Role for coronin 1 in mouse NK cell function.

Coronin 1, a member of the evolutionary conserved WD repeat protein family of coronin proteins is expressed in all leukocytes, but a role for coronin 1 in natural killer (NK) cell homeostasis and function remains unclear. Here, we have analyzed the number and functionality of NK cells in the presence and absence of coronin 1. In coronin 1-deficient mice, absolute NK cell numbers and phenotype were comparable to wild type mice in blood, spleen and liver. Following in vitro stimulation of the activating NK cell receptors NK1.1, NKp46, Ly49D and NKG2D, coronin 1-deficient NK cells were functional with respect to interferon-γ production, degranulation and intracellular Ca2+ mobilization. Also, both wild type as well as coronin 1-deficient NK cells showed comparable cytotoxic activity. Furthermore, activation and functionality of NK cells following Vesicular Stomatitis Virus (VSV) infection was similar between wild type and coronin 1-deficient mice. Taken together these data suggest that coronin 1 is dispensable for mouse NK cell homeostasis and function.

Pharmacological characterization of tachykinin tetrabranched derivatives.

BACKGROUND AND PURPOSE: Peptide welding technology (PWT) is a novel chemical strategy that allows the synthesis of multibranched peptides with high yield, purity and reproducibility. Using this technique, we have synthesized and pharmacologically characterized the tetrabranched derivatives of the tachykinins, substance P (SP), neurokinin A (NKA) and B (NKB). EXPERIMENTAL APPROACH: The following in vitro assays were used: calcium mobilization in cells expressing human recombinant NK receptors, BRET studies of G-protein - NK1 receptor interaction, guinea pig ileum and rat urinary bladder bioassays. Nociceptive behavioural response experiments were performed in mice following intrathecal injection of PWT2-SP. KEY RESULTS: In calcium mobilization studies, PWT tachykinin derivatives behaved as full agonists at NK receptors with a selectivity profile similar to that of the natural peptides. NK receptor antagonists display similar potency values when tested against PWT2 derivatives and natural peptides. In BRET and bioassay experiments PWT2-SP mimicked the effects of SP with similar potency, maximal effects and sensitivity to aprepitant. After intrathecal administration in mice, PWT2-SP mimicked the nociceptive effects of SP, but with higher potency and a longer-lasting action. Aprepitant counteracted the effects of PWT2-SP in vivo. CONCLUSIONS AND IMPLICATIONS: The present study has shown that the PWT technology can be successfully applied to the peptide sequence of tachykinins to generate tetrabranched derivatives characterized with a pharmacological profile similar to the native peptides. In vivo, PWT2-SP displayed higher potency and a marked prolongation of action, compared with SP.

Soluble ligands for NK cell receptors promote evasion of chronic lymphocytic leukemia cells from NK cell anti-tumor activity.

Natural killer (NK) cells are a major component of the anti-tumor immune response. NK cell dysfunctions have been reported in various hematologic malignancies, including chronic lymphocytic leukemia (CLL). Here we investigated the role of tumor cell-released soluble and exosomal ligands for NK cell receptors that modulate NK cell activity. Soluble CLL plasma factors suppressed NK cell cytotoxicity and down-regulated the surface receptors CD16 and CD56 on NK cells of healthy donors. The inhibition of NK cell cytotoxicity was attributed to the soluble ligand BAG6/BAT3 that engages the activating receptor NKp30 expressed on NK cells. Soluble BAG6 was detectable in the plasma of CLL patients, with the highest levels at the advanced disease stages. In contrast, NK cells were activated when BAG6 was presented on the surface of exosomes. The latter form was induced in non-CLL cells by cellular stress via an nSmase2-dependent pathway. Such cells were eliminated by lymphocytes in a xenograft tumor model in vivo. Here, exosomal BAG6 was essential for tumor cell killing because BAG6-deficient cells evaded immune detection. Taken together, the findings show that the dysregulated balance of exosomal vs soluble BAG6 expression may cause immune evasion of CLL cells.

Enzymatic synthesis of dimeric glycomimetic ligands of NK cell activation receptors.

This work reveals new structural relationships in the complex process of the interaction between activation receptors of natural killer cells (rat NKR-P1, human CD69) and novel bivalent carbohydrate glycomimetics. The length, glycosylation pattern and linker structure of receptor ligands were examined with respect to their ability to precipitate the receptor protein from solution, which simulates the in vivo process of receptor aggregation during NK cell activation. It was found that di-LacdiNAc triazole compounds show optimal performance, reaching up to 100% precipitation of the present protein receptors, and achieving high immunostimulatory activities without any tendency to trigger activation-induced apoptosis. In the synthesis of the compounds tested, two enzymatic approaches were applied. Whereas a ß-N-acetylhexosaminidase could only glycosylate one of the two acceptor sites available with yields below 10%, the Y284L mutant of human placental ß1,4-galactosyltransferase-1 worked as a perfect synthetic tool, accomplishing even quantitative glycosylation at both acceptor sites and with absolute regioselectivity for the C-4 position. This work insinuates new directions for further ligand structure optimisation and demonstrates the strong synthetic potential of the mutant human placental ß1,4-galactosyltransferase-1 in the synthesis of multivalent glycomimetics and glycomaterials.