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

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.

Exhibit Altered Cellular Localization in the Presence of HLA-B27 and Codistribute with Endo-Reticular Membrane.

() induce and require unfolded protein response (UPR) pathways for intracellular replication. infections can lead to reactive arthritis (ReA), which can exhibit associations with Human Leucocyte Antigen (HLA)-B27 : 05. normally reside in a juxtanuclear position to the Golgi apparatus, representing the formation and residence within the -containing vacuole (SCV).

Intrinsic Folding Properties of the HLA-B27 Heavy Chain Revealed by Single Chain Trimer Versions of Peptide-Loaded Class I Major Histocompatibility Complex Molecules.

Peptide-loaded Major Histocompatibility Complex (pMHC) class I molecules can be expressed in a single chain trimeric (SCT) format, composed of a specific peptide fused to the light chain beta-2 microglobulin (β2m) and MHC class I heavy chain (HC) by flexible linker peptides. pMHC SCTs have been used as effective molecular tools to investigate cellular immunity and represent a promising vaccine platform technology, due to their intracellular folding and assembly which is apparently independent of host cell folding pathways and chaperones. However, certain MHC class I HC molecules, such as the Human Leukocyte Antigen B27 (HLA-B27) allele, present a challenge due to their tendency to form HC aggregates.

Tumour-associated antigenic peptides are present in the HLA class I ligandome of cancer cell line derived extracellular vesicles.

The recent success of monoclonal antibody checkpoint inhibitor therapies that enhance the ability of CD8 T cells to detect cancer-related antigenic peptides has refocused the need to fully understand the repertoire of peptides being presented to the immune system. Whilst the peptide ligandome presented by cell surface human leucocyte antigen class I (HLA-I) molecules on cancer cells has been studied extensively, the ligandome of extracellular vesicles (EVs) remains poorly defined. Here, we report the HLA-I ligandome of both the cell surface and EVs from eight breast cancer cell lines (MCF7, MDA-MB-231, MDA-MB-361, MDA-MB-415, MDA-MB-453, HCC 1806, HCC 1395, and HCC 1954), and additionally the melanoma cell line ESTDAB-056 and the multiple myeloma line RPMI 8226.

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.

Real-time imaging of cellular forces using optical interference.

Important dynamic processes in mechanobiology remain elusive due to a lack of tools to image the small cellular forces at play with sufficient speed and throughput. Here, we introduce a fast, interference-based force imaging method that uses the illumination of an elastic deformable microcavity with two rapidly alternating wavelengths to map forces. We show real-time acquisition and processing of data, obtain images of mechanical activity while scanning across a cell culture, and investigate sub-second fluctuations of the piconewton forces exerted by macrophage podosomes.

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

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.

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 CD56CD3 natural killer (NK) cells which mediate cytotoxicity against tumors. Most cases are observed in children and adolescents with recurrent viral infections and cancer. and mutations are found in NKD patients with cancer.

Label-free optical hemogram of granulocytes enhanced by artificial neural networks.

An outstanding challenge for immunology is the classification of immune cells in a label-free fashion with high speed. For this purpose, optical techniques such as Raman spectroscopy or digital holographic microscopy have been used successfully to identify immune cell subsets. To achieve high accuracy, these techniques require a post-processing step using linear methods of multivariate processing, such as principal component analysis.

Measuring Synthesis and Degradation of MHC Class I Molecules.

Major histocompatibility complex (MHC) class I molecules function to present pathogen derived peptides to cytotoxic T cells and act as ligands for Natural Killer cells, thus alerting the immune system to the presence of invading pathogens. However, some MHC class I molecules, most notably HLA-B27, can be strongly associated with autoimmune diseases. In addition, the MHC class I pathway is a target for numerous viral evasion strategies Understanding not only the antigen presenting functions, but also the biosynthesis and the degradation pathways of MHC class I molecules has therefore become important in determining their role in pathogen and autoimmune related diseases.

Quantitative proteomic changes in LPS-activated monocyte-derived dendritic cells: A SWATH-MS study.

Dendritic cells are key immune cells that respond to pathogens and co-ordinate many innate and adaptive immune responses. Quantitative mass spectrometry using Sequential Window Acquisition of all THeoretical fragment-ion spectra-Mass Spectrometry (SWATH-MS) was performed here to determine the global alterations in monocyte-derived dendritic cells (moDCs) in response to stimulation with lipopolysaccharide (LPS). A moDC library of 4,666 proteins was generated and proteins were quantified at 0, 6 and 24 h post-LPS stimulation using SWATH-MS.

Non-obstructive intracellular nanolasers.

Molecular dyes, plasmonic nanoparticles and colloidal quantum dots are widely used in biomedical optics. Their operation is usually governed by spontaneous processes, which results in broad spectral features and limited signal-to-noise ratio, thus restricting opportunities for spectral multiplexing and sensing. Lasers provide the ultimate spectral definition and background suppression, and their integration with cells has recently been demonstrated.

exploits HLA-B27 and host unfolded protein responses to promote intracellular replication.

Objective: infections can lead to Reactive Arthritis (ReA), which can exhibit an association with human leucocyte antigen (HLA)-B*27:05, a molecule prone to misfolding and initiation of the unfolded protein response (UPR). This study examined how HLA-B*27:05 expression and the UPR affect the life-cycle within epithelial cells.

Methods: Isogenic epithelial cell lines expressing two copies of either HLA-B*27:05 and a control HLA-B*35:01 heavy chain (HC) were generated to determine the effect on the infection life-cycle.

The major histocompatibility complex class I immunopeptidome of extracellular vesicles.

Extracellular vesicles (EVs) are released by most cell types and have been associated with multiple immunomodulatory functions. MHC class I molecules have crucial roles in antigen presentation and in eliciting immune responses and are known to be incorporated into EVs. However, the MHC class I immunopeptidome of EVs has not been established.

Long-term imaging of cellular forces with high precision by elastic resonator interference stress microscopy.

Cellular forces are crucial for many biological processes but current methods to image them have limitations with respect to data analysis, resolution and throughput. Here, we present a robust approach to measure mechanical cell-substrate interactions in diverse biological systems by interferometrically detecting deformations of an elastic micro-cavity. Elastic resonator interference stress microscopy (ERISM) yields stress maps with exceptional precision and large dynamic range (2 nm displacement resolution over a >1 μm range, translating into 1 pN force sensitivity).

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

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.

Lasing in Live Mitotic and Non-Phagocytic Cells by Efficient Delivery of Microresonators.

Reliable methods to individually track large numbers of cells in real time are urgently needed to advance our understanding of important biological processes like cancer metastasis, neuronal network development and wound healing. It has recently been suggested to introduce microscopic whispering gallery mode lasers into the cytoplasm of cells and to use their characteristic, size-dependent emission spectrum as optical barcode but so far there is no evidence that this approach is generally applicable. Here, we describe a method that drastically improves intracellular delivery of resonators for several cell types, including mitotic and non-phagocytic cells.

Skin colour changes during experimentally-induced sickness.

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 2ng/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 8h in terms of lightness (L), redness (a) and yellowness (b) in a manner that is consistent with human colour perception.

Polymorphisms in the F Pocket of HLA-B27 Subtypes Strongly Affect Assembly, Chaperone Interactions, and Heavy-Chain Misfolding.

Objective: HLA-B27 is associated with the inflammatory spondyloarthritides (SpA), although subtypes HLA-B*27:06 and HLA-B*27:09 are not. These subtypes differ from the HLA-B*27:05 disease-associated allele primarily at residues 114 and 116 of the heavy chain, part of the F pocket of the antigen-binding groove. Dimerization of HLA-B27 during assembly has been implicated in disease onset.

Lasing within Live Cells Containing Intracellular Optical Microresonators for Barcode-Type Cell Tagging and Tracking.

We report on a laser that is fully embedded within a single live cell. By harnessing natural endocytosis of the cell, we introduce a fluorescent whispering gallery mode (WGM) microresonator into the cell cytoplasm. On pumping with nanojoule light pulses, green laser emission is generated inside the cells.

The use of wavelength modulated Raman spectroscopy in label-free identification of T lymphocyte subsets, natural killer cells and dendritic cells.

Determining the identity of cells of the immune system usually involves destructive fixation and chemical staining, or labeling with fluorescently labeled antibodies recognising specific cell surface markers. Completely label-free identification would be a significant advantage in conditions where untouched cells are a priority. We demonstrate here the use of Wavelength Modulated Raman Spectroscopy, to achieve label-free identification of purified, unfixed and untouched populations of major immune cell subsets isolated from healthy human donors.

CCCTC-binding factor recruitment to the early region of the human papillomavirus 18 genome regulates viral oncogene expression.

Unlabelled: Host cell differentiation-dependent regulation of human papillomavirus (HPV) gene expression is required for productive infection. The host cell CCCTC-binding factor (CTCF) functions in genome-wide chromatin organization and gene regulation. We have identified a conserved CTCF binding site in the E2 open reading frame of high-risk HPV types.

Endoplasmic reticulum degradation-enhancing α-mannosidase-like protein 1 targets misfolded HLA-B27 dimers for endoplasmic reticulum-associated degradation.

Objective: HLA-B27 forms misfolded heavy chain dimers, which may predispose individuals to inflammatory arthritis by inducing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). This study was undertaken to define the role of the UPR-induced ER-associated degradation (ERAD) pathway in the disposal of HLA-B27 dimeric conformers.

Methods: HeLa cell lines expressing only 2 copies of a carboxy-terminally Sv5-tagged HLA-B27 were generated.