Regulation of the mannose 6-phosphate/IGF II receptor expression at the cell surface by mannose 6-phosphate, insulin like growth factors and epidermal growth factor.

Mannose 6-phosphate, insulin like growth factors I and II (IGF I, IGF II), insulin and epidermal growth factor (EGF) induce a 1.5- to 2-fold increase of mannose 6-phosphate binding sites at the cell surface of human skin fibroblasts. The increase is completed within 10-15 min, is dose and temperature dependent, reversible and transient even in the presence of the effectors. It is due to a redistribution of mannose 6-phosphate/IGF II receptors from internal membranes to the cell surface, while the affinity of the receptors is not affected. Combinations of mannose 6-phosphate with IGF I, IGF II or EGF stimulate the redistribution of the receptor to the cell surface in an additive manner, while combinations of the growth factors result in a non-additive stimulation of redistribution. The redistribution is not dependent on extracellular calcium and appears also to be independent of changes of free intracellular calcium. Pre-treatment of fibroblasts with cholera toxin or pertussis toxin increases the number of cell surface receptors 2- and 1.5-fold, respectively. Neither of the toxins affects the redistribution of mannose 6-phosphate/IGF II receptors induced by the growth factors, while both toxins abolish the receptor redistribution induced by mannose 6-phosphate. These results suggest a multiple regulation of the cell surface expression of mannose 6-phosphate/IGF II receptors by Gs- and Gi-like proteins sensitive to cholera toxin and pertussis toxin and by stimulation of mannose 6-phosphate/IGF II, IGF I and EGF receptors.(ABSTRACT TRUNCATED AT 250 WORDS

Generation of Th1-polarizing dendritic cells using the TLR7/8 agonist CL075.

In this paper, we describe a new method for preparation of human dendritic cells (DCs) that secrete bioactive IL-12(p70) using synthetic immunostimulatory compounds as TLR7/8 agonists. Monocyte-derived DCs were generated using a procedure that provided mature cells within 3 d. Several maturation mixtures that contained various cytokines, IFN-gamma, different TLR agonists, and PGE(2) were compared for impact on cell recovery, phenotype, cytokine secretion, migration, and lymphocyte activation. Mixtures that included the TLR7/8 agonists R848 or CL075, combined with the TLR3 agonist polyinosinic:polycytidylic acid, yielded 3-d mature DCs that secreted high levels of IL-12(p70), showed strong chemotaxis to CCR7 ligands, and had a positive costimulatory potential. They also had excellent capacity to activate NK cells, effectively polarized CD4(+) and CD8(+) T cells to secrete IFN-gamma and to induce T cell-mediated cytotoxic function. Thereby, mature DCs prepared within 3 d using such maturation mixtures displayed optimal functions required for vaccine development

Effective and long-term control of EBV PTLD after transfer of peptide-selected T cells.

Posttransplantation lymphoproliferative disease (PTLD) associated with Epstein-Barr virus (EBV) is a life-threatening complication after allogeneic hematopoietic stem cell transplantation. PTLD is efficiently prevented by adoptive transfer of EBV-specific T cells from the donor. To make EBV-specific T cells available in urgent clinical situations, we developed a rapid protocol for their isolation by overnight stimulation of donor blood cells with peptides derived from 11 EBV antigens, interferon-gamma surface capture, and immunomagnetic separation. Six patients with PTLD received 1 transfusion of EBV-specific T cells. No response was seen in 3 patients who had late-stage disease with multiorgan dysfunction at the time of T-cell transfer. In 3 patients who received T cells at an earlier stage of disease, we observed complete and stable remission of PTLD. Two patients have remained free from EBV-associated disease for more than 2 years. CD8(+) T cells specific for EBV early antigens rapidly expanded after T-cell transfer, temporarily constituted greater than 20% of all peripheral blood lymphocytes, and were maintained throughout the observation period. Thus, a rapid and sustained reconstitution of a protective EBV-specific T-cell memory occurred after the infusion of small numbers of directly isolated EBV-specific T cells