Instability of the HLA-E peptidome of HIV presents a major barrier to therapeutic targeting.

Naturally occurring T cells that recognize microbial peptides via HLA-E, a nonpolymorphic HLA class Ib molecule, could provide the foundation for new universal immunotherapeutics. However, confidence in the biological relevance of putative ligands is crucial, given that the mechanisms by which pathogen-derived peptides can access the HLA-E presentation pathway are poorly understood. We systematically interrogated the HIV proteome using immunopeptidomic and bioinformatic approaches, coupled with biochemical and cellular assays. No HIV HLA-E peptides were identified by tandem mass spectrometry analysis of HIV-infected cells. In addition, all bioinformatically predicted HIV peptide ligands (>80) were characterized by poor complex stability. Furthermore, infected cell elimination assays using an affinity-enhanced T cell receptor bispecific targeted to a previously reported HIV Gag HLA-E epitope demonstrated inconsistent presentation of the peptide, despite normal HLA-E expression on HIV-infected cells. This work highlights the instability of the HIV HLA-E peptidome as a major challenge for drug development.

Promiscuous recognition of MR1 drives self-reactive mucosal-associated invariant T cell responses.

Mucosal-associated invariant T (MAIT) cells use canonical semi-invariant T cell receptors (TCR) to recognize microbial riboflavin precursors displayed by the antigen-presenting molecule MR1. The extent of MAIT TCR crossreactivity toward physiological, microbially unrelated antigens remains underexplored. We describe MAIT TCRs endowed with MR1-dependent reactivity to tumor and healthy cells in the absence of microbial metabolites. MAIT cells bearing TCRs crossreactive toward self are rare but commonly found within healthy donors and display T-helper-like functions in vitro. Experiments with MR1-tetramers loaded with distinct ligands revealed significant crossreactivity among MAIT TCRs both ex vivo and upon in vitro expansion. A canonical MAIT TCR was selected on the basis of extremely promiscuous MR1 recognition. Structural and molecular dynamic analyses associated promiscuity to unique TCRß-chain features that were enriched within self-reactive MAIT cells of healthy individuals. Thus, self-reactive recognition of MR1 represents a functionally relevant indication of MAIT TCR crossreactivity, suggesting a potentially broader role of MAIT cells in immune homeostasis and diseases, beyond microbial immunosurveillance.

The conserved C2 phospholipid-binding domain in Delta contributes to robust Notch signalling.

Accurate Notch signalling is critical for development and homeostasis. Fine-tuning of Notch-ligand interactions has substantial impact on signalling outputs. Recent structural studies have identified a conserved N-terminal C2 domain in human Notch ligands which confers phospholipid binding in vitro. Here, we show that Drosophila ligands Delta and Serrate adopt the same C2 domain structure with analogous variations in the loop regions, including the so-called ß1-2 loop that is involved in phospholipid binding. Mutations in the ß1-2 loop of the Delta C2 domain retain Notch binding but have impaired ability to interact with phospholipids in vitro. To investigate its role in vivo, we deleted five residues within the ß1-2 loop of endogenous Delta. Strikingly, this change compromises ligand function. The modified Delta enhances phenotypes produced by Delta loss-of-function alleles and suppresses that of Notch alleles. As the modified protein is present on the cell surface in normal amounts, these results argue that C2 domain phospholipid binding is necessary for robust signalling in vivo fine-tuning the balance of trans and cis ligand-receptor interactions.

Immune-Mobilizing Monoclonal T Cell Receptors Mediate Specific and Rapid Elimination of Hepatitis B-Infected Cells.

BACKGROUND AND AIMS: Therapies for chronic hepatitis B virus (HBV) infection are urgently needed because of viral integration, persistence of viral antigen expression, inadequate HBV-specific immune responses, and treatment regimens that require lifelong adherence to suppress the virus. Immune mobilizing monoclonal T Cell receptors against virus (ImmTAV) molecules represent a therapeutic strategy combining an affinity-enhanced T Cell receptor with an anti-CD3 T Cell-activating moiety. This bispecific fusion protein redirects T cells to specifically lyse infected cells expressing the target virus-derived peptides presented by human leukocyte antigen (HLA). APPROACH AND RESULTS: ImmTAV molecules specific for HLA-A*02:01-restricted epitopes from HBV envelope, polymerase, and core antigens were engineered. The ability of ImmTAV-Env to activate and redirect polyclonal T cells toward cells containing integrated HBV and cells infected with HBV was assessed using cytokine secretion assays and imaging-based killing assays. Elimination of infected cells was further quantified using a modified fluorescent hybridization of viral RNA assay. Here, we demonstrate that picomolar concentrations of ImmTAV-Env can redirect T cells from healthy and HBV-infected donors toward hepatocellular carcinoma (HCC) cells containing integrated HBV DNA resulting in cytokine release, which could be suppressed by the addition of a corticosteroid in vitro. Importantly, ImmTAV-Env redirection of T cells induced cytolysis of antigen-positive HCC cells and cells infected with HBV in vitro, causing a reduction of hepatitis B e antigen and specific loss of cells expressing viral RNA. CONCLUSIONS: The ImmTAV platform has the potential to enable the elimination of infected cells by redirecting endogenous non-HBV-specific T cells, bypassing exhausted HBV-specific T cells. This represents a promising therapeutic option in the treatment of chronic hepatitis B, with our lead candidate now entering trials.

Structural Insights into Notch Receptor-Ligand Interactions.

Pioneering cell aggregation experiments from the Artavanis-Tsakonas group in the late 1980's localized the core ligand recognition sequence in the Drosophila Notch receptor to epidermal growth factor-like (EGF) domains 11 and 12. Since then, advances in protein expression, structure determination methods and functional assays have enabled us to define the molecular basis of the core receptor/ligand interaction and given new insights into the architecture of the Notch complex at the cell surface. We now know that Notch EGF11 and 12 interact with the Delta/Serrate/LAG-2 (DSL) and C2 domains of ligand and that membrane-binding, together with additional protein-protein interactions outside the core recognition domains, are likely to fine-tune generation of the Notch signal. Furthermore, structure determination of O-glycosylated variants of Notch alone or in complex with receptor fragments, has shown that these sugars contribute directly to the binding interface, as well as to stabilizing intra-molecular domain structure, providing some mechanistic insights into the observed modulatory effects of O-glycosylation on Notch activity.Future challenges lie in determining the complete extracellular architecture of ligand and receptor in order to understand (i) how Notch/ligand complexes may form at the cell surface in response to physiological cues, (ii) the role of lipid binding in stabilizing the Notch/ligand complex, (iii) the impact of O-glycosylation on binding and signalling and (iv) to dissect the different pathologies that arise as a consequence of mutations that affect proteins involved in the Notch pathway.

Structural and functional dissection of the interplay between lipid and Notch binding by human Notch ligands.

Recent data have expanded our understanding of Notch signalling by identifying a C2 domain at the N-terminus of Notch ligands, which has both lipid- and receptor-binding properties. We present novel structures of human ligands Jagged2 and Delta-like4 and human Notch2, together with functional assays, which suggest that ligand-mediated coupling of membrane recognition and Notch binding is likely to be critical in establishing the optimal context for Notch signalling. Comparisons between the Jagged and Delta family show a huge diversity in the structures of the loops at the apex of the C2 domain implicated in membrane recognition and Jagged1 missense mutations, which affect these loops and are associated with extrahepatic biliary atresia, lead to a loss of membrane recognition, but do not alter Notch binding. Taken together, these data suggest that C2 domain binding to membranes is an important element in tuning ligand-dependent Notch signalling in different physiological contexts.

Structural basis for the binding of tryptophan-based motifs by δ-COP.

Coatomer consists of two subcomplexes: the membrane-targeting, ADP ribosylation factor 1 (Arf1):GTP-binding ßγδζ-COP F-subcomplex, which is related to the adaptor protein (AP) clathrin adaptors, and the cargo-binding αß'ε-COP B-subcomplex. We present the structure of the C-terminal µ-homology domain of the yeast δ-COP subunit in complex with the WxW motif from its binding partner, the endoplasmic reticulum-localized Dsl1 tether. The motif binds at a site distinct from that used by the homologous AP µ subunits to bind YxxΦ cargo motifs with its two tryptophan residues sitting in compatible pockets. We also show that the Saccharomyces cerevisiae Arf GTPase-activating protein (GAP) homolog Gcs1p uses a related WxxF motif at its extreme C terminus to bind to δ-COP at the same site in the same way. Mutations designed on the basis of the structure in conjunction with isothermal titration calorimetry confirm the mode of binding and show that mammalian δ-COP binds related tryptophan-based motifs such as that from ArfGAP1 in a similar manner. We conclude that δ-COP subunits bind Wxn(1-6)[WF] motifs within unstructured regions of proteins that influence the lifecycle of COPI-coated vesicles; this conclusion is supported by the observation that, in the context of a sensitizing domain deletion in Dsl1p, mutating the tryptophan-based motif-binding site in yeast causes defects in both growth and carboxypeptidase Y trafficking/processing.

A novel HFE mutation (c.del478) results in nonsense-mediated decay of the mutant transcript in a hemochromatosis patient.

On admission to hospital Caucasian 61 year old male with jaundice was found to have unexplained increased serum iron indices. He had bilateral peripheral arthritis. On further investigation he had grade II hepatocellular iron staining and a hepatic index of 5.4 leading to a diagnosis of hereditary hemochromatosis. He lacked the common C282Y HFE mutation. We sequenced the complete HFE gene and found that he was heterozygous for a novel single nucleotide deletion (c.del478) in exon 3 of HFE. He lacks any other mutation in HFE or HJV, TFR2, HAMP and SLC40A1. The HFE mutation causes a frameshift (p.P160fs) that introduces a premature termination codon leading to mRNA degradation by nonsense-mediated decay. Haploinsufficiency of HFE may be one possible explanation for hemochromatosis in this patient.

dE2F2-independent rescue of proliferation in cells lacking an activator dE2F1.

In Drosophila melanogaster, the loss of activator de2f1 leads to a severe reduction in cell proliferation and repression of E2F targets. To date, the only known way to rescue the proliferation block in de2f1 mutants was through the inactivation of dE2F2. This suggests that dE2F2 provides a major contribution to the de2f1 mutant phenotype. Here, we report that in mosaic animals, in addition to de2f2, the loss of a DEAD box protein Belle (Bel) also rescues proliferation of de2f1 mutant cells. Surprisingly, the rescue occurs in a dE2F2-independent manner since the loss of Bel does not relieve dE2F2-mediated repression. In the eye disc, bel mutant cells fail to undergo a G1 arrest in the morphogenetic furrow, delay photoreceptor recruitment and differentiation, and show a reduction of the transcription factor Ci155. The down-regulation of Ci155 is important since it is sufficient to partially rescue proliferation of de2f1 mutant cells. Thus, mutation of bel relieves the dE2F2-mediated cell cycle arrest in de2f1 mutant cells through a novel Ci155-dependent mechanism without functional inactivation of the dE2F2 repressor.