A role for the thiol-dependent reductase ERp57 in the assembly of MHC class I molecules

AbstractAn important mammalian defence strategy against intracellular pathogens is the presentation of cytoplasmically derived short peptides by major histocompatibility complex (MHC) class I molecules to cytotoxic T lymphocytes. MHC class I molecules assemble in the endoplasmic reticulum (ER) with chaperones, including calnexin and calreticulin, before binding to the transporter associated with antigen processing (TAP). We show here that the thiol-dependent reductase ERp57 (also known as ER60 protease) is involved in MHC class I assembly. ERp57 co-purified with the rat TAP complex (comprising TAP1 and TAP2), and associated with MHC class I molecules at an early stage in their biosynthesis. This association was sensitive to castanospermine, which inhibits the processing of glycoproteins. Human MHC class I molecules were also found to associate with ERp57. We conclude that ERp57 is a newly identified component of the MHC class I pathway, and that it appears to interact with MHC class I molecules before they associate with TAP

Suppression of MHC class I surface expression by calreticulin's P-domain in a calreticulin deficient cell line

AbstractCalreticulin (CRT) is an important chaperone protein, comprising an N-domain, P-domain and C-domain. It is involved in the folding and assembly of multi-component protein complexes in the endoplasmic reticulum, and plays a critical role in MHC class I antigen processing and presentation. To dissect the functional role and molecular basis of individual domains of the protein, we have utilized individual domains to rescue impaired protein assembly in a CRT deficient cell line. Unexpectedly, both P-domain fragment and NP domain of CRT not only failed to rescue defective cell surface expression of MHC class I molecules but further inhibited their appearance on the surface of cells. Formation of the TAP-associated peptide-loading complex and trafficking of the few detectable MHC class I molecules were not significantly impaired. Instead, this further suppression of MHC class I molecules on the cell surface appears due to the complex missing antigenic peptides, the third member of fully assembled MHC class I molecules. Therefore the P-domain of calreticulin appears to play a significant role in antigen presentation by MHC class I molecules

What is the role of HLA-I on cancer derived extracellular vesicles? Defining the challenges in characterisation and potential uses of this ligandome

The Human Leukocyte Antigen class I (HLA-I) system is an essential part of the immune system that is fundamental to the successful activation of cytotoxic lymphocytes, and an effective subsequent immune attack against both pathogen-infected and cancer cells. The importance of cytotoxic T cell activity and ability to detect foreign cancer-related antigenic peptides has recently been highlighted by the successful application of monoclonal antibody-based checkpoint inhibitors as novel immune therapies. Thus, there is an increased interest in fully characterising the repertoire of peptides that are being presented to cytotoxic CD8+ T cells by cancer cells. However, HLA-I is also known to be present on the surface of extracellular vesicles, which are released by most if not all cancer cells. Whilst the peptide ligandome presented by cell surface HLA class I molecules on cancer cells has been studied extensively, the ligandome of extracellular vesicles remains relatively poorly defined. Here, we will describe the current understanding of the HLA-I peptide ligandome and its role on cancer-derived extracellular vesicles, and evaluate the aspects of the system that have the potential to advance immune-based therapeutic approaches for the effective treatment of cancer.Publisher PDFPeer reviewe

Deep learning enabled laser speckle wavemeter with a high dynamic range

Funding: This work was supported by a Medical Research Scotland PhD studentship PhD 873-2015 awarded to R.K.G, and grant funding from Leverhulme Trust (RPG-2017-197) and UK Engineering and Physical Sciences Research Council (grant EP/P030017/1).The speckle pattern produced when a laser is scattered by a disordered medium has recently been shown to give a surprisingly accurate or broadband measurement of wavelength. Here it is shown that deep learning is an ideal approach to analyse wavelength variations using a speckle wavemeter due to its ability to identify trends and overcome low signal to noise ratio in complex datasets. This combination enables wavelength measurement at high precision over a broad operating range in a single step, with a remarkable capability to reject instrumental and environmental noise, which has not been possible with previous approaches. It is demonstrated that the noise rejection capabilities of deep learning provide attometre-scale wavelength precision over an operating range from 488 nm to 976 nm. This dynamic range is six orders of magnitude beyond the state of the art.Publisher PDFPeer reviewe

Does natural killer cell deficiency (NKD) increase the risk of cancer? NKD may increase the risk of some virus induced cancer

Natural killer cell deficiency (NKD) is a primary immunodeficiency where the main defect lies in CD56+CD3− natural killer (NK) cells which mediate cytotoxicity against tumors. Most cases are observed in children and adolescents with recurrent viral infections and cancer. GATA2 and MCM4 mutations are found in NKD patients with cancer. However, the question remains unclear whether NKD increases the risk of cancer. Mutations in the second zinc finger of GATA2 cause both NKD and haematopoietic malignancies. MCM4 splice site mutations are found in NKD patients and they increase susceptibility to DNA instability during replication. IRF8, RTEL1, and FCGR3A mutations are associated with NKD but their associations with cancer are unknown. Based on the studies, it is hypothesized that genetic mutations alone are sufficient to cause cancer. However, a number of NKD patients developed oncogenic viral infections which progressed into cancer. Here, we review the evidence of genetic mutations responsible for both NKD and cancer to identify whether NKD contributes to development of cancer. The findings provide insights into the role of NK cells in the prevention of cancer and the significance of assessing NK cell functions in susceptible individuals.Publisher PDFPeer reviewe

Multimodal discrimination of immune cells using a combination of Raman spectroscopy and digital holographic microscopy

This work was supported by the UK Engineering and Physical Sciences Research Council under grant EP/J01771X/1, A European Union FAMOS project (FP7 ICT, 317744), and the ’BRAINS’ 600th anniversary appeal, and Dr. E. Killick. We would also like to thank The RS Macdonald Charitable Trust for funding support. KD acknowledges support of a Royal Society Leverhulme Trust Senior Fellowship. This work was also supported by the PreDiCT-TB consortium [IMI Joint undertaking grant agreement number 115337, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in kind contribution (www.imi.europa.eu)]The ability to identify and characterise individual cells of the immune system under label-free conditions would be a significant advantage in biomedical and clinical studies where untouched and unmodified cells are required. We present a multi-modal system capable of simultaneously acquiring both single point Raman spectra and digital holographic images of single cells. We use this combined approach to identify and discriminate between immune cell populations CD4+ T cells, B cells and monocytes. We investigate several approaches to interpret the phase images including signal intensity histograms and texture analysis. Both modalities are independently able to discriminate between cell subsets and dual-modality may therefore be used a means for validation. We demonstrate here sensitivities achieved in the range of 86.8% to 100%, and specificities in the range of 85.4% to 100%. Additionally each modality provides information not available from the other providing both a molecular and a morphological signature of each cell.Publisher PDFPeer reviewe

Induction of HLA-B27 heavy chain homodimer formation after activation in dendritic cells

Introduction Ankylosing spondylitis (AS) is a severe, chronic inflammatory arthritis, with a strong association to the human major histocompatibilty complex (MHC) class I allele human leucocyte antigen (HLA) B27. Disulfide-linked HLA-B27 heavy-chain homodimers have been implicated as novel structures involved in the aetiology of AS. We have studied the formation of HLA-B27 heavy-chain homodimers in human dendritic cells, which are key antigen-presenting cells and regulators of mammalian immune responses. Method Both an in vitro dendritic-like cell line and monocyte-derived dendritic cells from peripheral blood were studied. The KG-1 dendritic-like cell line was transfected with HLA-B27 cDNA constructs, and the cellular distribution, intracellular assembly and ability of HLA-B27 to form heavy-chain homodimers was compared with human monocyte-derived dendritic cells after stimulation with bacterial lipopolysaccharide (LPS). Results Immature KG-1 cells expressing HLA-B27 display an intracellular source of MHC class I heavy-chain homodimers partially overlapping with the Golgi bodies, but not the endoplasmic reticulum, which is lost at cell maturation with phorbyl-12-myristate-13-acetate (PMA) and ionomycin. Significantly, the formation of HLA-B27 homodimers in transfected KG-1 cells is induced by maturation, with a transient induction also seen in LPS-stimulated human monocyte-derived dendritic cells expressing HLA-B27. The weak association of wildtype HLA-B*2705 with the transporter associated with antigen processing could also be enhanced by mutation of residues at position 114 and 116 in the peptide-binding groove to those present in the HLA-B*2706 allele. Conclusion We have demonstrated that HLA-B27 heavy-chain homodimer formation can be induced by dendritic cell activation, implying that these novel structures may not be displayed to the immune system at all times. Our data suggests that the behaviour of HLA-B27 on dendritic cells may be important in the study of inflammatory arthritis.</p

Skin colour changes during experimentally-induced sickness

This project was supported by Swedish foundation for humanities and social sciences and a British Academy Wolfson Foundation Research Professorship grant. AH is supported by a studentship from the Biotechnology and Biological Sciences Research Council.Skin colour may be an important cue to detect sickness in humans but how skin colour changes with acute sickness is currently unknown. To determine possible colour changes, 22 healthy Caucasian participants were injected twice, once with lipopolysaccharide (LPS, at a dose of 2 ng/kg body weight) and once with placebo (saline), in a randomised cross-over design study. Skin colour across 3 arm and 3 face locations was recorded spectrophotometrically over a period of 8 hours in terms of lightness (L∗), redness (a∗) and yellowness (b∗) in a manner that is consistent with human colour perception. In addition, carotenoid status was assessed as we predicted that a decrease it skin yellowness would reflect a drop in skin carotenoids. We found an early change in skin colouration 1-3 hours post LPS injection with facial skin becoming lighter and less red whilst arm skin become darker but also less red and less yellow. The LPS injection also caused a drop in plasma carotenoids from 3 hours onwards. However, the timing of the carotenoid changes was not consistent with the skin colour changes suggesting that other mechanisms, such as a reduction of blood perfusion, oxygenation or composition. This is the first experimental study characterising skin colour associated with acute illness, and shows that changes occur early in the development of the sickness response. Colour changes may serve as a cue to health, prompting actions from others in terms of care-giving or disease avoidance. Specific mechanisms underlying these colour changes require further investigation.PostprintPeer reviewe

High-content screening image dataset and quantitative image analysis of Salmonella infected human cells

This work was supported by the Medical Research Council Core funding the MRC LMCB (MC_U12266B) (JKV) and the EU FP7 Marie-Curie International Reintegration Grant PIRG08-GA-2010-276811 (JKV). ANA was funded by ARUK Fellowships Non-Clinical Career Development Fellowship Ref No: 18440. ANA and SJP were also in part funded by ARUK (Grant 21261).Objectives Salmonella bacteria can induce the unfolded protein response, a cellular stress response to misfolding proteins within the endoplasmic reticulum. Salmonella can exploit the host unfolded protein response leading to enhanced bacterial replication which was in part mediated by the induction and/or enhanced endo-reticular membrane synthesis. We therefore wanted to establish a quantitative confocal imaging assay to measure endo-reticular membrane expansion following Salmonella infections of host cells. Data description High-content screening confocal fluorescence microscopic image set of Salmonella infected HeLa cells is presented. The images were collected with a PerkinElmer Opera LX high-content screening system in seven 96-well plates, 50 field-of-views and DAPI, endoplasmic reticulum tracker channels and Salmonella mCherry protein in each well. Totally 93,300 confocal fluorescence microscopic images were published in this dataset. An ImageJ high-content image analysis workflow was used to extract features. Cells were classified as infected and non-infected, the mean intensity of endoplasmic reticulum tracker under Salmonella bacteria was calculated. Statistical analysis was performed by an R script, quantifying infected and non-infected cells for wild-type and ΔsifA mutant cells. The dataset can be further used by researchers working with big data of endoplasmic reticulum fluorescence microscopic images, Salmonella bacterial infection images and human cancer cells.Publisher PDFPeer reviewe

Characterising the HLA-I Immunopeptidome of plasma-derived extracellular vesicles in patients with melanoma

This work was funded by grants from Breast Cancer Now UK (2018JulPR1086), and the Melville Trust for the Care and Cure of Cancer UK (XCT014). We also gratefully acknowledge funding from the EPSRC via EP/L017008/1 for TEM imaging infrastructure, and EP/R023751/1 and EP/T019298/1.Extracellular vesicles (EVs) frequently express human leukocyte antigen class I (HLA-I) molecules. The immunopeptidomes presented on EV HLA-I are being mapped to provide key information on both specific cancer-related peptides, and for larger immunopeptidomic signatures associated with disease. Utilizing HLA-I immunoisolation and mass spectrometry, we characterised the HLA-I immunopeptidome of EVs derived from the melanoma cancer cell line, ESTDAB-026, and the plasma of 12 patients diagnosed with advanced stage melanoma, alongside 11 healthy controls. The EV HLA-I immunopeptidome derived from melanoma cells features T cell epitopes with known immunogenicity and peptides derived from known tumour associated antigens (TAAs). Both T cell epitopes with known immunogenicity and peptides derived from known TAAs were also identifiable in the melanoma patient samples. Patient stratification into two distinct groups with varying immunological profiles was also observed. The data obtained in this study suggests for the first time that the HLA-I immunopeptidome of EVs derived from blood may aid in the detection of important diagnostic or prognostic biomarkers and also provide new immunotherapy targets.Peer reviewe