Abstract

Among the costimulatory members of the tumour necrosis factor superfamily, the OX40 molecule and its ligand OX40L play a decisive role in the formation and the regulation of an efficient immune response upon antigen encounter. In mammals, their interaction allows for a sustained T cell response by providing anti-apoptotic signals as well as by promoting cytokine production, cell proliferation and differentiation. In chickens, however, the information on costimulatory signals accompanying TCR stimulation remains scarce. This study aimed at the characterisation of chicken OX40 and OX40L to gain more information about the complex process of T cell activation in chickens. We identifed the genes of chicken OX40 and OX40L in the chicken genome and confirmed their correct annotation by virtue of shared synteny with respective flanking genes in the genomes of human and mouse. The obtained sequences were further analysed for their phylogenetic and structural relationship to other members of the TNF family in birds and mammals. The analyses revealed a tight topological relation of chicken OX40 and OX40L to their respective homologues in other species. The conservation of a functionally relevant motif among species suggests that the OX40-OX40L system preserved its functions during evolution. After the cloning and the recombinant expression of soluble and cell-associated variants, we verified the mutual interaction of chicken OX40 and OX40L via ELISA and flow cytometric analyses and demonstrated that both soluble recombinant proteins tend to dimerise. As only few antibodies are available to characterise chicken cell types, we proved the application of recombinant OX40-Fc in staining of chicken cells. While OX40L could not be detected on non-activated cells, OX40L was upregulated on αβ and γδ T cells upon activation by IL-2 and IL-12. These findings confirm the existence of the OX40-OX40L system in chickens and suggest their involvement in the activation process of chicken T cells. Eventually, soluble and cell-bound variants of OX40 and OX40L, generated within this study, may serve as new tools to further characterise the involvement of the OX40-OX40L system in chicken T cell response.