated to NKG2A upregulation on CD8 tumor-infiltrating T lymphocytes (TIL) but not on CD4 TILs. CD94/NKG2A is exclusively expressed on PD-1 TILs while lacking intratumoral CD103 expression. We also found that the presence of CD94/NKG2A on human tumor-specific T cells impairs IL2 receptor-dependent proliferation, which affects IFNγ-mediated responses and antitumor cytotoxicity. These functionalities recover following antibody-mediated blockade and Our results suggest that enriched HLA-E:CD94/NKG2A inhibitory interaction can impair survival of PD-1 TILs in the tumor microenvironment.
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http://dx.doi.org/10.1158/2326-6066.CIR-18-0885DOI Listing
August 2019

Addendum: Precise tuning of gene expression levels in mammalian cells.

Nat Commun 2019 06 10;10(1):2622. Epub 2019 Jun 10.

Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, OX3 9DS, UK.

Following re-sequencing of the miSFIT constructs used in the paper, two of the construct variants inserted into the 3'UTR of PD-1, namely '12C' and '17A, 18G', have been found to contain additional insertions not present in the other construct variants. The data points corresponding to these constructs in Figs. 2c, f and Supplementary Fig. 9 are therefore no longer valid. However the overall conclusion that step-wise control over gene expression levels using the miSFIT constructs remains unaffected by these errors. Updated versions of Fig. 2 and Supplementary Fig. 9 are presented in the accompanying Addendum.
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http://dx.doi.org/10.1038/s41467-019-10615-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6557827PMC
June 2019

NOD2 and TLR2 Signal via TBK1 and PI31 to Direct Cross-Presentation and CD8 T Cell Responses.

Front Immunol 2019 30;10:958. Epub 2019 Apr 30.

MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom.

NOD2 and TLR2 recognize components of bacterial cell wall peptidoglycan and direct defense against enteric pathogens. CD8 T cells are important for immunity to such pathogens but how NOD2 and TLR2 induce antigen specific CD8 T cell responses is unknown. Here, we define how these pattern recognition receptors (PRRs) signal in primary dendritic cells (DCs) to influence MHC class I antigen presentation. We show NOD2 and TLR2 phosphorylate PI31 via TBK1 following activation in DCs. PI31 interacts with TBK1 and Sec16A at endoplasmic reticulum exit sites (ERES), which positively regulates MHC class I peptide loading and immunoproteasome stability. Following NOD2 and TLR2 stimulation, depletion of PI31 or inhibition of TBK1 activity impairs DC cross-presentation and CD8 T cell activation. DCs from Crohn's patients expressing polymorphisms show dysregulated cross-presentation and CD8 T cell responses. Our findings reveal unidentified mechanisms that underlie CD8 T cell responses to bacteria in health and in Crohn's.
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http://dx.doi.org/10.3389/fimmu.2019.00958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6503738PMC
September 2020

Behaviour and neuropathology in mice injected with human contactin-associated protein 2 antibodies.

Brain 2019 Jul;142(7):2000-2012

Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK.

Serum antibodies that bind to the surface of neurons or glia are associated with a wide range of rare but treatable CNS diseases. In many, if not most instances, the serum levels are higher than CSF levels yet most of the reported attempts to reproduce the human disease in mice have used infusion of antibodies into the mouse cerebral ventricle(s) or intrathecal space. We used the intraperitoneal route and injected purified plasma IgG from either a CASPR2-antibody-positive patient (n = 10 mice) or healthy individual (n = 9 mice) daily for 8 days. Lipopolysaccharide was injected intraperitoneally on Day 3 to cause a temporary breach in the blood brain barrier. A wide range of baseline behaviours, including tests of locomotion, coordination, memory, anxiety and social interactions, were established before the injections and tested from Day 5 until Day 11. At termination, brain tissue was analysed for human IgG, CASPR2 and c-fos expression, lymphocyte infiltration, and neuronal, astrocytic and microglial markers. Mice exposed to CASPR2-IgG, compared with control-IgG injected mice, displayed reduced working memory during the continuous spontaneous alternation test with trends towards reduced short-term and long-term memories. In the open field tests, activities were not different from controls, but in the reciprocal social interaction test, CASPR2-IgG injected mice showed longer latency to start interacting, associated with more freezing behaviour and reduced non-social activities of rearing and grooming. At termination, neuropathology showed more IgG deposited in the brains of CASPR2-IgG injected mice, but a trend towards increased CASPR2 expression; these results were mirrored in short-term in vitro experiments where CASPR2-IgG binding to hippocampal neurons and to CASPR2-transfected HEK cells led to some internalization of the IgG, but with a trend towards higher surface CASPR2 expression. Despite these limited results, in the CASPR2-IgG injected mouse brains there was increased c-fos expression in the piriform-entorhinal cortex and hypothalamus, and a modest loss of Purkinje cells. There was also increased microglia density, morphological changes in both microglia and astrocytes and raised complement C3 expression on astrocytes, all consistent with glial activation. Patients with CASPR2 antibodies can present with a range of clinical features reflecting central, autonomic and peripheral dysfunction. Although the behavioural changes in mice were limited to social interactions and mild working-memory defects, the neuropathological features indicate potentially widespread effects of the antibodies on different brain regions.
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http://dx.doi.org/10.1093/brain/awz119DOI Listing
July 2019

Cytoskeletal Control of Antigen-Dependent T Cell Activation.

Cell Rep 2019 03;26(12):3369-3379.e5

MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Headley Way, Oxford OX3 9DS, UK; Kennedy Institute for Rheumatology, University of Oxford, Roosevelt Drive, Oxford OX3 7LF, UK. Electronic address:

Cytoskeletal actin dynamics is essential for T cell activation. Here, we show evidence that the binding kinetics of the antigen engaging the T cell receptor influences the nanoscale actin organization and mechanics of the immune synapse. Using an engineered T cell system expressing a specific T cell receptor and stimulated by a range of antigens, we found that the peak force experienced by the T cell receptor during activation was independent of the unbinding kinetics of the stimulating antigen. Conversely, quantification of the actin retrograde flow velocity at the synapse revealed a striking dependence on the antigen unbinding kinetics. These findings suggest that the dynamics of the actin cytoskeleton actively adjusted to normalize the force experienced by the T cell receptor in an antigen-specific manner. Consequently, tuning actin dynamics in response to antigen kinetics may thus be a mechanism that allows T cells to adjust the lengthscale and timescale of T cell receptor signaling.
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http://dx.doi.org/10.1016/j.celrep.2019.02.074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6426652PMC
March 2019

Publisher Correction: Clonal analysis of Salmonella-specific effector T cells reveals serovar-specific and cross-reactive T cell responses.

Nat Immunol 2019 Apr;20(4):514

MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

In the version of this article initially published, the first affiliation lacked 'MRC'; the correct name of the institution is 'MRC Weatherall Institute of Molecular Medicine'. Two designations (SP110Y and ST110H) were incorrect in the legend to Fig. 6f,h,i. The correct text is as follows: for panel f, "...loaded with either the CdtB(105-125)SP110Y (DRB4*SP110Y) or the CdtB(105-125)ST110H (DRB4*ST110H) peptide variants..."; for panel h, "...decorated by the DRB4*SP110Y tetramer (lower-right quadrant), the DRB4*ST110H (upper-left quadrant)..."; and for panel i, "...stained ex vivo with DRB4*SP110Y, DRB4*ST110H...". In Fig. 8e, the final six residues (LTEAFF) of the sequence in the far right column of the third row of the table were missing; the correct sequence is 'CASSYRRTPPLTEAFF'. In the legend to Fig. 8d, a designation (HLyE) was incorrect; the correct text is as follows: "(HlyE?)." Portions of the Acknowledgements section were incorrect; the correct text is as follows: "This work was supported by the UK Medical Research Council (MRC) (MR/K021222/1) (G.N., M.A.G., A.S., V.C., A.J.P.),...the Oxford Biomedical Research Centre (A.J.P., V.C.),...and core funding from the Singapore Immunology Network (SIgN) (E.W.N.) and the SIgN immunomonitoring platform (E.W.N.)." Finally, a parenthetical element was phrased incorrectly in the final paragraph of the Methods subsection "T cell cloning and live fluorescence barcoding"; the correct phrasing is as follows: "...(which in all cases included HlyE, CdtB, Ty21a, Quailes, NVGH308, and LT2 strains and in volunteers T5 and T6 included PhoN)...". Also, in Figs. 3c and 4a, the right outlines of the plots were not visible; in the legend to Fig. 3, panel letter 'f' was not bold; and in Fig. 8f, 'ND' should be aligned directly beneath DRB4 in the key and 'ND' should be removed from the diagram at right, and the legend should be revised accordingly as follows: "...colors indicate the HLA class II restriction (gray indicates clones for which restriction was not determined (ND)). Clonotypes are grouped on the basis of pathogen selectivity (continuous line), protein specificity (dashed line) and epitope specificity; for ten HlyE-specific clones (pixilated squares), the epitope specificity was not determined...". The errors have been corrected in the HTML and PDF versions of the article.
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http://dx.doi.org/10.1038/s41590-019-0357-6DOI Listing
April 2019

Discovery of trehalose phospholipids reveals functional convergence with mycobacteria.

J Exp Med 2019 04 25;216(4):757-771. Epub 2019 Feb 25.

Department of Infectious Diseases and Immunology, School of Veterinary Medicine, Utrecht University, Utrecht, Netherlands

species are among the world's most prevalent pathogens. Because the cell wall interfaces with the host, we designed a lipidomics approach to reveal pathogen-specific cell wall compounds. Among the molecules differentially expressed between Paratyphi and Typhi, we focused on lipids that are enriched in Typhi, because it causes typhoid fever. We discovered a previously unknown family of trehalose phospholipids, 6,6'-diphosphatidyltrehalose (diPT) and 6-phosphatidyltrehalose (PT). Cardiolipin synthase B (ClsB) is essential for PT and diPT but not for cardiolipin biosynthesis. Chemotyping outperformed homology analysis in evaluating synthesis of diPT. DiPT is restricted to a subset of Gram-negative bacteria: large amounts are produced by Typhi, lower amounts by other pathogens, and variable amounts by strains. DiPT activates Mincle, a macrophage activating receptor that also recognizes mycobacterial cord factor (6,6'-trehalose dimycolate). Thus, Gram-negative bacteria show convergent function with mycobacteria. Overall, we discovered a previously unknown immunostimulant that is selectively expressed among medically important bacterial species.
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http://dx.doi.org/10.1084/jem.20181812DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446866PMC
April 2019

Precise tuning of gene expression levels in mammalian cells.

Nat Commun 2019 02 18;10(1):818. Epub 2019 Feb 18.

Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, OX3 9DS, UK.

Precise, analogue regulation of gene expression is critical for cellular function in mammals. In contrast, widely employed experimental and therapeutic approaches such as knock-in/out strategies are more suitable for binary control of gene activity. Here we report on a method for precise control of gene expression levels in mammalian cells using engineered microRNA response elements (MREs). First, we measure the efficacy of thousands of synthetic MRE variants under the control of an endogenous microRNA by high-throughput sequencing. Guided by this data, we establish a library of microRNA silencing-mediated fine-tuners (miSFITs) of varying strength that can be employed to precisely control the expression of user-specified genes. We apply this technology to tune the T-cell co-inhibitory receptor PD-1 and to explore how antigen expression influences T-cell activation and tumour growth. Finally, we employ CRISPR/Cas9 mediated homology directed repair to introduce miSFITs into the BRCA1 3'UTR, demonstrating that this versatile tool can be used to tune endogenous genes.
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http://dx.doi.org/10.1038/s41467-019-08777-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379387PMC
February 2019

NKG2A, a New Kid on the Immune Checkpoint Block.

Cell 2018 12;175(7):1720-1722

MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, UK.

One of the major breakthroughs of cancer immunotherapy has come from blocking immune checkpoint molecules on tumor-reactive T cells. Now, two studies examine targeting of a novel immune checkpoint, NKG2A, that can be expressed on both NK cells and on CD8 T cells, either combined with a tumor-targeting antibody or with a tumor-specific vaccine.
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http://dx.doi.org/10.1016/j.cell.2018.11.048DOI Listing
December 2018