T-cell libraries allow simple parallel generation of multiple peptide-specific human T-cell clones

Isolation of peptide-specific T-cell clones is highly desirable for determining the role of T-cells in human disease, as well as for the development of therapies and diagnostics. However, generation of monoclonal T-cells with the required specificity is challenging and time-consuming. Here we describe a library-based strategy for the simple parallel detection and isolation of multiple peptide-specific human T-cell clones from CD8+ or CD4+ polyclonal T-cell populations. T-cells were first amplified by CD3/CD28 microbeads in a 96U-well library format, prior to screening for desired peptide recognition. T-cells from peptide-reactive wells were then subjected to cytokine-mediated enrichment followed by single-cell cloning, with the entire process from sample to validated clone taking as little as 6 weeks. Overall, T-cell libraries represent an efficient and relatively rapid tool for the generation of peptide-specific T-cell clones, with applications shown here in infectious disease (Epstein–Barr virus, influenza A, and Ebola virus), autoimmunity (type 1 diabetes) and cancer

Distortion of the major histocompatibility complex class I binding groove to accommodate an insulin-derived 10-Mer peptide

The non-obese diabetic mouse model of type 1 diabetes continues to be an important tool for delineating the role of T-cell-mediated destruction of pancreatic β-cells. However, little is known about the molecular mechanisms that enable this disease pathway. We show that insulin reactivity by a CD8+ T-cell clone, known to induce type 1 diabetes, is characterized by weak T-cell antigen receptor binding to a relatively unstable peptide-MHC. The structure of the native 9- and 10-mer insulin epitopes demonstrated that peptide residues 7 and 8 form a prominent solvent-exposed bulge that could potentially be the main focus of T-cell receptor binding. The C terminus of the peptide governed peptide-MHC stability. Unexpectedly, we further demonstrate a novel mode of flexible peptide presentation in which the MHC peptide-binding groove is able to “open the back door” to accommodate extra C-terminal peptide residues

Hotspot autoimmune T cell receptor binding underlies pathogen and insulin peptide cross-reactivity

The cross-reactivity of T cells with pathogen- and self-derived peptides has been implicated as a pathway involved in the development of autoimmunity. However, the mechanisms that allow the clonal T cell antigen receptor (TCR) to functionally engage multiple peptide–major histocompatibility complexes (pMHC) are unclear. Here, we studied multiligand discrimination by a human, preproinsulin reactive, MHC class-I–restricted CD8+ T cell clone (1E6) that can recognize over 1 million different peptides. We generated high-resolution structures of the 1E6 TCR bound to 7 altered peptide ligands, including a pathogen-derived peptide that was an order of magnitude more potent than the natural self-peptide. Evaluation of these structures demonstrated that binding was stabilized through a conserved lock-and-key–like minimal binding footprint that enables 1E6 TCR to tolerate vast numbers of substitutions outside of this so-called hotspot. Highly potent antigens of the 1E6 TCR engaged with a strong antipathogen-like binding affinity; this engagement was governed though an energetic switch from an enthalpically to entropically driven interaction compared with the natural autoimmune ligand. Together, these data highlight how T cell cross-reactivity with pathogen-derived antigens might break self-tolerance to induce autoimmune disease

TCR‐induced alteration of primary MHC peptide anchor residue

The HLA‐A*02:01‐restricted decapeptide EAAGIGILTV, derived from melanoma antigen recognized by T‐cells‐1 (MART‐1) protein, represents one of the best‐studied tumor associated T‐cell epitopes, but clinical results targeting this peptide have been disappointing. This limitation may reflect the dominance of the nonapeptide, AAGIGILTV, at the melanoma cell surface. The decapeptide and nonapeptide are presented in distinct conformations by HLA‐A*02:01 and TCRs from clinically relevant T‐cell clones recognize the nonapeptide poorly. Here, we studied the MEL5 TCR that potently recognizes the nonapeptide. The structure of the MEL5‐HLA‐A*02:01‐AAGIGILTV complex revealed an induced fit mechanism of antigen recognition involving altered peptide–MHC anchoring. This “flexing” at the TCR–peptide–MHC interface to accommodate the peptide antigen explains previously observed incongruences in this well‐studied system and has important implications for future therapeutic approaches. Finally, this study expands upon the mechanisms by which molecular plasticity can influence antigen recognition by T cells

A long in situ section of the lower ocean crust: results of {ODP} Leg 176 drilling at the Southwest Indian Ridge

Ocean Drilling Program Leg 176 deepened Hole 735B in gabbroic lower ocean crust by 1 km to 1.5 km. The section has the physical properties of seismic layer 3, and a total magnetization sufficient by itself to account for the overlying lineated sea-surface magnetic anomaly. The rocks from Hole 735B are principally olivine gabbro, with evidence for two principal and many secondary intrusive events. There are innumerable late small ferrogabbro intrusions, often associated with shear zones that cross-cut the olivine gabbros. The ferrogabbros dramatically increase upward in the section. Whereas there are many small patches of ferrogabbro representing late iron- and titanium-rich melt trapped intragranularly in olivine gabbro, most late melt was redistributed prior to complete solidification by compaction and deformation. This, rather than in situ upward differentiation of a large magma body, produced the principal igneous stratigraphy. The computed bulk composition of the hole is too evolved to mass balance mid-ocean ridge basalt back to a primary magma, and there must be a significant mass of missing primitive cumulates. These could lie either below the hole or out of the section. Possibly the gabbros were emplaced by along-axis intrusion of moderately differentiated melts into the near-transform environment. Alteration occurred in three stages. High-temperature granulite- to amphibolite-facies alteration is most important, coinciding with brittle-ductile deformation beneath the ridge. Minor greenschist-facies alteration occurred under largely static conditions, likely during block uplift at the ridge transform intersection. Late post-uplift low-temperature alteration produced locally abundant smectite, often in previously unaltered areas. The most important features of the high- and low-temperature alteration are their respective associations with ductile and cataclastic deformation, and an overall decrease downhole with hydrothermal alteration generally =<5% in the bottom kilometer. Hole 735B provides evidence for a strongly heterogeneous lower ocean crust, and for the inherent interplay of deformation, alteration and igneous processes at slow-spreading ridges. It is strikingly different from gabbros sampled from fast-spreading ridges and at most well-described ophiolite complexes. We attribute this to the remarkable diversity of tectonic environments where crustal accretion occurs in the oceans and to the low probability of a section of old slow-spread crust formed near a major large-offset transform being emplaced on-land compared to sections of young crust from small ocean basins

Mutation of recombinant complement component C9 reveals the significance of the N-terminal region for polymerization

Complement component C9 binds to C5b-8 sites on target cells and polymerizes to form the membrane attack complex (MAC). The aim of the work reported here was to discover which region within C9 was responsible for protecting the globular protein against self-polymerization. Computer prediction modelling highlighted the domain at the N-terminus of C9, which was then investigated by site-directed mutagenesis. The mutated proteins were expressed using insect cells infected with baculovirus. Removal of 16, 20 or 23 amino acids at the N-terminus of C9 resulted in inactivation due to self-polymerization. In contrast, removal of 4, 8 or 12 amino acids resulted in a C9 that did not polymerize spontaneously, had two to threefold enhanced lytic activity on erythrocytes, and had increased binding to C5b-8 sites on rat neutrophils. These results suggest that the domain within the first 16 amino acids at the N-terminus of C9 is crucial in preventing the self-polymerization of the globular protein. We have also found that C9 contains a motif (27WSEWS31) common to a family of cytokine receptors that is similar to a tryptophan-rich motif (WEWWR) of the membrane pore formers, thiol-activated cytolysins. Mutation of this motif in C9 resulted in polymerized protein, consistent with this site keeping the N-terminus in a protected conformation and preventing premature self-polymerization

Digital living - People-centred innovation and strategy

This paper provides a summary of a research programme at BTexact Technologies which is aimed at helping a technology innovation company to ground its innovations, to see opportunities for the exploitation of its technologies, and to create sociotechnical visions which can help to drive technological innovation itself. As a by-product, the programme has also created strategic knowledge that is of critical importance to public and private policy/decision makers alike. This research is a key part of BTexact Technologies' approach to the creation of and response to disruptive technologies. Understanding 'usage by people' is absolutely critical to figuring out what is disruptive about technologies, why this is so, and therefore how to make money out of them. Since this is critical to several of BTexact's core competencies (and to those of its customers), the value of the research reported here is self-evident both to BTexact and to its customers. Without it, they will only ever make money by accident, a strategy that shareholders do not seem to find amusing.</p

More tricks with tetramers: a practical guide to staining T cells with peptide-MHC multimers

Analysis of antigen-specific T-cell populations by flow cytometry with peptide-MHC (pMHC) multimers is now commonplace. These reagents allow the tracking and phenotyping of T cells during infection, autoimmunity and cancer, and can be particularly revealing when used for monitoring therapeutic interventions. In 2009, we reviewed a number of 'tricks' that could be used to improve this powerful technology. More recent advances have demonstrated the potential benefits of using higher order multimers and of 'boosting' staining by inclusion of an antibody against the pMHC multimer. These developments now allow staining of T cells where the interaction between the pMHC and the T-cell receptor is over 20-fold weaker (K(D) > 1 mm) than could previously be achieved. Such improvements are particularly relevant when using pMHC multimers to stain anti-cancer or autoimmune T-cell populations, which tend to bear lower affinity T-cell receptors. Here, we update our previous work to include discussion of newer tricks that can produce substantially brighter staining even when using log-fold lower concentrations of pMHC multimer. We further provide a practical guide to using pMHC multimers that includes a description of several common pitfalls and how to circumvent them