Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma.

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when stimulated by T lymphocytes, oncogenic B cells survive and expand autonomously in undefined organ niches. Mantle cell lymphoma (MCL) is one such B cell disorder, where the median survival rate of patients is 4 - 5 years. This calls for the need of effective mechanisms by which the homing and engraftment of these cells are blocked in order to increase the survival and longevity of patients. Therefore, the effort to develop a xenograft mouse model to study the efficacy of MCL therapeutics by blocking the homing mechanism in vivo is of utmost importance. Development of animal recipients for human cell xenotransplantation to test early stage drugs have long been pursued, as relevant preclinical mouse models are crucial to screen new therapeutic agents. This animal model is developed to avoid human graft rejection and to establish a model for human diseases, and it may be an extremely useful tool to study disease progression of different lymphoma types and to perform preclinical testing of candidate drugs for hematologic malignancies, like MCL. We established a xenograft mouse model that will serve as an excellent resource to study and develop novel therapeutic approaches for MCL.

Anti-JAM-C therapy eliminates tumor engraftment in a xenograft model of mantle cell lymphoma.

Junctional adhesion molecule (JAM)-C is a member of the JAM family, expressed by a variety of different cell types, including human B lymphocytes and some B-cell lymphoma subtypes-in particular, mantle cell lymphoma (MCL). Treatment with anti-JAM-C pAbs reduces homing of human B cells to lymphoid organs in a NOD/SCID mouse model. In the present study, the role of JAM-C in the engraftment of human lymphoma B cells in mice was investigated. Administration of novel anti-JAM-C mAbs reduced tumor growth of JAM-C+ MCL cells in bone marrow, spleen, liver, and lymph nodes of mice. Treatment with anti-JAM-C antibodies significantly reduced the proliferation of JAM-C-expressing lymphoma B cells. Moreover, the binding of anti-JAM-C antibodies inhibited the phosphorylation of ERK1/2, without affecting other signaling pathways. The results identify for the first time the intracellular MAPK cascade as the JAM-C-driven signaling pathway in JAM-C+ B cells. Targeting JAM-C could constitute a new therapeutic strategy reducing lymphoma B-cell proliferation and their capacity to reach supportive lymphoid microenvironments.

Homing of human B cells to lymphoid organs and B-cell lymphoma engraftment are controlled by cell adhesion molecule JAM-C.

Junctional adhesion molecule C (JAM-C) is expressed by vascular endothelium and human but not mouse B lymphocytes. The level of JAM-C expression defines B-cell differentiation stages and allows the classification of marginal zone-derived (JAM-C-positive) and germinal center-derived (JAM-C-negative) B-cell lymphomas. In the present study, we investigated the role of JAM-C in homing of human B cells, using a xenogeneic nonobese diabetic/severe combined immunodeficient mouse model. Treatment with anti-JAM-C antibodies in short-term experiments reduced migration of normal and malignant JAM-C-expressing B cells to bone marrow, lymph nodes, and spleen. Blocking homing to the spleen is remarkable, as most other antiadhesion antibodies reduce homing of B cells only to bone marrow and lymph nodes. Long-term administration of anti-JAM-C antibodies prevented engraftment of JAM-Cpos lymphoma cells in bone marrow, spleen, and lymph nodes of mice. Plasmon resonance studies identified JAM-B as the major ligand for JAM-C, whereas homotypic JAM-C interactions remained at background levels. Accordingly, anti-JAM-C antibodies blocked adhesion of JAM-C-expressing B cells to their ligand JAM-B, and immunofluorescence analysis showed the expression of JAM-B on murine and human lymphatic endothelial cells. Targeting JAM-C could thus constitute a new therapeutic strategy to prevent lymphoma cells from reaching supportive microenvironments not only in the bone marrow and lymph nodes but also in the spleen.

RNA-Seq reveals an integrated immune response in nucleated erythrocytes.

BACKGROUND: Throughout the primary literature and within textbooks, the erythrocyte has been tacitly accepted to have maintained a unique physiological role; namely gas transport and exchange. In non-mammalian vertebrates, nucleated erythrocytes are present in circulation throughout the life cycle and a fragmented series of observations in mammals support a potential role in non-respiratory biological processes. We hypothesised that nucleated erythrocytes could actively participate via ligand-induced transcriptional re-programming in the immune response. METHODOLOGY/PRINCIPAL FINDINGS: Nucleated erythrocytes from both fish and birds express and regulate specific pattern recognition receptor (PRR) mRNAs and, thus, are capable of specific pathogen associated molecular pattern (PAMP) detection that is central to the innate immune response. In vitro challenge with diverse PAMPs led to de novo specific mRNA synthesis of both receptors and response factors including interferon-alpha (IFNα) that exhibit a stimulus-specific polysomal shift supporting active translation. RNA-Seq analysis of the PAMP (Poly (I:C), polyinosinic:polycytidylic acid)-erythrocyte response uncovered diverse cohorts of differentially expressed mRNA transcripts related to multiple physiological systems including the endocrine, reproductive and immune. Moreover, erythrocyte-derived conditioned mediums induced a type-1 interferon response in macrophages thus supporting an integrative role for the erythrocytes in the immune response. CONCLUSIONS/SIGNIFICANCE: We demonstrate that nucleated erythrocytes in non-mammalian vertebrates spanning significant phylogenetic distance participate in the immune response. RNA-Seq studies highlight a mRNA repertoire that suggests a previously unrecognized integrative role for the erythrocytes in other physiological systems.

The effects of immunostimulation through dietary manipulation in the rainbow trout; evaluation of mucosal immunity.

Immunostimulant-containing diets are commonly used in aquaculture to enhance the resistance of cultured fish to disease and stress. Although widespread in use, there have been conflicting results published, and surprisingly little is known about the regulation of immune response-related genes in tissues key to mucosal immunity induced by immunostimulant dietary feeding. Using a salmonid-specific microarray platform enriched with immune-related genes and in situ hybridization, we investigated dietary acclimation in two organs relevant to mucosal immunity, the gills and the intestine, in the rainbow trout (Oncorhynchus mykiss). Immunostimulant diets significantly changed gene expression profiles and gene distribution in a tissue-specific manner: genes and functional Gene Ontology categories involved in immunity were differently expressed at portals of entry where significant changes in genes and functional groups related to remodeling processes and antigen presentation were observed. Furthermore, genes involved in chemotaxis, cell differentiation, antigen-presenting capacity and tissue remodeling were localized in both organs.

Characterization and expression of NADPH oxidase in LPS-, poly(I:C)- and zymosan-stimulated trout (Oncorhynchus mykiss W.) macrophages.

In vertebrates, the generation of superoxide reactive oxygen species (ROS) via activation of the Nox/Duox family of NADPH oxidases is a prototypical feature of the pathogen-induced defensive responses of activated professional phagocytes. To understand the role of the rainbow trout (Oncorhynchus mykiss) Phox oxidase from a phylogenetic and functional perspective we describe the cloning, sequencing and expression analysis of multiple NADPH components in cultured macrophages. Phylogenetic analyses support the notion of the emergence of Phox-related components before the diversification of basal euteleosts and add to the limited collection of teleost NADPH oxidases. Expression studies using lipopolysaccharide, polyinosine-polycytidylic acid and zymosan to mimic the onset of inflammatory responses in trout macrophages suggest differences in regulation of the NADPH complex throughout the maturation/differentiation period of culture and between different treatments.

Characterization and expression of the transcription factor PU.1 during LPS-induced inflammation in the rainbow trout (Oncorhynchus mykiss).

The transcription factor PU.1 plays a key role in hematopoietic lineage development and therefore in determining immune cell fate. A full length cDNA transcript of 1237 nucleotides encoding a highly conserved putative protein of 293 amino acids was identified by EST analysis in lipopolysaccharide (LPS) activated trout macrophages. Phylogenetic analyses highlight the significant level of structural conservation of the PU.1 transcription factor reinforcing the importance of this molecule in animal immunity. In trout, the PU.1 mRNA shows a tissue-specific expression pattern and is induced in vivo by LPS in muscle, liver, intestine and brain. Furthermore PU.1 is highly expressed in trout macrophages in primary culture. In situ expression analysis in the head kidney describes a large number of PU.1+ve cells distributed through the tissue in both LPS-treated and control animals. Cellular proliferation examined by BrdU immunohistochemistry (IHC) shows that LPS regulates hematopoietic processes in adult fish by stimulating cellular proliferation 3 days after treatment. These studies provide initial insights into hematopoietic/cellular processes in the head kidney of rainbow trout after in vivo LPS challenge.