Characterisation of chicken OX40 and OX40L.

The Tumour Necrosis Factor superfamilies of receptors and ligands play a crucial role in the regulation of effective immune responses against pathogens and malignant cells. In chickens, only few members have been identified. Here, we characterise the chicken homologues for mammalian costimulatory molecules OX40 and OX40L, which are involved in sustaining T cell responses. Both genes were identified by virtue of their genomic localisation close to highly conserved genes and their structural relationship to their mammalian homologues. Following cloning and expression of soluble and cell-associated chicken OX40 and OX40L, we confirmed their mutual interaction via ELISA and flow cytometric analyses. In addition, we showed the application of soluble OX40-Fc in staining of chicken cells. Whereas non-activated cells did not express OX40L, activation by IL-2 and IL-12 resulted in upregulation of OX40L on αß and γδ T cell populations. Our results demonstrate the existence of the costimulatory OX40-OX40L system in the chicken and provide the basis for further investigations of chicken T cell responses.

Identification of Chicken GITR and GITR Ligand, Proof of Their Mutual Interaction, and Analysis of Chicken GITR Tissue Distribution by a Novel Antibody That Reveals Expression on Activated T Cells and Erythrocytes.

Glucocorticoid-induced TNFR (GITR) and its ligand, GITRL, belong to the costimulatory members of the TNF superfamily and are crucially involved in the formation and modulation of an effective immune response, comprising innate as well as adaptive mechanisms. In this study, we identify and describe chicken GITR and GITRL, and provide an initial characterization of the newly developed chGITR-specific mAb 9C5. Structural analyses of the putative chicken molecules GITR and GITRL confirmed the conservation of classic topological features compared with their mammalian homologs and suggested the ability of mutual interaction, which was verified via flow cytometry. Whereas only minute populations of native lymphocytes isolated from spleen, bursa, and thymus expressed GITR, it was strongly upregulated upon activation on αß and γδ T cells, comprising CD4+ as well as CD8+ subsets. In blood, a fraction of CD4+CD25+ T cells constitutively expressed GITR. In addition, virtually all chicken erythrocytes displayed high levels of GITR. Our results verify the existence of both GITR and its ligand, GITRL, in chickens; they provide the basis and novel tools to further characterize their impact within the immune response and reveal the so-far unrecognized expression of GITR on erythrocytes.

Regulation of neurite morphogenesis by interaction between R7 regulator of G protein signaling complexes and G protein subunit Gα13.

The R7 regulator of G protein signaling family (R7-RGS) critically regulates nervous system development and function. Mice lacking all R7-RGS subtypes exhibit diverse neurological phenotypes, and humans bearing mutations in the retinal R7-RGS isoform RGS9-1 have vision deficits. Although each R7-RGS subtype forms heterotrimeric complexes with Gß5 and R7-RGS-binding protein (R7BP) that regulate G protein-coupled receptor signaling by accelerating deactivation of Gi/o α-subunits, several neurological phenotypes of R7-RGS knock-out mice are not readily explained by dysregulated Gi/o signaling. Accordingly, we used tandem affinity purification and LC-MS/MS to search for novel proteins that interact with R7-RGS heterotrimers in the mouse brain. Among several proteins detected, we focused on Gα13 because it had not been linked to R7-RGS complexes before. Split-luciferase complementation assays indicated that Gα13 in its active or inactive state interacts with R7-RGS heterotrimers containing any R7-RGS isoform. LARG (leukemia-associated Rho guanine nucleotide exchange factor (GEF)), PDZ-RhoGEF, and p115RhoGEF augmented interaction between activated Gα13 and R7-RGS heterotrimers, indicating that these effector RhoGEFs can engage Gα13·R7-RGS complexes. Because Gα13/R7-RGS interaction required R7BP, we analyzed phenotypes of neuronal cell lines expressing RGS7 and Gß5 with or without R7BP. We found that neurite retraction evoked by Gα12/13-dependent lysophosphatidic acid receptors was augmented in R7BP-expressing cells. R7BP expression blunted neurite formation evoked by serum starvation by signaling mechanisms involving Gα12/13 but not Gαi/o These findings provide the first evidence that R7-RGS heterotrimers interact with Gα13 to augment signaling pathways that regulate neurite morphogenesis. This mechanism expands the diversity of functions whereby R7-RGS complexes regulate critical aspects of nervous system development and function.