Fine Mapping the Interaction Between Dendritic Cell-Specific Intercellular Adhesion Molecule (ICAM)-3-Grabbing Nonintegrin and the Cytomegalovirus Envelope Glycoprotein B.

Background: Human cytomegalovirus (HCMV) is a leading cause of virally induced congenital disorders and morbidities in immunocompromised individuals, ie, transplant, cancer, or acquired immune deficiency syndrome patients. Human cytomegalovirus infects virtually all cell types through the envelope glycoprotein complex gH/gL/gO with or without a contribution of the pentameric gH/gL/pUL128L. Together with gH/gL, the HCMV envelope glycoprotein B (gB) contributes to the viral fusion machinery. Methods: We previously showed that gB is a ligand for the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) contributing to HCMV attachment to and infection of DC-SIGN-expressing cells. However, the features of the DC-SIGN/gB interaction remain unclear. To address this point, the role of glycans on gB and the consequences of mutagenesis and antibody-mediated blockades on both partners were examined in this study. Results: We identified DC-SIGN amino acid residues involved in this interaction through an extensive mutagenesis study. We also showed the importance of high-mannose N-glycans decorating the asparagine residue at position 208, demonstrating that the antigenic domain 5 on gB is involved in the interaction with DC-SIGN. Finally, antibody-mediated blockades allowed us to identify DC-SIGN as a major HCMV attachment receptor on monocyte-derived dendritic cells. Conclusions: Taken together, these results have permitted us to fine-map the interaction between DC-SIGN and HCMV gB.

AF4p12, a human homologue to the furry gene of Drosophila, as a novel MLL fusion partner.

More than 35 different partner genes with the mixed lineage leukemia (MLL) gene have been cloned from leukemia cells with translocations involving chromosome 11 band q23. In this study, we report on a novel fusion partner of the MLL gene, AF4p12, which we have identified as the human homologue to the furry gene of Drosophila. AF4p12, highly conserved in evolution, encodes a large protein of 3,105 amino acids. The expression of AF4p12 has been preferentially detected in colon, placenta, and brain tissues and in tumor cells of lymphoid origin. We show that the t(4;11)(p12;q23) translocation results in the creation of a chimeric RNA encoding a putative fusion protein containing 1,362 amino acids from the NH2-terminal part of MLL and 712 amino acids from the COOH-terminal part of AF4p12. FLT3 and HOXA9 genes are overexpressed in this leukemia. We found that the COOH-terminal part of AF4p12 fused to MLL contains a leucine zipper motif and exhibits transcriptional activation properties when fused to Gal4 DNA-binding domains in transient transfection assays. The AF4p12 fragment fused to MLL may contribute to the oncogenic activation of MLL, possibly through specific recruitment of the transcriptional machinery.

Neutralizing and Targeting Properties of a New Set of α4ß7-Specific Antibodies Are Influenced by Their Isotype.

The homing of lymphocytes to the mucosa is mainly controlled by α4ß7 integrin, and it is amplified during gut chronic inflammation, as occurs with HIV and/or inflammatory bowel diseases. We designed and applied an improved immunization strategy based on an innovative selection process to isolate new α4ß7 lymphocyte-specific monoclonal antibodies that are able to prevent their migration into inflamed gut tissues and/or to counteract HIV infection in vitro. First, 5 monoclonal antibodies (1 IgA, 1 IgM, and 4 IgGs) were selected based on their capacity to recognize α4 or ß7 homodimers and α4ß7 heterodimers in transfected human cells. Their ability to block gp120/α4ß7 or MAdCAM-1/α4ß7 interactions was then measured in vitro with human T and B lymphocytes. In vitro, the anti-α4ß7 IgA isotype was found to have the highest affinity for the α4ß7 heterodimer, and it significantly reduced HIV replication in retinoic acid-treated α4ß7 CD4 human T cells. This α4ß7-specific IgA also displayed a high avidity for human and mouse α4ß7 lymphocytes in both mouse and human inflammatory colitis tissues. These new antibodies, and in particular those with mucosa-targeting isotypes such as IgA, could therefore be potential novel therapeutic tools for treating HIV and inflammatory bowel disease.

Characterization of a novel canine T-cell line established from a spontaneously occurring aggressive T-cell lymphoma with large granular cell morphology.

Dogs with lymphoma are established as good model for human non-Hodgkin lymphoma studies. Canine cell lines derived from lymphomas may be valuable tools for testing new therapeutic drugs. In this context, we established a canine T-cell line, PER-VAS, from a primary aggressive T-cell lymphoma with large granular morphology. Flow cytometric analysis revealed a stable immunophenotype: PER-VAS cells were positively labelled for CD5, CD45, MHC II and TLR3, and were negative for CD3, CD4 and CD8 expression. Although unstable along the culture process, IL-17 and MMP12 proteins were detectable as late as at passages 280 and 325i.e. respectively 24 and 29 months post isolation. At passage 325, PER-VAS cells maintained the expression of IL-17, CD3, CD56, IFNγ and TNFα mRNAs as shown by RT-PCR analysis. Stable rearrangement of the TCRγ gene has been evidenced by PCR. PER-VAS cells have a high proliferation index with a doubling time of 16.5h and were tumorigenic in Nude mice. Compared to the canine cell lines already reported, PER-VAS cells display an original expression pattern, close to NKT cells, which makes them valuable tools for in vitro comparative research on lymphomas.