Simon J. Powis

HLA-E is expressed on trophoblast and interacts with CD94/NKG2 receptors on decidual NK cells

Non‐classical MHC class I molecule HLA‐E is the ligand for CD94 / NKG2 NK cell receptors. Surface expression of HLA‐E requires binding of specific HLA class I leader sequences. The uterine mucosa in early pregnancy (decidua) is infiltrated by large numbers of NK cells, which are closely associated with placental trophoblast cells. In this study we demonstrate that trophoblast cells express HLA‐E on their cell surface in addition to the previously reported expression of HLA‐G and HLA‐C. Furthermore, we show that the vast majority of decidual NK cells bind to HLA‐E tetrameric complexes and this binding is inhibited by mAb to CD94. Thus, recognition of fetal HLA‐E by decidual NK cells may play a key role in regulation of placentation. The functional consequences of decidual NK cell interaction were investigated in cytotoxicity assays using polyclonal decidual NK cells. The overall effect of CD94 / NKG2 interaction with HLA‐E is inhibition of cytotoxicity by decidual NK cells. However, since decidual NK cells are unable to kill trophoblast even in the presence of mAb to MHC class I molecules and NK cell receptors, HLA‐E interaction with CD94 / NKG2 receptors may regulate other functions besides cytolysis during implantation.

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

An 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.

The rat cim effect: TAP allele-dependent changes in a class I MHC anchor motif and evidence against C-terminal trimming of peptides in the ER.

Functional polymorphism in the rat peptide transporter associated with antigen processing (TAP) changes the peptide pool available for binding and presentation by a class I MHC allele, RT1.Aa. The peptide binding motif for RT1.Aa, determined by stabilization with synthetic peptides, included a strong preference for arginine at the peptide C terminus. Analysis of natural peptides bound to RT1.Aa by both pool sequencing and anhydrotrypsin chromatography revealed that TAP polymorphism determined the presence or absence of arginine as the peptide C-terminal residue. This result highlights the in vivo impact of TAP-peptide selectivity, and provides evidence against a high rate of generation of new C termini by protease activity in the endoplasmic reticulum.

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.

The oxidoreductase ERp57 efficiently reduces partially folded in preference to fully folded MHC class I molecules

The oxidoreductase ERp57 is an integral component of the peptide loading complex of major histocompatibility complex (MHC) class I molecules, formed during their chaperone‐assisted assembly in the endoplasmic reticulum. Misfolded MHC class I molecules or those denied suitable peptides are retrotranslocated and degraded in the cytosol. The presence of ERp57 during class I assembly suggests it may be involved in the reduction of intrachain disulfides prior to retrotranslocation. We have studied the ability of ERp57 to reduce MHC class I molecules in vitro. Recombinant ERp57 specifically reduced partially folded MHC class I molecules, whereas it had little or no effect on folded and peptide‐loaded MHC class I molecules. Reductase activity was associated with cysteines at positions 56 and 405 of ERp57, the N‐terminal residues of the active CXXC motifs. Our data suggest that the reductase activity of ERp57 may be involved during the unfolding of MHC class I molecules, leading to targeting for degradation.

Assembly and export of MHC class I peptide ligands

During the assembly of MHC class I molecules with peptide, a series of transient interactions are made with endoplasmic reticulum-resident chaperones and MHC class I-specific accessory molecules. These interactions culminate in the trafficking of MHC class I molecules to the cell surface and presentation of peptide to CD8+ T lymphocytes. Recent studies have revealed just how important these early interactions are, and how they influence the quality control of assembly and the optimisation of peptide–ligand binding.

LMP2+ proteasomes are required for the presentation of specific antigens to cytotoxic T lymphocytes

BACKGROUND Major histocompatibility complex (MHC) class I molecules present short peptides generated by intracellular protein degradation to cytotoxic T lymphocytes (CTL). The multisubunit, non-lysosomal proteinases known as proteasomes have been implicated in the generation of these peptides. Two interferon-gamma (IFN-gamma)-inducible proteasome subunits, LMP2 and LMP7, are encoded within the MHC gene cluster in a region associated with antigen presentation. The incorporation of these LMP subunits into proteasomes may alter their activity so as to favour the generation of peptides able to bind to MHC class I molecules. It has been difficult, however, to demonstrate a specific requirement for LMP2 or LMP7 in the presentation of peptide epitopes to CTL. RESULTS We describe a T-cell lymphoma, termed SP3, that displays a novel selective defect in MHC class I-restricted presentation of influenza virus antigens. Of the MHC-encoded genes implicated in the class I pathway, only LMP2 is underexpressed in SP3 cells. Expression of IFN-gamma in transfected SP3 cells simultaneously restores LMP2 expression and antigen presentation to CTL. Expression of antisense-LMP2 mRNA in these IFN-gamma-transfected cells selectively represses antigen recognition and the induction of surface class I MHC expression. Moreover, the expression of this antisense-LMP2 mRNA in L929 fibroblast cells, which constitutively express LMP2 and have no presentation defect, blocks the presentation of the same influenza virus antigens that SP3 cells are defective in presenting. CONCLUSIONS Our results show that the LMP2 proteasome subunit can directly influence both MHC class I-restricted antigen presentation and class I surface expression.

Regulation of exosome release from mammary epithelial and breast cancer cells - a new regulatory pathway

PURPOSE Exosomes are small 50-100nm sized extracellular vesicles released from normal and tumour cells and are a source of a new intercellular communication pathway. Tumour exosomes promote tumour growth and progression. What regulates the release and homoeostatic levels of exosomes, in cancer, in body fluids remains undefined. METHODS We utilised a human mammary epithelial cell line (HMEC B42) and a breast cancer cell line derived from it (B42 clone 16) to investigate exosome production and regulation. Exosome numbers were quantified using a Nanosight LM10 and measured in culture supernatants in the absence and presence of exosomes in the medium. Concentrated suspensions of exosomes from the normal mammary epithelial cells, the breast cancer cells and bladder cancer cells were used. The interaction of exosomes with tumour cells was also investigated using fluorescently labelled exosomes. RESULTS Exosome release from normal human mammary epithelial cells and breast cancer cells is regulated by the presence of exosomes, derived from their own cells, in the extracellular environment of the cells. Exosomes from normal mammary epithelial cells also inhibit exosome secretion by breast cancer cells, which occurs in a tissue specific manner. Labelled exosomes from mammary epithelial cells are internalised into the tumour cells implicating a dynamic equilibrium and suggesting a mechanism for feedback control. CONCLUSIONS These data suggest a previously unknown novel feedback regulatory mechanism for controlling exosome release, which may highlight a new therapeutic approach to controlling the deleterious effects of tumour exosomes. This regulatory mechanism is likely to be generic to other tumours.

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.

Aberrant NF-KappaB Expression in Autism Spectrum Condition: A Mechanism for Neuroinflammation

Autism spectrum condition (ASC) is recognized as having an inflammatory component. Post-mortem brain samples from patients with ASC display neuroglial activation and inflammatory markers in cerebrospinal fluid, although little is known about the underlying molecular mechanisms. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a protein found in almost all cell types and mediates regulation of immune response by inducing the expression of inflammatory cytokines and chemokines, establishing a feedback mechanism that can produce chronic or excessive inflammation. This article describes immunodetection and immunofluorescence measurements of NF-κB in human post-mortem samples of orbitofrontal cortex tissue donated to two independent centers: London Brain Bank, Kings College London, UK (ASC: n = 3, controls: n = 4) and Autism Tissue Program, Harvard Brain Bank, USA (ASC: n = 6, controls: n = 5). The hypothesis was that concentrations of NF-κB would be elevated, especially in activated microglia in ASC, and pH would be concomitantly reduced (i.e., acidification). Neurons, astrocytes, and microglia all demonstrated increased extranuclear and nuclear translocated NF-κB p65 expression in brain tissue from ASC donors relative to samples from matched controls. These between-groups differences were increased in astrocytes and microglia relative to neurons, but particularly pronounced for highly mature microglia. Measurement of pH in homogenized samples demonstrated a 0.98-unit difference in means and a strong (F = 98.3; p = 0.00018) linear relationship to the expression of nuclear translocated NF-κB in mature microglia. Acridine orange staining localized pH reductions to lysosomal compartments. In summary, NF-κB is aberrantly expressed in orbitofrontal cortex in patients with ASC, as part of a putative molecular cascade leading to inflammation, especially of resident immune cells in brain regions associated with the behavioral and clinical symptoms of ASC.