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.

Splenic γδ T cell subsets can be separated by a novel mab specific for two CD45 isoforms.

CD45 isoforms have been identified in a variety of different species and mab against various isoforms have been instrumental to define cellular subsets. In the process of generating novel mab against chicken γδ T cells two mab with specificity for CD45 were identified and characterized. The analysis of the chicken CD45 genomic structure suggested three exons being involved in alternative splicing. We cloned and expressed the full length CD45 isoform and three shorter isoforms. While the 7D12 mab reacted with all of these isoforms, the 8B1 mab selectively reacted with two short isoforms lacking either exons 3 and 5 or exons 3, 5 and 6. As expected, the reactivity of 7D12 included all leukocyte subsets, also including thrombocytes. In contrast, the 8B1 mab only reacted with lymphocytes and monocytes. 8B1 expression was found on almost all blood αß T cells, while a γδ T cell subset and virtually all B cells lacked 8B1 reactivity. The fraction of 8B1- αß and γδ cells was larger in splenocytes as compared to PBL and there was also a population of 8B1+ splenic B cells. CD3 stimulation of splenic T cells resulted in upregulation of the 8B1 antigen on all T cells. Three-color immunofluorescence revealed differences in CD28 expression between the 8B1⁺ and 8B1¯ γδ T cell subsets with a higher CD28 expression level on 8B1¯ cells. The CD28 antigen was upregulated upon stimulation of the cells with IL-2 and IL-12. This novel mab will be a useful tool to further analyze chicken γδ T cells in more detail.