Emulsion technologies for multicellular tumour spheroid radiation assays

A major limitation with current in vitro technologies for testing anti-cancer therapies at the pre-clinical level is the use of 2D cell culture models which provide a poor reflection of the tumour physiology in vivo. Three dimensional cell culture models, such as the multicellular spheroid, provide instead a more accurate representation. However, existing spheroid-based assessment methods are generally labour-intensive and low-throughput. Emulsion based technologies offer enhanced mechanical stability during multicellular tumour spheroid formation and culture and are scalable to enable higher-throughput assays. The aim of this study was to investigate the characteristics of emulsion-based techniques for the formation and long term culture of multicellular UVW glioma cancer spheroids and apply these findings to assess the cytotoxic effect of radiation on spheroids. Our results showed that spheroids formed within emulsions had similar morphological and growth characteristics to those formed using traditional methods. Furthermore, we have identified the effects produced on the proliferative state of the spheroids due to the compartmentalised nature of the emulsions and applied this for mimicking tumour growth and tumour quiescence. Finally, proof of concept results are shown to demonstrate the scalability potential of the technology for developing high-throughput screening assays

Inhibition of Poly(ADP-Ribose) polymerase enhances the toxicity of 131I-Metaiodobenzylguanidine/Topotecan combination therapy to cells and xenografts that express the noradrenaline transporter

Targeted radiotherapy using [131I]meta-iodobenzylguanidine ([131I]MIBG) has produced remissions in some neuroblastoma patients. We previously reported that combining [131I]MIBG with the topoisomerase I (Topo-I) inhibitor topotecan induced long-term DNA damage and supra-additive toxicity to NAT-expressing cells and xenografts. This combination treatment is undergoing clinical evaluation. This present study investigated the potential of PARP-1 inhibition, in vitro and in vivo, to further enhance [131I]MIBG/topotecan efficacy

Lysosomotropism depends on glucose : a chloroquine resistance mechanism

There has been long-standing interest in targeting pro-survival autophagy as a combinational cancer therapeutic strategy. Clinical trials are in progress testing Chloroquine (CQ) or its derivatives in combination with chemo- or radio-therapy for solid and haematological cancers. While CQ has shown efficacy in pre-clinical models, its mechanism of action remains equivocal. Here, we tested how effectively CQ sensitises metastatic breast cancer cells to further stress conditions such as ionising irradiation, doxorubicin, PI3K-Akt inhibition and serum withdrawal. Contrary to the conventional model, the cytotoxic effects of CQ were found to be autophagy-independent, since genetic targeting of ATG7 or the ULK1/2 complex could not sensitise cells, like CQ, to serum depletion. Interestingly, while CQ combined with serum starvation was robustly cytotoxic, further glucose starvation under these conditions led to a full rescue of cell viability. Inhibition of hexokinase using 2-deoxyglucose (2DG) similarly led to CQ resistance. As this form of cell death did not resemble classical caspase-dependent apoptosis, we hypothesised that CQ-mediated cytotoxicity was primarily via a lysosome-dependent mechanism. Indeed, CQ treatment led to marked lysosomal swelling and recruitment of Galectin3 to sites of membrane damage. Strikingly, glucose starvation or 2DG prevented CQ from inducing lysosomal damage and subsequent cell death. Importantly, we found that the related compound, amodiaquine, was more potent than CQ for cell killing and not susceptible to interference from glucose starvation. Taken together, our data indicate that CQ effectively targets the lysosome to sensitise towards cell death but is prone to a glucose-dependent resistance mechanism, thus providing rationale for the related compound amodiaquine (currently used in humans) as a better therapeutic option for cancer

The Shaping of T Cell Receptor Recognition by Self-Tolerance

SummaryDuring selection of the T cell repertoire, the immune system navigates the subtle distinction between self-restriction and self-tolerance, yet how this is achieved is unclear. Here we describe how self-tolerance toward a trans-HLA (human leukocyte antigen) allotype shapes T cell receptor (TCR) recognition of an Epstein-Barr virus (EBV) determinant (FLRGRAYGL). The recognition of HLA-B8-FLRGRAYGL by two archetypal TCRs was compared. One was a publicly selected TCR, LC13, that is alloreactive with HLA-B44; the other, CF34, lacks HLA-B44 reactivity because it arises when HLA-B44 is coinherited in trans with HLA-B8. Whereas the alloreactive LC13 TCR docked at the C terminus of HLA-B8-FLRGRAYGL, the CF34 TCR docked at the N terminus of HLA-B8-FLRGRAYGL, which coincided with a polymorphic region between HLA-B8 and HLA-B44. The markedly contrasting footprints of the LC13 and CF34 TCRs provided a portrait of how self-tolerance shapes the specificity of TCRs selected into the immune repertoire

Natural HLA Class I Polymorphism Controls the Pathway of Antigen Presentation and Susceptibility to Viral Evasion

HLA class I polymorphism creates diversity in epitope specificity and T cell repertoire. We show that HLA polymorphism also controls the choice of Ag presentation pathway. A single amino acid polymorphism that distinguishes HLA-B*4402 (Asp116) from B*4405 (Tyr116) permits B*4405 to constitutively acquire peptides without any detectable incorporation into the transporter associated with Ag presentation (TAP)-associated peptide loading complex even under conditions of extreme peptide starvation. This mode of peptide capture is less susceptible to viral interference than the conventional loading pathway used by HLA-B*4402 that involves assembly of class I molecules within the peptide loading complex. Thus, B*4402 and B*4405 are at opposite extremes of a natural spectrum in HLA class I dependence on the PLC for Ag presentation. These findings unveil a new layer of MHC polymorphism that affects the generic pathway of Ag loading, revealing an unsuspected evolutionary trade-off in selection for optimal HLA class I loading versus effective pathogen evasion

A Naturally Selected Dimorphism within the HLA-B44 Supertype Alters Class I Structure, Peptide Repertoire, and T Cell Recognition

HLA-B*4402 and B*4403 are naturally occurring MHC class I alleles that are both found at a high frequency in all human populations, and yet they only differ by one residue on the α2 helix (B*4402 Asp156→B*4403 Leu156). CTLs discriminate between HLA-B*4402 and B*4403, and these allotypes stimulate strong mutual allogeneic responses reflecting their known barrier to hemopoeitic stem cell transplantation. Although HLA-B*4402 and B*4403 share >95% of their peptide repertoire, B*4403 presents more unique peptides than B*4402, consistent with the stronger T cell alloreactivity observed toward B*4403 compared with B*4402. Crystal structures of B*4402 and B*4403 show how the polymorphism at position 156 is completely buried and yet alters both the peptide and the heavy chain conformation, relaxing ligand selection by B*4403 compared with B*4402. Thus, the polymorphism between HLA-B*4402 and B*4403 modifies both peptide repertoire and T cell recognition, and is reflected in the paradoxically powerful alloreactivity that occurs across this “minimal” mismatch. The findings suggest that these closely related class I genes are maintained in diverse human populations through their differential impact on the selection of peptide ligands and the T cell repertoire

Allelic polymorphism in the T cell receptor and its impact on immune responses

In comparison to human leukocyte antigen (HLA) polymorphism, the impact of allelic sequence variation within T cell receptor (TCR) loci is much less understood. Particular TCR loci have been associated with autoimmunity, but the molecular basis for this phenomenon is undefined. We examined the T cell response to an HLA-B*3501-restricted epitope (HPVGEADYFEY) from Epstein-Barr virus (EBV), which is frequently dominated by a TRBV9*01 public TCR (TK3). However, the common allelic variant TRBV9*02, which differs by a single amino acid near the CDR2β loop (Gln55→His55), was never used in this response. The structure of the TK3 TCR, its allelic variant, and a nonnaturally occurring mutant (Gln55→Ala55) in complex with HLA-B*3501 revealed that the Gln55→His55 polymorphism affected the charge complementarity at the TCR-peptide-MHC interface, resulting in reduced functional recognition of the cognate and naturally occurring variants of this EBV peptide. Thus, polymorphism in the TCR loci may contribute toward variability in immune responses and the outcome of infection

The Long-Baseline Neutrino Experiment: Exploring Fundamental Symmetries of the Universe

The preponderance of matter over antimatter in the early Universe, the dynamics of the supernova bursts that produced the heavy elements necessary for life and whether protons eventually decay --- these mysteries at the forefront of particle physics and astrophysics are key to understanding the early evolution of our Universe, its current state and its eventual fate. The Long-Baseline Neutrino Experiment (LBNE) represents an extensively developed plan for a world-class experiment dedicated to addressing these questions. LBNE is conceived around three central components: (1) a new, high-intensity neutrino source generated from a megawatt-class proton accelerator at Fermi National Accelerator Laboratory, (2) a near neutrino detector just downstream of the source, and (3) a massive liquid argon time-projection chamber deployed as a far detector deep underground at the Sanford Underground Research Facility. This facility, located at the site of the former Homestake Mine in Lead, South Dakota, is approximately 1,300 km from the neutrino source at Fermilab -- a distance (baseline) that delivers optimal sensitivity to neutrino charge-parity symmetry violation and mass ordering effects. This ambitious yet cost-effective design incorporates scalability and flexibility and can accommodate a variety of upgrades and contributions. With its exceptional combination of experimental configuration, technical capabilities, and potential for transformative discoveries, LBNE promises to be a vital facility for the field of particle physics worldwide, providing physicists from around the globe with opportunities to collaborate in a twenty to thirty year program of exciting science. In this document we provide a comprehensive overview of LBNE's scientific objectives, its place in the landscape of neutrino physics worldwide, the technologies it will incorporate and the capabilities it will possess.Comment: Major update of previous version. This is the reference document for LBNE science program and current status. Chapters 1, 3, and 9 provide a comprehensive overview of LBNE's scientific objectives, its place in the landscape of neutrino physics worldwide, the technologies it will incorporate and the capabilities it will possess. 288 pages, 116 figure

Drugs and drug-like molecules can modulate the function of mucosal-associated invariant T cells

The major-histocompatibility-complex-(MHC)-class-I-related molecule MR1 can present activating and non-activating vitamin-B-based ligands to mucosal-associated invariant T cells (MAIT cells). Whether MR1 binds other ligands is unknown. Here we identified a range of small organic molecules, drugs, drug metabolites and drug-like molecules, including salicylates and diclofenac, as MR1-binding ligands. Some of these ligands inhibited MAIT cells ex vivo and in vivo, while others, including diclofenac metabolites, were agonists. Crystal structures of a T cell antigen receptor (TCR) from a MAIT cell in complex with MR1 bound to the non-stimulatory and stimulatory compounds showed distinct ligand orientations and contacts within MR1, which highlighted the versatility of the MR1 binding pocket. The findings demonstrated that MR1 was able to capture chemically diverse structures, spanning mono- and bicyclic compounds, that either inhibited or activated MAIT cells. This indicated that drugs and drug-like molecules can modulate MAIT cell function in mammals