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 αβ T cells, while a γδ T cell subset and virtually all B cells lacked 8B1 reactivity. The fraction of 8B1^- αβ 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.