Human IgG1 antibodies antagonizing activating receptor NKG2D on natural killer cells.

NKG2D is a surface receptor expressed on NK cells but also on CD8(+) T cells, gammadelta T cells, and auto-reactive CD4(+)/CD28(-) T cells of patients with rheumatoid arthritis. Various studies suggested that NKG2D plays a critical role in autoimmune diseases, e.g., in diabetes, celiac disease and rheumatoid arthritis (RA), rendering the activating receptor a potential target for antibody-based therapies. Here, we describe the generation and characteristics of a panel of human, high-affinity anti-NKG2D IgG1 monoclonal antibodies (mAbs) derived by phage display. The lead molecule mAb E4 bound with an affinity (KD) of 2.7 +/- 1.4 x 10(-11) M to soluble and membrane-bound human NKG2D, and cross-reacted with NKG2D from cynomolgus macaque, indicating potential suitability for studies in a relevant primate model. MAb E4 potently antagonized the cytolytic activity of NKL cells against BaF/3-MICA cells expressing NKG2D ligand, and blocked the NKG2D ligand-induced secretion of TNFalpha, IFNgamma and GM-CSF, as well as surface expression of CRTAM by NK cells cultured on immobilized MICA or ULBP-1 ligands. The antibody did not show a detectable loss of binding to NKG2D after seven days in human serum at 37 degrees C, and resisted thermal inactivation up to 70 degrees C. Based on these results, anti-human NKG2D mAb E4 provides an ideal candidate for development of a novel therapeutic agent antagonizing a key receptor of NK and cytotoxic T cells with implications in autoimmune diseases.

Biogenesis of Leishmania major-harboring vacuoles in murine dendritic cells.

In mammalian hosts, Leishmania sp. parasites are obligatory intracellular organisms that invade macrophages and dendritic cells (DC), where they reside in endocytic organelles termed parasitophorous vacuoles (PV). Most of the present knowledge of the characteristics of PV harboring Leishmania sp. is derived from studies with infected macrophages. Since DC play a key role in host resistance to leishmaniasis, there is a need to understand the properties and biogenesis of PV in Leishmania sp.-infected DC. Therefore, we determined the acquisition of endosomal and lysosomal molecules by Leishmania major-containing compartments in DC at different maturation stages, using fluorescence labeling and confocal microscopy. The results show that newly formed phagosomes in DC rapidly develop into late endosomal compartments. However, the small GTPase Rab7, which regulates late fusion processes, was found only in PV of mature bone marrow-derived DC (BMDC); it was absent in immature BMDC, suggesting an arrest of their PV biogenesis at the stage of late endosomes. Indeed, fusion assays with endocytic tracers demonstrated that the fusion activity of L. major-harboring PV toward lysosomes is higher in mature BMDC than in immature BMDC. The inhibition of PV-lysosome fusion in DC is dependent upon the viability and life cycle stage of the parasite, because live promastigotes blocked the fusion almost completely, whereas killed organisms and amastigotes induced a considerable level of fusion activity. The differences in the fusion competences of immature and mature DC may be relevant for their distinct functional activities in the uptake, transport, and presentation of parasite antigens.