Tumor necrosis factor-alpha regulates the expression of inducible costimulator receptor ligand on CD34(+) progenitor cells during differentiation into antigen presenting cells.

The inducible costimulator receptor (ICOS) is a third member of the CD28 receptor family that regulates T cell activation and function. ICOS binds to a newly identified ligand on antigen presenting cells different from the CD152 ligands CD80 and CD86. We used soluble ICOSIg and a newly developed murine anti-human ICOS ligand (ICOSL) monoclonal antibody to further characterize the ICOSL during ontogeny of antigen presenting cells. In a previous study, we found that ICOSL is expressed on monocytes, dendritic cells, and B cells. To define when ICOSL is first expressed on myeloid antigen presenting cells, we examined ICOSL expression on CD34(+) cells in bone marrow. We found that CD34(bright) cells regardless of their myeloid commitment were ICOSL(-), whereas ICOSL was first expressed when CD34 expression diminished and the myeloid marker CD33 appeared. However, acute myeloid leukemia cells were ICOSL-negative, whereas among B-cell malignancies only some cases of the most mature tumors such as prolymphocytic leukemia and hairy cell leukemia were positive. Next, we investigated purified CD34(+) hematopoietic progenitor cells that did not constitutively express ICOSL but were induced to express ICOSL within 12 h after granulocyte/macrophage colony-stimulating factor/tumor necrosis factor alpha (TNF-alpha) stimulation. Interestingly, ICOSL was induced prior to CD80/CD86 induction on CD34(+) cells so that ICOSL was expressed in the absence of CD80/CD86. This suggests that ICOSL is an early differentiation marker along the monocytic/dendritic maturation pathway. Induction of ICOSL was dependent on TNF-alpha and was regulated via NF-kappa B as revealed by use of inhibitors specific for I kappa B alpha phosphorylation such as BAY 11-7082 and BAY 11-7085. The antigen presenting capacity of TNF-alpha stimulated CD34(+) cells was strongly inhibited by ICOSIg fusion proteins or by NF-kappa B inhibition. Thus, TNF-alpha-induced ICOSL expression seemed to be functionally important for the costimulatory capacity of CD34(+) hematopoietic progenitor cells.

Retroviral interleukin-7 gene transfer into human dendritic cells enhances T cell activation.

Tumor vaccination with dendritic cells (DC) presenting tumor antigens to T cells is a promising approach in immunotherapy. The aim of this study was to enhance T cell stimulatory ability of human DC by retroviral expression of the interleukin-7 (IL-7) gene. IL-7 has been shown to provide a potent costimulatory signal for the proliferation of T cells and the generation of cytotoxic T cells (CTL). DC were generated from human peripheral blood mononuclear cells (PBMC). DC were analyzed by light- and electron-microscopy, immunophenotype (CD1a+, CD14-, CD80+, CD86+, HLA-DR+) and functional assays. According to these criteria, 75-85% of the cells were DC. The cells did not produce measurable amounts of IL-7 spontaneously nor did they express the IL-7 receptor. A retroviral IL-7 expression vector was constructed. Retroviral infection was performed with either the LXSN-hIL-7 vector of its variant LXSN. Using the LXSN-hIL-7 vector, IL-7 production of 2296 pg/10(6) cells/24 h could be achieved on average. Transduction of DC was confirmed by RT-PCR in a CD1a-enriched cell fraction. Transduction efficiency by a control virus coding for beta-galactosidase was about 30%. In autologous mixed lymphocyte reaction (MLR), IL-7 transduced DC augmented T cell proliferation by a factor of two compared with unmodified or mock-transfected DC, and in allogeneic MLR there was a 2.7-fold increase in T cell proliferation. The increase in T cell proliferation could be correlated to IL-7 secretion by DC. Dendritic cells that have been simultaneously peptide-loaded and gene-modified to secrete IL-7 are a potential tool to amplify activation of tumor-specific T cells.

Successful retroviral mediated transduction of a reporter gene in human dendritic cells: feasibility of therapy with gene-modified antigen presenting cells.

In this study we have analyzed the feasibility of gene transfer in human dendritic cells (DCs). DCs were generated from T and B cell-depleted peripheral blood mononuclear cells cultured for 7 days in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). The cells showed morphologic and immunophenotypical features typical of DCs, including expression of major histocompatibility complex (MHC) class I and II molecules, CD1a, CD80, CD86, CD13, CD33, CD40, and CD54. The cells showed high stimulatory activity in both allogeneic and autologous mixed lymphocyte reaction (MLR). The bacterial reporter gene lacZ coding for beta-galactosidase (beta-gal) was introduced in DCs by three sequential cycles of infection using a MFG retroviral vector system. After 7 days of culture 35-67% of the cells showed high expression of beta-gal activity, proving successful gene transfer. Stable integration of the lacZ gene was demonstrated by genomic DNA-polymerase chain reaction (PCR) up to 20 days after gene transfer. The percentage of transduction was similar when DCs were further purified by immunomagnetic separation according to CD1a-expression. We conclude that human DCs can be efficiently gene modified, further broadening the spectrum of possible DC-based clinical applications.

Demonstration of the humoral immune response of horses to Babesia caballi by western blotting.

Babesia caballi-infected or normal equine erythrocytes were solubilized in sodium dodecyl sulfate (SDS) buffer and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Antigens were allowed to react with sera from horses experimentally or field-infected with B. caballi and with sera from non-infected horses. Major babesial antigens recognized by immune sera had apparent mol. wts of 141, 112, 70, 50, 48, 34, and 30 kDa. The polypeptides at 50 and 48 kDa were recognized earliest and throughout infection, but also weakly by 3/100 equine sera tested negative and 1/33 sera tested false positive by the complement fixation test (CFT) and immunofluorescence antibody test (IFAT). Thus, further characterization and purification of B. caballi antigens are required to identify target antigens for an improved enzyme immuno assay. Until such an assay is available, Western blotting can provide a specific tool for the diagnosis of B. caballi infections, particularly in cases of contradicting CFT and IFAT results.