Chicken CD56 defines NK cell subsets in embryonic spleen and lung

NK cells have been widely investigated in various mammalian species, but their characterization in non-mammalian vertebrates has been hampered by the lack of appropriate markers. Here, we tested the suitability of a novel anti-chicken CD56 mab as a marker on NK cells. The entire CD56 extracellular domain was cloned into a vector providing a FLAG epitope as well as the CD8 transmembrane and CD3ζ cytoplasmic domain. This construct was used to stably transfect the chicken 2D8 B cell line as monitored by anti-FLAG staining. BALB/c mice were repeatedly immunized with this cell line before generation of hybridomas and screening of supernatants on the transfected cell line versus untransfected cells. The 4B5 hybridoma was further selected due to its reactivity with transfected cells only. Staining of various cell preparations isolated from blood, spleen, embryonic spleen, lung and intestine revealed a CD56 positive subpopulation in lung and embryonic spleen, whereas no reactivity could be observed with other cells. In two colour immunofluorescence, some of the CD56(+) lung cells coexpressed CD8, while they were largely negative for the markers 28-4 and 20E5. CD8 expression was also observed for a subset of CD56(+) embryonic splenocytes and 28-4 was found to be coexpressed on about half of the CD56(+) embryonic splenocytes. CD56 is therefore expressed on a subset of chicken NK cells predominantly found in embryonic spleen and lung.