Re-evaluation of the involvement of NK cells and C-type lectin-like NK receptors in modulation of immune responses by multivalent GlcNAc-terminated oligosaccharides

Abstract

Recognition of glycosylation patterns is one of the basic features of innate immunity. Ability of C-type lectin-like receptors such as NKR-P1 to bind saccharide moieties has become recently a controversial issue. In the present study, binding assay with soluble fluorescently labeled recombinant rat NKR-P1A and mouse NKR-P1C proteins revealed apparently no affinity to the various neoglycoproteins. Lack of functional linkage between NKR-P1 and previously described saccharide binder was supported by the fact, that synthetic N-acetyl-D-glucosamine octabranched dendrimer on polyamidoamine scaffold (GN8P) did not change gene expression of NKR-P1 isoforms in C57BL/6 and BALB/c mice divergent in the NK gene complex (both in vitro and in vivo). Surprisingly, N-acetyl-D-glucosamine-coated tetrabranched polyamido-amine dendrimer specifically binds to NKT cells and macrophages but not to NK cells (consistently with changes in cytokine patterns). Despite the fact that GN8P has been tested as an immunomodulator in anti-cancer treatment animal models for many years, surprisingly no changes in cytokine profiles in serum relevant to anti-cancer responses using B16F10 and CT26 harboring mouse strains C57BL/6 and BALB/c are observed. Our results indicate possible indirect involvement of NK cells in GN8P mediated immune responses.

Keywords: Anti-tumor immunity; C-type lectin related protein; Carbohydrate dendrimer; Clr; GN4P-A: GlcNAc4-PAMAM-ATTO 565; GN4P-NH(2)-GlcNAc(4)-PAMAM; GN4P: GlcNAc4-PAMAM; GN8P: GlcNAc8-PAMAM; GlcNAc; Gzmb; Macrophages; N-acetyl-d-glucosamine; N-acetyl-d-glucosamine-coated octabranched polyamidoamine dendrimer; N-acetyl-d-glucosamine-coated tetrabranched polyamidoamine dendrimer; N-acetyl-d-glucosamine-coated tetrabranched polyamidoamine dendrimer fluorescently labeled with ATTO 565; N-acetyl-d-glucosamine-coated tetrabranched polyamidoamine dendrimer with free NH(2) group; NK cells; NKG2D; NKR-P1; NKR-P1 receptors; NKT cells; PAMAM dendrimer; PMA; Prf; SBA; SMC; granzyme B; natural killer group 2, member D; natural killer receptor protein 1; perforin; phorbol 12-myristate 13-acetate; polyamidoamine dendrimer; soybean agglutinin; spleen mononuclear cell.