EULAR study group on 'MHC-I-opathy': identifying disease-overarching mechanisms across disciplines and borders.

The 'MHC-I (major histocompatibility complex class I)-opathy' concept describes a family of inflammatory conditions with overlapping clinical manifestations and a strong genetic link to the MHC-I antigen presentation pathway. Classical MHC-I-opathies such as spondyloarthritis, Behçet's disease, psoriasis and birdshot uveitis are widely recognised for their strong association with certain MHC-I alleles and gene variants of the antigen processing aminopeptidases ERAP1 and ERAP2 that implicates altered MHC-I peptide presentation to CD8+T cells in the pathogenesis. Progress in understanding the cause and treatment of these disorders is hampered by patient phenotypic heterogeneity and lack of systematic investigation of the MHC-I pathway.Here, we discuss new insights into the biology of MHC-I-opathies that strongly advocate for disease-overarching and integrated molecular and clinical investigation to decipher underlying disease mechanisms. Because this requires transformative multidisciplinary collaboration, we introduce the EULAR study group on MHC-I-opathies to unite clinical expertise in rheumatology, dermatology and ophthalmology, with fundamental and translational researchers from multiple disciplines such as immunology, genomics and proteomics, alongside patient partners. We prioritise standardisation of disease phenotypes and scientific nomenclature and propose interdisciplinary genetic and translational studies to exploit emerging therapeutic strategies to understand MHC-I-mediated disease mechanisms. These collaborative efforts are required to address outstanding questions in the etiopathogenesis of MHC-I-opathies towards improving patient treatment and prognostication.

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. Utilizing HLA-I immunoisolation and mass spectrometry, we detected a total of 6574 peptides from the cell surface and 2461 peptides from the EVs of the cell lines studied. Within the EV HLA-I ligandome, we identified 150 peptides derived from tumour associated antigenic proteins, of which 19 peptides have been shown to elicit T-cell responses in previous studies. Our data thus show the prevalence of clinically relevant tumour-associated antigenic peptides in the HLA-I ligandome presented on EV.

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.

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

OBJECTIVE: Salmonella enterica 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 Salmonella 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 Salmonella infection life-cycle. A cell line expressing HLA-B*27:05.HC physically linked to the light chain beta-2-microglobulin and a specific peptide (referred to as a single chain trimer, SCT) was also generated to determine the effects of HLA-B27 folding status on S.enterica life-cycle. XBP-1 venus and AMP dependent Transcription Factor (ATF6)-FLAG reporters were used to monitor UPR activation in infected cells. Triacin C was used to inhibit de novo lipid synthesis during UPR, and confocal imaging of ER tracker stained membrane allowed quantification of glibenclamide-associated membrane. RESULTS: S.enterica demonstrated enhanced replication with an altered cellular localisation in the presence of HLA-B*27:05.HC but not in the presence of HLA-B*27:05.SCT or HLA-B*35:01. HLA-B*27:05.HC altered the threshold for UPR induction. Salmonella activated the UPR and required XBP-1 for replication, which was associated with endoreticular membrane expansion and lipid metabolism. CONCLUSIONS: HLA-B27 misfolding and a UPR cellular environment are associated with enhanced Salmonella replication, while Salmonella itself can activate XBP-1 and ATF6. These data provide a potential mechanism linking the life-cycle of Salmonella with the physicochemical properties of HLA-B27 and cellular events that may contribute to ReA pathogenesis. Our observations suggest that the UPR pathway maybe targeted for future therapeutic intervention.

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. Here we demonstrate for the first time a comparison between artificial neural networks and principal component analysis (PCA) to classify the key granulocyte cell lineages of neutrophils and eosinophils using both digital holographic microscopy and Raman spectroscopy. Artificial neural networks can offer advantages in terms of classification accuracy and speed over a PCA approach. We conclude that digital holographic microscopy with convolutional neural networks based analysis provides a route to a robust, stand-alone and high-throughput hemogram with a classification accuracy of 91.3 % at a throughput rate of greater than 100 cells per second.

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. Here, using a small-scale immunoisolation of the antigen serotypes HLA-A*02:01 and HLA-B*27:05 expressed on the Epstein-Barr virus-transformed B cell line Jesthom and MS of the eluted peptides from both cells and EVs, we identified 516 peptides that bind either HLA-A*02:01 or HLA-B*27:05. Of importance, the predicted serotype-binding affinities and peptide-anchor motifs did not significantly differ between the peptide pools isolated from cells or EVs, indicating that during EV biogenesis, no obvious editing of the MHC class I immunopeptidome occurs. These results, for the first time, establish EVs as a source of MHC class I peptides that can be used for the study of the immunopeptidome and in the discovery of potential neoantigens for immunotherapies.

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. 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 1-3h 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 3h 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.

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. Using organotypic raft cultures of primary human keratinocytes containing high-risk HPV18 genomes, we show that CTCF recruitment to this conserved site regulates viral gene expression in differentiating epithelia. Mutation of the CTCF binding site increases the expression of the viral oncoproteins E6 and E7 and promotes host cell proliferation. Loss of CTCF binding results in a reduction of a specific alternatively spliced transcript expressed from the early gene region concomitant with an increase in the abundance of unspliced early transcripts. We conclude that high-risk HPV types have evolved to recruit CTCF to the early gene region to control the balance and complexity of splicing events that regulate viral oncoprotein expression. IMPORTANCE: The establishment and maintenance of HPV infection in undifferentiated basal cells of the squamous epithelia requires the activation of a subset of viral genes, termed early genes. The differentiation of infected cells initiates the expression of the late viral transcripts, allowing completion of the virus life cycle. This tightly controlled balance of differentiation-dependent viral gene expression allows the virus to stimulate cellular proliferation to support viral genome replication with minimal activation of the host immune response, promoting virus productivity. Alternative splicing of viral mRNAs further increases the complexity of viral gene expression. In this study, we show that the essential host cell protein CTCF, which functions in genome-wide chromatin organization and gene regulation, is recruited to the HPV genome and plays an essential role in the regulation of early viral gene expression and transcript processing. These data highlight a novel virus-host interaction important for HPV pathogenicity.

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. Our approach can be applied to different cell types, and cells with microresonators remain viable for weeks under standard conditions. The characteristics of the lasing spectrum provide each cell with a barcode-type label which enables uniquely identifying and tracking individual migrating cells. Self-sustained lasing from cells paves the way to new forms of cell tracking, intracellular sensing, and adaptive imaging.

Monitoring the Rab27 associated exosome pathway using nanoparticle tracking analysis.

Exosomes are secreted by many cell types and display multiple biological functions. The ability to both rapidly detect and quantify exosomes in biological samples would assist in the screening of agents that interfere with their release, and which may therefore be of clinical relevance. Nanoparticle tracking analysis, which detects the size and concentration of exosomes, was used to monitor the inhibition of exosome secretion from MDA-MB-231 breast cancer cells expressing inhibitory RNA targeted for Rab27a, a known component of the exosome pathway. Inhibition of both Rab27a and Rab27b was observed, resulting in alterations to intracellular CD63+ compartments and the release of fewer exosomes into the culture medium, as determined by nanoparticle tracking analysis and confirmed by immunoblotting and protein quantification. These data show that nanoparticle tracking analysis can be used effectively and rapidly to monitor the disruption of exosome secretion.

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. 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.

The multi-faceted nature of HLA class I dimer molecules.

The canonical role of major histocompatibility complex class I (MHCI) molecules in antigen presentation involves the recognition of a short peptide of intracellular origin, bound to the upper surface of the class I molecule, by CD8(+) T lymphocytes. Assembly and loading of the MHCI is a highly regulated, chaperone-mediated process and only when the fully folded MHCI molecule is correctly loaded with peptide is it released from the endoplasmic reticulum for trafficking to the cell surface. Current models of the interactions of MHCI molecules with their cognate receptors visualize them functioning as monomeric entities. However, in recent years, new data have revealed MHCI molecules with the ability to form disulphide-linked dimeric structures, with several distinct dimer entities being elucidated. We describe here three types of MHCI dimers; HLA-B27 dimers formed predominantly through the possession of an unpaired cysteine within the peptide-binding groove; HLA-G dimers, which form through a cysteine on its external surface; and a novel population we term redox-induced dimers, which can form between cysteine residues in the cytoplasmic tail domains. The characteristics of these dimeric MHCI molecules and their role in both normal immune responses and in disease pathogenesis are reviewed in this article.

MHC class I dimer formation by alteration of the cellular redox environment and induction of apoptosis.

Many MHC class I molecules contain unpaired cysteine residues in their cytoplasmic tail domains, the function of which remains relatively uncharacterized. Recently, it has been shown that in the small secretory vesicles known as exosomes, fully folded MHC class I dimers can form through a disulphide bond between the cytoplasmic tail domain cysteines, induced by the low levels of glutathione in these extracellular vesicles. Here we address whether similar MHC class I dimers form in whole cells by alteration of the redox environment. Treatment of the HLA-B27-expressing Epstein-Barr virus-transformed B-cell line Jesthom, and the leukaemic T-cell line CEM transfected with HLA-B27 with the strong oxidant diamide, and the apoptosis-inducing and glutathione-depleting agents hydrogen peroxide and thimerosal, induced MHC class I dimers. Furthermore, induction of apoptosis by cross-linking FasR/CD95 on CEM cells with monoclonal antibody CH-11 also induced MHC class I dimers. As with exosomal MHC class I dimers, the formation of these structures on cells is controlled by the cysteine at position 325 in the cytoplasmic tail domain of HLA-B27. Therefore, the redox environment of cells intimately controls induction of MHC class I dimers, the formation of which may provide novel structures for recognition by the immune system.

Nanoparticle tracking analysis monitors microvesicle and exosome secretion from immune cells.

Nanoparticle tracking analysis permits the determination of both the size distribution and relative concentration of microvesicles, including exosomes, in the supernatants of cultured cells and biological fluids. We have studied the release of microvesicles from the human lymphoblastoid T-cell lines Jurkat and CEM. Unstimulated, both cell lines release microvesicles in the size range 70-90 nm, which can be depleted from the supernatant by ultracentrifugation at 100 000 g, and by anti-CD45 magnetic beads, and which by immunoblotting also contain the exosome-associated proteins Alix and Tsg101. Incubation with known potentiators of exosome release, the ionophores monensin and A23187, resulted in a significant increase in microvesicle release that was both time and concentration dependent. Mass spectrometric analysis of proteins isolated from ultracentrifuged supernatants of A23187-treated cells revealed the presence of exosome-associated proteins including heat-shock protein 90, tubulin, elongation factor α1, actin and glyceraldehyde 3-phosphate dehydrogenase. Additionally, treatment of peripheral blood monocyte-derived dendritic cells with bacterial lipopolysaccharide displayed an increase in secreted microvesicles. Consequently, nanoparticle tracking analysis can be effectively applied to monitor microvesicle release from cells of the immune system.

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

Salmonella enteritica (S. enteritica) induce and require unfolded protein response (UPR) pathways for intracellular replication. Salmonella infections can lead to reactive arthritis (ReA), which can exhibit associations with Human Leucocyte Antigen (HLA)-B∗27 : 05. S. enteritica normally reside in a juxtanuclear position to the Golgi apparatus, representing the formation and residence within the Salmonella-containing vacuole (SCV). Changes in cellular localization of infecting Salmonella can alter their ability to replicate. We therefore used isogenic epithelial cell lines expressing physiological levels of HLA-B∗27 : 05 heavy chain (HC) and a control HLA-B allele, HLA-B∗35 : 01.HC to determine any changes in Salmonella localization within epithelial cells. Expression of HLA-B∗27 : 05 but not HLA-B∗35 : 01 was associated with a quantifiable change in S. enteritica cellular distribution away from the Golgi apparatus. Furthermore, the Salmonella requirements for UPR induction and the consequences of the concomitant endoplasmic reticulum (ER) membrane expansion were determined. Using confocal imaging, S. enteritica bacteria exhibited a significant and quantifiable codistribution with endo-reticular membrane as determined by ER tracker staining. Isogenic S. enterica Typhimurium mutant strains, which can infect but exhibit impaired intracellular growth, demonstrated that the activation of the UPR was dependent on an integral intracellular niche. Therefore, these data identify cellular changes accompanying Salmonella induction of the UPR and in the presence of HLA-B27.

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 (ß2m) 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. We constructed a series of single chain trimeric molecules to determine the behaviour of the HLA-B27 HC in a scenario that usually allows for efficient MHC class I molecule folding. When stably expressed, a pMHC SCT incorporating HLA-B27 HC formed chaperone-bound homodimers within the endoplasmic reticulum (ER). A series of HLA-B27 SCT substitution mutations revealed that the F pocket and antigen binding groove regions of the HLA-B27 HC defined the folding and dimerisation of the single chain complex, independently of the peptide sequence. Furthermore, pMHC SCTs can demonstrate variability in their association with the intracellular antigen processing machinery.

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

Calreticulin (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.

Expression of MHC class I dimers and ERAP1 in an ankylosing spondylitis patient cohort.

The human leucocyte antigen HLA-B27 is strongly associated with ankylosing spondylitis, a form of seronegative inflammatory arthritis. In this study aspects related to several hypothesized mechanisms of disease pathogenesis have been investigated. Blood monocyte-derived dendritic cells (DC) from a small patient cohort of 29 patients with ankylosing spondylitis and one with reactive arthritis, were compared with DC from 34 healthy control subjects, of whom four were found to be HLA-B27 positive. The ability of HLA-B27 to form heavy-chain dimers reactive with monoclonal antibody HC10 was tested, along with the induction of endoplasmic reticulum (ER) stress, assessed by splicing xbp1 mRNA and immunoblotting of Immunoglobulin Binding Protein (BiP). Additionally, the protein expression levels of the ER resident aminopeptidase gene ERAP1 in patients with ankylosing spondylitis was also determined, following its recent identification as a novel disease-associated gene. No significant difference was noted in the global levels of HC10-reactive MHC class I dimers formed in either the patient or control DC populations. Stress on the ER, as determined by xbp1 mRNA splicing, was not detected but lower levels of BiP were observed in the DC from patients. Of further potential interest, in this patient cohort the expression of ERAP1 appeared to be higher in a number of patient DC samples when compared with controls, suggesting over-expression of ERAP1 as a mechanism promoting ankylosing spondylitic pathogenesis.

Novel MHC class I structures on exosomes.

Exosomes are nanometer-sized vesicles released by a number of cell types including those of the immune system, and often contain numerous immune recognition molecules including MHC molecules. We demonstrate in this study that exosomes can display a significant proportion of their MHC class I (MHC I) content in the form of disulfide-linked MHC I dimers. These MHC I dimers can be detected after release from various cell lines, human monocyte-derived dendritic cells, and can also be found in human plasma. Exosome-associated dimers exhibit novel characteristics which include 1) being composed of folded MHC I, as detected by conformational-dependent Abs, and 2) dimers forming between two different MHC I alleles. We show that dimer formation is mediated through cysteine residues located in the cytoplasmic tail domains of many MHC I molecules, and is associated with a low level of glutathione in exosomes when compared with whole cell lysates. We propose these exosomal MHC I dimers as novel structures for recognition by immune receptors.

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. We also demonstrate force imaging of beating neonatal cardiomyocytes at 100 fps which reveals mechanical aspects of spontaneous oscillatory contraction waves in between the main contraction cycles. These examples illustrate the wider potential of our technique for monitoring cellular forces with high throughput and excellent temporal resolution.